Enhanced fat oxidizing mechanisms -
Their oxidation generates the highest energy yield for ATP or heat production of all common energy substrates. They are indispensable components of membrane lipids, and FA anchors enable peripheral membrane proteins to be tethered to biological membranes.
Additionally, specific FAs act as highly bioactive signalling molecules or serve as their precursors. Lipotoxicity results from the ability of FAs to act as detergents, to affect acid—base homeostasis and to generate highly bioactive lipids, such as ceramides and diradylglycerols diglycerides, DGs.
Together, these processes cause cellular stress and dysfunction, leading to various forms of cell death 1. Essentially every cell type can store TGs to some degree in intracellular organelles termed lipid droplets LDs 2. In mammals and many other vertebrates, the majority of TGs is deposited in adipocytes of adipose tissue.
While TGs represent an efficient, inert form of FAs for storage and transport, they are unable to traverse cell membranes. Accordingly, TG transport into or out of cells either requires their hydrolytic breakdown into FAs and glycerol or their specialized-vesicle-mediated transport across cell membranes.
The latter process comprises secretion and uptake of TG-rich lipoproteins or TG transport by extracellular vesicles EVs. The hydrolysis of TGs is catalysed by lipases in a process called lipolysis. Extracellular lipolysis in the gastrointestinal tract and the vascular system degrades TGs to provide FAs and monoradylglycerols monoglycerides, MGs to the underlying parenchymal tissues.
Conversely, intracellular lipolysis releases FAs from LD-associated TGs in the cytoplasm or lipoprotein-associated TGs in lysosomes. Extracellular lipases of the digestive tract and the vascular system mostly belong to the pancreatic lipase gene family.
Accordingly, the two intracellular canonical pathways responsible for TG degradation are termed neutral and acid lipolysis Fig. In accordance with the fundamental function of lipases, their dysfunction or deficiency often causes severe pathologies including dyslipidaemias and lipid-storage diseases.
More indirectly, lipase activities and lipolytic processes affect many aspects of homeostasis and have been associated with the pathogenesis of obesity, type 2 diabetes, nonalcoholic fatty liver disease NAFLD , cancer, heart disease, cachexia, infectious diseases and others. Consecutive hydrolysis of TGs by ATGL, HSL and MGL or ABHD6, generating FAs and glycerol G.
Transesterification of DGs or MGs with DG, generating TGs, is catalysed by ATGL. In acid lipolysis, TGs are sequestered from LDs via lipophagy and are subsequently hydrolysed by LAL in lysosomes Lys , generating FAs and glycerol. Recognition that enzyme-catalysed TG hydrolysis must precede membrane passage was achieved early in the 20th century 3 , yet it took more than 50 years to identify and characterize the first intracellular TG hydrolase that participates in the process.
In , Steinberg and colleagues described hormone-sensitive lipase HSL as the key hydrolase in the degradation of TGs and DGs and the role of monoglyceride lipase MGL in MG hydrolysis in adipocytes 4. HSL was considered rate-limiting for TG hydrolysis for the following four decades, but observations that HSL-deficient mice maintained hormone-induced FA release in adipose tissue, lacked obesity and accumulated DGs 5 , 6 finally argued for the involvement of alternative enzymes and mechanisms in TG hydrolysis.
The biochemical characteristics and enzymatic activities of ATGL, HSL and MGL are summarized in Table 1 and Fig. Human adipose triglyceride lipase ATGL is a amino-acid protein.
It harbours a patatin domain named after a structural unit in patatin, which is a relatively weak phospholipase present in potato tubers 7. The human genome carries nine genes encoding patatin-domain-containing proteins, which are designated patatin-like phospholipase domain 1—9 PNPLA1 — PNPLA9 PNPLA2 encodes ATGL.
It contains a catalytic dyad consisting of S47 and D Interestingly, the carboxy-terminal half of the enzyme is dispensable for enzyme activity on lipid emulsions but essential for LD binding and TG hydrolysis in cells The molecular basis for the requirement of this C-terminal region for enzyme binding to LDs is poorly understood.
It may involve a hydrophobic stretch amino acids to and two potential phosphorylation sites S, S However, the protein kinases thought to be involved and the impact of ATGL phosphorylation on its LD localization and activity remain controversial 12 , 13 and require further study.
Long-chain FA-containing TGs represent by far the best substrates for ATGL, while FA esters in DGs, glycerophospholipids or retinylesters are only poorly hydrolysed. Interestingly, however, the enzyme exhibits transacylase activity leading to the formation of TG and MG 8 , 14 , 15 from two DG molecules in a disproportionation reaction.
Whether this anabolic function of ATGL is physiologically relevant remains unanswered, as is the question of whether ATGL acts as a transacylase with alternative FA-donor or FA-acceptor substrates.
ATGL shares transacylation activity with other members of the PNPLA family, including PNPLA1 and PNPLA3 refs. Overall, despite a high potential for physiological relevance, this coenzyme-A-independent esterification reaction and its role in lipid remodelling remain elusive.
Specific transport mechanisms guide ATGL from the endoplasmic reticulum ER membrane to LDs. The structural features within ATGL that determine its effective trafficking are still insufficiently understood.
More is known about the role of factors involved in ATGL transport. Deletion of key transport vesicle components or effectors, including ADP ribosylation factor-1 ARF-1 , small GTP-binding protein-1 SAR-1 or Golgi-Brefeldin A resistance factor GBF-1 , as well as deficiency of the coat protein complex-I COP-I , prevent ATGL translocation and lead to defective TG hydrolysis and LD accumulation 17 , The list of factors that affect ATGL translocation was recently extended by oxysterol-binding protein like-2 OSBP-2 and family with sequence similarity , member B FAMB.
OSBP-2 forms a complex with the COP-I subunit COP-B1 and binds ATGL, directing the enzyme to LDs FAMB is a Golgi-associated protein that regulates the vesicular transport of ATGL and other proteins to LDs via Arf-related protein-1 ARFRP1 Loss-of-function mutations in the PNPLA2 gene cause neutral lipid storage disease with myopathy NLSDM in humans Currently, approximately people with the condition with more than 30 different mutations have been identified.
Onset and severity of disease are highly variable, arguing for gene—gene and gene—environment interactions that contribute to the clinical presentation of the disease Progressive skeletal myopathy is common in people with NLSDM and leads to pronounced motor impairment.
More than half of all affected individuals have severe dilated cardiomyopathy, and many require heart transplantation for survival ATGL deficiency in mice phenocopies many of the clinical findings in people with NLSDM, but the murine condition is more serious, leading to cardiomyopathy and death without exception when the animals are 3 to 4 months old ATGL deficiency in the heart leads not only to a severe TG accumulation in cardiomyocytes, but also to a striking defect in the activation of the transcription factor peroxisome-proliferator-activated receptor-α PPARα , reduced mitochondrial biogenesis and function, and defective respiration, which eventually causes lethality Restoring ATGL expression in the heart of ATGL-deficient mice rescues the cardiac phenotype, prevents premature lethality 25 and may therefore provide a potential treatment strategy for people with NLSDM.
These initial studies in ATGL-knockout ATGL-KO mice highlighted the crucial role of the enzyme for lipolysis not only in adipose tissue, but also in major energy-consuming organs, such as the heart. They also demonstrated that, in addition to its important role for the provision of energy substrates, ATGL-mediated lipolysis also participates in the regulation of major signalling pathways that regulate energy homeostasis.
These pathways include insulin signalling 26 , 27 and nuclear receptor signalling via PPARα refs. The lipolysis-dependent regulation of PPAR activation involves transcriptional 33 and post-transcriptional mechanisms In fact, the closest structural relatives of HSL are found in prokaryotes, archaea and plants.
Tissue-specific protein isoforms, ranging from amino acids in most tissues to 1, amino acids in testis , result from alternative transcription start sites and exon usage.
HSL exhibits broad substrate specificity with highest activity against DGs and cholesteryl esters CEs , followed by TGs, MGs, retinyl esters and short-chain acyl esters. The enzyme harbours five serine phosphorylation sites, which are targeted by multiple protein kinases and have important regulatory functions affecting enzyme activity see section on regulation of lipolysis.
Important insights into the physiological role of the enzyme in humans were revealed by clinical characterization of homozygous individuals with HSL deficiency and heterozygous carriers of loss-of-function LIPE gene mutations 36 , HSL deficiency manifests in a more benign phenotype than does ATGL deficiency.
It is characterized by relatively mild forms of dyslipidaemia, hepatic steatosis, partial lipodystrophy and type 2 diabetes. HSL-deficient adipose tissue consists of small, insulin-resistant adipocytes, which exhibit increased inflammation and impaired glycerol release upon lipolytic stimulation Partial lipodystrophy results from a downregulation of PPAR-γ and its downstream target genes when HSL is lacking, leading to reduced adipogenesis and insulin sensitivity.
Notably, HSL also regulates the transcriptional activity of another nuclear receptor, carbohydrate responsive element binding protein CHREBP Independent of its hydrolytic activity, HSL binds to CHREBP and thereby prevents its translocation into the nucleus, reduces target gene expression and leads to insulin resistance.
Conversely, blocking HSL binding to CHREBP increases target gene expression and insulin sensitivity However, the fact that people with reduced or no HSL expression are generally more insulin resistant than are individuals with normal HSL expression 36 , 37 suggests that the HSL-mediated regulation of insulin sensitivity via CHREBP is not a predominant regulatory mechanism of insulin sensitivity in humans.
Mice deficient in HSL phenocopy ATGL-deficient mice with PPARγ-dependent lipodystrophy in adipose tissue 5 , 30 , 39 , 40 , suggesting that the regulation of these nuclear receptors by lipolysis is not enzyme-specific.
HSL has a prominent role in DG hydrolysis within the lipolytic cascade as mice lacking HSL accumulate DGs in various tissues 6. Finally, in contrast to humans, HSL-deficient mice are sterile owing to defective spermatogenesis, sperm motility and fertility 5 , The molecular basis for this species-specific difference of enzyme function in spermatogenesis remains to be discovered.
Monoacylglycerol lipase MGL was the first characterized MG hydrolase Table 1 42 , The enzyme hydrolyses both sn -1 MG and sn -2 MG, in which FAs are esterified to the terminal or middle hydroxyl group of the glycerol backbone, respectively, to generate glycerol and FAs.
Although the preferred substrate for MGL are MGs, the enzyme was also shown to hydrolyse prostaglandin-glycerol esters and FA-ethyl ethers 44 , 45 , MGL expression is subject to nutritional 47 and PPARα-dependent regulation 48 , and MGL protein is targeted for proteasomal degradation involving Staphylococcal nuclease and tudor domain-containing 1 SND1 To date, no post-translational modifications of the enzyme are known to affect enzyme activity.
In addition to MGL, several other enzymes are reported to hydrolyse MGs. ABHD6 comprises amino acids with an active site composed of S, D and H It is localized to the inner leaflet of the plasma membrane and preferentially hydrolyses FAs at the sn -1 position over the sn -2 position of MGs.
ABHD6 also hydrolyses other substrates, including lysophospholipids 50 and bis monoacylglycerol -phosphate 51 , The highest expression levels of ABHD6 are observed in liver, kidney, intestine, ovary, brown adipose tissue and pancreatic beta cells.
In some tissues for example pancreas and cancer cell lines that have low MGL expression, ABHD6 becomes the main MG hydrolase 53 and has been associated with the development of insulin resistance 50 and cancer progression 54 , Since the catabolic pathways of TGs and glycerophospholipids PLs converge in the formation of MGs, it is difficult to assign specific phenotypes resulting from enzyme overexpression or deficiency to changes in TG-derived versus PL-derived MGs.
MGL deficiency in mice causes MG accumulation in various tissues, minor changes in plasma very-low-density lipoprotein VLDL metabolism, impaired intestinal lipid absorption and a moderate protection from diet-induced obesity and hepatic steatosis 56 , 57 , Most severe accumulation of MGs is observed in brain with high levels of 2-arachidonoylglycerol 2-AG 59 , In mice, MGL deficiency ameliorates neuroinflammation and cancer malignancy owing to diminished 2-AG hydrolysis and reduced arachidonic acid availability 61 , 62 , 63 , Pharmacological inhibition or genetic deletion of ABHD6 also increases sn -1 and sn -2 MG concentrations in different tissues, thereby altering multiple aspects of metabolism and energy homeostasis 53 , ABHD6 inhibition induces insulin secretion, adipose tissue browning and brown adipose tissue activation, and exerts neuroprotective and anti-inflammatory effects.
The different phenotypes arising from MGL or ABHD6 inhibition remain poorly understood and may be explained by their different cellular localization and expression patterns. The subsequent release of FAs from white adipose tissue induces a metabolic switch in many energy-consuming tissues from glucose to FA utilization.
Simultaneously, the upregulation of hepatic gluconeogenesis and glucose secretion provides glucose to glucose-dependent cells and tissues. Numerous endocrine, paracrine and autocrine factors, including hormones, cytokines and neurotransmitters, trigger the switch between basal and induced lipolysis by regulating the major lipolytic enzymes ATGL and HSL on multiple levels, including gene transcription, post-translational protein modifications and, in the case of ATGL, regulation by enzyme coactivators and inhibitors.
The classical activation pathway in adipocytes involves catecholamines, which activate both enzymes ATGL and HSL via hormonal epinephrine or sympathetic-neuronal norepinephrine stimulation. Other activating hormones and cytokines include glucocorticoids, thyroid hormone, eicosanoids, atrial natriuretic peptides, growth hormone, interleukins, tumour necrosis factor α, leptin and many others Insulin represents the classical inhibitory hormone for ATGL and HSL.
Regulation of lipase gene transcription is a major mechanism controlling lipolysis summarized in Fig. Unfortunately, the transcriptional control elements in the promoters of the genes coding for ATGL Pnpla2 and HSL Lipe have not been sufficiently characterized. Evidence suggests that both genes are direct targets for the PPAR family of nuclear receptor transcription factors 67 , 68 , Other members of the nuclear receptor family, RXR, LXR-α and steroidogenic factor-1, are specific for Lipe and do not regulate Pnpla2 transcription 70 , The Lipe promoter also contains a sterol regulatory element rendering Lipe gene expression subject to sterol regulatory element binding proteins SREBPs Several transcription factors, including the PPAR family of nuclear receptor transcription factors, SP1, TFE3 and CEBPα, regulate the transcription of Pnpla2 and Lipe.
Other members of the nuclear receptor family, RXR, LXR-α and SF1, as well as SREBPs are specific for Lipe , but do not regulate Pnpla2 transcription.
Pnpla2 expression is activated by FOXO1, STAT5, EGR1, and inhibited by SNAIL1. STAT5 is phosphorylated and activated by JAK2, and is dependent on GHR activation.
MAP also activates ERK-3 to increase FOXO1-mediated ATGL transcription. On the contrary, IR activation inhibits Pnpla2 transcription by the PKB- and CDK1-catalysed phosphorylation of FOXO1, which leads to protein interaction and cytoplasmic retention of the transcription factor.
Neutral lipolysis is further regulated by mTORC1 and mTORC2, enabling cells to react to nutrient availability. mTORC1 regulates Pnpla2 transcription by inhibiting EGR1, while mTORC2 inhibits lipolysis by activating PKB-mediated FOXO1 phosphorylation.
Rapamycin-insensitive companion of mTOR Rictor inhibits SIRT-mediated deacetylation of FOXO1 and Pnpla2 expression through a yet unknown mechanism. While catecholamines exert their prolipolytic effects predominantly on the level of enzyme activities, other lipolysis-activating hormones and cytokines act primarily via transcriptional control of lipase expression.
For example, growth hormone GH induces Pnpla2 transcription via two distinct mechanisms. The predominant pathway involves GH binding to its receptor and subsequent activation of the transcription factor signal transducer and activator of transcription-5 STAT5 through Janus kinase-2 JAK2 -catalysed phosphorylation.
Phospho-Stat-5 directly activates Pnpla2 transcription in white and brown adipocytes 75 , Consistent with this finding, JAK2 or STAT5 deletion impairs lipase expression 75 , This combination of effects results in a robust induction of lipolysis.
The MAP kinase pathway was recently also shown to activate ERK-3 in response to β-adrenergic stimulation. This atypical member of the MAP kinase family increases forkhead box protein O-1 FOXO1 -mediated ATGL transcription and stimulates lipolysis GDF-3 and activin B bind to activin-receptor-1c also called activin receptor like kinase 7, Alk7 leading to the phosphorylation of SMAD transcription factors, which in turn, inhibit CEBPα and PPARγ expression and thereby decrease lipase gene transcription Concomitantly, the Alk7-dependent pathway also downregulates β3-adrenergic receptor expression leading to a reduction of catecholamine-induced lipolysis In contrast to GDF-3 and activin B, myostatin inhibits lipolysis in adipose tissue via binding to activin type-II receptors.
Attenuation of lipolysis by myostatin appears to be due to a general impairment of anabolic pathways in lipid metabolism, decreased adipocyte differentiation as well as proliferation 85 , The molecular mechanisms of other classical prolipolytic inflammatory cytokines, such as tumour necrosis factor-α TNFα , interleukin-1β IL-1β or interleukin-6 IL-6 , remain poorly characterized and mostly affect lipolysis by indirect mechanisms Transcription factor FOXO1 is the key factor in insulin-mediated downregulation of Pnpla2 gene expression FOXO1 binds directly to the promoter of Pnpla2 to induce its expression The transactivating function of FOXO1 is predominantly controlled by its modification status: phosphorylation determines its intracellular localization and acetylation regulates its activity.
Insulin inhibits Pnpla2 transcription by the protein kinase B PKB -catalysed phosphorylation of FOXO1, which leads to protein interaction and cytoplasmic retention of the transcription factor In hepatocytes, CDK1-dependent phosphorylation of FOXO1 similarly suppresses ATGL expression by cytoplasmic retention of FOXO1.
Consistent with this finding, hepatocyte-specific CDK1 deletion induces ATGL-mediated hepatic fat degradation In contrast, interaction of proteins with FOXO1 is inhibited by the AMPK-dependent phosphorylation of Ser22 of FOXO1, which enables translocation of this protein to the nucleus A reciprocal feedback is plausible given the fact that SIRT1 regulates ATGL via FOXO1, and ATGL also activates SIRT1, thereby affecting the transcription of genes involved in autophagy 32 , In addition to FOXO1, insulin inhibits lipolysis via the Zn-finger transcription factor SNAIL1.
In adipocytes, insulin increases SNAIL1 expression, which binds to E2-box sequences in the Pnpla2 promoter and represses its transcription mTOR is embedded in two larger protein complexes named mTORC1 and mTORC2.
mTORC1 activity is predominantly regulated by essential amino acids and induces cell growth and proliferation by the induction of protein, lipid and nucleic acid biosynthesis. mTORC1 inhibition by rapamycin or deletion of the regulatory-associated protein of mTOR Raptor induces lipolysis in an ATGL-dependent manner in cells and mice , , The underlying mechanisms include an upregulation of ATGL transcription by the transcription factor early growth response protein-1 EGR1 as well as post-transcriptional mechanisms mTORC2 inhibits lipolysis in adipose tissue and amplifies the antilipolytic function of insulin by phosphorylating AKT , Moreover, and independently of the canonical mTORC2—AKT axis, rapamycin-insensitive companion of mTOR Rictor deletion leads to an induction of lipolysis via SIRT6-mediated deacetylation of FOXO1, and induction of ATGL expression Further studies are needed to clarify how mTORC2 affects SIRT6 activity.
The canonical activation pathway of lipolysis in adipocytes involves the binding of catecholamines to β-adrenergic G-protein-coupled receptors. Upon hormone binding, the α-subunit of the receptor-coupled trimeric G s protein dissociates and stimulates adenylate cyclase, resulting in cAMP synthesis Very recently, a new G-protein-coupled receptor was identified that constitutively activates adenylate cyclase in brown adipose tissue independently of ligand binding cAMP activates protein kinase A PKA , which phosphorylates HSL at residues S, S and S and perilipin-1, a key player in the regulation of lipolysis at residues S81, S, S, S, S and S PKA is not the only protein kinase phosphorylating HSL and perilipin Atrial natriuretic peptides via guanyl-cyclase-derived cGMP activate protein kinase G, which phosphorylates both proteins at the same sites as PKA Perilipin-1 phosphorylation is required for HSL translocation, but also triggers its dissociation from CGI, thereby enabling CGI to interact with ATGL and to activate enzyme activity Fig.
The central role of perilipin-1 in the regulation of ATGL in humans became evident when Gandotra et al. This inability of perilipin-1 to buffer CGI during basal lipolysis results in the constitutive hyperactivation of ATGL, unrestrained lipolysis, hyperlipidemia and fatty liver disease , Lipolysis is stimulated by activation of β-adrenergic G-protein-coupled receptors β-AR.
Binding of noradrenalin NA , adrenocorticotropic hormone ACTH or secretin leads to dissociation of the receptor-coupled trimeric G s protein and stimulation of adenylate cyclase AC , resulting in cAMP synthesis.
cAMP activates protein kinase A PKA , which phosphorylates HSL and PLIN1. PLIN1 sequesters CGI, which is released upon PKA- or PKG-mediated phosphorylation to stimulate ATGL activity. PKG is activated by atrial natriuretic peptides ANPs via GC-derived cGMP.
Enzymatic activity of ATGL is enhanced by CGI and inhibited by G0S2 and HILPDA. FABP4 interacts with CGI to further stimulate ATGL activity. Lipolysis is inhibited by insulin or insulin-like growth factor-1 IGF-1 via insulin receptor or IGF receptor.
Ligand binding subsequently activates insulin receptor substrate-1, phosphatidylinositolkinase, PDK1 and PKB, which activates PDE3B, resulting in the hydrolysis of cAMP.
Lipolysis is also inhibited by adenosine, which decreases cAMP levels by activation of α-adrenergic receptors AA 1 R coupled with G i proteins, inhibiting adenylate cyclase and cAMP synthesis.
In cell types with high oxidation rates, such as hepatocytes or cardiomyocytes, perilipin-5 coordinates the interaction of ATGL with CGI Perilipin-5, unlike perilipin-1, is able to bind both ATGL and CGI, but the binding is mutually exclusive , According to current understanding, two perilipin-5 molecules bound to ATGL and CGI, respectively, need to oligomerize to enable ATGL activation Perilipin-5 interaction with ATGL depends on the PKA-mediated phosphorylation of S in perilipin-5 refs.
Interestingly, hormone-stimulated ATGL binding to perilipin-5 also promotes the translocation of FAs to the nucleus, where they act as allosteric activators of SIRT1 ref. Overall, however, the impact of perilipin-5 on lipolysis appears more modulatory than necessary, since periplipindeficient mice exhibit elevated lipolysis , while overexpression of perilipin-5 leads to reduced lipolysis and a steatotic phenotype in transgenic mice In contrast to perilipin-1 and perilipin-5, the impact of perilipin-2, perilipin-3 and perilipin-4 on regulation of hormone-stimulated lipolysis is not well understood and may be less prominent , Insulin and IGFs represent the predominant inhibitory hormones of lipolysis.
They diminish both activity and gene expression of lipolytic enzymes The dominant pathway to inhibit enzyme activities involves the deactivation of the cAMP—PKA—pathway. Interestingly, this process requires PDE3B interaction with ABHD15 ref. Another mechanism inhibiting lipolysis involves the activation of α-adrenergic receptors coupled with G i -proteins by adenosine, which inhibits adenylate cyclase and cAMP synthesis Fig.
High cellular AMP concentrations during fasting or prolonged exercise induce ATGL gene transcription via AMPK dependent phosphorylation of FOXO1 see section on transcriptional control.
Whether AMPK also activates lipolysis by directly phosphorylating ATGL and HSL remains controversial. Both enzymes are AMPK phosphorylation targets ATGL-S and HSL-S However, while AMPK-catalysed ATGL phosphorylation increases its activity 12 , HSL phosphorylation has an inhibitory effect.
Whether AMPK stimulates or inhibits lipolysis may therefore strongly depend on specific experimental conditions leading to different results in various studies Also, the finding that adipose-specific deletion of AMPK has no effect on TG hydrolysis argues against a prominent role of AMPK in the regulation of lipolysis Notably, however, a recent report demonstrated that lipolysis activates AMPK Under hormonal stimulation, excessive lipolysis leads to increased FA re-esterification within a futile cycle of TG hydrolysis and resynthesis.
This process is quite energy demanding and results in accumulated AMP and the activation of AMPK. This, in turn, causes mTORC1 inactivation and cellular growth arrest, a mechanism that may explain the inhibitory effect of lipolysis on cancer cell growth , These results are also consistent with the finding that enhancing AMPK activity by an AMPK-stabilizing peptide reduces adipose tissue atrophy in cancer-associated cachexia Human CGI is a protein of amino acids that belongs to a family of 17 ABHD proteins, most of which are lipid hydrolases CGI is an exception because it lacks the nucleophilic amino acid residue in the catalytic triad of the active site that is essential for functional hydrolases Unfortunately, molecular details concerning the actual activation mechanism are still unknown and will likely require three-dimensional structural information for both proteins.
Phylogenetic and mutational analyses revealed that the highly conserved amino acid residues R and G in human CGI are essential for ATGL activation Importantly, the Grannemann group developed a small-molecule inhibitor that disrupts the interaction of perilipin-1 and CGI In the presence of this inhibitor, CGI constitutively activates ATGL even in the absence of hormonal stimulation, which leads to unrestrained TG degradation Absence of this phenotype in ATGL-deficient humans and mice argues strongly for an ATGL-independent epidermal function for CGI In humans, mutations in the gene for CGI cause an autosomal recessive skin disease designated NLSD with Ichthyosis NLSDI 21 , Since its original discovery in the s, more than cases of NLSDI have been reported.
The disease is characterized by the accumulation of TGs in many tissues and severe ichthyosis Independent of ATGL, CGI in keratinocytes interacts with and activates PNPLA1 transacylase activity driving acylceramide synthesis in the skin see section on PNPLA1 , CGI also interacts with PNPLA3, the closest relative of ATGL within the PNPLA family see below in section on PNPLA3 Other ATGL-independent functions of CGI include an acyltransferase- and HDAC4-specific protease activity Whether these activities contribute to normal skin barrier function and the pathogenesis of NLSDI is currently not known.
Like CGI, G0S2 turned out to be a multifunctional protein involved in cell proliferation, mitochondrial ATP production, apoptosis and cancer development Its best-characterized function, however, relates to its interaction and inhibition of ATGL , Liu and colleagues elegantly demonstrated that G0S2 in mice and humans interacts via a hydrophobic stretch within the patatin domain of ATGL, thereby suppressing enzyme activity Non-cooperativity of G0S2 and CGI suggest that the coregulators bind to ATGL at different sites.
Oberer and colleagues developed a amino-acid peptide ranging from K20 to A52 of human G0S2, including the hydrophobic ATGL interaction region, which inhibited ATGL with similar potency as full-length G0S2 ref. Both full-length G0S2 and the G0S2-derived peptide noncompetitively inhibit ATGL with a half-maximal inhibitory concentration IC 50 in the nanomolar range.
G0S2 specifically inhibits ATGL but fails to inhibit other lipid hydrolases of the PNPLA family or lipolytic enzymes In addition to its antilipolytic function, G0S2 may also have lysophosphatidyl-acyltransferase activity G0S2 protein abundance is highest in adipose and relatively low in most other tissues.
Consistent with the strong inhibitory effect of G0S2 on ATGL activity, its tissue-specific overexpression leads to steatosis in cardiac muscle or liver in mice , Somewhat unexpectedly, G0S2-deficient mice exhibited a relatively modest phenotype with a slight increase in lipolysis and minor alterations in lipid and energy metabolism, as well as adipose tissue morphology , The effect of G0S2 deficiency was more pronounced in the liver, resulting in decreased liver fat and resistance to high-fat-diet-induced hepatosteatosis The relatively low expression levels of G0S2 in mice may explain the lack of a major phenotype upon its deletion.
Hypoxia-induced lipid droplet-associated protein HILPDA , also called hypoxia-induced gene-2 HIG2 , is a G0S2-related peptide of 63 amino acids that also inhibits ATGL activity. HILPDA was originally identified as an LD-binding protein that is highly expressed in response to hypoxia The promoter of the Hilpda gene harbours a number of hypoxia-responsive elements that are targeted by the transcription factors HIF-1 and HIF-2 ref.
Like G0S2 , Hilpda is a PPARα target gene HILPDA is abundantly expressed in immune cells, adipose tissue, liver and lung The protein binds to LDs and interacts with the N-terminal region of ATGL, thereby inhibiting ATGL activity in the absence or presence of CGI refs.
Compared with G0S2, however, human HILPDA is a much less potent ATGL inhibitor Independently of its effects on ATGL, murine HILPDA also interacts with the TG-synthesizing enzyme diacylglycerol-acyltransferase-1 DGAT1 and promotes lipid synthesis in hepatocytes and adipocytes Despite these pronounced activities of HILPDA in vitro, the in vivo consequences of its deficiency in adipose tissue are very moderate.
HILPDA deficiency affected neither plasma FA, glycerol or TG concentrations nor adipose tissue mass during fasting or cold exposure, apparently ruling out the possibility that HILPDA is a crucial regulator of lipolysis , The generation of double-knockout mouse lines may clarify whether the benign phenotype of G0S2 or HILPDA deficiency results from mutual compensatory functions.
Lipase—protein interaction studies revealed a large number of factors that interact with ATGL, HSL or their respective coregulators. In addition to the already mentioned factors ABHD-5, G0S2, HILPDA, ATGL-trafficking proteins, perilipins and CHREBP , at least a dozen factors have been described to affect lipolysis by direct lipase or coregulator binding.
An overview of these factors was recently published by Hofer et al. They include the ATGL regulatory factors FSP27, pigment epithelium-derived factor PEDF and proteins or the HSL interaction partners fatty acid binding protein-4 FABP4 , vimentin and cavin FABP4 also interacts with CGI and plays an important role in the export of lipolysis-derived FAs or their transport to the cell nucleus 34 , , The only known neutral lipid hydrolase which is active under acidic conditions in lysosomes is LAL.
This enzyme, originally discovered in ref. LAL, encoded by the Lipa gene, is highly glycosylated and ubiquitously expressed, with highest levels observed in hepatocytes and macrophages.
Several transcript variants of LAL have been described, of which a amino-acid, kDa protein exhibits the highest enzymatic activity The three-dimensional structure of the human enzyme was recently solved and revealed strong structural similarities to gastric lipase, another member of the acid lipase family Regulation of LAL activity is less complex than that of neutral lipases and involves primarily transcriptional mechanisms.
Established transcription factors that activate LAL expression include PPARs, FOXO1 and the E-box transcription factors TFEB and TFE3 ref. Additionally, LAL is released from cells into the interstitium by a mechanism called exophagy, where the lysosomal membrane fuses with the plasma membrane, liberating the cargo The function of LAL with a pH optimum of 4.
Conversely, cells can also internalize extracellular LAL. LAL reaction products include non-esterified cholesterol and FAs. Cholesterol is subsequently released from lysosomes by an NPC1-dependent process and suppresses cholesterol de novo synthesis by multiple mechanisms , Excess cholesterol is re-esterified and stored as CE in cytoplasmic LDs.
The export mechanism for FAs is not well understood. After their liberation, they contribute to hepatic VLDL synthesis , induce the alternative activation of macrophages , represent precursors for highly bioactive lipid mediators and drive thermogenesis in brown adipose tissue The role of lysosomes in the degradation of cargo derived from various endocytosed lipoproteins was recognized many decades ago.
This function was extended when, in , lipophagy was identified as a distinct form of autophagy and a quantitatively relevant mechanism for the hydrolysis of TGs and CEs in hepatocytes and macrophages , , Three types of autophagic pathways contribute to TG degradation: macrolipophagy, microlipophagy, and chaperone-mediated autophagy CMA Fig.
During macrolipophagy, autophagosomes sequester and engulf parts of LDs and subsequently fuse with lysosomes to form autolysosomes In contrast, microlipophagy does not involve enclosure of LD components within autophagosomes. Microlipophagy is a well-established process in yeast but has only recently been discovered in mammalian cells Interestingly, knockdown of ATGL in hepatocytes leads to reduced microlipophagy, indicating a connection between microlipophagy and neutral lipolysis A plausible explanation for this dependency is that ATGL activity reduces LD size, thereby rendering LDs more accessible for microlipophagy , ATGL also plays an important role in CMA, in which the autophagosomal degradation of perilipins allows ATGL to access the surface of LDs and thereby stimulates lipolysis Heat shock protein 70 HSP70 directly interacts with perilipin-2 and perilipin-3 on LDs by recognizing a pentapeptide motif It subsequently delivers them to lysosomes and facilitates their uptake and degradation, mediated by lysosome-associated membrane 2 ref.
Mutation of Hsp70 binding motifs on perilipins leads to reduced CMA and LD accumulation in cultured cells and mouse livers in vivo, despite unaltered macroautophagy Three types of autophagic pathways contribute to the degradation of cytosolic LDs. During microlipophagy, Ras-related in brain RAB proteins facilitate the flux of lipids and proteins from LDs to the lysosome.
Lysosomal acid lipase LAL is the only known lipase capable of hydrolysing neutral lipids under acidic conditions. Independent of LAL, chaperone-mediated authophagy facilitates a HSPmediated transfer and lysosome-associated membrane 2 LAMP2 dependent lysosomal uptake and degradation of perilipins to increase the accessibility of cytosolic lipases to LDs.
LAL deficiency in mice leads to massive accumulation of CEs and TGs in the liver, intestine, adrenal glands and macrophages , In humans, complete absence of LAL activity also provokes severe hepatosteatosis and hepato-splenomegaly.
Unlike the murine model, however, the human deficiency, known as Wolman disease, is lethal within the first year after birth A more benign variant of the disease is called cholesteryl ester storage disease CESD.
People with CESD have a higher risk for atherosclerosis and coronary heart disease This phenotype is explained by increased lysosomal retention of CE in macrophages owing to low LAL activities leading to insufficient suppression of cholesterol de novo synthesis and foam-cell formation Several GWAS studies identified common polymorphisms in the Lipa gene that associate with cardiovascular diseases However, their impact on LAL function remains controversial.
The canonical lipases ATGL, HSL, MGL and LAL are the best-characterized enzymes with regard to hydrolysis of cytoplasmic TG stores. In non-adipose tissues, however, substantial TG hydrolase activities are observed even in the absence of ATGL and HSL.
In addition to a significant contribution of acid lipolysis, several alternative neutral lipases of diverse protein families have been connected to TG catabolism in the past decade summarized in Table 1. Although the critical role of ATGL in lipolysis is undisputed, the distinct biochemical functions of its most closely related family members—PNPLA1, PNPLA3, PNPLA4 and PNPLA5—remain equivocal.
While an unambiguous assignment of their primary physiological substrates is still missing, some of these enzymes are directly involved in disease development. PNPLA1 is a amino-acid protein specifically expressed in differentiated keratinocytes of the skin , It is an established causative gene for autosomal recessive congenital ichthyosis ARCI , which is characterized by a severe defect in the development of the epidermal corneocyte lipid envelope CLE and the transepithelial water barrier in the skin in humans and dogs The clinical presentation essentially phenocopies the deficiency of CGI in the skin , arguing for a common involvement of both proteins in the formation of the CLE.
PNPLA1 is a transacylase that specifically transfers linoleic acid from TG to ω-hydroxy ceramide, thus giving rise to ω- O -acylceramides , These skin-specific lipids are essential for the generation of the CLE and an intact skin permeability barrier.
Transacylation of FAs requires a hydrolysis reaction that is coupled with an esterification reaction. CGI interacts with PNPLA1 and recruits the enzyme onto cytosolic LDs where PNPLA1 utilizes TG-derived FAs for ω-hydroxy ceramide acylation , PNPLA1 mutations leading to ARCI may additionally impair autophagy, arguing for a potential role of PNPLA1 in lipophagy-mediated degradation of LDs In humans, PNPLA3 is less abundant in adipocytes, but highly expressed in hepatocytes Key transcription factors responsible for the expression of PNPLA3 include CHREBP in response to carbohydrates and SREBP1c in response to insulin , PNPLA3 localizes to LDs and exhibits various enzymatic activities including TG-, DG-, MG- and retinyl ester hydrolase activity, PLA 2 activity and transacylase activity 8 , , , It remains unclear which of these activities are physiologically relevant in adipose tissue and in the liver The fact that PNPLA3-KO mice have essentially no abnormal phenotype with normal plasma and hepatic TG concentrations , argued against a major role of PNPLA3 in lipid homeostasis.
This view changed dramatically when Hobbs and colleagues reported that a single amino acid exchange at position from isoleucine to methionine in PNPLA3 p.
IM strongly associates with NAFLD This association was replicated in numerous studies and extended to associations of the mutation with steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma Important progress concerning the function of PNPLA3 was achieved by recent studies suggesting that excess PNPLA3 on the surface of LDs competes with ATGL for its coactivator CGI refs.
PNPLA3-IM has higher affinity to CGI than does wild-type PNPLA3 ref. This leads to reduced ATGL activity, which may explain the fatty liver disease in people carrying the variant.
Additionally, the IM variant may promote increased TG synthesis via a transacylation reaction PNPLA4 was originally identified as gene sequence 2 GS2 in the human genome and is also found in other mammalian species but not in mice The PNPLA4 gene is located on the X chromosome between the genes encoding steroid sulfatase and Kallmann Syndrome The human PNPLA4 protein comprises amino acids and is expressed in numerous tissues, including adipose tissue, skeletal and cardiac muscle, kidney, liver and skin , Similar to PNPLA3, various enzymatic activities have been attributed to PNPLA4, including TG and retinyl ester hydrolase, phospholipase and transacylase activities 8 , , Genetic analyses have suggested that PNPLA4 is involved in the development of two rare congenital disorders: combined oxidative phosphorylation deficiency COXPD and X-linked intellectual disability , COXPD arises from a hemizygous nonsense mutation resulting in a C-terminally-truncated protein Rter and defective assembly of complexes I, III and IV of the respiratory electron transport chain.
Whether the enzymatic activity of PNPLA4 is involved in the pathogenesis of COXPD is unknown. Fortunately, small-molecule inhibitors have recently been developed that may help to elucidate the biochemical function of PNPL4 and its role in patho- physiology PNPLA5 also named GS2-like is a amino-acid protein that is ubiquitously expressed and, unlike PNPLA4, is present in both mice and humans PNPLA5 exerts hydrolytic activities towards multiple lipid substrates, including TGs and retinyl esters, but has also been shown to exhibit transacylase activity , Notably, PNPLA5-dependent hydrolysis of LD-associated TGs has been suggested to affect autophagosome biogenesis Furthermore, rare variants of PNPLA5 strongly associate with LDL cholesterol levels in humans They catalyse the hydrolysis of a wide range of endogenous substrates and xenobiotics CES family members that exhibit neutral TG hydrolase activity include human CES1 also called human cholesterylesterhydrolase-1, hCEH and CES2, and mouse Ces1d also called Ces3 or TG hydrolase-1 , Ces1g also called Ces1 or esterase-X and Ces1c.
The previously confusing nomenclature of human and murine CES enzymes has recently been revised for more clarity The carboxylesterase 1 CES1 gene in the human genome encodes a kDa protein with high abundance in liver and intestine and lower abundance in kidney, adipose tissue, heart and macrophages , CES1 preferably hydrolyses CEs and TGs , Hepatocyte-specific overexpression of human CES1 in transgenic mice increases hepatic TG hydrolase activity; lowers TG, FA and cholesterol content; and reduces reactive oxygen species ROS , apoptosis and inflammation, which cumulatively protect mice from western-diet- or alcohol-induced steatohepatitis Both hepatocyte- and macrophage-specific overexpression of CES1 in mice decrease atherosclerosis susceptibility in LDL-receptor-deficient mice and, consistent with this finding, treatment of THP-1 macrophages with a CES1 inhibitor caused accumulation of CE Hence, the role of human CES1 in lipid metabolism remains controversial.
Ces1d is highly expressed in adipose tissue and liver where it localizes to the lumen of the ER In adipose tissue, Ces1d is additionally associated with LDs and may contribute to basal lipolysis , In mouse hepatocytes, transgenic overexpression or pharmacological inhibition of Ces1d increased or decreased VLDL assembly and secretion, respectively , , In accordance with these findings, liver-specific and global Ces1d deficiency in mice lowers VLDL production and plasma TG and CE concentrations , Despite strong evidence for a role of Ces1d in hepatic VLDL assembly and secretion, it remains unclear how an enzyme residing in the lumen of the ER actually contributes mechanistically to lipoprotein synthesis.
Interestingly, while global Ces1d deficiency leads to TG accumulation in isolated hepatocytes, conditional Ces1d knockout in liver cells does not. In fact, they are actually protected from high-fat-diet-induced hepatic steatosis , The protective effect of Ces1d deficiency in the liver might be due to reduced FA-synthase activity and increased hepatic FA oxidation , Moreover, mice with global, but not liver-specific, Ces1d deficiency exhibit improved insulin sensitivity and glucose tolerance , The recent finding that Ces1d deficiency does not affect CE hydrolysis or bile-acid synthesis in mice argues against an important role for CES1 enzymes in cholesterol homeostasis see previous paragraph.
The second murine ortholog to human CES1 with established TG hydrolase activity is Ces1g. Overexpression of Ces1g increases TG hydrolase activity and reduces TG content in cultured rat hepatoma cells and livers of transgenic mice , Conversely, global- and liver-specific Ces1g-KO mice exhibit hepatic steatosis and hyperlipidemia , Restoration of hepatic Ces1g expression reverses hepatic steatosis, hyperlipidemia and insulin resistance in global Ces1g-deficient mice Several studies suggest that the lipid phenotype observed in Ces1g-deficient mice results from diminished FA signalling to restrain SREBP1c activation, leading to reduced FA oxidation and increased de novo lipogenesis , , CES2 preferably hydrolyses esters with a large alcohol group and a small acyl group, and as for Ces2c, TG and DG hydrolase activities have been reported , CES2 is abundantly expressed in the small intestine, colon and liver and is decreased in mice and humans with obesity and NAFLD , , Adenoviral overexpression of human CES2 in the liver of mice prevents genetic- and diet-induced steatohepatitis by increasing TG hydrolase activity and FA oxidation and reduces SREBP1c mediated lipogenesis, inflammation, apoptosis and fibrosis.
Murine Ces2c is a highly abundant TG, DG and MG hydrolase in liver and intestine , Its expression is decreased in livers of mice with diet- or genetically induced obesity , Loss of hepatic Ces2c in chow- or western-diet-fed mice causes hepatic steatosis Conversely, increasing hepatic Ces2c expression ameliorated obesity and hepatic steatosis, and improved glucose tolerance and insulin sensitivity lean ].
lean ] during Incr between L and O. A t test or Mann—Whitney rank sum test for nonparametric values was used to identify differences in the parameters Fat max , Fat max zone and MFO and in the SIN model variables dilatation, symmetry and translation of the whole-body fat oxidation kinetics obtained during Incr.
These tests were also used to determine differences in anthropometric and physical characteristics between O and L. There was no significant difference in age and height between the two groups Table 1. Weight, BMI, fat mass FM and FFM were significantly higher in O compared with L Table 1.
Fasting glucose was similar in O and L, whereas fasting insulin and HOMA-IR were significantly higher in O compared with L Table 1. RER was significantly lower in O than in L 0. FORs, expressed in g. FORs, expressed in mg. Whole-body fat oxidation kinetics were characterized by similar translation, significantly lower dilatation and left-shift symmetry in O compared with L Table 2 , Figure 2D.
MFO was similar in O and in L, and Fat max , Fat max zone and RERFat max were significantly lower in O compared with L Table 2. DE was similar in O and L and respiratory equivalent showed no significant main group effect and no significant interaction effect data not shown.
Values are the means±SE. There were no significant main group and interaction effects for plasma glucose and lactate concentrations data not shown. PPO: peak power output. There were no significant main group or interaction effects for plasma NE and ANP concentrations Figures 4B and 4C , respectively.
The results of this study showed that O presented a lower Fat max , a left-shifted and less dilated curve and a lower reliance on fat oxidation at high but not at low or moderate exercise intensities.
Moreover, MFO was similar in the two groups. Despite the blunted lipolysis, O presented higher NEFA availability, most likely due to larger amounts of FM.
Therefore, contrary to our hypothesis, these results suggest that the decreased FORs in O relative to L at high exercise intensities are not associated with decreased plasma NEFA availability, but most likely linked to impaired muscular capacity to oxidize NEFA.
The absolute values were similar between O and L counterparts and are in line with previous studies that tested, on cycle-ergometer, individuals with similar class of obesity [6] , [8] , [26] , [27]. Moreover, it has been suggested that may also be indicative of a true maximal oxygen consumption in lean [28] and in obese individuals [29] and previous studies compared FORs in obese and lean individuals reporting FORs as a function of [7] , [10].
Moreover, this allows us to compare our results to previous findings [7] , [10]. Our results, according to previous findings [6] , [30] , showed that RER was lower in obese compared to lean counterparts, indicating a greater proportion of energy derived from fat during low-to-moderate exercise intensities.
Indeed, the fat oxidation kinetics, expressed in g. Contrary to Ara et al. However, as our O presented higher FFM values, these observations report only indications of the global fat oxidation irrespective of differences in FFM between the two groups.
Therefore, expressing the FORs in mg. Moreover, the significant interaction effect clearly demonstrates that the pattern of fat oxidation kinetics was different in the two groups, substantially confirming the findings of FORs expressed in g.
This comparison showed that obese individuals presented a left-shifted and less dilated curve compared to lean individuals, associated with a lower reliance on fat oxidation at high exercise intensities.
This finding may be explained by the lower E concentrations at moderate intensities in O than in L and by the significant interaction effect for E between L and O Figure 4A. In fact, it has been recently shown that E, and not NE, is a determinant of exercise-induced lipid mobilization in human subcutaneous AT [32].
Moreover, as E concentrations were not significantly different at rest and during low exercise intensities between the two groups, higher insulin concentrations may also contribute to the blunted lipolysis in O [8]. In addition, although previous studies suggested that the plasma ANP a stimulator of lipolysis in AT [33] is lower in obese than in lean individuals [34] most likely linked to a higher expression of natriuretic peptide clearance receptors in AT with obesity [35] , our results showed no difference between O and L at Rest and during exercise.
Indeed, it has been suggested that ANP receptor expression in AT was reduced in obese hypertensive but not in non-hypertensive obese individuals [35] , suggesting that the alteration of ANP-induced cardio-metabolic actions may be related to hypertension [36].
However, because our O were not hypertensive, it is difficult to relate the blunted lipolysis in O with lower ANP receptor expression in AT. Interestingly, higher absolute concentrations of NEFA and glycerol were found during all exercise intensities in O, suggesting that higher NEFA availability may be due to larger amounts of FM in these individuals [37].
Moreover, the NEFA concentration profiles display similar kinetics in both groups, indicating that NEFA availability cannot explain the different patterns of fat oxidation kinetics with regard to exercise intensities between the 2 groups. Therefore, contrary to our hypothesis, the lower FORs in O relative to L at high exercise intensities were not due to decreased plasma NEFA availability.
Furthermore, despite a continuous increase in lipolysis Figure 3 , the stable plasma NEFA concentrations observed during Incr may suggest an enhanced NEFA uptake with respect to exercise intensities by skeletal muscle cells in both groups.
The reduced FORs at high exercise intensity may be linked to the decreased activity of the muscle carnitine palmitoyltransferase CPT-1 and muscle citrate synthase CS an index of mitochondrial content in obese subjects relative to lean controls [1] , [38] , [39].
Moreover, as previously reported [8] , [9] , O can maintain total lipid oxidation at rest and during moderate exercise only if they can compensate for the reduction in plasma NEFA oxidation with enhanced intra-muscular triglyceride IMTG oxidation.
The higher and similar FORs in O compared with L during low and moderate exercise intensities, respectively, seem to confirm this mechanism. However, our results may indicate that this IMTG compensation may not be possible at high exercise intensities, leading to decreased FORs in O compared with L.
In fact, it has been shown that IMTG oxidation decreases to a greater extent than plasma NEFA oxidation as the exercise intensity increases from moderate to high [41]. Thus, plasma NEFA oxidation, which represents a more important part of the total lipid oxidation during high exercise intensities, may exert a more substantial limiting effect and thus decrease the total lipid oxidation at these intensities in O.
Therefore, we suggest that O may have a muscular defect in the ability to oxidize lipids [38] , [39] at high exercise intensities, most likely due to decreased plasma NEFA oxidation [6] , [9]. Our results are in contrast with those of Ara et al.
who found higher FORs during all exercise intensities and higher Fat max and MFO in obese compared with lean individuals. Therefore, we created a sub-group of 8 O BMI: This suggests that the lower reliance to fat oxidation at high exercise intensities may be directly associated with obesity and not with differences in aerobic fitness between the 2 groups.
In the sub-groups matched for aerobic fitness, O present similar MFO O: 6. Our results may also be relevant from a clinical standpoint and for exercise prescription in O.
In fact, Fat max was found to be lower in O than in L, and its values were similar to those reported in the literature in lean [5] , [31] and obese individuals [4] , [7] , [10].
Training programs in class II and III O are rare, but targeting the training intensity in the zone that elicits MFO appears to be appropriate [4]. Some methodological limitations exist and need to be addressed. Firstly, our O were studied during a lifestyle education program, and therefore this condition may interfere with our findings and not be completely representative of the general obese population.
However, the testing session was conducted at the end of the hospitalization program, when the weight changes were minimal.
In addition, contrary to previous studies that performed only one day of diet control before the trial [5] , [7] , [10] , our O followed a 3-week balanced diet before the testing session. Moreover, although our O may present a favourable condition to promote fat oxidation [42] , [43] , the decreased FORs observed during high exercise intensities and the lower dilatation may suggest that obese individuals really suffer from an impaired capacity to oxidize lipids.
Secondly, although indirect calorimetry is extensively used to determine substrate oxidation during exercise, changes in the size of the bicarbonate pool may interfere with calculations of substrate oxidation at higher intensities [44].
Moreover, our results of and respiratory equivalent, represented as a function of exercise intensity, showed that there was no difference between groups, suggesting that indirect calorimetry may be accurately used to assess and compare substrate oxidation in the two groups. In summary, this study showed that O with high BMI presented a left-shifted and less dilated curve and a lower reliance on fat oxidation at high but not at low or moderate exercise intensities.
Despite the blunted lipolysis, O presented higher NEFA availability most likely due to larger amounts of FM , suggesting that the decreased FORs in O at high exercise intensities are most likely linked to impaired muscular capacity to oxidize lipids.
In addition, the different pattern of fat oxidation kinetics between the two groups may be directly associated with obesity and not with differences in aerobic fitness. The narrowing of the FORs and the lower Fat max and Fat max zone may have important implications for the appropriate exercise intensity prescription in training programs designed to optimize fat oxidation in O.
We thank Dr. Paolo Fanari for his helpful assistance in the laboratory, Ivana Di Sabato for blood analysis and Dr. Francesca Amati for her helpful suggestions and criticism.
Conceived and designed the experiments: SL FC MC SM AS AB DM. Performed the experiments: SL FC MC SM. Analyzed the data: SL SM AS AB DM.
Wrote the paper: SL DM. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Article Authors Metrics Comments Media Coverage Reader Comments Figures. Abstract This study aimed to compare fat oxidation, hormonal and plasma metabolite kinetics during exercise in lean L and obese O men.
Funding: The authors have no support or funding to report. Introduction Obesity is associated with a variety of health-related risks, such as hypertension and type 2 diabetes, all of which may center around insulin resistance [1]. Download: PPT. Preliminary testing All subjects underwent dual-energy X-ray absorptiometry DEXA for measurements of body composition DPX-IQ X-ray bone densitometer version 4.
Experimental protocol Seven days after the preliminary test, the experimental trial was performed in the morning between hours after a minimum h overnight fast. The intensity of exercise and the amount of weekly exercise performed, but not changes in fat mass, were correlated with the change in fasting RQ Table 3.
We examined, initially using bivariate analyses, potential correlates of the improvement in insulin-stimulated glucose metabolism induced by the weight loss and physical activity intervention.
We also examined whether there were baseline preintervention characteristics that were predictive of the intervention-induced change in insulin sensitivity Table 4. Those who were more insulin resistant before the intervention had greater improvements in insulin sensitivity, as did those who had low fasting rates of fat oxidation at baseline.
This may imply that those who need the intervention the most, i. It is possible, however, that this was influenced by regression to the mean, resulting in an overestimation of the association between baseline and change values. Initial patterns of adipose tissue distribution or level of physical fitness did not predict improved insulin sensitivity.
Next, we examined whether changes induced in body composition, fitness, and fasting metabolism predicted the improvement in insulin sensitivity Table 4. The loss of total body fat, subcutaneous abdominal fat, and subfascial thigh fat, but not the selective loss of visceral fat or subcutaneous thigh fat, was associated with the improved insulin sensitivity.
Changes in V o 2max and exercise energy expenditure tended to be associated with the improved insulin sensitivity. However, the strongest simple correlate with improved insulin sensitivity was increased fasting rates of fat oxidation and, accordingly, the reduced fasting RQ, as shown in Table 3 and Fig.
Similar associations were observed with respect to the nonoxidative component of insulin sensitivity. Stepwise multivariate regression analysis was then used to examine the interaction and interdependence of these changes in physiologic and body composition parameters.
After accounting for increases in fasting rates of fat oxidation, the selective loss of subfascial thigh fat emerged next, followed by the loss in total body fat as independent predictors of this improvement. Enhanced postabsorptive fat oxidation remained a significant correlate of improved insulin sensitivity after adjusting for the loss of body fat or improved physical fitness in the model.
To further examine whether the addition of physical activity provided more metabolic benefit than weight loss alone, we compared the findings from the current research volunteers with that attained in a separate group of obese subjects BMI Figure 3 illustrates the improvements in insulin sensitivity in a subset of men and women 9 women and 7 men who completed the exercise and caloric restriction intervention, as compared with those who either completed a program of caloric restriction only 29 10 women and 6 men or exercise without weight loss 4 women and 3 men.
The subset of individuals from the two groups were matched for fat mass lost: 7. Subjects in these groups were of similar age 36 ± 5, 38 ± 6, and 39 ± 5 years for exercise-only, diet-only, and exercise plus diet groups, respectively. Identical protocols, including timing of the glucose clamps following intervention, were followed in each of these three groups, although it is possible that the separate interventions were confounded by time effects.
Physical activity and moderate sustained weight loss are advocated for the treatment of obesity, the insulin resistance syndrome, and the prevention of type 2 diabetes. It is not clear how physical activity and weight loss interact in achieving these effects.
Even a single session of moderate to strenuous intensity exercise can induce a transient improvement in insulin sensitivity 13 , effects that appear to wane within a few days Exercise also increases oxidative enzyme activity in skeletal muscle and induces related biochemical and morphologic changes that would seem to confer a metabolic basis for improved insulin sensitivity.
However, in clinical interventions carefully controlled with respect to matching energy balance, exercise in the absence of weight loss has been found to have modest 17 , 30 or no 16 effect on insulin sensitivity. These findings have suggested that exercise might be of lesser value than weight loss as an intervention for the insulin resistance syndrome.
We sought to address this apparent conundrum in two ways. First, we employed exercise interventions in conjunction with weight loss, an intervention model that more appropriately matches clinical recommendations than either intervention in isolation.
The improvement in insulin sensitivity in these obese subjects was greater than those observed for other studies examining exercise or weight loss alone 16 , Similarly, Bogardus et al.
Ross et al. Therefore, these results suggest a synergistic effect of weight loss and exercise to improve insulin resistance. The timing of the insulin sensitivity measure after the last exercise bout could also affect the magnitude of the improvements in insulin sensitivity across studies. It is important to account for the influence of acute exercise on insulin sensitivity.
We chose 36—48 h after the last exercise bout to perform the glucose clamp to avoid both the acute exercise effects and potential de-training effects. This likely contributed to greater improvements with exercise training compared with other studies that performed the insulin sensitivity measure 4—6 days following the last exercise bout 16 , 17 , Improved insulin sensitivity through either weight loss or exercise has been associated with the loss of abdominal visceral fat 17 , Obese subjects in the present study lost a significant amount of fat from several region-specific depots, including abdominal fat and subfascial thigh fat.
In multivariate analysis, only the loss of subfascial thigh fat was independently correlated with the improvement in insulin sensitivity after adjusting for the change in systemic fatty acid oxidation. The attenuation of muscle on CT as a marker of muscle lipid 26 did not change, although muscle attenuation can increase with diet-induced weight loss However, lipid within muscle can be higher in endurance-trained athletes 23 , raising the possibility that weight loss and exercise have counterbalancing effects on muscle lipid, perhaps explaining the lack of change in muscle attenuation.
Our findings indicate that the combination of exercise and weight loss enhances not only the insulin-stimulated capacity for glucose utilization, but also enhances the capacity for fat oxidation during fasting conditions.
The improvement in fat oxidation during fasting conditions was associated with characteristics specific for the exercise intervention, namely intensity and duration of physical activity, but was not associated significantly with overall or regional loss of adiposity.
In turn, the magnitude of improvement in insulin-stimulated glucose metabolism was strongly related to the concomitant increase in fat oxidation during fasting conditions.
Impaired insulin-stimulated glucose metabolism is the most well-established manifestation of skeletal muscle insulin resistance, but it is also recognized that another very important facet of skeletal muscle insulin resistance is altered patterns of fat oxidation.
Normally, insulin effectively suppresses fat oxidation 31 , but this suppression of fat oxidation is impaired in insulin resistance Additionally, it has long been noted that in those with normal insulin sensitivity, skeletal muscle has a high reliance upon fat oxidation during fasting conditions In contrast, among those with obesity and insulin resistance, and in those with type 2 diabetes, fasting rates of fat oxidation are reduced in skeletal muscle In prior cross-sectional studies, we observed that impaired fat oxidation during fasting predicts severity of insulin-resistant glucose metabolism We had also noted that following diet-induced weight loss, but without changes in patterns of physical activity, there was improvement in insulin-stimulated glucose metabolism but there was not a significant change in fasting rates of fat oxidation Thus, in weight loss without exercise, there appeared to be a separation in the effects on those aspects of insulin resistance related to insulin-stimulated glucose metabolism from those aspects regulating rates of fat oxidation.
The current study sought to probe whether the addition of exercise might address insulin resistance in a manner different from weight loss alone. There are several conceptual reasons to postulate that physical activity might modify the aspects of insulin resistance related to the capacity for fat oxidation.
First, exercise training at moderate intensity is typically associated with induction of a higher capacity for, and reliance upon, fat oxidation during the exercise session Second, chronic exercise effects, namely increased activity of oxidative enzymes and increased capillary density, might facilitate fatty acid utilization both at rest and during physical activity.
In contrast, during physical activity, muscle generates a negative energy balance and is comprised of consumption of muscle glycogen and muscle triglyceride 24 , In contrast to the decrease in energy expenditure induced by weight loss through reductions in caloric intake 18 , the lack of change in resting energy expenditure despite weight loss was likely due to the exercise component.
Therefore, the current findings that an exercise intervention increases the reliance on fat oxidation while maintaining resting energy expenditure are in accord with these prior observations.
The novel finding of the current study that helps to extend the clinical implications of these prior data is that the augmentation of resting rates of fat oxidation are a specific metabolic correlate of the amplitude of improvement in insulin sensitivity.
In summary, the improvement in insulin sensitivity resulting from a program combining exercise and diet is associated with an increased reliance on fat oxidation during fasted conditions. This enhanced fat oxidation is likely due to exercise and not caloric restriction. Therefore, the greater improvements in insulin sensitivity in obese subjects who perform regular exercise with weight loss, as compared with those who lose weight without exercise, are likely mediated by changes in skeletal muscle fatty acid metabolism.
Improvements in insulin sensitivity with combined diet and exercise. Relation of fatty acid oxidation to improvement in insulin sensitivity. Improvements in insulin sensitivity by weight loss with exercise, weight loss without exercise, or exercise without weight loss.
Changes in body composition and physical fitness during combined exercise and caloric restriction. CT data were obtained and analyzed for 17 volunteers. HU, Hounsfield units.
Changes in systemic energy expenditure and substrate oxidation during insulin-stimulated and fasting conditions. Simple correlation coefficients r determined using simple linear regression analysis. Exercise intensity and energy expenditure were estimated from individual heart rate- V o 2 relationships during each exercise session.
Δ, change as a continuous variable from pre- to postintervention. Exercise energy expenditure was estimated from individual heart rate- V o 2 relationships during each exercise session. This work was funded by R01DK to D. K , K24 DK to D.
Fat oxidation is Ehanced process in which the body Snacks for sustained energy before a game Sweet potato and black bean enchiladas lipids, releasing energy mechwnisms fuel far performance. But why is using Fitness as a fuel important for endurance performance? Oxidizimg does oxidizlng body decide Optimal Recovery Nutrition use fats Sweet potato and black bean enchiladas than sugars? And how can you develop your fat oxidation capacity to boost your fuel efficiency and your power output? In this article, we will take a dive into what fat oxidation is and how to make your body burn more fats than sugars during exercise. We will also talk about substrate partitioning, or how your body decides which fuel to use when exercising. Finally, we will look at different types of training interventions and what their actual effects are on fat utilisation.Fatty acid metabolism consists of various oxirizing processes involving Enanced closely related to fatty acidsa family Hydration for workouts molecules classified within the Enhnced macronutrient category.
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The decarboxylation reactions occur before malate is formed in the cycle. However, acetyl-CoA can be converted to acetoacetate, which can decarboxylate to acetone either spontaneously, or catalyzed by acetoacetate decarboxylase. Acetol can be converted to propylene glycol.
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The glycerol released into the blood during the lipolysis of triglycerides in adipose tissue can only be taken up by the liver. Here it is converted into glycerol 3-phosphate by the action of glycerol kinase which hydrolyzes one molecule of ATP per glycerol molecule which is phosphorylated.
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Fatty acids are an integral part of the phospholipids that make up the bulk of the plasma membranesor cell membranes, of cells. These phospholipids can be cleaved into diacylglycerol DAG and inositol trisphosphate IP 3 through hydrolysis of the phospholipid, phosphatidylinositol 4,5-bisphosphate PIP 2by the cell membrane bound enzyme phospholipase C PLC.
One product of fatty acid metabolism are the prostaglandinscompounds having diverse hormone -like effects in animals. Prostaglandins have been found in almost every tissue in humans and other animals.
They are enzymatically derived from arachidonic acid, a carbon polyunsaturated fatty acid. Every prostaglandin therefore contains 20 carbon atoms, including a 5-carbon ring.
They are a subclass of eicosanoids and form the prostanoid class of fatty acid derivatives. The prostaglandins are synthesized in the cell membrane by the cleavage of arachidonate from the phospholipids that make up the membrane.
This is catalyzed either by phospholipase A 2 acting directly on a membrane phospholipid, or by a lipase acting on DAG diacyl-glycerol. The arachidonate is then acted upon by the cyclooxygenase component of prostaglandin synthase.
This forms a cyclopentane ring roughly in the middle of the fatty acid chain. The reaction also adds 4 oxygen atoms derived from two molecules of O 2. The resulting molecule is prostaglandin G 2which is converted by the hydroperoxidase component of the enzyme complex into prostaglandin H 2.
This highly unstable compound is rapidly transformed into other prostaglandins, prostacyclin and thromboxanes. If arachidonate is acted upon by a lipoxygenase instead of cyclooxygenase, Hydroxyeicosatetraenoic acids and leukotrienes are formed. They also act as local hormones. Prostaglandins have two derivatives: prostacyclins and thromboxanes.
Prostacyclins are powerful locally acting vasodilators and inhibit the aggregation of blood platelets. Through their role in vasodilation, prostacyclins are also involved in inflammation. They are synthesized in the walls of blood vessels and serve the physiological function of preventing needless clot formation, as well as regulating the contraction of smooth muscle tissue.
Their name comes from their role in clot formation thrombosis. A significant proportion of the fatty acids in the body are obtained from the diet, in the form of triglycerides of either animal or plant origin.
The fatty acids in the fats obtained from land animals tend to be saturated, whereas the fatty acids in the triglycerides of fish and plants are often polyunsaturated and therefore present as oils. These triglycerides cannot be absorbed by the intestine. The activated complex can work only at a water-fat interface.
Therefore, it is essential that fats are first emulsified by bile salts for optimal activity of these enzymes. the fat soluble vitamins and cholesterol and bile salts form mixed micellesin the watery duodenal contents see diagrams on the right.
The contents of these micelles but not the bile salts enter the enterocytes epithelial cells lining the small intestine where they are resynthesized into triglycerides, and packaged into chylomicrons which are released into the lacteals the capillaries of the lymph system of the intestines.
This means that the fat-soluble products of digestion are discharged directly into the general circulation, without first passing through the liver, unlike all other digestion products.
The reason for this peculiarity is unknown. The chylomicrons circulate throughout the body, giving the blood plasma a milky or creamy appearance after a fatty meal.
The fatty acids are absorbed by the adipocytes [ citation needed ]but the glycerol and chylomicron remnants remain in the blood plasma, ultimately to be removed from the circulation by the liver. The free fatty acids released by the digestion of the chylomicrons are absorbed by the adipocytes [ citation needed ]where they are resynthesized into triglycerides using glycerol derived from glucose in the glycolytic pathway [ citation needed ].
These triglycerides are stored, until needed for the fuel requirements of other tissues, in the fat droplet of the adipocyte. The liver absorbs a proportion of the glucose from the blood in the portal vein coming from the intestines. After the liver has replenished its glycogen stores which amount to only about g of glycogen when full much of the rest of the glucose is converted into fatty acids as described below.
These fatty acids are combined with glycerol to form triglycerides which are packaged into droplets very similar to chylomicrons, but known as very low-density lipoproteins VLDL.
These VLDL droplets are processed in exactly the same manner as chylomicrons, except that the VLDL remnant is known as an intermediate-density lipoprotein IDLwhich is capable of scavenging cholesterol from the blood.
This converts IDL into low-density lipoprotein LDLwhich is taken up by cells that require cholesterol for incorporation into their cell membranes or for synthetic purposes e. the formation of the steroid hormones.
The remainder of the LDLs is removed by the liver. Adipose tissue and lactating mammary glands also take up glucose from the blood for conversion into triglycerides. This occurs in the same way as in the liver, except that these tissues do not release the triglycerides thus produced as VLDL into the blood.
All cells in the body need to manufacture and maintain their membranes and the membranes of their organelles. Whether they rely entirely on free fatty acids absorbed from the blood, or are able to synthesize their own fatty acids from blood glucose, is not known.
The cells of the central nervous system will almost certainly have the capability of manufacturing their own fatty acids, as these molecules cannot reach them through the blood brain barrier. Much like beta-oxidationstraight-chain fatty acid synthesis occurs via the six recurring reactions shown below, until the carbon palmitic acid is produced.
The diagrams presented show how fatty acids are synthesized in microorganisms and list the enzymes found in Escherichia coli. FASII is present in prokaryotesplants, fungi, and parasites, as well as in mitochondria.
In animals as well as some fungi such as yeast, these same reactions occur on fatty acid synthase I FASIa large dimeric protein that has all of the enzymatic activities required to create a fatty acid. FASI is less efficient than FASII; however, it allows for the formation of more molecules, including "medium-chain" fatty acids via early chain termination.
by transferring fatty acids between an acyl acceptor and donor. They also have the task of synthesizing bioactive lipids as well as their precursor molecules. Elongation, starting with stearateis performed mainly in the endoplasmic reticulum by several membrane-bound enzymes.
The enzymatic steps involved in the elongation process are principally the same as those carried out by fatty acid synthesisbut the four principal successive steps of the elongation are performed by individual proteins, which may be physically associated.
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Regulation of carnitine palmitoyltransferase CPT I during fasting in rainbow trout Oncorhynchus mykiss promotes increased mitochondrial fatty acid oxidation. Physiol Biochem Zool. McClelland GB, Dalziel AC, Fragoso NM, Moyes CD. Muscle remodeling in relation to blood supply: implications for seasonal changes in mitochondrial enzymes. J Exp Biol. Houle-Leroy P, Garland Jr T, Swallow JG, Guderley H. Effects of voluntary activity and genetic selection on muscle metabolic capacities in house mice Mus domesticus. J Appl Physiol CAS Google Scholar. Heather LC, Cole MA, Lygate CA, Evans RD, Stuckey DJ, Murray AJ, et al. Fatty acid transporter levels and palmitate oxidation rate correlate with ejection fraction in the infarcted rat heart. Cardiovasc Res. Download references. AJMu thanks the Research Councils UK for supporting his academic fellowship and the WYNG Foundation of Hong Kong for supporting work in his laboratory. LDR is supported by the Medical Research Council-Human Nutrition Research Elsie Widdowson Fellowship. AJMo thanks the Natural Sciences and Engineering Research Council for supporting her postdoctoral fellowship. MF acknowledges support from the Medical Research Council G Tom Ashmore, Andrea J. Morash, Aleksandra O. Kotwica, John Finnerty, Cristina Branco, Andrew S. Cowburn, Randall S. Department of Biochemistry, University of Cambridge, Cambridge, UK. Tom Ashmore, Lee D. Roberts, James A. West, Steven A. MRC-Human Nutrition Research, University of Cambridge, Cambridge, UK. You can also search for this author in PubMed Google Scholar. Correspondence to Andrew J. TA, LDR, AJMo, MF, JLG and AJMu designed the protocols, reviewed and edited all data and wrote the paper; TA, LDR, AJMo, AOK, JAW, JF, SAM, BOF, CB, ASC and AJMu conducted the study and performed data analysis. KC provided materials. All authors reviewed the paper. All authors read and approved the final manuscript. Muscle differentiation marker expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of 0, 50 and μM nitrate, and A in the presence and absence of sGC i 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalinone ODQ , 1 μM and B in the presence and absence of PGK i KT, 1 μM. PDF 52 kb. Ucp3 , Acadl and Cpt1b expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of μM nitrate. PDF 24 kb. Open Access This article is distributed under the terms of the Creative Commons Attribution 4. Reprints and permissions. Ashmore, T. et al. Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism. BMC Biol 13 , Download citation. Received : 16 July Accepted : 10 December Published : 22 December Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Download PDF. Research article Open access Published: 22 December Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism Tom Ashmore 1 , 2 , Lee D. Roberts 2 , 3 , Andrea J. Morash 1 , Aleksandra O. Kotwica 1 , John Finnerty 1 , James A. West 2 , Steven A. Murfitt 2 , Bernadette O. Fernandez 4 , Cristina Branco 1 , Andrew S. Cowburn 1 , Kieran Clarke 5 , Randall S. Johnson 1 , Martin Feelisch 4 , Julian L. Murray 1 Show authors BMC Biology volume 13 , Article number: Cite this article Accesses 34 Citations 10 Altmetric Metrics details. Abstract Background Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid FA oxidation in response to increased lipid supply. Results Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. Conclusions Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle. Background As the largest insulin-sensitive tissue in the body, skeletal muscle plays a vital role in maintaining glucose homeostasis via the uptake, storage and oxidation of carbohydrate during the postprandial period. Full size image. Table 1 The effects of 18 days supplementation with low 0. Discussion Herein, we report that moderate doses of dietary inorganic nitrate increase the capacity for FA oxidation in skeletal muscle. Conclusions We have found that a moderate dose of dietary nitrate enhances skeletal muscle FA oxidation capacity by promoting intra-mitochondrial pathways of FA oxidation and, at higher doses, mitochondrial biogenesis. Methods All procedures involving live animals were carried out by a licence holder in accordance with UK Home Office regulations, and underwent review by the University of Cambridge Animal Welfare and Ethical Review Committee. Nitrate and nitrite levels Nitrate was quantified using a dedicated HPLC system ENO, Eicom; Tokyo, Japan , employing sequential ion chromatography, online reduction to nitrite using a cadmium column, and post-column-derivatization with a modified Griess reagent, as described in previously published protocols [ 47 ]. Metabolic profiling Aqueous and organic metabolites were extracted from soleus muscle as described previously [ 23 ]. Respirometry Respirometry was performed on 2—5 mg saponin- permeabilized soleus muscle fibre bundles at 37 °C using Clark-type oxygen electrodes Strathkelvin Instruments, Strathkelvin, UK , essentially as described [ 23 ]. CPT1 activity and palmitate oxidation assays Mitochondria were isolated from soleus muscle from rats according to published protocols [ 48 ]. Enzyme analysis Frozen tissues were powdered under liquid nitrogen with a mortar and pestle and homogenized in potassium phosphate buffer mM KH 2 PO 4 , 5 mM EDTA, 0. PPAR binding assays Frozen tissue was crushed using a liquid nitrogen-cooled pestle and mortar, and homogenates prepared from which the nuclear subcellular fraction was isolated using a commercial kit Cayman Chemical Company, MI, USA. Immunoblotting Immunoblotting for citrate synthase, PGC-1α and malonyl-CoA decarboxylase was performed on soleus muscle lysates according to published protocols [ 56 ]. Availability of supporting data All data supporting the results of this article are available in an online Additional file 3. Abbreviations cAMP: cycline adenosine monophosphate cGMP: cyclic guanosine monophosphate CPT: carnitine palmitoyl-transferase CS: citrate synthase eNOS: endothelial nitric oxide synthase FA: fatty acid HIF: hypoxia-inducible factor HOAD: 3-hydroxyacyl CoA dehydrogenase HRP: horseradish peroxidase MCD: malonyl-CoA decarboxylase NO: nitric oxide OXPHOS: oxidative phosphorylation PGC-1α: PPARγ coactivator 1α PKG: cGMP activated protein kinase PPAR: peroxisome proliferator-activated receptor sGC: soluble guanylyl cyclase SQC: standardised quality controlled UCP: uncoupling protein WAT: white adipose tissue. References Kelley DE. Article PubMed CAS PubMed Central Google Scholar Ukropcova B, McNeil M, Sereda O, de Jonge L, Xie H, Bray GA, et al. Article PubMed CAS PubMed Central Google Scholar Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, et al. Article PubMed CAS Google Scholar Simoneau JA, Veerkamp JH, Turcotte LP, Kelley DE. PubMed CAS Google Scholar Shulman GI. 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CAS Google Scholar Heather LC, Cole MA, Lygate CA, Evans RD, Stuckey DJ, Murray AJ, et al. Article PubMed CAS Google Scholar Download references. Murray Department of Biochemistry, University of Cambridge, Cambridge, UK Tom Ashmore, Lee D. Griffin MRC-Human Nutrition Research, University of Cambridge, Cambridge, UK Lee D. View author publications. Additional information Competing interests The authors declare that they have no competing interests. Lee D. Therefore, in the current study, we sought to determine whether the addition of physical activity to a weight loss program would influence fasting patterns of lipid oxidation and contribute to improved insulin resistance. To provide an appropriate context for assessing the additional effect of physical activity to those of weight loss, the current study also included several measures of body composition with emphasis upon regional fat distribution and, in particular, accumulation of fat within the abdomen and skeletal muscle. An additional seven normal weight volunteers four women and three men completed an identical physical activity program but without weight loss. Baseline characteristics, including BMI, insulin sensitivity, and age, were similar in these two obese groups. Volunteers were weight stable ±2 kg body wt for at least 6 months before the study. None of the volunteers had type 2 diabetes, nor were they participating in any regular exercise before the study. Individuals with treated or untreated hypertension were excluded. The protocol was approved by the University of Pittsburgh Institutional Review Board, and all volunteers gave written informed consent. Whole-body fat mass FM and fat-free mass FFM were assessed by dual-energy X-ray absorptiometry Lunar model DPX-L; Lunar, Madison, WI using software version 1. Cross-sectional areas and location of adipose tissue within the abdomen and thigh were determined using computed tomography CT imaging CT scanner; General Electric, Milwaukee, WI and commercially available software Sice-O-Matic; Tomovision, Montreal, Canada. Abdominal subcutaneous and visceral adipose tissue were measured in one image acquired at the L4-L5 vertebral disc space using an established method Muscle area was further characterized by its mean attenuation value within that range, representing a marker of muscle lipid content such that lower attenuation values reflect higher lipid content Thigh adipose tissue was further distinguished by manual tracings as intermuscular thigh adipose tissue, subfascial adipose tissue, and subcutaneous adipose tissue, as described previously Maximal aerobic capacity V o 2max was measured using an incremental protocol on an electronically braked cycle ergometer Sensormedics, Yorba Linda, CA. Heart rate, blood pressure, and electrocardiogram were recorded before, during, and immediately following this test. Oxygen consumption V o 2 was calculated via direct calorimetry Sensormedics The heart rate- V o 2 relationship was plotted for each person in order to provide individualized exercise intensity prescriptions and also to estimate energy expenditure during their exercise sessions. Plasma glucose and insulin were measured before glucose ingestion and at 30, 60, 90, and min following glucose ingestion. Total area under the oral glucose tolerance test OGTT curve for glucose was computed using a trapezoid approximation procedure, using zero as the baseline. Subjects were instructed to consume a weight-maintaining diet containing at least g carbohydrate for at least 3 days before measurements of insulin sensitivity and to avoid strenuous activity for 36—48 h preceding these studies. Postintervention metabolic assessments were performed 36—48 h following the last exercise session. A catheter was placed in a forearm vein for the insulin infusion Humulin; Eli Lilly, Indianapolis, IN , and an additional catheter was inserted into a radial artery for blood sampling. No tracer was administered to determine glucose disposal since hepatic glucose production was expected to be completely suppressed at this insulin infusion rate in these nondiabetic volunteers. Plasma glucose was determined at 5-min intervals during the clamp. Whole-body indirect calorimetry was performed in the postabsorptive state and during the last 30 min of insulin infusion, using an open-circuit spirometry metabolic monitor system DeltaTrac, Anaheim, CA , in order to calculate fat and glucose oxidation from respiratory gas exchange A week program of exercise training was conducted after completion of baseline metabolic and body composition assessments. Subjects were asked to participate in a minimum of four and a maximum of six exercise sessions weekly. At least one exercise session per week was supervised for each participant to assure that the target exercise intensity and duration was achieved. Subjects were instructed on the proper use of a wireless heart rate monitors Polar, Kempele, Finland to record exercise duration and mean heart rate for estimation of weekly caloric expenditure. Logs of exercise sessions were kept, including exercise duration and average heart rate. At week 8, volunteers performed a submaximal V o 2 exercise test on a cycle ergometer to reestablish the heart rate-energy expenditure relationship. During weeks 5—8, exercise sessions were increased to 40 min at the same intensity. All subjects kept detailed 7-day food records for the entire week intervention. The week caloric restriction-induced weight loss program conducted in the subset of obese individuals has been described previously Plasma glucose during the OGTT and glucose clamp were measured using an automated glucose oxidase reaction Glucose Analyzer; YSI, Yellow Springs, OH. Serum insulin was determined using commercially available radioimmunoassay kits Pharmacia, Uppsala, Sweden. Data are presented as mean ± SE, unless otherwise indicated. Changes in whole-body fatty acid oxidation, insulin sensitivity R d , physical fitness V o 2max , and body composition were compared using paired t tests. Differences in insulin sensitivity among the intervention groups were examined using a two-way ANOVA group × time. Bivariate and multivariate linear regression analysis was used to determine whether the changes in physical fitness, fatty acid oxidation, or body composition were associated with improvements in insulin sensitivity. All statistics were performed using JMP version 3. Most of this was comprised of the loss of fat mass 5. There was a modest decrease in FFM. All volunteers underwent CT imaging for regional fat distribution, but unfortunately CT data for eight subjects were lost due to irreparable damage to a storage disk. Mean muscle attenuation values did not change. Of the changes in regional fat depots, only the change in visceral fat was different in men and women; men lost significantly more visceral fat than women 35 vs. Physical fitness V o 2max increased on average by The average intensity per exercise session was 7. Although the exercise prescription was based on uniform guidelines, there was considerable variation among participants in their average duration and intensity of exercise. Of the 25 volunteers who completed the intervention, only 2 subjects failed to exhibit improvements in insulin sensitivity. There was no change in rates of insulin-stimulated glucose oxidation Fig. Systemic energy expenditure during insulin-stimulated conditions was not altered by the intervention Table 2. The rate of systemic energy expenditure expressed per kilogram FFM did not change following intervention Table 2 , though the absolute rate of energy expenditure was reduced, reflecting a reduction in FFM Table 1. Correspondingly, postabsorptive glucose oxidation was significantly reduced Table 2. The contribution of protein oxidation during postabsorptive conditions was minor 7. The intensity of exercise and the amount of weekly exercise performed, but not changes in fat mass, were correlated with the change in fasting RQ Table 3. We examined, initially using bivariate analyses, potential correlates of the improvement in insulin-stimulated glucose metabolism induced by the weight loss and physical activity intervention. We also examined whether there were baseline preintervention characteristics that were predictive of the intervention-induced change in insulin sensitivity Table 4. Those who were more insulin resistant before the intervention had greater improvements in insulin sensitivity, as did those who had low fasting rates of fat oxidation at baseline. This may imply that those who need the intervention the most, i. It is possible, however, that this was influenced by regression to the mean, resulting in an overestimation of the association between baseline and change values. Initial patterns of adipose tissue distribution or level of physical fitness did not predict improved insulin sensitivity. Next, we examined whether changes induced in body composition, fitness, and fasting metabolism predicted the improvement in insulin sensitivity Table 4. The loss of total body fat, subcutaneous abdominal fat, and subfascial thigh fat, but not the selective loss of visceral fat or subcutaneous thigh fat, was associated with the improved insulin sensitivity. Changes in V o 2max and exercise energy expenditure tended to be associated with the improved insulin sensitivity. However, the strongest simple correlate with improved insulin sensitivity was increased fasting rates of fat oxidation and, accordingly, the reduced fasting RQ, as shown in Table 3 and Fig. Similar associations were observed with respect to the nonoxidative component of insulin sensitivity. Stepwise multivariate regression analysis was then used to examine the interaction and interdependence of these changes in physiologic and body composition parameters. After accounting for increases in fasting rates of fat oxidation, the selective loss of subfascial thigh fat emerged next, followed by the loss in total body fat as independent predictors of this improvement. Enhanced postabsorptive fat oxidation remained a significant correlate of improved insulin sensitivity after adjusting for the loss of body fat or improved physical fitness in the model. To further examine whether the addition of physical activity provided more metabolic benefit than weight loss alone, we compared the findings from the current research volunteers with that attained in a separate group of obese subjects BMI Figure 3 illustrates the improvements in insulin sensitivity in a subset of men and women 9 women and 7 men who completed the exercise and caloric restriction intervention, as compared with those who either completed a program of caloric restriction only 29 10 women and 6 men or exercise without weight loss 4 women and 3 men. The subset of individuals from the two groups were matched for fat mass lost: 7. Subjects in these groups were of similar age 36 ± 5, 38 ± 6, and 39 ± 5 years for exercise-only, diet-only, and exercise plus diet groups, respectively. Identical protocols, including timing of the glucose clamps following intervention, were followed in each of these three groups, although it is possible that the separate interventions were confounded by time effects. Physical activity and moderate sustained weight loss are advocated for the treatment of obesity, the insulin resistance syndrome, and the prevention of type 2 diabetes. It is not clear how physical activity and weight loss interact in achieving these effects. Even a single session of moderate to strenuous intensity exercise can induce a transient improvement in insulin sensitivity 13 , effects that appear to wane within a few days Exercise also increases oxidative enzyme activity in skeletal muscle and induces related biochemical and morphologic changes that would seem to confer a metabolic basis for improved insulin sensitivity. However, in clinical interventions carefully controlled with respect to matching energy balance, exercise in the absence of weight loss has been found to have modest 17 , 30 or no 16 effect on insulin sensitivity. These findings have suggested that exercise might be of lesser value than weight loss as an intervention for the insulin resistance syndrome. We sought to address this apparent conundrum in two ways. First, we employed exercise interventions in conjunction with weight loss, an intervention model that more appropriately matches clinical recommendations than either intervention in isolation. The improvement in insulin sensitivity in these obese subjects was greater than those observed for other studies examining exercise or weight loss alone 16 , Similarly, Bogardus et al. Ross et al. Therefore, these results suggest a synergistic effect of weight loss and exercise to improve insulin resistance. The timing of the insulin sensitivity measure after the last exercise bout could also affect the magnitude of the improvements in insulin sensitivity across studies. It is important to account for the influence of acute exercise on insulin sensitivity. We chose 36—48 h after the last exercise bout to perform the glucose clamp to avoid both the acute exercise effects and potential de-training effects. This likely contributed to greater improvements with exercise training compared with other studies that performed the insulin sensitivity measure 4—6 days following the last exercise bout 16 , 17 , Improved insulin sensitivity through either weight loss or exercise has been associated with the loss of abdominal visceral fat 17 , Obese subjects in the present study lost a significant amount of fat from several region-specific depots, including abdominal fat and subfascial thigh fat. In multivariate analysis, only the loss of subfascial thigh fat was independently correlated with the improvement in insulin sensitivity after adjusting for the change in systemic fatty acid oxidation. The attenuation of muscle on CT as a marker of muscle lipid 26 did not change, although muscle attenuation can increase with diet-induced weight loss However, lipid within muscle can be higher in endurance-trained athletes 23 , raising the possibility that weight loss and exercise have counterbalancing effects on muscle lipid, perhaps explaining the lack of change in muscle attenuation. Our findings indicate that the combination of exercise and weight loss enhances not only the insulin-stimulated capacity for glucose utilization, but also enhances the capacity for fat oxidation during fasting conditions. The improvement in fat oxidation during fasting conditions was associated with characteristics specific for the exercise intervention, namely intensity and duration of physical activity, but was not associated significantly with overall or regional loss of adiposity. In turn, the magnitude of improvement in insulin-stimulated glucose metabolism was strongly related to the concomitant increase in fat oxidation during fasting conditions. Impaired insulin-stimulated glucose metabolism is the most well-established manifestation of skeletal muscle insulin resistance, but it is also recognized that another very important facet of skeletal muscle insulin resistance is altered patterns of fat oxidation. Normally, insulin effectively suppresses fat oxidation 31 , but this suppression of fat oxidation is impaired in insulin resistance Additionally, it has long been noted that in those with normal insulin sensitivity, skeletal muscle has a high reliance upon fat oxidation during fasting conditions In contrast, among those with obesity and insulin resistance, and in those with type 2 diabetes, fasting rates of fat oxidation are reduced in skeletal muscle In prior cross-sectional studies, we observed that impaired fat oxidation during fasting predicts severity of insulin-resistant glucose metabolism We had also noted that following diet-induced weight loss, but without changes in patterns of physical activity, there was improvement in insulin-stimulated glucose metabolism but there was not a significant change in fasting rates of fat oxidation Thus, in weight loss without exercise, there appeared to be a separation in the effects on those aspects of insulin resistance related to insulin-stimulated glucose metabolism from those aspects regulating rates of fat oxidation. The current study sought to probe whether the addition of exercise might address insulin resistance in a manner different from weight loss alone. There are several conceptual reasons to postulate that physical activity might modify the aspects of insulin resistance related to the capacity for fat oxidation. First, exercise training at moderate intensity is typically associated with induction of a higher capacity for, and reliance upon, fat oxidation during the exercise session Second, chronic exercise effects, namely increased activity of oxidative enzymes and increased capillary density, might facilitate fatty acid utilization both at rest and during physical activity. In contrast, during physical activity, muscle generates a negative energy balance and is comprised of consumption of muscle glycogen and muscle triglyceride 24 , In contrast to the decrease in energy expenditure induced by weight loss through reductions in caloric intake 18 , the lack of change in resting energy expenditure despite weight loss was likely due to the exercise component. |
| Top bar navigation | Ohno, Y. The promoter oxidizibg the Hilpda gene ocidizing a number Enhanced fat oxidizing mechanisms hypoxia-responsive elements that are targeted by the transcription factors HIF-1 EEnhanced HIF-2 ref. In this article, mechainsms will take a dive into mevhanisms fat oxidation Enhznced and how to make your body burn more fats than sugars during exercise. This association was replicated in numerous studies and extended to associations of the mutation with steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma Consistent with the strong inhibitory effect of G0S2 on ATGL activity, its tissue-specific overexpression leads to steatosis in cardiac muscle or liver in mice The longer you train, the more you deplete glycogen and once those stores are depleted, you will switch to burning fat for fuel. |
| Recent Posts | This study aimed to compare Enhanced fat oxidizing mechanisms oxidation, hormonal and plasma metabolite kinetics during exercise in mechanismz L Enhqnced obese O men. Enoyl-ACP reductase. Gillilan, R. If we want to burn fat, what are the best methods to do this? The human genome carries nine genes encoding patatin-domain-containing proteins, which are designated patatin-like phospholipase domain 1—9 PNPLA1 — PNPLA9 Zimmermann, R. |
Enhanced fat oxidizing mechanisms -
The column was re-equilibrated for a further 3 min at cessation of the gradient. Malonyl-CoA was normalised to a universally 13 C- and 15 N-labelled glutamate internal standard Cambridge Isotope Laboratories Inc. cGMP data were normalised to the same internal standard and compared with an 8-point cGMP calibration line with concentrations ranging from 1 nM to 50 μM.
cAMP data were normalised to the same internal standard and compared with an 8-point cAMP calibration line with concentrations ranging from 1 nM to 50 μM. Organic extracts were reconstituted in μL chloroform:methanol and mixed thoroughly.
When fully dissolved, μL of each sample were transferred to a 3. This derivatisation procedure converts FAs to fatty acid methyl esters. The vials were then vortexed, and incubated for 90 min at 80 °C.
After cooling, μL HPLC-grade water and 1 mL hexane were added, and the vials thoroughly mixed by vortexing. The upper, organic, layer was transferred to a 2 mL GC vial and allowed to dry overnight in a fume hood. FAME samples were reconstituted in μL hexane and a 2 μL injection run on a Trace GC Ultra coupled to a Trace DSQ II single quadrupolar mass spectrometer Thermo Scientific.
The transfer line from the oven to the mass spectrometer was heated to °C and the inlet to °C. Chromatograms were processed using the Xcalibur software suite version 2.
Individual peaks were integrated and subsequently normalised to total peak intensity. Peaks were assigned based on fragmentation patterns and matched to the National Institute of Standards and Technology NIST, USA library.
Respirometry was performed on 2—5 mg saponin- permeabilized soleus muscle fibre bundles at 37 °C using Clark-type oxygen electrodes Strathkelvin Instruments, Strathkelvin, UK , essentially as described [ 23 ]. Briefly, 0. Muscle fibres were recovered from the electrode chamber to allow normalisation of oxygen consumption rates to dry muscle mass.
For C2C12 studies, cells suspended in μL of respiration medium were added to the Clark-type electrode chambers for analysis, along with 0. Mitochondria were isolated from soleus muscle from rats according to published protocols [ 48 ].
CPT1 activity was determined in soleus muscle mitochondrial isolates using 3 H-carnitine according to previously published protocols [ 49 ]. The assay buffer contained mM Tris—HCl, 0. The reaction was initiated with the addition of 20 μL of mitochondrial homogenates and incubated for 8 min at 37 °C.
The reaction was then terminated by the addition of 60 μL of HCl. The palmitoyl- 3 H-carnitine formed during the reaction was separated according to previously published protocols [ 50 , 51 ] and the radioactivity counted to determine CPT1 activity.
Palmitate oxidation rates were measured in soleus muscle mitochondrial isolates using 14 C-palmitate according to previously published methods [ 52 , 53 ]. Briefly, modified Krebs-Ringer buffer mM NaCl, 2. A suspended microcentrifuge tube containing μL of benzethonium hydroxide were placed inside the vial to trap the 14 CO 2 produced during the reaction.
Mitochondria were added to the system, which was then sealed with a rubber cap. The reaction was initiated by the addition of palmitate:BSA complex containing 1 μCi of 14 C-palmitate to a final palmitate concentration of μM via a syringe through the rubber cap.
The vial was incubated for 30 min at 37 °C before termination with 50 μL of HClO 4. The microcentrifuge tube containing the benzethonium hydroxide and trapped 14 CO 2 was then removed, transferred to a scintillation vial, and the radioactivity counted.
Frozen tissues were powdered under liquid nitrogen with a mortar and pestle and homogenized in potassium phosphate buffer mM KH 2 PO 4 , 5 mM EDTA, 0. HOAD activity was assayed according to a published protocol [ 54 ].
The assay buffer contained 50 mM imidazole pH 7. NADH absorbance was monitored at nm for 3 min. CS was assayed according to a previously published protocol [ 55 ].
The assay buffer contained 20 mM Tris pH 8. The reaction was initiated by the addition of 0. Frozen tissue was crushed using a liquid nitrogen-cooled pestle and mortar, and homogenates prepared from which the nuclear subcellular fraction was isolated using a commercial kit Cayman Chemical Company, MI, USA.
Ice-cold hypotonic buffer was added to crushed tissue in a 5 μL:1 mg ratio. The solution was homogenised using a polytron before being incubated on ice for 15 min.
The pellet was resuspended in μL hypotonic buffer and incubated on ice. The pellet was resuspended in 50 μL ice-cold extraction buffer, vortexed vigorously for 15 s and subsequently rocked on a shaking platform for 15 min on ice.
A small aliquot was used to quantify protein concentration using a spectrophotometer. The wells of a plate were coated by a double-stranded DNA dsDNA sequence containing the peroxisome proliferator response element.
By utilising nuclear extracts, only binding of proteins within the nucleus are quantified, which effectively represents activated PPARs. Nuclear extracts and all kit reagents were allowed to equilibrate to room temperature before use.
To the blank and non-specific binding wells μL of transcription factor assay buffer were added. In the competitor dsDNA wells, 80 μL transcription factor assay buffer were added followed by 10 μL PPAR competitor dsDNA. In the positive control wells, 90 μL of transcription factor assay buffer were added followed by 90 μL of positive control.
Finally, 90 μL of transcription factor assay buffer were added to each sample well followed by 10 μL nuclear extract. The plate was covered and incubated overnight at 4 °C without agitation.
The wells were then emptied and washed five times with μL of wash buffer, with care taken on the final wash to remove residual buffer.
The wash step was repeated as above and any residual wash buffer carefully removed. To each well, except the blanks, μL of horseradish peroxidase HRP -conjugated secondary antibody were added and the plate covered and incubated for 1 h at room temperature without agitation.
The wells were washed with μL of wash buffer as above, before μL of developing solution was added and the plate incubated for 30 min at room temperature with gentle shaking.
After incubation, μL stop solution were added to each well and the absorbance read at nm within 5 min. RNA concentration was quantified at nm using a SmartSpecPlus spectrophotometer Bio-Rad.
For analysis of steady-state mRNA levels, the relative abundance of transcripts of interest was assessed by quantitative-PCR in SYBR Green FastStart Universal Master Mix Applied Biosystems with a StepOnePlus detection system Applied Biosystems.
QuantiTect primer assays for rat Ppara and Pparbd were obtained from QIAgen. For gene analysis in C2C12 cells, RNA extraction was performed as above, with the single difference being that μL of lysis buffer were added directly to the wells, and subsequently pipetted onto the spin columns for purification.
Production of cDNA and RT-qPCR analysis of Myod , Tnni1 , Tnni2 , CptIb , Acadl , Hadh , Ucp3 , Cycs , and Ndufs1 expression proceeded as above, using QuantiTect primer assays obtained from QIAgen.
Immunoblotting for citrate synthase, PGC-1α and malonyl-CoA decarboxylase was performed on soleus muscle lysates according to published protocols [ 56 ]. Membranes were incubated in primary antibody solution containing rabbit polyclonal IgG raised against CS, PGC-1α or MCD all Abcam, UK for 2 h at room temperature CS and PGC-1α or overnight at 4 °C MCD.
After washing with TBS-T for 2 h with a solution change every 15 min, membranes were incubated in secondary antibody solution containing goat anti-rabbit IgG, conjugated to HRP for 1 h, before visualisation using ECL-plus and quantification as previously described [ 56 ].
Samples diluted 20 times and standards were incubated in duplicate on a well plate containing an immobilised antibody to mouse CPT1B for 2 h at 37 °C.
After unbound substances were washed away, biotin conjugated to a secondary antibody raised against CPT1B was added, and the plate incubated for 1 h at 37 °C.
After further washes, a streptavidin-HRP conjugate was added and incubated for 1 h at 37 °C, followed by further washes to remove unbound conjugate. A substrate solution was then added to the wells and incubated, protected from light, for 30 min at 37 °C.
Diluted HCl was added to stop the enzymatic reaction, and finally the optical density was measured at nm with readings at nm subtracted to account for optical imperfections in the plate. Data were collated and normalised to total protein in the original sample.
Analysis of variance ANOVA was used to determine significant differences across the four groups of the hypoxia and dose—response studies. Data were collated in Excel before 1- or 2-way analysis of variance ANOVA was used to determine significant differences across experimental groups Graphpad, Instat.
Bonferroni post-hoc analysis was used for multiple analysis of selected groups, where appropriate. All data supporting the results of this article are available in an online Additional file 3. Kelley DE. Skeletal muscle fat oxidation: timing and flexibility are everything.
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Cardiovasc Res. Download references. AJMu thanks the Research Councils UK for supporting his academic fellowship and the WYNG Foundation of Hong Kong for supporting work in his laboratory.
LDR is supported by the Medical Research Council-Human Nutrition Research Elsie Widdowson Fellowship. AJMo thanks the Natural Sciences and Engineering Research Council for supporting her postdoctoral fellowship.
MF acknowledges support from the Medical Research Council G Tom Ashmore, Andrea J. Morash, Aleksandra O. Kotwica, John Finnerty, Cristina Branco, Andrew S. Cowburn, Randall S.
Department of Biochemistry, University of Cambridge, Cambridge, UK. Tom Ashmore, Lee D. Roberts, James A. West, Steven A. MRC-Human Nutrition Research, University of Cambridge, Cambridge, UK.
You can also search for this author in PubMed Google Scholar. Correspondence to Andrew J. TA, LDR, AJMo, MF, JLG and AJMu designed the protocols, reviewed and edited all data and wrote the paper; TA, LDR, AJMo, AOK, JAW, JF, SAM, BOF, CB, ASC and AJMu conducted the study and performed data analysis.
KC provided materials. All authors reviewed the paper. All authors read and approved the final manuscript. Muscle differentiation marker expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of 0, 50 and μM nitrate, and A in the presence and absence of sGC i 1H-[1,2,4] oxadiazolo[4,3-a]quinoxalinone ODQ , 1 μM and B in the presence and absence of PGK i KT, 1 μM.
PDF 52 kb. Ucp3 , Acadl and Cpt1b expression in C2C12 myoblasts cultured and differentiated over 6 days in the presence of μM nitrate. PDF 24 kb. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.
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Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Skip to main content. Search all BMC articles Search. Prostaglandins have been found in almost every tissue in humans and other animals.
They are enzymatically derived from arachidonic acid, a carbon polyunsaturated fatty acid. Every prostaglandin therefore contains 20 carbon atoms, including a 5-carbon ring. They are a subclass of eicosanoids and form the prostanoid class of fatty acid derivatives. The prostaglandins are synthesized in the cell membrane by the cleavage of arachidonate from the phospholipids that make up the membrane.
This is catalyzed either by phospholipase A 2 acting directly on a membrane phospholipid, or by a lipase acting on DAG diacyl-glycerol. The arachidonate is then acted upon by the cyclooxygenase component of prostaglandin synthase. This forms a cyclopentane ring roughly in the middle of the fatty acid chain.
The reaction also adds 4 oxygen atoms derived from two molecules of O 2. The resulting molecule is prostaglandin G 2 , which is converted by the hydroperoxidase component of the enzyme complex into prostaglandin H 2.
This highly unstable compound is rapidly transformed into other prostaglandins, prostacyclin and thromboxanes. If arachidonate is acted upon by a lipoxygenase instead of cyclooxygenase, Hydroxyeicosatetraenoic acids and leukotrienes are formed. They also act as local hormones.
Prostaglandins have two derivatives: prostacyclins and thromboxanes. Prostacyclins are powerful locally acting vasodilators and inhibit the aggregation of blood platelets. Through their role in vasodilation, prostacyclins are also involved in inflammation. They are synthesized in the walls of blood vessels and serve the physiological function of preventing needless clot formation, as well as regulating the contraction of smooth muscle tissue.
Their name comes from their role in clot formation thrombosis. A significant proportion of the fatty acids in the body are obtained from the diet, in the form of triglycerides of either animal or plant origin.
The fatty acids in the fats obtained from land animals tend to be saturated, whereas the fatty acids in the triglycerides of fish and plants are often polyunsaturated and therefore present as oils.
These triglycerides cannot be absorbed by the intestine. The activated complex can work only at a water-fat interface. Therefore, it is essential that fats are first emulsified by bile salts for optimal activity of these enzymes.
the fat soluble vitamins and cholesterol and bile salts form mixed micelles , in the watery duodenal contents see diagrams on the right.
The contents of these micelles but not the bile salts enter the enterocytes epithelial cells lining the small intestine where they are resynthesized into triglycerides, and packaged into chylomicrons which are released into the lacteals the capillaries of the lymph system of the intestines.
This means that the fat-soluble products of digestion are discharged directly into the general circulation, without first passing through the liver, unlike all other digestion products. The reason for this peculiarity is unknown.
The chylomicrons circulate throughout the body, giving the blood plasma a milky or creamy appearance after a fatty meal. The fatty acids are absorbed by the adipocytes [ citation needed ] , but the glycerol and chylomicron remnants remain in the blood plasma, ultimately to be removed from the circulation by the liver.
The free fatty acids released by the digestion of the chylomicrons are absorbed by the adipocytes [ citation needed ] , where they are resynthesized into triglycerides using glycerol derived from glucose in the glycolytic pathway [ citation needed ].
These triglycerides are stored, until needed for the fuel requirements of other tissues, in the fat droplet of the adipocyte. The liver absorbs a proportion of the glucose from the blood in the portal vein coming from the intestines.
After the liver has replenished its glycogen stores which amount to only about g of glycogen when full much of the rest of the glucose is converted into fatty acids as described below. These fatty acids are combined with glycerol to form triglycerides which are packaged into droplets very similar to chylomicrons, but known as very low-density lipoproteins VLDL.
These VLDL droplets are processed in exactly the same manner as chylomicrons, except that the VLDL remnant is known as an intermediate-density lipoprotein IDL , which is capable of scavenging cholesterol from the blood. This converts IDL into low-density lipoprotein LDL , which is taken up by cells that require cholesterol for incorporation into their cell membranes or for synthetic purposes e.
the formation of the steroid hormones. The remainder of the LDLs is removed by the liver. Adipose tissue and lactating mammary glands also take up glucose from the blood for conversion into triglycerides. This occurs in the same way as in the liver, except that these tissues do not release the triglycerides thus produced as VLDL into the blood.
All cells in the body need to manufacture and maintain their membranes and the membranes of their organelles. Whether they rely entirely on free fatty acids absorbed from the blood, or are able to synthesize their own fatty acids from blood glucose, is not known.
The cells of the central nervous system will almost certainly have the capability of manufacturing their own fatty acids, as these molecules cannot reach them through the blood brain barrier. Much like beta-oxidation , straight-chain fatty acid synthesis occurs via the six recurring reactions shown below, until the carbon palmitic acid is produced.
The diagrams presented show how fatty acids are synthesized in microorganisms and list the enzymes found in Escherichia coli. FASII is present in prokaryotes , plants, fungi, and parasites, as well as in mitochondria. In animals as well as some fungi such as yeast, these same reactions occur on fatty acid synthase I FASI , a large dimeric protein that has all of the enzymatic activities required to create a fatty acid.
FASI is less efficient than FASII; however, it allows for the formation of more molecules, including "medium-chain" fatty acids via early chain termination. by transferring fatty acids between an acyl acceptor and donor. They also have the task of synthesizing bioactive lipids as well as their precursor molecules.
Elongation, starting with stearate , is performed mainly in the endoplasmic reticulum by several membrane-bound enzymes. The enzymatic steps involved in the elongation process are principally the same as those carried out by fatty acid synthesis , but the four principal successive steps of the elongation are performed by individual proteins, which may be physically associated.
Abbreviations: ACP — Acyl carrier protein , CoA — Coenzyme A , NADP — Nicotinamide adenine dinucleotide phosphate. Note that during fatty synthesis the reducing agent is NADPH , whereas NAD is the oxidizing agent in beta-oxidation the breakdown of fatty acids to acetyl-CoA.
This difference exemplifies a general principle that NADPH is consumed during biosynthetic reactions, whereas NADH is generated in energy-yielding reactions. The source of the NADPH is two-fold.
NADPH is also formed by the pentose phosphate pathway which converts glucose into ribose, which can be used in synthesis of nucleotides and nucleic acids , or it can be catabolized to pyruvate. In humans, fatty acids are formed from carbohydrates predominantly in the liver and adipose tissue , as well as in the mammary glands during lactation.
The pyruvate produced by glycolysis is an important intermediary in the conversion of carbohydrates into fatty acids and cholesterol.
However, this acetyl CoA needs to be transported into cytosol where the synthesis of fatty acids and cholesterol occurs.
This cannot occur directly. To obtain cytosolic acetyl-CoA, citrate produced by the condensation of acetyl CoA with oxaloacetate is removed from the citric acid cycle and carried across the inner mitochondrial membrane into the cytosol.
The oxaloacetate is returned to mitochondrion as malate and then converted back into oxaloacetate to transfer more acetyl-CoA out of the mitochondrion. Acetyl-CoA is formed into malonyl-CoA by acetyl-CoA carboxylase , at which point malonyl-CoA is destined to feed into the fatty acid synthesis pathway.
Acetyl-CoA carboxylase is the point of regulation in saturated straight-chain fatty acid synthesis, and is subject to both phosphorylation and allosteric regulation. Regulation by phosphorylation occurs mostly in mammals, while allosteric regulation occurs in most organisms.
Allosteric control occurs as feedback inhibition by palmitoyl-CoA and activation by citrate. When there are high levels of palmitoyl-CoA, the final product of saturated fatty acid synthesis, it allosterically inactivates acetyl-CoA carboxylase to prevent a build-up of fatty acids in cells.
Citrate acts to activate acetyl-CoA carboxylase under high levels, because high levels indicate that there is enough acetyl-CoA to feed into the Krebs cycle and produce energy. High plasma levels of insulin in the blood plasma e. after meals cause the dephosphorylation and activation of acetyl-CoA carboxylase, thus promoting the formation of malonyl-CoA from acetyl-CoA, and consequently the conversion of carbohydrates into fatty acids, while epinephrine and glucagon released into the blood during starvation and exercise cause the phosphorylation of this enzyme, inhibiting lipogenesis in favor of fatty acid oxidation via beta-oxidation.
Disorders of fatty acid metabolism can be described in terms of, for example, hypertriglyceridemia too high level of triglycerides , or other types of hyperlipidemia. These may be familial or acquired. Familial types of disorders of fatty acid metabolism are generally classified as inborn errors of lipid metabolism.
These disorders may be described as fatty acid oxidation disorders or as a lipid storage disorders , and are any one of several inborn errors of metabolism that result from enzyme or transport protein defects affecting the ability of the body to oxidize fatty acids in order to produce energy within muscles, liver, and other cell types.
When a fatty acid oxidation disorder affects the muscles, it is a metabolic myopathy. Moreover, cancer cells can display irregular fatty acid metabolism with regard to both fatty acid synthesis [44] and mitochondrial fatty acid oxidation FAO [45] that are involved in diverse aspects of tumorigenesis and cell growth.
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Download as PDF Printable version. Set of biological processes. Main article: Fatty acid synthesis. Main article: Citric acid cycle § Glycolytic end products are used in the conversion of carbohydrates into fatty acids.
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For more information about PLOS Mecanisms Enhanced fat oxidizing mechanisms, click here. This study Enhanceed to compare fat oxidation, hormonal and plasma Weight management inspiration kinetics Enyanced exercise in lean L and Enbanced O men. Sixteen L and 16 O men [Body Mass Index BMI : Fat oxidation rates FORs were determined using indirect calorimetry. A sinusoidal model, including 3 independent variables dilatation, symmetry, translationwas used to describe fat oxidation kinetics and determine the intensity Fat max eliciting maximal fat oxidation. Blood samples were drawn for the hormonal and plasma metabolite determination at each step of Incr.
BMC Biology volume 13 oxidizinb, Article number: Cite this article. Oixdizing Enhanced fat oxidizing mechanisms. Insulin sensitivity in skeletal muscle oxidizjng associated with metabolic flexibility, including oxodizing high capacity oxidizimg increase fatty acid FA oxidation Citrus bioflavonoids for respiratory health response Enhanced fat oxidizing mechanisms increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms.
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