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1.  Muscle Mitochondrial ATP Synthesis and Glucose Transport/Phosphorylation in Type 2 Diabetes 
PLoS Medicine  2007;4(5):e154.
Muscular insulin resistance is frequently characterized by blunted increases in glucose-6-phosphate (G-6-P) reflecting impaired glucose transport/phosphorylation. These abnormalities likely relate to excessive intramyocellular lipids and mitochondrial dysfunction. We hypothesized that alterations in insulin action and mitochondrial function should be present even in nonobese patients with well-controlled type 2 diabetes mellitus (T2DM).
Methods and Findings
We measured G-6-P, ATP synthetic flux (i.e., synthesis) and lipid contents of skeletal muscle with 31P/1H magnetic resonance spectroscopy in ten patients with T2DM and in two control groups: ten sex-, age-, and body mass-matched elderly people; and 11 younger healthy individuals. Although insulin sensitivity was lower in patients with T2DM, muscle lipid contents were comparable and hyperinsulinemia increased G-6-P by 50% (95% confidence interval [CI] 39%–99%) in all groups. Patients with diabetes had 27% lower fasting ATP synthetic flux compared to younger controls (p = 0.031). Insulin stimulation increased ATP synthetic flux only in controls (younger: 26%, 95% CI 13%–42%; older: 11%, 95% CI 2%–25%), but failed to increase even during hyperglycemic hyperinsulinemia in patients with T2DM. Fasting free fatty acids and waist-to-hip ratios explained 44% of basal ATP synthetic flux. Insulin sensitivity explained 30% of insulin-stimulated ATP synthetic flux.
Patients with well-controlled T2DM feature slightly lower flux through muscle ATP synthesis, which occurs independently of glucose transport /phosphorylation and lipid deposition but is determined by lipid availability and insulin sensitivity. Furthermore, the reduction in insulin-stimulated glucose disposal despite normal glucose transport/phosphorylation suggests further abnormalities mainly in glycogen synthesis in these patients.
Michael Roden and colleagues report that even patients with well-controlled insulin-resistant type 2 diabetes have altered mitochondrial function.
Editors' Summary
Diabetes mellitus is an increasingly common chronic disease characterized by high blood sugar (glucose) levels. In normal individuals, blood sugar levels are maintained by the hormone insulin. Insulin is released by the pancreas when blood glucose levels rise after eating (glucose is produced by the digestion of food) and “instructs” insulin-responsive muscle and fat cells to take up glucose from the bloodstream. The cells then use glucose as a fuel or convert it into glycogen, a storage form of glucose. In type 2 diabetes, the commonest type of diabetes, the muscle and fat cells become nonresponsive to insulin (a condition called insulin resistance) and consequently blood glucose levels rise. Over time, this hyperglycemia increases the risk of heart attacks, kidney failure, and other life-threatening complications.
Why Was This Study Done?
Insulin resistance is often an early sign of type 2 diabetes, sometimes predating its development by many years, so understanding its causes might provide clues about how to stop the global diabetes epidemic. One theory is that mitochondria—cellular structures that produce the energy (in the form of a molecule called ATP) needed to keep cells functioning—do not work properly in people with insulin resistance. Mitochondria change (metabolize) fatty acids into energy, and recent studies have revealed that fat accumulation caused by poorly regulated fatty acid metabolism blocks insulin signaling, thus causing insulin resistance. Other studies using magnetic resonance spectroscopy (MRS) to study mitochondrial function noninvasively in human muscle indicate that mitochondria are dysfunctional in people with insulin resistance by showing that ATP synthesis is impaired in such individuals. In this study, the researchers have examined both baseline and insulin-stimulated mitochondrial function in nonobese patients with well-controlled type 2 diabetes and in normal controls to discover more about the relationship between mitochondrial dysfunction and insulin resistance.
What Did the Researchers Do and Find?
The researchers determined the insulin sensitivity of people with type 2 diabetes and two sets of people (the “controls”) who did not have diabetes: one in which the volunteers were age-matched to the people with diabetes, and the other containing younger individuals (insulin resistance increases with age). To study insulin sensitivity in all three groups, the researchers used a “hyperinsulinemic–euglycemic clamp.” For this, after an overnight fast, the participants' insulin levels were kept high with a continuous insulin infusion while blood glucose levels were kept normal using a variable glucose infusion. In this situation, the glucose infusion rate equals glucose uptake by the body and therefore measures tissue sensitivity to insulin. Before and during the clamp, the researchers used MRS to measure glucose-6-phosphate (an indicator of how effectively glucose is taken into cells and phosphorylated), ATP synthesis, and the fat content of the participants' muscle cells. Insulin sensitivity was lower in the patients with diabetes than in the controls, but muscle lipid content was comparable and hyperinsulinemia increased glucose-6-phosphate levels similarly in all the groups. Patients with diabetes and the older controls had lower fasting ATP synthesis rates than the young controls and, whereas insulin stimulation increased ATP synthesis in all the controls, it had no effect in the patients with diabetes. In addition, fasting blood fatty acid levels were inversely related to basal ATP synthesis, whereas insulin sensitivity was directly related to insulin-stimulated ATP synthesis.
What Do These Findings Mean?
These findings indicate that the impairment of muscle mitochondrial ATP synthesis in fasting conditions and after insulin stimulation in people with diabetes is not due to impaired glucose transport/phosphorylation or fat deposition in the muscles. Instead, it seems to be determined by lipid availability and insulin sensitivity. These results add to the evidence suggesting that mitochondrial function is disrupted in type 2 diabetes and in insulin resistance, but also suggest that there may be abnormalities in glycogen synthesis. More work is needed to determine the exact nature of these abnormalities and to discover whether they can be modulated to prevent the development of insulin resistance and type 2 diabetes. For now, though, these findings re-emphasize the need for people with type 2 diabetes or insulin resistance to reduce their food intake to compensate for the reduced energy needs of their muscles and to exercise to increase the ATP-generating capacity of their muscles. Both lifestyle changes could improve their overall health and life expectancy.
Additional Information.
Please access these Web sites via the online version of this summary at
The MedlinePlus encyclopedia has pages on diabetes
The US National Institute of Diabetes and Digestive and Kidney Diseases provides information for patients on diabetes and insulin resistance
The US Centers for Disease Control and Prevention has information on diabetes for patients and professionals
American Diabetes Association provides information for patients on diabetes and insulin resistance
Diabetes UK has information for patients and professionals on diabetes
PMCID: PMC1858707  PMID: 17472434
2.  Mesenchymal stem cell transplantation for the infarcted heart: therapeutic potential for insulin resistance beyond the heart 
This study aimed to evaluate the efficacy of mesenchymal stem cell (MSC) transplantation to mitigate abnormalities in cardiac-specific and systemic metabolism mediated by a combination of a myocardial infarction and diet-induced insulin resistance.
C57BL/6 mice were high-fat fed for eight weeks prior to induction of a myocardial infarction via chronic ligation of the left anterior descending coronary artery. MSCs were administered directly after myocardial infarction induction through a single intramyocardial injection. Echocardiography was performed prior to the myocardial infarction as well as seven and 28 days post-myocardial infarction. Hyperinsulinemic-euglycemic clamps coupled with 2-[14C]deoxyglucose were employed 36 days post-myocardial infarction (13 weeks of high-fat feeding) to assess systemic insulin sensitivity and insulin-mediated, tissue-specific glucose uptake in the conscious, unrestrained mouse. High-resolution respirometry was utilized to evaluate cardiac mitochondrial function in saponin-permeabilized cardiac fibers.
MSC administration minimized the decline in ejection fraction following the myocardial infarction. The greater systolic function in MSC-treated mice was associated with increased in vivo cardiac glucose uptake and enhanced mitochondrial oxidative phosphorylation efficiency. MSC therapy promoted reductions in fasting arterial glucose and fatty acid concentrations. Additionally, glucose uptake in peripheral tissues including skeletal muscle and adipose tissue was elevated in MSC-treated mice. Enhanced glucose uptake in these tissues was associated with improved insulin signalling as assessed by Akt phosphorylation and prevention of a decline in GLUT4 often associated with high-fat feeding.
These studies provide insight into the utility of MSC transplantation as a metabolic therapy that extends beyond the heart exerting beneficial systemic effects on insulin action.
PMCID: PMC3847505  PMID: 24007410
Diabetes; Glucose uptake; Isotopic tracer; Mitochondria; Myocardial infarction
3.  The relative contribution of intramyocellular lipid to whole body fat oxidation is reduced with age, but subsarcolemmal lipid accumulation and insulin resistance are only associated with overweight individuals 
Diabetes  2016;65(4):840-850.
Insulin resistance is closely related to intramyocellular lipid (IMCL) accumulation, and both are associated with increasing age. It remains to be determined to what extent perturbations in IMCL metabolism are related to the ageing process per se. On two separate occasions whole-body and muscle insulin sensitivity (euglycaemic hyperinsulinaemic clamp with 2-deoxyglucose) and fat utilisation during 1 h of exercise at 50% VO2max ([U-13C]palmitate infusion combined with electron microscopy of IMCL) were determined in young lean (YL), old lean (OL), and old overweight (OO) males. OL displayed comparable IMCL content and insulin sensitivity to YL, whereas OO were markedly insulin resistant and had over 2-fold greater IMCL in the subsarcolemmal (SSL) region. Indeed, whereas the plasma free fatty acid rate of appearance and disappearance was twice that of YL in both OL and OO, SSL only increased during exercise in OO. Thus, skeletal muscle insulin resistance and lipid accumulation often observed in older individuals are likely due to lifestyle factors, rather than inherent ageing of skeletal muscle as usually reported. However, age per se appears to cause exacerbated adipose tissue lipolysis, suggesting that strategies to reduce muscle lipid delivery and improve adipose tissue function may be warranted in older overweight individuals.
The global prevalence of type 2 diabetes is most apparent in older people (1), and it is estimated that the number of people over 65 years of age with diabetes will have increased 4.5 fold by 2050 (2). Gaining mechanistic insight of age related insulin resistance and strategies to improve insulin sensitivity with age are clearly warranted. Although ageing is associated with insulin resistance, age per se does not appear to cause insulin resistance (3, 4, 5). Several factors that likely contribute to age related insulin resistance include increased abdominal adiposity and reduced physical activity (3, 4), along with declines in muscle mass (6, 7). Of note, intramyocellular lipid (IMCL) accumulates with age, particularly in subsarcolemmal (SSL) regions (8), and has been strongly associated with insulin resistance (9, 10, 11, 12). Indeed, SSL lipid accumulation has been linked to the accumulation of metabolites, such as diacylglycerol (DAG) and ceramide, thought by some (13, 14, 15), but not others (16), to contribute to impaired insulin-stimulated muscle glucose uptake. Nevertheless, it remains contentious as to which factors associated with age influence IMCL accumulation.
The accumulation of IMCL and associated metabolites likely result from an imbalance between muscle lipid delivery and oxidation. Indeed, studies have demonstrated reduced free fatty acid (FFA) oxidation in older people compared to young, despite whole-body lipolysis and plasma FFA availability being greater at rest and during exercise at the same absolute and relative intensities (17, 18). Linked to this, several studies have suggested age related blunting of FFA oxidation and increased IMCL accumulation are a result of reduced muscle mitochondrial content (8) and function (3, 19, 20) with age. However, increased adiposity and reduced habitual levels of physical activity also affect FFA flux and oxidation in older individuals (21), and studies to date have not controlled for these factors when investigating changes in muscle IMCL metabolism with age. Therefore, we investigated the effect of ageing on whole-body and skeletal muscle lipid metabolism, with parallel characterization of muscle insulin sensitivity, in lean young and older individuals matched for estimated habitual physical activity levels and body composition. To determine the effect of adiposity and reduced physical activity on the ageing process, the older lean individuals were also compared to a group of older overweight individuals matched for lean mass. We hypothesized that an age-associated imbalance between FFA delivery and oxidation in skeletal muscle during exercise would only be observed in older overweight individuals, which would manifest as reduced IMCL oxidation and increased IMCL storage, particularly in the SSL region, and be associated with skeletal muscle insulin resistance.
PMCID: PMC4894456  PMID: 26740597
4.  Role of the glucosamine pathway in fat-induced insulin resistance. 
To examine whether the hexosamine biosynthetic pathway might play a role in fat-induced insulin resistance, we monitored the effects of prolonged elevations in FFA availability both on skeletal muscle levels of UDP-N-acetyl-hexosamines and on peripheral glucose disposal during 7-h euglycemic-hyperinsulinemic (approximately 500 microU/ml) clamp studies. When the insulin-induced decrease in the plasma FFA levels (to approximately 0.3 mM) was prevented by infusion of a lipid emulsion in 15 conscious rats (plasma FFA approximately 1.4 mM), glucose uptake (5-7 h = 32.5+/-1.7 vs 0-2 h = 45.2+/-2.8 mg/kg per min; P < 0.01) and glycogen synthesis (P < 0.01) were markedly decreased. During lipid infusion, muscle UDP-N-acetyl-glucosamine (UDP-GlcNAc) increased by twofold (to 53.4+/-1.1 at 3 h and to 55.5+/-1.1 nmol/gram at 7 h vs 20.4+/-1.7 at 0 h, P < 0.01) while glucose-6-phosphate (Glc-6-P) levels were increased at 3 h (475+/-49 nmol/gram) and decreased at 7 h (133+/-7 vs 337+/-28 nmol/gram at 0 h, P < 0.01). To discern whether such an increase in the skeletal muscle UDP-GlcNAc concentration could account for the development of insulin resistance, we generated similar increases in muscle UDP-GlcNAc using three alternate experimental approaches. Euglycemic clamps were performed after prolonged hyperglycemia (18 mM, n = 10), or increased availability of either glucosamine (3 micromol/kg per min; n = 10) or uridine (30 micromol/kg per min; n = 4). These conditions all resulted in very similar increases in the skeletal muscle UDP-GlcNAc (to approximately 55 nmol/gram) and markedly impaired glucose uptake and glycogen synthesis. Thus, fat-induced insulin resistance is associated with: (a) decreased skeletal muscle Glc-6-P levels indicating defective transport/phosphorylation of glucose; (b) marked accumulation of the endproducts of the hexosamine biosynthetic pathway preceding the onset of insulin resistance. Most important, the same degree of insulin resistance can be reproduced in the absence of increased FFA availability by a similar increase in skeletal muscle UDP-N-acetyl-hexosamines. In conclusion, our results support the hypothesis that increased FFA availability induces skeletal muscle insulin resistance by increasing the flux of fructose-6-phosphate into the hexosamine pathway.
PMCID: PMC508047  PMID: 9151789
5.  SIRT3 Is Crucial for Maintaining Skeletal Muscle Insulin Action and Protects Against Severe Insulin Resistance in High-Fat–Fed Mice 
Diabetes  2015;64(9):3081-3092.
Protein hyperacetylation is associated with glucose intolerance and insulin resistance, suggesting that the enzymes regulating the acetylome play a role in this pathological process. Sirtuin 3 (SIRT3), the primary mitochondrial deacetylase, has been linked to energy homeostasis. Thus, it is hypothesized that the dysregulation of the mitochondrial acetylation state, via genetic deletion of SIRT3, will amplify the deleterious effects of a high-fat diet (HFD). Hyperinsulinemic-euglycemic clamp experiments show, for the first time, that mice lacking SIRT3 exhibit increased insulin resistance due to defects in skeletal muscle glucose uptake. Permeabilized muscle fibers from HFD-fed SIRT3 knockout (KO) mice showed that tricarboxylic acid cycle substrate–based respiration is decreased while fatty acid–based respiration is increased, reflecting a fuel switch from glucose to fatty acids. Consistent with reduced muscle glucose uptake, hexokinase II (HKII) binding to the mitochondria is decreased in muscle from HFD-fed SIRT3 KO mice, suggesting decreased HKII activity. These results show that the absence of SIRT3 in HFD-fed mice causes profound impairments in insulin-stimulated muscle glucose uptake, creating an increased reliance on fatty acids. Insulin action was not impaired in the lean SIRT3 KO mice. This suggests that SIRT3 protects against dietary insulin resistance by facilitating glucose disposal and mitochondrial function.
PMCID: PMC4542443  PMID: 25948682
6.  Lower Intrinsic ADP-Stimulated Mitochondrial Respiration Underlies In Vivo Mitochondrial Dysfunction in Muscle of Male Type 2 Diabetic Patients 
Diabetes  2008;57(11):2943-2949.
OBJECTIVE—A lower in vivo mitochondrial function has been reported in both type 2 diabetic patients and first-degree relatives of type 2 diabetic patients. The nature of this reduction is unknown. Here, we tested the hypothesis that a lower intrinsic mitochondrial respiratory capacity may underlie lower in vivo mitochondrial function observed in diabetic patients.
RESEARCH DESIGN AND METHODS—Ten overweight diabetic patients, 12 first-degree relatives, and 16 control subjects, all men, matched for age and BMI, participated in this study. Insulin sensitivity was measured with a hyperinsulinemic-euglycemic clamp. Ex vivo intrinsic mitochondrial respiratory capacity was determined in permeabilized skinned muscle fibers using high-resolution respirometry and normalized for mitochondrial content. In vivo mitochondrial function was determined by measuring phosphocreatine recovery half-time after exercise using 31P-magnetic resonance spectroscopy.
RESULTS—Insulin-stimulated glucose disposal was lower in diabetic patients compared with control subjects (11.2 ± 2.8 vs. 28.9 ± 3.7 μmol · kg−1 fat-free mass · min−1, respectively; P = 0.003), with intermediate values for first-degree relatives (22.1 ± 3.4 μmol · kg−1 fat-free mass · min−1). In vivo mitochondrial function was 25% lower in diabetic patients (P = 0.034) and 23% lower in first-degree relatives, but the latter did not reach statistical significance (P = 0.08). Interestingly, ADP-stimulated basal respiration was 35% lower in diabetic patients (P = 0.031), and fluoro-carbonyl cyanide phenylhydrazone–driven maximal mitochondrial respiratory capacity was 31% lower in diabetic patients (P = 0.05) compared with control subjects with intermediate values for first-degree relatives.
CONCLUSIONS—A reduced basal ADP-stimulated and maximal mitochondrial respiratory capacity underlies the reduction in in vivo mitochondrial function, independent of mitochondrial content. A reduced capacity at both the level of the electron transport chain and phosphorylation system underlies this impaired mitochondrial capacity.
PMCID: PMC2570390  PMID: 18678616
7.  Effects of Dietary n-3 Fatty Acids on Hepatic and Peripheral Insulin Sensitivity in Insulin-Resistant Humans 
Diabetes Care  2015;38(7):1228-1237.
Dietary n-3 polyunsaturated fatty acids, including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), prevent insulin resistance and stimulate mitochondrial biogenesis in rodents, but the findings of translational studies in humans are thus far ambiguous. The aim of this study was to evaluate the influence of EPA and DHA on insulin sensitivity, insulin secretion, and muscle mitochondrial function in insulin-resistant, nondiabetic humans using a robust study design and gold-standard measurements.
Thirty-one insulin-resistant adults received 3.9 g/day EPA+DHA or placebo for 6 months in a randomized double-blind study. Hyperinsulinemic-euglycemic clamp with somatostatin was used to assess hepatic and peripheral insulin sensitivity. Postprandial glucose disposal and insulin secretion were measured after a meal. Measurements were performed at baseline and after 6 months of treatment. Abdominal fat distribution was evaluated by MRI. Muscle oxidative capacity was measured in isolated mitochondria using high-resolution respirometry and noninvasively by magnetic resonance spectroscopy.
Compared with placebo, EPA+DHA did not alter peripheral insulin sensitivity, postprandial glucose disposal, or insulin secretion. Hepatic insulin sensitivity, determined from the suppression of endogenous glucose production by insulin, exhibited a small but significant improvement with EPA+DHA compared with placebo. Muscle mitochondrial function was unchanged by EPA+DHA or placebo.
This study demonstrates that dietary EPA+DHA does not improve peripheral glucose disposal, insulin secretion, or skeletal muscle mitochondrial function in insulin-resistant nondiabetic humans. There was a modest improvement in hepatic insulin sensitivity with EPA+DHA, but this was not associated with any improvements in clinically meaningful outcomes.
PMCID: PMC4477338  PMID: 25852206
8.  Opening of the mitochondrial permeability transition pore links mitochondrial dysfunction to insulin resistance in skeletal muscle☆ 
Molecular Metabolism  2013;3(2):124-134.
Insulin resistance is associated with mitochondrial dysfunction, but the mechanism by which mitochondria inhibit insulin-stimulated glucose uptake into the cytoplasm is unclear. The mitochondrial permeability transition pore (mPTP) is a protein complex that facilitates the exchange of molecules between the mitochondrial matrix and cytoplasm, and opening of the mPTP occurs in response to physiological stressors that are associated with insulin resistance. In this study, we investigated whether mPTP opening provides a link between mitochondrial dysfunction and insulin resistance by inhibiting the mPTP gatekeeper protein cyclophilin D (CypD) in vivo and in vitro. Mice lacking CypD were protected from high fat diet-induced glucose intolerance due to increased glucose uptake in skeletal muscle. The mitochondria in CypD knockout muscle were resistant to diet-induced swelling and had improved calcium retention capacity compared to controls; however, no changes were observed in muscle oxidative damage, insulin signaling, lipotoxic lipid accumulation or mitochondrial bioenergetics. In vitro, we tested 4 models of insulin resistance that are linked to mitochondrial dysfunction in cultured skeletal muscle cells including antimycin A, C2-ceramide, ferutinin, and palmitate. In all models, we observed that pharmacological inhibition of mPTP opening with the CypD inhibitor cyclosporin A was sufficient to prevent insulin resistance at the level of insulin-stimulated GLUT4 translocation to the plasma membrane. The protective effects of mPTP inhibition on insulin sensitivity were associated with improved mitochondrial calcium retention capacity but did not involve changes in insulin signaling both in vitro and in vivo. In sum, these data place the mPTP at a critical intersection between alterations in mitochondrial function and insulin resistance in skeletal muscle.
PMCID: PMC3953683  PMID: 24634818
MPTP, mitochondrial permeability transition pore; CYPD, cyclophilin D; HFD, high fat diet; LFD, low fat diet; WT, wild type; KO, knockout; CSA, cyclosporin A; BKA, bongkrekic acid; O2•, superoxide; [3H]-2-DOG, [3H]-2-deoxyglucose; Rg′, rate of glucose transport; FFA, free fatty acid; DAG, diacylglycerol; TEM, transmission electron microscopy; PDH, pyruvate dehydrogenase; PDHa, active PDH; PDHt, total PDH; MCAD, medium chain acyl-CoA dehydrogenase; β-HAD, β-hydroxyacyl-CoA dehydrogenase; PM, plasma membrane; ANT, adenine nucleotide translocator; VDAC, voltage-dependent anion channel; HK2, hexokinase 2; ETC, electron transport chain; OXPHOS, oxidative phosphorylation; MnSOD, mitochondrial manganese superoxide dismutase; MIRKO, muscle insulin receptor knockout; MHC, myosin heavy chain; TBARS, thiobarbituric acid reactive substances; Glucose; Insulin resistance; Mitochondrial dysfunction; Mitochondrial permeability transition pore; Cyclophilin D; Skeletal muscle
9.  Overfeeding Reduces Insulin Sensitivity and Increases Oxidative Stress, without Altering Markers of Mitochondrial Content and Function in Humans 
PLoS ONE  2012;7(5):e36320.
Mitochondrial dysfunction and increased oxidative stress are associated with obesity and type 2 diabetes. High fat feeding induces insulin resistance and increases skeletal muscle oxidative stress in rodents, but there is controversy as to whether skeletal muscle mitochondrial biogenesis and function is altered.
Methodology and Principal Findings
Forty (37±2 y) non-obese (25.6±0.6 kg/m2) sedentary men (n = 20) and women (n = 20) were overfed (+1040±100 kcal/day, 46±1% of energy from fat) for 28 days. Hyperinsulinemic-euglycemic clamps were performed at baseline and day 28 of overfeeding and skeletal muscle biopsies taken at baseline, day 3 and day 28 of overfeeding in a sub cohort of 26 individuals (13 men and 13 women) that consented to having all 3 biopsies performed. Weight increased on average in the whole cohort by 0.6±0.1 and 2.7±0.3 kg at days 3 and 28, respectively (P<0.0001, without a significant difference in the response between men and women (P = 0.4). Glucose infusion rate during the hyperinsulinemic-euglycemic clamp decreased from 54.8±2.8 at baseline to 50.3±2.5 µmol/min/kg FFM at day 28 of overfeeding (P = 0.03) without a significant difference between men and women (P = 0.4). Skeletal muscle protein carbonyls and urinary F2-isoprostanes increased with overfeeding (P<0.05). Protein levels of muscle peroxisome proliferator-activated receptor gamma coactivator-1α (PGC1α) and subunits from complex I, II and V of the electron transport chain were increased at day 3 (all P<0.05) and returned to basal levels at day 28. No changes were detected in muscle citrate synthase activity or ex vivo CO2 production at either time point.
Peripheral insulin resistance was induced by overfeeding, without reducing any of the markers of mitochondrial content that were examined. Oxidative stress was however increased, and may have contributed to the reduction in insulin sensitivity observed.
Trial Registration NCT00562393
PMCID: PMC3346759  PMID: 22586466
10.  Butyrate Improves Insulin Sensitivity and Increases Energy Expenditure in Mice 
Diabetes  2009;58(7):1509-1517.
We examined the role of butyric acid, a short-chain fatty acid formed by fermentation in the large intestine, in the regulation of insulin sensitivity in mice fed a high-fat diet.
In dietary-obese C57BL/6J mice, sodium butyrate was administrated through diet supplementation at 5% wt/wt in the high-fat diet. Insulin sensitivity was examined with insulin tolerance testing and homeostasis model assessment for insulin resistance. Energy metabolism was monitored in a metabolic chamber. Mitochondrial function was investigated in brown adipocytes and skeletal muscle in the mice.
On the high-fat diet, supplementation of butyrate prevented development of insulin resistance and obesity in C57BL/6 mice. Fasting blood glucose, fasting insulin, and insulin tolerance were all preserved in the treated mice. Body fat content was maintained at 10% without a reduction in food intake. Adaptive thermogenesis and fatty acid oxidation were enhanced. An increase in mitochondrial function and biogenesis was observed in skeletal muscle and brown fat. The type I fiber was enriched in skeletal muscle. Peroxisome proliferator–activated receptor-γ coactivator-1α expression was elevated at mRNA and protein levels. AMP kinase and p38 activities were elevated. In the obese mice, supplementation of butyrate led to an increase in insulin sensitivity and a reduction in adiposity.
Dietary supplementation of butyrate can prevent and treat diet-induced insulin resistance in mouse. The mechanism of butyrate action is related to promotion of energy expenditure and induction of mitochondria function.
PMCID: PMC2699871  PMID: 19366864
11.  High Fat Diet-Induced Changes in Mouse Muscle Mitochondrial Phospholipids Do Not Impair Mitochondrial Respiration Despite Insulin Resistance 
PLoS ONE  2011;6(11):e27274.
Type 2 diabetes mellitus and muscle insulin resistance have been associated with reduced capacity of skeletal muscle mitochondria, possibly as a result of increased intake of dietary fat. Here, we examined the hypothesis that a prolonged high-fat diet consumption (HFD) increases the saturation of muscle mitochondrial membrane phospholipids causing impaired mitochondrial oxidative capacity and possibly insulin resistance.
C57BL/6J mice were fed an 8-week or 20-week low fat diet (10 kcal%; LFD) or HFD (45 kcal%). Skeletal muscle mitochondria were isolated and fatty acid (FA) composition of skeletal muscle mitochondrial phospholipids was analyzed by thin-layer chromatography followed by GC. High-resolution respirometry was used to assess oxidation of pyruvate and fatty acids by mitochondria. Insulin sensitivity was estimated by HOMA-IR.
Principal Findings
At 8 weeks, mono-unsaturated FA (16∶1n7, 18∶1n7 and 18∶1n9) were decreased (−4.0%, p<0.001), whereas saturated FA (16∶0) were increased (+3.2%, p<0.001) in phospholipids of HFD vs. LFD mitochondria. Interestingly, 20 weeks of HFD descreased mono-unsaturated FA while n-6 poly-unsaturated FA (18∶2n6, 20∶4n6, 22∶5n6) showed a pronounced increase (+4.0%, p<0.001). Despite increased saturation of muscle mitochondrial phospholipids after the 8-week HFD, mitochondrial oxidation of both pyruvate and fatty acids were similar between LFD and HFD mice. After 20 weeks of HFD, the increase in n-6 poly-unsaturated FA was accompanied by enhanced maximal capacity of the electron transport chain (+49%, p = 0.002) and a tendency for increased ADP-stimulated respiration, but only when fuelled by a lipid-derived substrate. Insulin sensitivity in HFD mice was reduced at both 8 and 20 weeks.
Our findings do not support the concept that prolonged HF feeding leads to increased saturation of skeletal muscle mitochondrial phospholipids resulting in a decrease in mitochondrial fat oxidative capacity and (muscle) insulin resistance.
PMCID: PMC3225362  PMID: 22140436
12.  Paradoxical Coupling of Triglyceride Synthesis and Fatty Acid Oxidation in Skeletal Muscle Overexpressing DGAT1 
Diabetes  2009;58(11):2516-2524.
Transgenic expression of diacylglycerol acyltransferase-1 (DGAT1) in skeletal muscle leads to protection against fat-induced insulin resistance despite accumulation of intramuscular triglyceride, a phenomenon similar to what is known as the “athlete paradox.” The primary objective of this study is to determine how DGAT1 affects muscle fatty acid oxidation in relation to whole-body energy metabolism and insulin sensitivity.
We first quantified insulin sensitivity and the relative tissue contributions to the improved whole-body insulin sensitivity in muscle creatine kisase (MCK)-DGAT1 transgenic mice by hyperinsulinemic-euglycemic clamps. Metabolic consequences of DGAT1 overexpression in skeletal muscles were determined by quantifying triglyceride synthesis/storage (anabolic) and fatty acid oxidation (catabolic), in conjunction with gene expression levels of representative marker genes in fatty acid metabolism. Whole-body energy metabolism including food consumption, body weights, oxygen consumption, locomotor activity, and respiration exchange ratios were determined at steady states.
MCK-DGAT1 mice were protected against muscle lipoptoxicity, although they remain susceptible to hepatic lipotoxicity. While augmenting triglyceride synthesis, DGAT1 overexpression also led to increased muscle mitochondrial fatty acid oxidation efficiency, as compared with wild-type muscles. On a high-fat diet, MCK-DGAT1 mice displayed higher basal metabolic rates and 5–10% lower body weights compared with wild-type littermates, whereas food consumption was not different.
DGAT1 overexpression in skeletal muscle led to parallel increases in triglyceride synthesis and fatty acid oxidation. Seemingly paradoxical, this phenomenon is characteristic of insulin-sensitive myofibers and suggests that DGAT1 plays an active role in metabolic “remodeling” of skeletal muscle coupled with insulin sensitization.
PMCID: PMC2768165  PMID: 19675136
13.  A Combination of Nutriments Improves Mitochondrial Biogenesis and Function in Skeletal Muscle of Type 2 Diabetic Goto–Kakizaki Rats 
PLoS ONE  2008;3(6):e2328.
Recent evidence indicates that insulin resistance in skeletal muscle may be related to reduce mitochondrial number and oxidation capacity. However, it is not known whether increasing mitochondrial number and function improves insulin resistance. In the present study, we investigated the effects of a combination of nutrients on insulin resistance and mitochondrial biogenesis/function in skeletal muscle of type 2 diabetic Goto–Kakizaki rats.
Methodology/Principal Findings
We demonstrated that defect of glucose and lipid metabolism is associated with low mitochondrial content and reduced mitochondrial enzyme activity in skeletal muscle of the diabetic Goto-Kakizaki rats. The treatment of combination of R-α-lipoic acid, acetyl-L-carnitine, nicotinamide, and biotin effectively improved glucose tolerance, decreased the basal insulin secretion and the level of circulating free fatty acid (FFA), and prevented the reduction of mitochondrial biogenesis in skeletal muscle. The nutrients treatment also significantly increased mRNA levels of genes involved in lipid metabolism, including peroxisome proliferator–activated receptor-α (Pparα), peroxisome proliferator–activated receptor-δ (Pparδ), and carnitine palmitoyl transferase-1 (Mcpt-1) and activity of mitochondrial complex I and II in skeletal muscle. All of these effects of mitochondrial nutrients are comparable to that of the antidiabetic drug, pioglitazone. In addition, the treatment with nutrients, unlike pioglitazone, did not cause body weight gain.
These data suggest that a combination of mitochondrial targeting nutrients may improve skeletal mitochondrial dysfunction and exert hypoglycemic effects, without causing weight gain.
PMCID: PMC2391295  PMID: 18523557
14.  Effects of nonesterified fatty acid availability on tissue-specific glucose utilization in rats in vivo. 
Journal of Clinical Investigation  1988;82(1):293-299.
The pathophysiological significance of the glucose-fatty acid cycle in skeletal muscle in vivo is uncertain. We have examined the short term effects of increased availability of nonesterified FFA on tissue-specific glucose uptake and storage in rat tissues in vivo basally and during a hyperinsulinemic (150 mU/liter) euglycemic clamp. Circulating FFA were elevated to 2 mmol/liter (FFA 1) or 4 mmol/liter (FFA 2). Elevated FFA produced a dose-dependent inhibition of myocardial glucose utilization in both basal (FFA1, 42%; FFA2, 68%; P less than 0.001, by analysis of variance) and clamp groups (FFA1, 39%; FFA2, 49%; P less than 0.001) and also suppressed brown adipose tissue glucose utilization during the clamp (-42%, P less than 0.001). In contrast to heart, glucose utilization in skeletal muscle was suppressed by FFA only in the FFA1 basal group (-36%, P less than 0.001); in other groups (e.g., FFA2 clamp) elevated FFA produced increased skeletal muscle glucose utilization (+68%, P less than 0.001) that was directed toward glycogen (+175%, P less than 0.05) and lipid deposition (+125%, P less than 0.005). FFA stimulated basal glucose utilization in white (e.g., FFA2, +220%, P less than 0.005) and brown adipose tissue (e.g., FFA2, +200%, P less than 0.005). Thus elevated FFA can acutely inhibit glucose utilization in skeletal muscle in addition to cardiac muscle in vivo supporting a possible role for the glucose-fatty acid cycle in skeletal muscle in acute insulin resistance. However, at high levels or with elevated insulin, FFA stimulates glucose utilization and storage in skeletal muscle. By promoting accumulation of glucose storage products, chronic elevation of FFA may lead to skeletal muscle (and therefore whole body) insulin resistance.
PMCID: PMC303508  PMID: 3292587
15.  Control of Muscle Mitochondria by Insulin Entails Activation of Akt2-mtNOS Pathway: Implications for the Metabolic Syndrome 
PLoS ONE  2008;3(3):e1749.
In the metabolic syndrome with hyperinsulinemia, mitochondrial inhibition facilitates muscle fat and glycogen accumulation and accelerates its progression. In the last decade, nitric oxide (NO) emerged as a typical mitochondrial modulator by reversibly inhibiting citochrome oxidase and oxygen utilization. We wondered whether insulin-operated signaling pathways modulate mitochondrial respiration via NO, to alternatively release complete glucose oxidation to CO2 and H2O or to drive glucose storage to glycogen.
Methodology/Principal Findings
We illustrate here that NO produced by translocated nNOS (mtNOS) is the insulin-signaling molecule that controls mitochondrial oxygen utilization. We evoke a hyperinsulinemic-normoglycemic non-invasive clamp by subcutaneously injecting adult male rats with long-lasting human insulin glargine that remains stable in plasma by several hours. At a precise concentration, insulin increased phospho-Akt2 that translocates to mitochondria and determines in situ phosphorylation and substantial cooperative mtNOS activation (+4–8 fold, P<.05), high NO, and a lowering of mitochondrial oxygen uptake and resting metabolic rate (−25 to −60%, P<.05). Comparing in vivo insulin metabolic effects on gastrocnemius muscles by direct electroporation of siRNA nNOS or empty vector in the two legs of the same animal, confirmed that in the silenced muscles disrupted mtNOS allows higher oxygen uptake and complete (U-14C)-glucose utilization respect to normal mtNOS in the vector-treated ones (respectively 37±3 vs 10±1 µmolO2/h.g tissue and 13±1 vs 7.2±1 µmol 3H2O/h.g tissue, P<.05), which reciprocally restricted glycogen-synthesis by a half.
These evidences show that after energy replenishment, insulin depresses mitochondrial respiration in skeletal muscle via NO which permits substrates to be deposited as macromolecules; at discrete hyperinsulinemia, persistent mtNOS activation could contribute to mitochondrial dysfunction with insulin resistance and obesity and therefore, to the progression of the metabolic syndrome.
PMCID: PMC2258147  PMID: 18335029
16.  Chronic Reduction of Plasma Free Fatty Acid Improves Mitochondrial Function and Whole-Body Insulin Sensitivity in Obese and Type 2 Diabetic Individuals 
Diabetes  2014;63(8):2812-2820.
Insulin resistance and dysregulation of free fatty acid (FFA) metabolism are core defects in type 2 diabetic (T2DM) and obese normal glucose tolerant (NGT) individuals. Impaired muscle mitochondrial function (reduced ATP synthesis) also has been described in insulin-resistant T2DM and obese subjects. We examined whether reduction in plasma FFA concentration with acipimox improved ATP synthesis rate and altered reactive oxygen species (ROS) production. Eleven NGT obese and 11 T2DM subjects received 1) OGTT, 2) euglycemic insulin clamp with muscle biopsy, and 3) 1H-magnetic resonance spectroscopy of tibialis anterior muscle before and after acipimox (250 mg every 6 h for 12 days). ATP synthesis rate and ROS generation were measured in mitochondria isolated from muscle tissue ex vivo with chemoluminescence and fluorescence techniques, respectively. Acipimox 1) markedly reduced the fasting plasma FFA concentration and enhanced suppression of plasma FFA during oral glucose tolerance tests and insulin clamp in obese NGT and T2DM subjects and 2) enhanced insulin-mediated muscle glucose disposal and suppression of hepatic glucose production. The improvement in insulin sensitivity was closely correlated with the decrease in plasma FFA in obese NGT (r = 0.81) and T2DM (r = 0.76) subjects (both P < 0.001). Mitochondrial ATP synthesis rate increased by >50% in both obese NGT and T2DM subjects and was strongly correlated with the decrease in plasma FFA and increase in insulin-mediated glucose disposal (both r > 0.70, P < 0.001). Production of ROS did not change after acipimox. Reduction in plasma FFA in obese NGT and T2DM individuals improves mitochondrial ATP synthesis rate, indicating that the mitochondrial defect in insulin-resistant individuals is, at least in part, reversible.
PMCID: PMC4113069  PMID: 24353180
17.  Altered Skeletal Muscle Fatty Acid Handling in Subjects with Impaired Glucose Tolerance as Compared to Impaired Fasting Glucose 
Nutrients  2016;8(3):164.
Altered skeletal muscle fatty acid (FA) metabolism contributes to insulin resistance. Here, we compared skeletal muscle FA handling between subjects with impaired fasting glucose (IFG; n = 12 (7 males)) and impaired glucose tolerance (IGT; n = 14 (7 males)) by measuring arterio-venous concentration differences across forearm muscle. [2H2]-palmitate was infused intravenously, labeling circulating endogenous triacylglycerol (TAG) and free fatty acids (FFA), whereas [U-13C]-palmitate was incorporated in a high-fat mixed-meal, labeling chylomicron-TAG. Skeletal muscle biopsies were taken to determine muscle TAG, diacylglycerol (DAG), FFA, and phospholipid content, their fractional synthetic rate (FSR) and degree of saturation, and gene expression. Insulin sensitivity was assessed using a hyperinsulinemic-euglycemic clamp. Net skeletal muscle glucose uptake was lower (p = 0.018) and peripheral insulin sensitivity tended to be reduced (p = 0.064) in IGT as compared to IFG subjects. Furthermore, IGT showed higher skeletal muscle extraction of VLDL-TAG (p = 0.043), higher muscle TAG content (p = 0.025), higher saturation of FFA (p = 0.004), lower saturation of TAG (p = 0.017) and a tendency towards a lower TAG FSR (p = 0.073) and a lower saturation of DAG (p = 0.059) versus IFG individuals. Muscle oxidative gene expression was lower in IGT subjects. In conclusion, increased liver-derived TAG extraction and reduced lipid turnover of saturated FA, rather than DAG content, in skeletal muscle accompany the more pronounced insulin resistance in IGT versus IFG subjects.
PMCID: PMC4808892  PMID: 26985905
skeletal muscle; lipid metabolism; insulin resistance; impaired glucose tolerance; impaired fasting glucose
18.  Impaired Mitochondrial Activity in the Insulin-Resistant Offspring of Patients with Type 2 Diabetes 
The New England journal of medicine  2004;350(7):664-671.
Insulin resistance appears to be the best predictor of the development of diabetes in the children of patients with type 2 diabetes, but the mechanism responsible is unknown.
We performed hyperinsulinemic–euglycemic clamp studies in combination with infusions of [6,6-2H2]glucose in healthy, young, lean, insulin-resistant offspring of patients with type 2 diabetes and insulin-sensitive control subjects matched for age, height, weight, and physical activity to assess the sensitivity of liver and muscle to insulin. Proton (1H) magnetic resonance spectroscopy studies were performed to measure intramyo-cellular lipid and intrahepatic triglyceride content. Rates of whole-body and subcutaneous fat lipolysis were assessed by measuring the rates of [2H5]glycerol turnover in combination with microdialysis measurements of glycerol release from subcutaneous fat. We performed 31P magnetic resonance spectroscopy studies to assess the rates of mitochondrial oxidative-phosphorylation activity in muscle.
The insulin-stimulated rate of glucose uptake by muscle was approximately 60 percent lower in the insulin-resistant subjects than in the insulin-sensitive control subjects (P<0.001) and was associated with an increase of approximately 80 percent in the intramyocellular lipid content (P=0.005). This increase in intramyocellular lipid content was most likely attributable to mitochondrial dysfunction, as reflected by a reduction of approximately 30 percent in mitochondrial phosphorylation (P=0.01 for the comparison with controls), since there were no significant differences in systemic or localized rates of lipolysis or plasma concentrations of tumor necrosis factor α, interleukin-6, resistin, or adiponectin.
These data support the hypothesis that insulin resistance in the skeletal muscle of insulin-resistant offspring of patients with type 2 diabetes is associated with dysregulation of intramyocellular fatty acid metabolism, possibly because of an inherited defect in mitochondrial oxidative phosphorylation.
PMCID: PMC2995502  PMID: 14960743
19.  Age, Obesity, and Sex Effects on Insulin Sensitivity and Skeletal Muscle Mitochondrial Function 
Diabetes  2009;59(1):89-97.
Reductions in insulin sensitivity in conjunction with muscle mitochondrial dysfunction have been reported to occur in many conditions including aging. The objective was to determine whether insulin resistance and mitochondrial dysfunction are directly related to chronological age or are related to age-related changes in body composition.
Twelve young lean, 12 young obese, 12 elderly lean, and 12 elderly obese sedentary adults were studied. Insulin sensitivity was measured by a hyperinsulinemic-euglycemic clamp, and skeletal muscle mitochondrial ATP production rates (MAPRs) were measured in freshly isolated mitochondria obtained from vastus lateralis biopsy samples using the luciferase reaction.
Obese participants, independent of age, had reduced insulin sensitivity based on lower rates of glucose infusion during a hyperinsulinemic-euglycemic clamp. In contrast, age had no independent effect on insulin sensitivity. However, the elderly participants had lower muscle MAPRs than the young participants, independent of obesity. Elderly participants also had higher levels inflammatory cytokines and total adiponectin. In addition, higher muscle MAPRs were also noted in men than in women, whereas glucose infusion rates were higher in women.
The results demonstrate that age-related reductions in insulin sensitivity are likely due to an age-related increase in adiposity rather than a consequence of advanced chronological age. The results also indicate that an age-related decrease in muscle mitochondrial function is neither related to adiposity nor insulin sensitivity. Of interest, a higher mitochondrial ATP production capacity was noted in the men, whereas the women were more insulin sensitive, demonstrating further dissociation between insulin sensitivity and muscle mitochondrial function.
PMCID: PMC2797949  PMID: 19833885
20.  Dynamic changes in fat oxidation in human primary myocytes mirror metabolic characteristics of the donor 
Journal of Clinical Investigation  2005;115(7):1934-1941.
Metabolic flexibility of skeletal muscle, that is, the preference for fat oxidation (FOx) during fasting and for carbohydrate oxidation in response to insulin, is decreased during insulin resistance. The aim of this study was to test the hypothesis that the capacity of myotubes to oxidize fat in vitro reflects the donor’s metabolic characteristics. Insulin sensitivity (IS) and metabolic flexibility of 16 healthy, young male subjects was determined by euglycemic hyperinsulinemic clamp. Muscle samples were obtained from vastus lateralis, cultured, and differentiated into myotubes. In human myotubes in vitro, we measured suppressibility (glucose suppression of FOx) and adaptability (an increase in FOx in the presence of high palmitate concentration). We termed these dynamic changes in FOx metabolic switching. In vivo, metabolic flexibility was positively correlated with IS and maximal oxygen uptake and inversely correlated with percent body fat. In vitro suppressibility was inversely correlated with IS and metabolic flexibility and positively correlated with body fat and fasting FFA levels. Adaptability was negatively associated with percent body fat and fasting insulin and positively correlated with IS and metabolic flexibility. The interindividual variability in metabolic phenotypes was preserved in human myotubes separated from their neuroendocrine environment, which supports the hypothesis that metabolic switching is an intrinsic property of skeletal muscle.
PMCID: PMC1159139  PMID: 16007256
21.  Skeletal muscle mitochondrial health and spinal cord injury 
World Journal of Orthopedics  2016;7(10):628-637.
Mitochondria are the main source of cellular energy production and are dynamic organelles that undergo biogenesis, remodeling, and degradation. Mitochondrial dysfunction is observed in a number of disease states including acute and chronic central or peripheral nervous system injury by traumatic brain injury, spinal cord injury (SCI), and neurodegenerative disease as well as in metabolic disturbances such as insulin resistance, type II diabetes and obesity. Mitochondrial dysfunction is most commonly observed in high energy requiring tissues like the brain and skeletal muscle. In persons with chronic SCI, changes to skeletal muscle may include remarkable atrophy and conversion of muscle fiber type from oxidative to fast glycolytic, combined with increased infiltration of intramuscular adipose tissue. These changes contribute to a proinflammatory environment, glucose intolerance and insulin resistance. The loss of metabolically active muscle combined with inactivity predisposes individuals with SCI to type II diabetes and obesity. The contribution of skeletal muscle mitochondrial density and electron transport chain activity to the development of the aforementioned comorbidities following SCI is unclear. A better understanding of the mechanisms involved in skeletal muscle mitochondrial dynamics is imperative to designing and testing effective treatments for this growing population. The current editorial will review ways to study mitochondrial function and the importance of improving skeletal muscle mitochondrial health in clinical populations with a special focus on chronic SCI.
PMCID: PMC5065669  PMID: 27795944
Mitochondria; Spinal cord injuries; Body composition; Diabetes mellitus; Obesity; Metabolism
22.  Duration of rise in free fatty acids determines salicylate's effect on hepatic insulin sensitivity 
The Journal of Endocrinology  2013;217(1):31-43.
We have shown in rats that sodium salicylate (SS), which inhibits IkBa kinase B (IKKB), prevents hepatic and peripheral insulin resistance caused by short-term (7 h) i.v. administration of Intralipid and heparin (IH). We wished to further determine whether this beneficial effect of SS persisted after prolonged (48 h) IH infusion, which better mimics the chronic free fatty acid (FFA) elevation of obesity. Hence, we performed hyperinsulinemic euglycemic clamps with tritiated glucose methodology to determine hepatic and peripheral insulin sensitivity in rats infused with saline, IH, IH and SS, or SS alone. SS prevented peripheral insulin resistance (P<0.05) caused by prolonged plasma FFA elevation; however, it did not prevent hepatic insulin resistance. In skeletal muscle, protein levels of phospho-IkBa were augmented by prolonged IH administration and this was prevented by SS, suggesting that IH activates while SS prevents the activation of IKKB. Markers of IKKB activation, namely protein levels of phospho-IkBa and IkBa, indicated that IKKB is not activated in the liver after prolonged FFA elevation. Phosphorylation of serine 307 at insulin receptor substrate (IRS)-1, which is a marker of proximal insulin resistance, was not altered by IH administration in the liver, suggesting that this is not a site of hepatic insulin resistance in the prolonged lipid infusion model. Our results suggest that the role of IKKB in fat-induced insulin resistance is time and tissue dependent and that hepatic insulin resistance induced by prolonged lipid elevation is not due to an IRS-1 serine 307 kinase.
PMCID: PMC3601809  PMID: 23328071
free fatty acids; insulin resistance; liver; glucose metabolism
23.  Differential regulation of PGC-1α expression in rat liver and skeletal muscle in response to voluntary running 
The beneficial actions of exercise training on lipid, glucose and energy metabolism and insulin sensitivity appear to be in part mediated by PGC-1α. Previous studies have shown that spontaneously exercised rats show at rest enhanced responsiveness to exogenous insulin, lower plasma insulin levels and increased skeletal muscle insulin sensitivity. This study was initiated to examine the functional interaction between exercise-induced modulation of skeletal muscle and liver PGC-1α protein expression, whole body insulin sensitivity, and circulating FFA levels as a measure of whole body fatty acid (lipid) metabolism.
Two groups of male Wistar rats (2 Mo of age, 188.82 ± 2.77 g BW) were used in this study. One group consisted of control rats placed in standard laboratory cages. Exercising rats were housed individually in cages equipped with running wheels and allowed to run at their own pace for 5 weeks. At the end of exercise training, insulin sensitivity was evaluated by comparing steady-state plasma glucose (SSPG) concentrations at constant plasma insulin levels attained during the continuous infusion of glucose and insulin to each experimental group. Subsequently, soleus and plantaris muscle and liver samples were collected and quantified for PGC-1α protein expression by Western blotting. Collected blood samples were analyzed for glucose, insulin and FFA concentrations.
Rats housed in the exercise wheel cages demonstrated almost linear increases in running activity with advancing time reaching to maximum value around 4 weeks. On an average, the rats ran a mean (Mean ± SE) of 4.102 ± 0.747 km/day and consumed significantly more food as compared to sedentary controls (P < 0.001) in order to meet their increased caloric requirement. Mean plasma insulin (P < 0.001) and FFA (P < 0.006) concentrations were lower in the exercise-trained rats as compared to sedentary controls. Mean steady state plasma insulin (SSPI) and glucose (SSPG) concentrations were not significantly different in sedentary control rats as compared to exercise-trained animals. Plantaris PGC-1α protein expression increased significantly from a 1.11 ± 0.12 in the sedentary rats to 1.74 ± 0.09 in exercising rats (P < 0.001). However, exercise had no effect on PGC-1α protein content in either soleus muscle or liver tissue. These results indicate that exercise training selectively up regulates the PGC-1α protein expression in high-oxidative fast skeletal muscle type such as plantaris muscle.
These data suggest that PGC-1α most likely plays a restricted role in exercise-mediated improvements in insulin resistance (sensitivity) and lowering of circulating FFA levels.
PMCID: PMC2874794  PMID: 20433743
24.  Relationships between Mitochondrial Function and Metabolic Flexibility in Type 2 Diabetes Mellitus 
PLoS ONE  2013;8(2):e51648.
Mitochondrial dysfunction, lipid accumulation, insulin resistance and metabolic inflexibility have been implicated in the etiology of type 2 diabetes (T2D), yet their interrelationship remains speculative. We investigated these interrelationships in a group of T2D and obese normoglycemic control subjects.
49 non-insulin dependent male T2D patients and 54 male control subjects were enrolled, and a hyperinsulinemic-euglycemic clamp and indirect calorimetry were performed. A muscle biopsy was taken and intramyocellular lipid (IMCL) was measured. In vivo mitochondrial function was measured by PCr recovery in 30 T2D patients and 31 control subjects.
Fasting NEFA levels were significantly elevated in T2D patients compared with controls, but IMCL was not different. Mitochondrial function in T2D patients was compromised by 12.5% (p<0.01). Whole body glucose disposal (WGD) was higher at baseline and lower after insulin stimulation. Metabolic flexibility (ΔRER) was lower in the type 2 diabetic patients (0.050±0.033 vs. 0.093±0.050, p<0.01). Mitochondrial function was the sole predictor of basal respiratory exchange ratio (RER) (R2 = 0.18, p<0.05); whereas WGD predicted both insulin-stimulated RER (R2 = 0.29, p<0.001) and metabolic flexibility (R2 = 0.40, p<0.001).
These results indicate that defects in skeletal muscle in vivo mitochondrial function in type 2 diabetic patients are only reflected in basal substrate oxidation and highlight the importance of glucose disposal rate as a determinant of substrate utilization in response to insulin.
PMCID: PMC3572106  PMID: 23418416
25.  Insulin and GLP-1 infusions demonstrate the onset of adipose-specific insulin resistance in a large fasting mammal: potential glucogenic role for GLP-1 
Physiological reports  2013;1(2):e00023.
Prolonged food deprivation increases lipid oxidation and utilization, which may contribute to the onset of the insulin resistance associated with fasting. Because insulin resistance promotes the preservation of glucose and oxidation of fat, it has been suggested to be an adaptive response to food deprivation. However, fasting mammals exhibit hypoinsulinemia, suggesting that the insulin resistance-like conditions they experience may actually result from reduced pancreatic sensitivity to glucose/capacity to secrete insulin. To determine whether fasting results in insulin resistance or in pancreatic dysfunction, we infused early- and late-fasted seals (naturally adapted to prolonged fasting) with insulin (0.065 U/kg), and a separate group of late-fasted seals with low (10 pM/kg) or high (100 pM/kg) dosages of glucagon-like peptide-1 (GLP-1) immediately following a glucose bolus (0.5g/kg), and measured the systemic and cellular responses. Because GLP-1 facilitates glucose-stimulated insulin secretion, these infusions provide a method to assess pancreatic insulin-secreting capacity. Insulin infusions increased the phosphorylation of insulin receptor and Akt in adipose and muscle of early and late fasted seals; however the timing of the signaling response was blunted in adipose of late fasted seals. Despite the dose-dependent increases in insulin and increased glucose clearance (high dose), both GLP-1 dosages produced increases in plasma cortisol and glucagon, which may have contributed to the glucogenic role of GLP-1. Results suggest that fasting induces adipose-specific insulin resistance in elephant seal pups, while maintaining skeletal muscle insulin sensitivity, and therefore suggests that the onset of insulin resistance in fasting mammals is an evolved response to cope with prolonged food deprivation.
PMCID: PMC3755502  PMID: 23997935
insulin sensitivity; glucose intolerance; GLP-1; elephant seal; fatty acids; adipose tissue

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