Skeletal Muscle Of Obese Humans Research Articles

Diabetes is accompanied by changes in the levels of proteins involved in endosomal GLUT4 trafficking in obese human skeletal muscle.

IntroductionThe regulated delivery of the glucose transporter GLUT4 from intracellular stores to the plasma membrane underpins insulin‐stimulated glucose transport. Insulin‐stimulated glucose transport is impaired in skeletal muscle of patients with type‐2 diabetes, and this may arise because of impaired intracellular trafficking of GLUT4. However, molecular details of any such impairment have not been described. We hypothesized that GLUT4 and/or levels of proteins involved in intracellular GLUT4 trafficking may be impaired in skeletal muscle in type‐2 diabetes and tested this in obese individuals without and without type‐2 diabetes.MethodsWe recruited 12 participants with type‐2 diabetes and 12 control participants. All were overweight or obese with BMI of 25–45 kg/m2. Insulin sensitivity was measured using an insulin suppression test (IST), and vastus lateralis biopsies were taken in the fasted state. Cell extracts were immunoblotted to quantify levels of a range of proteins known to be involved in intracellular GLUT4 trafficking.ResultsObese participants with type‐2 diabetes exhibited elevated fasting blood glucose and increased steady state glucose infusion rates in the IST compared with controls. Consistent with this, skeletal muscle from those with type‐2 diabetes expressed lower levels of GLUT4 (30%, p = .014). Levels of Syntaxin4, a key protein involved in GLUT4 vesicle fusion with the plasma membrane, were similar between groups. By contrast, we observed reductions in levels of Syntaxin16 (33.7%, p = 0.05), Sortilin (44%, p = .006) and Sorting Nexin‐1 (21.5%, p = .039) and −27 (60%, p = .001), key proteins involved in the intracellular sorting of GLUT4, in participants with type‐2 diabetes.ConclusionsWe report significant reductions of proteins involved in the endosomal trafficking of GLUT4 in skeletal muscle in obese people with type 2 diabetes compared with age‐ and weight‐matched controls. These abnormalities of intracellular GLUT4 trafficking may contribute to reduced whole body insulin sensitivity.

Vaspin promotes insulin sensitivity of elderly muscle and is upregulated in obesity.

Adipokines have emerged as central mediators of insulin sensitivity and metabolism, in part due to the known association of obesity with metabolic syndrome disorders such as type 2 diabetes. Recent studies in rodents have identified the novel adipokine vaspin, as playing a protective role in inflammatory metabolic diseases by functioning to promote insulin sensitivity during metabolic stress. However, at present the skeletal muscle and adipose tissue expression of vaspin in humans is poorly characterised. Furthermore, the functional role of vaspin in skeletal muscle insulin sensitivity has not been studied. Since skeletal muscle is the major tissue for insulin-stimulated glucose uptake understanding the functional role of vaspin in human muscle insulin signalling is critical in determining its role in glucose homeostasis. The objective of this study was to profile the skeletal muscle and subcutaneous adipose tissue expression of vaspin in humans of varying adiposity and to determine the functional role of vaspin in mediating insulin signalling and glucose uptake in human skeletal muscle. Our data shows that vaspin is secreted from both human subcutaneous adipose tissue and skeletal muscle, and is more highly expressed in obese older individuals compared to lean older individuals. Furthermore, we demonstrate that vaspin induces activation of the PI3K/AKT axis, independent of insulin receptor activation, promotes GLUT4 expression and translocation and sensitises older obese human skeletal muscle to insulin-mediated glucose uptake.

Skeletal Muscle–Specific Deletion of MKP-1 Reveals a p38 MAPK/JNK/Akt Signaling Node That Regulates Obesity-Induced Insulin Resistance

Stress responses promote obesity and insulin resistance, in part, by activating the stress-responsive mitogen-activated protein kinases (MAPKs), p38 MAPK, and c-Jun NH2-terminal kinase (JNK). Stress also induces expression of MAPK phosphatase-1 (MKP-1), which inactivates both JNK and p38 MAPK. However, the equilibrium between JNK/p38 MAPK and MKP-1 signaling in the development of obesity and insulin resistance is unclear. Skeletal muscle is a major tissue involved in energy expenditure and glucose metabolism. In skeletal muscle, MKP-1 is upregulated in high-fat diet-fed mice and in skeletal muscle of obese humans. Mice lacking skeletal muscle expression of MKP-1 (MKP1-MKO) showed increased skeletal muscle p38 MAPK and JNK activities and were resistant to the development of diet-induced obesity. MKP1-MKO mice exhibited increased whole-body energy expenditure that was associated with elevated levels of myofiber-associated mitochondrial oxygen consumption. miR-21, a negative regulator of PTEN expression, was upregulated in skeletal muscle of MKP1-MKO mice, resulting in increased Akt activity consistent with enhanced insulin sensitivity. Our results demonstrate that skeletal muscle MKP-1 represents a critical signaling node through which inactivation of the p38 MAPK/JNK module promotes obesity and insulin resistance.

Adipogenic progenitors from obese human skeletal muscle give rise to functional white adipocytes that contribute to insulin resistance.

Recent reports indicate that inter/intramuscular adipose tissue (IMAT), composed by adipocytes underneath the deep fascia of the muscles, is positively correlated with aging, obesity and insulin resistance in humans. However, no molecular/cellular evidence is available to support these interactions. The current study aimed to better characterize human skeletal muscle-derived adipogenic progenitors obtained from obese volunteers and investigate the impact of derived adipocytes on insulin action in primary skeletal muscle cells. Primary cultured stroma-vascular fraction (SVF) obtained from vastus lateralis muscle biopsies of middle-aged obese subjects was immunoseparated (magnetic beads or flow cytometry). The characteristics and/or metabolic phenotype of CD56(+), CD56(-) and CD56(-)CD15(+) cellular fractions were investigated by complementary approaches (flow cytometry, cytology, quantitative PCR and metabolic assays). The effects of conditioned media from CD56(-)CD15(+) cells differentiated into adipocytes on insulin action and signaling in human primary myotubes was also examined. Our data indicate that CD56(+) and CD56(-) cellular fractions isolated from cultured SVF of human muscle contain two distinct committed progenitors: CD56(+) cells (that is, satellite cells) as myogenic progenitors and CD15(+) cells as adipogenic progenitors, respectively. CD56(-)CD15(+)-derived adipocytes display the phenotype and metabolic properties of white adipocytes. Secretions of CD56(-)CD15(+) cells differentiated into functional white adipocytes reduced insulin-mediated non-oxidative glucose disposal (P=0.0002) and insulin signaling. Using in-vitro models, we show for the first time that secretions of skeletal muscle adipocytes are able to impair insulin action and signaling of muscle fibers. This paracrine effect could explain, at least in part, the negative association between high levels of IMAT and insulin sensitivity in obesity and aging.

Selective overexpression of Toll-like receptor-4 in skeletal muscle impairs metabolic adaptation to high-fat feeding.

Toll-like receptor-4 (TLR-4) is elevated in skeletal muscle of obese humans, and data from our laboratory have shown that activation of TLR-4 in skeletal muscle via LPS results in decreased fatty acid oxidation (FAO). The purpose of this study was to determine whether overexpression of TLR-4 in skeletal muscle alters mitochondrial function and whole body metabolism in the context of a chow and high-fat diet. C57BL/6J mice (males, 6-8 mo of age) with skeletal muscle-specific overexpression of the TLR-4 (mTLR-4) gene were created and used for this study. Isolated mitochondria and whole muscle homogenates from rodent skeletal muscle (gastrocnemius and quadriceps) were investigated. TLR-4 overexpression resulted in a significant reduction in FAO in muscle homogenates; however, mitochondrial respiration and reactive oxygen species (ROS) production did not appear to be affected on a standard chow diet. To determine the role of TLR-4 overexpression in skeletal muscle in response to high-fat feeding, mTLR-4 mice and WT control mice were fed low- and high-fat diets for 16 wk. The high-fat diet significantly decreased FAO in mTLR-4 mice, which was observed in concert with elevated body weight and fat, greater glucose intolerance, and increase in production of ROS and cellular oxidative damage compared with WT littermates. These findings suggest that TLR-4 plays an important role in the metabolic response in skeletal muscle to high-fat feeding.

Intermuscular and perimuscular fat expansion in obesity correlates with skeletal muscle T cell and macrophage infiltration and insulin resistance.

Background/ObjectivesLimited numbers of studies demonstrated obesity-induced macrophage infiltration in skeletal muscle (SM), but dynamics of immune cell accumulation and contribution of T cells to SM insulin resistance are understudied.Subjects/MethodsT cells and macrophage markers were examined in SM of obese humans by RT-PCR. Mice were fed high-fat diet (HFD) for 2–24 weeks, and time course of macrophage and T cell accumulation was assessed by flow cytometry and quantitative RT-PCR. Extramyocellular adipose tissue (EMAT) was quantified by high-resolution micro-CT, and correlation to T cell number in SM was examined. CD11a−/− mice and C57BL/6 mice were treated with CD11a-neutralizing antibody to determine the role of CD11a in T cell accumulation in SM. To investigate the involvement JAK/STAT, the major pathway for T helper I (TH1) cytokine IFNγ? in SM and adipose tissue inflammation and insulin resistance, mice were treated with a JAK1/JAK2 inhibitor, baricitinib.ResultsMacrophage and T cells markers were upregulated in SM of obese compared with lean humans. SM of obese mice had higher expression of inflammatory cytokines, with macrophages increasing by 2 weeks on HFD and T cells increasing by 8 weeks. The immune cells were localized in EMAT. Micro-CT revealed that EMAT expansion in obese mice correlated with T cell infiltration and insulin resistance. Deficiency or neutralization of CD11a reduced T cell accumulation in SM of obese mice. T cells polarized into a proinflammatory TH1 phenotype, with increased STAT1 phosphorylation in SM of obese mice. In vivo inhibition of JAK/STAT pathway with baricitinib reduced T cell numbers and activation markers in SM and adipose tissue and improved insulin resistance in obese mice.ConclusionsObesity-induced expansion of EMAT in SM was associated with accumulation and proinflammatory polarization of T cells, which may regulate SM metabolic functions through paracrine mechanisms. Obesity-associated SM “adiposopathy” may thus play an important role in development of insulin resistance and inflammation.

A novel method to detect in vivo oxidant production reveals microvascular endothelial dysfunction linked to NADPH oxidase in obese human skeletal muscle

Excessive production of reactive oxygen species (ROS) contributes to the development of endothelial dysfunction and progression of vascular diseases that are observed in obesity. Three microdialysis probes were inserted into the vastus lateralis of young, sedentary lean (LN; n=7), overweight (OW; n=8) and obese (OB; n=7) individuals, allowing sampling of extracellular fluid. To measure extracellular ROS, probes were perfused with saline containing 5 mM ethanol, 100 μM Amplex Ultrared, 1 U/ml horseradish peroxidase, and 10 U/ml superoxide dismutase, with or without 300 μM apocynin (Apo; an NADPH oxidase inhibitor). Dialysate fluorescence was measured at the outlet. Endothelial function was assessed with addition of acetylcholine (ACh) to the perfusate, with local blood flow calculated from the ethanol outflow:inflow ratio. Extracellular ROS was elevated in OB 3.2‐fold over OW, and 5.6‐fold over LN (p < 0.05), which was prevented by Apo (p < 0.01). ACh‐stimulated blood flow was depressed 60% in OB vs. OW, and 68% vs. LN (p = 0.01). Apo augmented ACh‐stimulated blood flow only in OB (p = 0.02). Microvascular endothelial dysfunction was evident in OB, which was largely reversed by NADPH oxidase inhibition. It can be concluded that the substantial elevation in ROS in OB was predominantly driven by NADPH oxidase.Supported by a doctoral student grant from the American College of Sports Medicine.

Deletion of Skeletal Muscle SOCS3 Prevents Insulin Resistance in Obesity

Obesity is associated with chronic low-grade inflammation that contributes to defects in energy metabolism and insulin resistance. Suppressor of cytokine signaling (SOCS)-3 expression is increased in skeletal muscle of obese humans. SOCS3 inhibits leptin signaling in the hypothalamus and insulin signal transduction in adipose tissue and the liver. Skeletal muscle is an important tissue for controlling energy expenditure and whole-body insulin sensitivity; however, the physiological importance of SOCS3 in this tissue has not been examined. Therefore, we generated mice that had SOCS3 specifically deleted in skeletal muscle (SOCS MKO). The SOCS3 MKO mice had normal muscle development, body mass, adiposity, appetite, and energy expenditure compared with wild-type (WT) littermates. Despite similar degrees of obesity when fed a high-fat diet, SOCS3 MKO mice were protected against the development of hyperinsulinemia and insulin resistance because of enhanced skeletal muscle insulin receptor substrate 1 (IRS1) and Akt phosphorylation that resulted in increased skeletal muscle glucose uptake. These data indicate that skeletal muscle SOCS3 does not play a critical role in regulating muscle development or energy expenditure, but it is an important contributing factor for inhibiting insulin sensitivity in obesity. Therapies aimed at inhibiting SOCS3 in skeletal muscle may be effective in reversing obesity-related glucose intolerance and insulin resistance.

Mitochondrial lipid oxidation is impaired in cultured myotubes from obese humans

The skeletal muscle of obese humans is characterized by an inability to appropriately respond to alterations in substrate availability. The purpose of this study was to determine if this metabolic inflexibility with obesity is retained in mitochondria of human skeletal muscle cells raised in culture (HSkMC) and to identify potential mechanisms involved. Mitochondrial respiration was measured in permeabilized myotubes cultured from lean and obese individuals before and after a 24-h lipid incubation. Mitochondrial respiration (state 3) in the presence of lipid substrate (palmitoyl carnitine) increased by almost twofold after lipid incubation in HSkMC from lean, but not obese subjects, indicative of metabolic inflexibility with obesity. The 24-h lipid incubation increased mitochondrial DNA (mtDNA) copy number in HSkMC from lean subjects by +16% (P<0.05); conversely, mtDNA copy number decreased in myotubes cultured from obese individuals (-13%, P=0.06). When respiration data were normalized to mtDNA copy number and other indices of mitochondrial content (COX-IV protein content and CS activity), the significant treatment effects of lipid incubation persisted in the lean subjects, suggesting concomitant alterations in mitochondrial function; no similar adjustment was evident in HSkMC from obese individuals. These data indicate that the skeletal muscle of obese individuals inherently lacks metabolic flexibility in response to lipid exposure, which consists of an inability to increase mitochondrial respiration in the presence of lipid substrate and perhaps by an inability to induce mitochondrial proliferation.

Expression of the Splicing Factor Gene SFRS10 Is Reduced in Human Obesity and Contributes to Enhanced Lipogenesis

Alternative mRNA splicing provides transcript diversity and may contribute to human disease. We demonstrate that expression of several genes regulating RNA processing is decreased in both liver and skeletal muscle of obese humans. We evaluated a representative splicing factor, SFRS10, downregulated in both obese human liver and muscle and in high-fat-fed mice, and determined metabolic impact of reduced expression. SFRS10-specific siRNA induces lipogenesis and lipid accumulation in hepatocytes. Moreover, Sfrs10 heterozygous mice have increased hepatic lipogenic gene expression, VLDL secretion, and plasma triglycerides. We demonstrate that LPIN1, a key regulator of lipid metabolism, is a splicing target of SFRS10; reduced SFRS10 favors the lipogenic β isoform of LPIN1. Importantly, LPIN1β-specific siRNA abolished lipogenic effects of decreased SFRS10 expression. Together, our results indicate that reduced expression of SFRS10, as observed in tissues from obese humans, alters LPIN1 splicing, induces lipogenesis, and therefore contributes to metabolic phenotypes associated with obesity.

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

Systemic Effects of White Adipose Tissue Dysregulation and Obesity‐Related Inflammation

The role of toll‐like receptor 4 in mitochondrial function and reactive oxygen species (ROS) production in skeletal muscle

Toll-like receptor 4 (TLR4) is elevated in skeletal muscle of obese humans, and data from our lab showed that activation of TLR4 in skeletal muscle with lipopplysaccharide (LPS) results in decreased fatty acid oxidation. The purpose of this study was to determine if TLR4 activation in skeletal muscle alters mitochondrial function and increases the production of ROS. C2C12 and human primary myotubes, and mitochondria isolated from rodent gastrocnemius muscle studied. TLR4 activation (LPS 50pg/ml for 2h) resulted in a significant reduction in maximal oxidative respiration in both C2C12 and human primary myotubes, which was observed in concert with an increase in production of ROS, and alterations in mitochondrial gene expression and protein content. These effects were blocked in the presence of the antioxidant, N-acetyl cysteine (NAC). Moreover, mitochondria isolated from skeletal muscle of mice 4 hours following an intraperitoneal injection of LPS (1mg/kg) displayed reduced respiratory control, ADP-stimulated respiration, and maximal respiration. These findings suggest that TLR4 activation causes mitochondrial dysfunction in skeletal muscle, which is partially dependent on the production of ROS.

Leptin receptor 170 kDa (OB‐R170) protein expression is reduced in obese human skeletal muscle: a potential mechanism of leptin resistance

To examine whether obesity-associated leptin resistance could be due to down-regulation of leptin receptors (OB-Rs) and/or up-regulation of suppressor of cytokine signalling 3 (SOCS3) and protein tyrosine phosphatase 1B (PTP1B) in skeletal muscle, which blunt janus kinase 2-dependent leptin signalling and signal transducer and activator of transcription 3 (STAT3) phosphorylation and reduce AMP-activated protein kinase (AMPK) and acetyl-coenzyme A carboxylase (ACC) phosphorylation. Deltoid and vastus lateralis muscle biopsies were obtained from 20 men: 10 non-obese control subjects (mean +/- s.d. age, 31 +/- 5 years; height, 184 +/- 9 cm; weight, 91 +/- 13 kg; and percentage body fat, 24.8 +/- 5.8%) and 10 obese (age, 30 +/- 7 years; height, 184 +/- 8 cm; weight, 115 +/- 8 kg; and percentage body fat, 34.9 +/- 5.1%). Skeletal muscle OB-R170 (OB-R long isoform) protein expression was 28 and 25% lower (both P < 0.05) in arm and leg muscles, respectively, of obese men compared with control subjects. In normal-weight subjects, SOCS3 protein expression, and STAT3, AMPKalpha and ACCbeta phosphorylation, were similar in the deltoid and vastus lateralis muscles. In obese subjects, the deltoid muscle had a greater amount of leptin receptors than the vastus lateralis, whilst SOCS3 protein expression was increased and basal STAT3, AMPKalpha and ACCbeta phosphorylation levels were reduced in the vastus lateralis compared with the deltoid muscle (all P < 0.05). In summary, skeletal muscle leptin receptors and leptin signalling are reduced in obesity, particularly in the leg muscles.

Tumor Necrosis Factor-α Induces Caspase-mediated Cleavage of Peroxisome Proliferator-activated Receptor γ in Adipocytes

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-dependent transcription factor that acts as a primary regulator of adipogenesis and controls adipocyte metabolism and insulin action. Increased expression of tumor necrosis factor (TNFalpha) in adipose tissue of obese subjects potently suppresses the expression of PPARgamma and attenuates adipocyte functions. Here we show that PPARgamma is a substrate of caspase-3 and caspase-6 during TNFalpha receptor signaling in adipocytes, and the consequent PPARgamma cleavage disrupts its nuclear localization. TNFalpha treatment of 3T3-L1 adipocytes decreases full-length PPARgamma while increasing the level of a 45-kDa immunoreactive PPARgamma fragment. Specific inhibitors of caspase-3 and caspase-6 attenuate the cleavage of PPARgamma protein in response to TNFalpha in cultured adipocytes. Incubation of nuclear fractions with recombinant caspase-3 and caspase-6 also generates a 45-kDa PPARgamma cleavage product. Dispersion of nuclear PPARgamma to the cytoplasm in response to TNFalpha treatment occurs in parallel with detection of activated caspase-3. We suggest that activation of the caspase cascade by TNFalpha down-regulates PPARgamma protein and PPARgamma-mediated metabolic processes in adipose cells.

Fatty acids increase toll‐like receptor‐4 gene expression, protein content in a saturation‐dependent manner in C2C12 skeletal muscle cells

Growing evidence suggests that obesity, and associated metabolic dysregulation, occurs in concert with chronic low‐grade inflammation. Our laboratory has reported that toll‐like receptor‐4 (TLR4) expression, an inflammatory mediator, is elevated in the skeletal muscle of obese humans and associated with reduced fatty acid oxidation and increased lipid synthesis. The purpose of this study was to examine the role of saturated vs. monounsaturated fatty acids on TLR4 transcription and function. Our hypothesis was that saturated fatty acids would sensitize the TLR4 pathway via increased receptor content. To this end, skeletal muscle cells were incubated in the presence of varying ratios of palmitate (16:0) and oleate (18:1) to examine the effects of fatty acid saturation on TLR4. Fatty acid exposure resulted in a significant and stepwise increase in TLR4 mRNA and protein content with increasing palmitate concentration. To determine if the fatty acid effect on TLR4 was translated into a functional consequence, we measured the effect of TLR4 activation on substrate metabolism. As the degree of fatty acid saturation was increased in the media, the effect of TLR4 activation on glucose and fat oxidation was exacerbated. In conclusion, exposure to a lipotoxic environment sensitizes the TLR4 pathway in skeletal muscle and suggests that this is accomplished via a mechanism that is fatty acid saturation specific.

Activation of toll‐like receptor‐4 modulates skeletal muscle substrate metabolism in a manner that mimics the metabolic phenotype observed in skeletal muscle of obese humans

Obesity is associated with chronic inflammation and the development of type 2 diabetes. Skeletal muscle from obese humans is resistant to insulin and characterized by abnormal substrate metabolism, i.e. increased glycolytic activity, reduced oxidative capacity, and elevated lipid accumulation. Toll‐like receptors (TLRs) are transmembrane receptors that are important in the induction of inflammatory responses. TLRs have been linked to lipid‐induced skeletal muscle insulin resistance and may contribute to reduced oxidative capacity and skeletal muscle lipid accumulation associated with obesity. In the current study, we demonstrate that the TLR4 signaling pathway is up regulated in skeletal muscle of obese humans and regulates substrate metabolism in a manner that reproduces the metabolic phenotype of skeletal muscle observed with obesity. TLR4 gene expression is increased in skeletal muscle of obese humans (p&lt;0.001) and significantly related to BMI (r=0.92, p&lt;0.001) and intramyocellular triglyceride synthesis (r=0.86, p&lt;0.006). In addition, treatment of skeletal muscle cells with Lipopolysaccharide results in a 15% reduction in fatty acid oxidation (p&lt;0.002), a 30% increase in glucose oxidation (p=0.04), and a 33% increase in triglyceride synthesis (p&lt;0.002). These results provide a potential mechanism linking innate immunity with abnormal substrate metabolism associated with obesity.Supported by the American Diabetes Association (Junior Faculty Award, MWH) and the National Institutes of Health (DK078765‐01, MWH).

Endothelin Limits Insulin Action in Obese/Insulin-Resistant Humans

The normal action of insulin to vasodilate and redistribute blood flow in support of skeletal muscle metabolism is impaired in insulin-resistant states. Increased endogenous endothelin contributes to endothelial dysfunction in obesity and diabetes. Here, we test the hypothesis that increased endogenous endothelin action also contributes to skeletal muscle insulin resistance via impairments in insulin-stimulated vasodilation. We studied nine lean and seven obese humans, measuring the metabolic and hemodynamic effects of insulin (300 mU . m(-2) . min(-1)) alone and during femoral artery infusion of BQ123 (an antagonist of type A endothelin receptors, 1 micromol/min). Endothelin antagonism augmented skeletal muscle responses to insulin in obese subjects through changes in both leg blood flow (LBF) and glucose extraction. Insulin-stimulated LBF was significantly increased in obese subjects only. These changes, combined with differential effects on glucose extraction, resulted in augmented insulin-stimulated leg glucose uptake in obese subjects (54.7 +/- 5.7 vs. 107.4 +/- 18.9 mg/min with BQ123), with no change in lean subjects (103.7 +/- 11.4 vs. 88.9 +/- 16.3, P = 0.04 comparing BQ123 across groups). BQ123 allowed augmented leg glucose extraction in obese subjects even in the face of NOS antagonism. These findings suggest that increased endogenous endothelin action contributes to insulin resistance in skeletal muscle of obese humans, likely through both vascular and tissue effects.

Expression of Mfn2, the Charcot-Marie-Tooth Neuropathy Type 2A Gene, in Human Skeletal Muscle

The primary gene mutated in Charcot-Marie-Tooth type 2A is mitofusin-2 (Mfn2). Mfn2 encodes a mitochondrial protein that participates in the maintenance of the mitochondrial network and that regulates mitochondrial metabolism and intracellular signaling. The potential for regulation of human Mfn2 gene expression in vivo is largely unknown. Based on the presence of mitochondrial dysfunction in insulin-resistant conditions, we have examined whether Mfn2 expression is dysregulated in skeletal muscle from obese or nonobese type 2 diabetic subjects, whether muscle Mfn2 expression is regulated by body weight loss, and the potential regulatory role of tumor necrosis factor (TNF)alpha or interleukin-6. We show that mRNA concentration of Mfn2 is decreased in skeletal muscle from both male and female obese subjects. Muscle Mfn2 expression was also reduced in lean or in obese type 2 diabetic patients. There was a strong negative correlation between the Mfn2 expression and the BMI in nondiabetic and type 2 diabetic subjects. A positive correlation between the Mfn2 expression and the insulin sensitivity was also detected in nondiabetic and type 2 diabetic subjects. To determine the effect of weight loss on Mfn2 mRNA expression, six morbidly obese subjects were subjected to weight loss by bilio-pancreatic diversion. Mean expression of muscle Mfn2 mRNA increased threefold after reduction in body weight, and a positive correlation between muscle Mfn2 expression and insulin sensitivity was again detected. In vitro experiments revealed an inhibitory effect of TNFalpha or interleukin-6 on Mfn2 expression in cultured cells. We conclude that body weight loss upregulates the expression of Mfn2 mRNA in skeletal muscle of obese humans, type 2 diabetes downregulates the expression of Mfn2 mRNA in skeletal muscle, Mfn2 expression in skeletal muscle is directly proportional to insulin sensitivity and is inversely proportional to the BMI, TNFalpha and interleukin-6 downregulate Mfn2 expression and may participate in the dysregulation of Mfn2 expression in obesity or type 2 diabetes, and the in vivo modulation of Mfn2 mRNA levels is an additional level of regulation for the control of muscle metabolism and could provide a molecular mechanism for alterations in mitochondrial function in obesity or type 2 diabetes.

Acute and Chronic Effects of Leptin on Skeletal Muscle Fatty Acid Metabolism

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Lipid oxidation is reduced in obese human skeletal muscle

The purpose of this study was to discern cellular mechanisms that contribute to the suppression of lipid oxidation in the skeletal muscle of obese individuals. Muscle was obtained from obese [body mass index (BMI), 38.3 +/- 3.1 kg/m(2)] and lean (BMI, 23.8 +/- 0.9 kg/m(2)) women, and fatty acid oxidation was studied by measuring (14)CO(2) production from (14)C-labeled fatty acids. Palmitate oxidation, which is at least partially dependent on carnitine palmitoyltransferase-1 (CPT-1) activity, was depressed (P < 0.05) by approximately 50% with obesity (6.8 +/- 2.2 vs. 13.7 +/- 1.4 nmole CO(2).g(-1).h(-1)). The CPT-1-independent event of palmitoyl carnitine oxidation was also depressed (P < 0.01) by approximately 45%. There were significant negative relationships (P < 0.05) for adiposity with palmitate (r = -0.76) and palmitoyl carnitine (r = -0.82) oxidation. Muscle CPT-1 and citrate synthase activity, an index of mitochondrial content, were also significantly (P < 0.05) reduced ( approximately 35%) with obesity. CPT-1 (r = -0.48) and citrate synthase (r = -0.65) activities were significantly (P < 0.05) related to adiposity. These data suggest that lesions at CPT-1 and post-CPT-1 events, such as mitochondrial content, contribute to the reduced reliance on fat oxidation evident in human skeletal muscle with obesity.