Delayed Skeletal Muscle Mitochondrial ADP Recovery in Youth With Type 1 Diabetes Relates to Muscle Insulin Resistance

Insulin resistance (IR) increases cardiovascular morbidity and is associated with mitochondrial dysfunction. IR is now recognized to be present in type 1 diabetes; however, its relationship with mitochondrial function is unknown. We determined the relationship between IR and muscle mitochondrial function in type 1 diabetes using the hyperinsulinemic-euglycemic clamp and ³¹P-MRS before, during, and after near-maximal isometric calf exercise. Volunteers included 21 nonobese adolescents with type 1 diabetes and 17 nondiabetic control subjects with similar age, sex, BMI, Tanner stage, and activity levels. We found that youths with type 1 diabetes were more insulin resistant (median glucose infusion rate 10.1 vs. 18.9 mg/kglean/min; P < 0.0001) and had a longer time constant of the curve of ADP conversion to ATP (23.4 ± 5.3 vs. 18.8 ± 3.9 s, P < 0.001) and a lower rate of oxidative phosphorylation (median 0.09 vs. 0.21 mmol/L/s, P < 0.001). The ADP time constant (β = −0.36, P = 0.026) and oxidative phosphorylation (β = 0.02, P < 0.038) were related to IR but not HbA_1c. Normal-weight youths with type 1 diabetes demonstrated slowed postexercise ATP resynthesis and were more insulin resistant than control subjects. The correlation between skeletal muscle mitochondrial dysfunction in type 1 diabetes and IR suggests a relationship between mitochondrial dysfunction and IR in type 1 diabetes.     

Activating HSP72 in Rodent Skeletal Muscle Increases Mitochondrial Number and Oxidative Capacity and Decreases Insulin Resistance

Induction of heat shock protein (HSP)72 protects against obesity-induced insulin resistance, but the underlying mechanisms are unknown. Here, we show that HSP72 plays a pivotal role in increasing skeletal muscle mitochondrial number and oxidative metabolism. Mice overexpressing HSP72 in skeletal muscle (HSP72Tg) and control wild-type (WT) mice were fed either a chow or high-fat diet (HFD). Despite a similar energy intake when HSP72Tg mice were compared with WT mice, the HFD increased body weight, intramuscular lipid accumulation (triacylglycerol and diacylglycerol but not ceramide), and severe glucose intolerance in WT mice alone. Whole-body VO₂, fatty acid oxidation, and endurance running capacity were markedly increased in HSP72Tg mice. Moreover, HSP72Tg mice exhibited an increase in mitochondrial number. In addition, the HSP72 coinducer BGP-15, currently in human clinical trials for type 2 diabetes, also increased mitochondrial number and insulin sensitivity in a rat model of type 2 diabetes. Together, these data identify a novel role for activation of HSP72 in skeletal muscle. Thus, the increased oxidative metabolism associated with activation of HSP72 has potential clinical implications not only for type 2 diabetes but also for other disorders where mitochondrial function is compromised.     

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.Obesity is associated with a chronic low-grade inflammatory response that induces defects in energy balance, insulin sensitivity, and lipid metabolism (1). The suppressor of cytokine signaling (SOCS) family of proteins (SOCS1–7), which bind via their SH2 domains to tyrosine phosphorylation sites on cytokine receptors, inhibit inflammatory signal transduction. In obesity, consistent with increases in inflammation, SOCS3 is upregulated in the hypothalamus (2), adipose tissue (3), and liver (4,5). SOCS3 expression is also increased in human and rodent skeletal muscle with obesity (6,7).Skeletal muscle is an important tissue contributing to basal metabolic rate and control of whole-body insulin sensitivity. A recent study has shown that the overexpression of SOCS3 in skeletal muscle by ∼150-fold disrupts calcineurin signaling, resulting in defects in muscle sarcoplasmic reticulum and mitochondria (8). As a result of impaired muscle development, transgenic SOCS3-overexpressing mice had reduced ambulatory activity levels. Although these data suggest a potentially intriguing role for SOCS3 in regulating muscle function, a major caveat of these studies involving the overexpression of SOCS3 is that, in the absence of overt inflammation, SOCS3 expression in muscle is low (9). SOCS3 also may play an important role in regulating energy balance because it inhibits leptin activation of Y985 within the leptin receptor (10,11). SOCS3 heterozygous mice (12) or those with SOCS3 deleted in hypothalamic neurons (13) have reduced appetite and are protected from development of diet-induced obesity attributable to enhanced hypothalamic leptin sensitivity within proopiomelanocortin-expressing neurons (11). Like the hypothalamus, we have shown that skeletal muscle also becomes resistant to leptin in obesity (14,15), which is characterized by an impaired ability of leptin to increase fatty acid oxidation via the AMP-activated protein kinase (AMPK) (16). In cultured muscle cells, the overexpression of SOCS3 inhibits leptin activation of AMPK and fatty acid oxidation (17). However, because leptin also activates AMPK in skeletal muscle via hypothalamic circuits (18), it is unknown whether physiological increases in SOCS3 expression in obesity (two- to threefold) may be of biological importance for regulating muscle function and energy balance.SOCS3 is an important negative regulator of insulin signaling (19). Genetic deletion of SOCS3 from mouse liver results in enhanced insulin signaling because of increased insulin receptor substrate 1 (IRS1) phosphorylation (20,21). However, when mice are fed a high-fat diet (HFD), the enhanced liver insulin sensitivity paradoxically promotes liver lipogenesis, exacerbating the development of nonalcoholic fatty liver disease, systemic inflammation, and the onset of obesity (21). These data, which are in contrast to transient partial reductions in SOCS3 expression using small interfering RNA (5,22), suggest that chronic inhibition of SOCS3 in the liver is not an appropriate treatment for insulin resistance in obesity. In skeletal muscle, SOCS3 has been shown to coimmunoprecipitate with both the insulin receptor (IR) and IRS1 (23); however, in contrast to reports in adipose tissue (24) and liver (5), the overexpression of SOCS3 in skeletal muscle is not associated with reduced IRS1 signaling or the development of insulin resistance (8).Given the importance of skeletal muscle in the regulation of energy metabolism and insulin sensitivity, we generated mice with muscle-specific deletion of SOCS3 (SOCS3 MKO). We demonstrate that deletion of SOCS3 in muscle does not alter muscle development, body mass, adiposity, or energy expenditure, but it causes substantial protection against the development of obesity-induced hyperinsulinemia and hyperglycemia attributable to enhanced skeletal muscle IRS1 phosphorylation and glucose uptake.     

Lipid-Induced Insulin Resistance Is Not Mediated by Impaired Transcapillary Transport of Insulin and Glucose in Humans

Increased lipid availability reduces insulin-stimulated glucose disposal in skeletal muscle, which is generally explained by fatty acid–mediated inhibition of insulin signaling. It remains unclear whether lipids also impair transcapillary transport of insulin and glucose, which could become rate controlling for glucose disposal. We hypothesized that lipid-induced insulin resistance is induced by inhibiting myocellular glucose uptake and not by interfering with the delivery of insulin or glucose. We measured changes in interstitial glucose and insulin in skeletal muscle of healthy volunteers during intravenous administration of triglycerides plus heparin or glycerol during physiologic and supraphysiologic hyperinsulinemia, by combining microdialysis with oral glucose tolerance tests and euglycemic-hyperinsulinemic clamps. Lipid infusion reduced insulin-stimulated glucose disposal by ∼70% (P < 0.05) during clamps and dynamic insulin sensitivity by ∼12% (P < 0.05) during oral glucose loading. Dialysate insulin and glucose levels were unchanged or even transiently higher (P < 0.05) during lipid than during glycerol infusion, whereas regional blood flow remained unchanged. These results demonstrate that short-term elevation of free fatty acids (FFAs) induces insulin resistance, which in skeletal muscle occurs primarily at the cellular level, without impairment of local perfusion or transcapillary transport of insulin and glucose. Thus, vascular effects of FFAs are not rate controlling for muscle insulin-stimulated glucose disposal.Skeletal muscle accounts for the majority of glucose uptake after a meal and almost all glucose disposal during hyperinsulinemic-euglycemic clamps (1). In type 2 diabetes (T2DM), muscle insulin resistance predicts postprandial hyperglycemia, but the underlying mechanisms are unclear. Insulin-resistant humans frequently present with increased plasma free fatty acids (FFAs) (2), which can give rise to myocellular diacylglycerols or ceramides and impair insulin signaling (3–5). Insulin increases muscle microvascular perfusion and facilitates delivery of nutrients and hormones to the interstitium (6). Animal models of lipid-induced insulin resistance suggest that insulin-mediated microvascular perfusion is already reduced in prediabetic states and relates to impaired insulin action (7,8). Preventing the access of glucose and insulin to myocytes could contribute to lower glucose disposal and place abnormal microvascular insulin action as an early event in the development of T2DM.We hypothesized that lipid-induced insulin resistance results from myocellular glucose uptake, but not from impaired delivery of insulin or glucose to the interstitium. We monitored changes of interstitial insulin and glucose in muscle of humans during intravenous triglycerides or glycerol administration under physiologic dynamic (oral glucose tolerance test [OGTT]) and supraphysiologic constant hyperinsulinemic (clamp) conditions.     

Age, Obesity, and Sex Effects on Insulin Sensitivity and Skeletal Muscle Mitochondrial Function

OBJECTIVE

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.

RESEARCH DESIGN AND METHODS

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.

RESULTS

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.

CONCLUSIONS

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.As the population ages, the prevalence of several chronic health problems such as obesity, type 2 diabetes, and cardiovascular disease has risen. Insulin resistance is recognized as a key factor contributing to the development of both type 2 diabetes and its related cardiometabolic disorders (1,2). Insulin resistance and impaired glucose tolerance are commonly observed phenomena among elderly adults. For example, the glucose excursion postprandially is substantially greater and remains elevated longer in nondiabetic elderly adults than in nondiabetic younger adults, which is indicative of age-related declines in insulin sensitivity and glucose tolerance (3). Aging is associated with detrimental changes in body composition, which persists even when elderly adults are matched to younger adults for BMI (4). Adiposity, in particular abdominal adiposity, is well accepted as a determinant of insulin resistance and therefore may be a key mediator for the development of age-related insulin resistance. Despite an inverse relationship between age and insulin sensitivity (4,5), it remains contentious whether chronological age is a primary determinant of insulin resistance or whether age-related elevations in adiposity and/or physical inactivity are the primary causes of age-related insulin resistance (6,7).Aging is also associated with reductions in skeletal muscle mitochondrial function. In particular, skeletal muscle mitochondrial ATP production rates (MAPRs) in elderly people are reduced in vivo in the resting state (8) as well as in vitro in the maximally stimulated state (3). These age-related reductions in MAPRs have also been associated with concomitant reductions in skeletal muscle mitochondrial enzyme activities (9), protein synthesis and expression (3,10), and mtDNA abundance in humans (3,11) and rodents (12). Of interest, insulin resistance is closely associated with skeletal muscle mitochondrial dysfunction in some (3,5,13,14) but not in all conditions (15,16). This close association between muscle mitochondrial dysfunction and insulin resistance has led to the hypothesis that mitochondrial dysfunction could be the basis of insulin resistance (5). Another equally plausible hypothesis is that insulin resistance causes muscle mitochondrial dysfunction (16,17). In support of the later hypothesis is the demonstration that, in type 2 diabetic people, muscle MAPR fails to increase in response to physiologically high insulin levels, unlike in nondiabetic people (18). However, it should be recognized that the association between insulin resistance and mitochondrial dysfunction are not consistent. For example, we recently reported that Asian Indians in comparison with Northern European Americans matched for age, sex, and BMI are severely insulin resistant, while having higher muscle MAPR and mitochondrial DNA copy numbers (19). Furthermore, a recent report also indicated that while a low-calorie diet substantially enhanced insulin sensitivity (e.g., ∼30% increase in insulin-stimulated glucose disposal), it failed to increase skeletal muscle mitochondrial function in the absence of exercise (19). In contrast, in rats, a high-fat diet caused insulin resistance while enhancing mitochondrial biogenesis (15). Together, the results from the above studies indicate that the close association between insulin sensitivity and muscle mitochondrial function can be uncoupled, arguing against the hypothesis that insulin resistance causes muscle mitochondrial dysfunction or vice versa.Age is not only associated with insulin resistance and muscle mitochondrial dysfunction but is also associated with changes in body composition, which likely contribute to the development of age-related insulin resistance (20). We therefore sought to determine whether the changes in insulin sensitivity and muscle mitochondrial function are secondary to age-related changes in body composition rather than being directly related to chronological age. We studied 48 lean and obese, young and elderly men and women. Insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp and skeletal muscle mitochondrial function by measuring MAPRs from freshly prepared mitochondria obtained from muscle biopsy samples. The studies demonstrated the impact of not only age and body weight, but also sex on insulin sensitivity and muscle mitochondrial function in humans.     

Alterations in Skeletal Muscle Fatty Acid Handling Predisposes Middle-Aged Mice to Diet-Induced Insulin Resistance

OBJECTIVE

Although advanced age is a risk factor for type 2 diabetes, a clear understanding of the changes that occur during middle age that contribute to the development of skeletal muscle insulin resistance is currently lacking. Therefore, we sought to investigate how middle age impacts skeletal muscle fatty acid handling and to determine how this contributes to the development of diet-induced insulin resistance.

RESEARCH DESIGN AND METHODS

Whole-body and skeletal muscle insulin resistance were studied in young and middle-aged wild-type and CD36 knockout (KO) mice fed either a standard or a high-fat diet for 12 weeks. Molecular signaling pathways, intramuscular triglycerides accumulation, and targeted metabolomics of in vivo mitochondrial substrate flux were also analyzed in the skeletal muscle of mice of all ages.

RESULTS

Middle-aged mice fed a standard diet demonstrated an increase in intramuscular triglycerides without a concomitant increase in insulin resistance. However, middle-aged mice fed a high-fat diet were more susceptible to the development of insulin resistance—a condition that could be prevented by limiting skeletal muscle fatty acid transport and excessive lipid accumulation in middle-aged CD36 KO mice.

CONCLUSION

Our data provide insight into the mechanisms by which aging becomes a risk factor for the development of insulin resistance. Our data also demonstrate that limiting skeletal muscle fatty acid transport is an effective approach for delaying the development of age-associated insulin resistance and metabolic disease during exposure to a high-fat diet.Over the past few decades, type 2 diabetes has increased in prevalence largely as a result of the obesity epidemic (1). Although it is widely accepted that skeletal muscle insulin resistance is a major determinant in both the onset and progression of type 2 diabetes (2), the exact cause of decreased insulin action in skeletal muscle is not known (3). That said, it is generally believed that skeletal muscle insulin resistance develops secondary to impaired mitochondrial fatty acid oxidation (4,5). However, several other studies have shown that lipid accumulation is not associated with skeletal muscle insulin resistance (6–8) or overall mitochondrial dysfunction (9–13). Consistent with this, a growing body of evidence has suggested that the cause of skeletal muscle insulin resistance may not result from impaired fatty acid oxidation but might actually result from excessive skeletal muscle mitochondrial fatty acid oxidation and ensuing mitochondrial stress (12,14). While it is not known which of these two processes are most relevant in the pathogenesis of skeletal muscle insulin resistance, it is clear that excessive entry of fatty acids into the skeletal muscle plays a central role in diet-induced insulin resistance.Because advanced age is a significant risk factor in the etiology of type 2 diabetes (15,16), the accompanying loss of mitochondrial function observed with normal aging has been proposed to contribute to the increased risk of type 2 diabetes in the elderly population (17). However, a clear understanding of the physiological changes that occur during the onset of middle age and the influence that this may have on the development of insulin resistance is currently lacking. This is particularly important given that the baby boomer generation, the largest population group in the Western world, is currently classified as middle-aged (18) as well as the fact that the prevalence of type 2 diabetes in the Western world is expected to increase dramatically over the next 5–10 years (16,18). The study herein was designed to investigate how middle age impacts whole-body glucose utilization, fatty acid handling, and triglyceride accumulation within skeletal muscle as well as the susceptibility of middle-aged mice to the development of diet-induced insulin resistance.     

FOXO1 Mediates Vitamin D Deficiency–Induced Insulin Resistance in Skeletal Muscle

Prospective epidemiological studies have consistently shown a relationship between vitamin D deficiency, insulin resistance, and type 2 diabetes mellitus (DM2). This is supported by recent trials showing that vitamin D supplementation in prediabetic or insulin‐resistant patients with inadequate vitamin D levels improves insulin sensitivity. However, the molecular mechanisms underlying vitamin D deficiency–induced insulin resistance and DM2 remain unknown. Skeletal muscle insulin resistance is a primary defect in the majority of patients with DM2. Although sustained activation of forkhead box O1 (FOXO1) in skeletal muscle causes insulin resistance, a relationship between vitamin D deficiency and FOXO1 activation in muscle is unknown. We generated skeletal muscle‐specific vitamin D receptor (VDR)‐null mice and discovered that these mice developed insulin resistance and glucose intolerance accompanied by increased expression and activity of FOXO1. We also found sustained FOXO1 activation in the skeletal muscle of global VDR‐null mice. Treatment of C2C12 muscle cells with 1,25‐dihydroxyvitamin D (VD3) reduced FOXO1 expression, nuclear translocation, and activity. The VD3‐dependent suppression of FOXO1 activation disappeared by knockdown of VDR, indicating that it is VDR‐dependent. Taken together, these results suggest that FOXO1 is a critical target mediating VDR‐null signaling in skeletal muscle. The novel findings provide the conceptual support that persistent FOXO1 activation may be responsible for insulin resistance and impaired glucose metabolism in vitamin D signaling‐deficient mice, as well as evidence for the utility of vitamin D supplementation for intervention in DM2. © 2015 American Society for Bone and Mineral Research.     

Chemerin Is a Novel Adipocyte-Derived Factor Inducing Insulin Resistance in Primary Human Skeletal Muscle Cells

OBJECTIVE

Chemerin is an adipokine that affects adipogenesis and glucose homeostasis in adipocytes and increases with BMI in humans. This study was aimed at investigating the regulation of chemerin release and its effects on glucose metabolism in skeletal muscle cells.

RESEARCH DESIGN AND METHODS

Human skeletal muscle cells were treated with chemerin to study insulin signaling, glucose uptake, and activation of stress kinases. The release of chemerin was analyzed from in vitro differentiated human adipocytes and adipose tissue explants from 27 lean and 26 obese patients.

RESULTS

Human adipocytes express chemerin and chemokine-like receptor 1 (CMKLR1) differentiation dependently and secrete chemerin (15 ng/ml from 10⁶ cells). This process is slightly but significantly increased by tumor necrosis factor-α and markedly inhibited by >80% by peroxisome proliferator–activated receptor-γ activation. Adipose tissue explants from obese patients are characterized by significantly higher chemerin secretion compared with lean control subjects (21 and 8 ng from 10⁷ cells, respectively). Chemerin release is correlated with BMI, waist-to-hip ratio, and adipocyte volume. Furthermore, higher chemerin release is associated with insulin resistance at the level of lipogenesis and insulin-induced antilipolysis in adipocytes. Chemerin induces insulin resistance in human skeletal muscle cells at the level of insulin receptor substrate 1, Akt and glycogen synthase kinase 3 phosphorylation, and glucose uptake. Furthermore, chemerin activates p38 mitogen-activated protein kinase, nuclear factor-κB, and extracellular signal–regulated kinase (ERK)-1/2. Inhibition of ERK prevents chemerin-induced insulin resistance, pointing to participation of this pathway in chemerin action.

CONCLUSIONS

Adipocyte-derived secretion of chemerin may be involved in the negative cross talk between adipose tissue and skeletal muscle contributing to the negative relationship between obesity and insulin sensitivity.Obesity is one of the most serious health hazards, especially in the Western world. Frequently, obesity is accompanied by metabolic disturbances, such as insulin resistance, hyperglycemia, dyslipidemia, hypertension, and other components of the metabolic syndrome (1,2). Insulin resistance is a hallmark of obesity, emerging early in the metabolic syndrome, and is highly associated with increased visceral adipose tissue mass. The concept of adipose tissue as a major secretory and endocrine active organ producing a variety of bioactive proteins that may regulate energy metabolism and insulin sensitivity is now widely accepted (3), and increased adipose tissue mass, especially in the visceral compartment, is now described as one of the major risk factors for the development of type 2 diabetes (4–6). Adipocytes from obese subjects are characterized by altered metabolic and endocrine function leading to an increased secretion of proinflammatory adipokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-6, angiotensinogen, and resistin (7,8). It is likely that some of these secreted molecules may be factors underlying the association of increased body fat to insulin resistance in peripheral organs, such as skeletal muscle. We previously demonstrated that skeletal muscle cells treated with conditioned medium from adipocytes or the adipokine monocyte chemotactic protein (MCP)-1 are characterized by an impairment of insulin signaling and glucose uptake (9,10) and could thereby define the mechanism of a negative cross talk between adipose tissue and skeletal muscle.Recently, the rapidly growing adipokine family was expanded by chemerin, a secreted chemoattractant protein. Initially discovered in body fluids associated with inflammatory processes (11), chemerin and its receptor, chemokine-like receptor 1 (CMKLR1, or ChemR23) are also highly expressed in adipose tissue (12,13). In adipocytes, chemerin and CMKLR1 are necessary for adipogenesis (13). In vivo data revealed that chemerin is elevated in adipose tissue of diabetic Psammomys obesus compared with control subjects (12). However, no difference in chemerin levels between diabetic and control patients could be observed, despite a correlation of chemerin levels with BMI, blood triglycerides, and blood pressure (12). Because skeletal muscle is the major postprandial glucose-uptaking organ, the current study was meant to describe effects of the novel adipokine chemerin on skeletal muscle insulin sensitivity in the context of the negative cross talk between adipose tissue and skeletal muscle.     

Heat Treatment Improves Glucose Tolerance and Prevents Skeletal Muscle Insulin Resistance in Rats Fed a High-Fat Diet

OBJECTIVE—Heat treatment and overexpression of heat shock protein 72 (HSP72) have been shown to protect against high-fat diet–induced insulin resistance, but little is known about the underlying mechanism or the target tissue of HSP action. The purpose of this study is to determine whether in vivo heat treatment can prevent skeletal muscle insulin resistance.RESEARCH DESIGN AND METHODS—Male Wistar rats were fed a high-fat diet (60% calories from fat) for 12 weeks and received a lower-body heat treatment (41°C for 20 min) once per week.RESULTS—Our results show that heat treatment shifts the metabolic characteristics of rats on a high-fat diet toward those on a standard diet. Heat treatment improved glucose tolerance, restored insulin-stimulated glucose transport, and increased insulin signaling in soleus and extensor digitorum longus (EDL) muscles from rats fed a high-fat diet. Heat treatment resulted in decreased activation of Jun NH₂-terminal kinase (JNK) and inhibitor of κB kinase (IKK-β), stress kinases implicated in insulin resistance, and upregulation of HSP72 and HSP25, proteins previously shown to inhibit JNK and IKK-β activation, respectively. Mitochondrial citrate synthase and cytochrome oxidase activity decreased slightly with the high-fat diet, but heat treatment restored these activities. Data from L6 cells suggest that one bout of heat treatment increases mitochondrial oxygen consumption and fatty acid oxidation.CONCLUSIONS—Our results indicate that heat treatment protects skeletal muscle from high-fat diet–induced insulin resistance and provide strong evidence that HSP induction in skeletal muscle could be a potential therapeutic treatment for obesity-induced insulin resistance.Insulin resistance is associated with many related health complications, including type 2 diabetes and heart disease. A recent study demonstrated induction of the natural defense system of the body, heat shock proteins (HSPs), protects against obesity-induced insulin resistance (1). Earlier studies in patients with type 2 diabetes showed that hot tub therapy improved glycemic control (2) and an inverse correlation between HSP72 mRNA expression and the degree of type 2 diabetes (3). Currently, several HSP-inducing drugs are under investigation or in clinical trials for diabetic neuropathy and neurodegenerative diseases (4,5) and could be considered for prevention of insulin resistance. However, little is known about the mechanism behind this newly discovered role of HSP72, whether other inducible HSPs could be protective against insulin resistance, or the primary target tissue of HSP action.Skeletal muscle is the major tissue responsible for whole-body insulin-mediated glucose uptake (6,7). HSPs are expressed in skeletal muscle and are strongly induced with exercise training (8,9). Overexpression of HSP72 has been shown to reduce skeletal muscle atrophy and oxidative stress with age (10). Therefore, skeletal muscle is a logical choice as the target tissue for the benefits of HSP overexpression. Previous studies indicate basal levels of HSPs differ between muscle fiber types with slow-twitch oxidative muscles having higher constitutive expression of HSPs than fast-twitch glycolytic muscles (11). In contrast, fast-twitch muscles possess greater capacity for HSP induction in response to physiological stressors and exercise (11,12). It is uncertain whether HSPs would be equally effective as mediators of insulin action in slow- and fast-twitch muscle.The purpose of the present study was to determine whether weekly in vivo heat treatment could prevent skeletal muscle insulin resistance in rats fed a high-fat diet and elucidate mechanisms of HSP function in skeletal muscle. We hypothesized that heat treatment allows skeletal muscle to adapt and resist the development of insulin resistance as a result of increased HSP expression. Our findings indicate that heat treatment prevents skeletal muscle insulin resistance and stress kinase activation, whereas increased oxygen consumption and fatty acid oxidation in L6 cells suggest that heat treatment can improve mitochondrial function.     

Ceramides Contained in LDL Are Elevated in Type 2 Diabetes and Promote Inflammation and Skeletal Muscle Insulin Resistance

Dysregulated lipid metabolism and inflammation are linked to the development of insulin resistance in obesity, and the intracellular accumulation of the sphingolipid ceramide has been implicated in these processes. Here, we explored the role of circulating ceramide on the pathogenesis of insulin resistance. Ceramide transported in LDL is elevated in the plasma of obese patients with type 2 diabetes and correlated with insulin resistance but not with the degree of obesity. Treating cultured myotubes with LDL containing ceramide promoted ceramide accrual in cells and was accompanied by reduced insulin-stimulated glucose uptake, Akt phosphorylation, and GLUT4 translocation compared with LDL deficient in ceramide. LDL-ceramide induced a proinflammatory response in cultured macrophages via toll-like receptor–dependent and –independent mechanisms. Finally, infusing LDL-ceramide into lean mice reduced insulin-stimulated glucose uptake, and this was due to impaired insulin action specifically in skeletal muscle. These newly identified roles of LDL-ceramide suggest that strategies aimed at reducing hepatic ceramide production or reducing ceramide packaging into lipoproteins may improve skeletal muscle insulin action.Obesity is associated with the development of chronic diseases, including dyslipidemia, nonalcoholic fatty liver disease, type 2 diabetes, and atherosclerosis. Insulin resistance is a central feature of the pathophysiology of these disorders and is defined as a subnormal response of tissues to the actions of insulin, resulting in decreased glucose uptake into skeletal muscle and impaired suppression of glucose production by the liver. Although the mechanisms responsible for the development of insulin resistance are not fully defined, there is compelling evidence that defective lipid metabolism (1) and consequent subclinical inflammation (2) plays a causative role.Dyslipidemia resulting from overnutrition and defective adipocyte lipolysis is postulated to be a major contributor to liver and skeletal muscle insulin resistance, at least in part by promoting the intracellular accumulation of lipid metabolites that impair insulin signal transduction (1). Ceramide has been postulated as a primary lipid mediator of skeletal muscle insulin resistance based on findings that intracellular ceramide is elevated in insulin-resistant states (3–5) and that pharmacological inhibition of de novo ceramide synthesis enhances insulin action in insulin-resistant rodents (6). Ceramide induces insulin resistance by inhibiting insulin signal transduction, principally at Akt (7), and possibly via activation of serine/threonine kinases such as Jun NH₂-terminal kinase (JNK) (8), which in turn inhibits activation of insulin receptor substrate proteins (9). Ceramide is also postulated to activate proinflammatory pathways in macrophages, perhaps via amplification of toll-like receptor 4 (TLR4)–mediated inflammation (10,11). Interestingly, activation of TLR4 can increase ceramide levels in macrophages (12), supporting a model whereby ceramide can both induce and amplify macrophage inflammation. Consequently, the activation of tissue-resident macrophages would be predicted to promote a proinflammatory milieu that impairs insulin action.Although the consequences of tissue ceramide accumulation have been extensively studied over the last decade, it is now apparent that ceramides are also increased in the plasma of obese, type 2 diabetic mice (6,13) and humans (14), and that weight loss induced by gastric bypass surgery (15) or lifestyle modification (16) reduces plasma ceramide. Clinical data indicate that circulating ceramides correlate with systemic insulin resistance and inflammation (14,17), and pharmacological inhibition of whole-body ceramide synthesis in obese mice decreases plasma ceramide, reduces inflammatory parameters, and improves insulin action (6,18). Notably, not all studies report an association between obesity/diabetes and elevated circulating ceramide levels (19–21), and to date, there is no evidence for a direct effect of circulating ceramide on peripheral insulin action and inflammation. In the current study, we show that ceramides contained in LDL are elevated in type 2 diabetes and establish a link between LDL-ceramide, skeletal muscle insulin resistance, inflammation, and impaired systemic insulin action.     

Downregulation of AMPK Accompanies Leucine- and Glucose-Induced Increases in Protein Synthesis and Insulin Resistance in Rat Skeletal Muscle

OBJECTIVE

Branched-chain amino acids, such as leucine and glucose, stimulate protein synthesis and increase the phosphorylation and activity of the mammalian target of rapamycin (mTOR) and its downstream target p70S6 kinase (p70S6K). We examined in skeletal muscle whether the effects of leucine and glucose on these parameters and on insulin resistance are mediated by the fuel-sensing enzyme AMP-activated protein kinase (AMPK).

RESEARCH DESIGN AND METHODS

Rat extensor digitorum longus (EDL) muscle was incubated with different concentrations of leucine and glucose with or without AMPK activators. Muscle obtained from glucose-infused rats was also used as a model.

RESULTS

In the EDL, incubation with 100 or 200 μmol/l leucine versus no added leucine suppressed the activity of the α2 isoform of AMPK by 50 and 70%, respectively, and caused concentration-dependent increases in protein synthesis and mTOR and p70S6K phosphorylation. Very similar changes were observed in EDL incubated with 5.5 or 25 mmol/l versus no added glucose and in muscle of rats infused with glucose in vivo. Incubation of the EDL with the higher concentrations of both leucine and glucose also caused insulin resistance, reflected by a decrease in insulin-stimulated Akt phosphorylation. Coincubation with the AMPK activators AICAR and α-lipoic acid substantially prevented all of those changes and increased the phosphorylation of specific sites of mTOR inhibitors raptor and tuberous sclerosis complex 2 (TSC2). In contrast, decreases in AMPK activity induced by leucine and glucose were not associated with a decrease in raptor or TSC2 phosphorylation.

CONCLUSIONS

The results indicate that both leucine and glucose modulate protein synthesis and mTOR/p70S6 and insulin signaling in skeletal muscle by a common mechanism. They also suggest that the effects of both molecules are associated with a decrease in AMPK activity and that AMPK activation prevents them.AMP-activated protein kinase (AMPK) is a fuel-sensing enzyme that has classically been defined in terms of its role in restoring ATP levels in energy-depleted cells. In skeletal muscle, AMPK is typically activated by such factors as glucose deprivation and contraction (exercise) (1,2). The activated AMPK in turn enhances processes that generate ATP, such as fatty acid oxidation and glucose transport, and downregulates others that consume ATP and can be diminished temporarily without jeopardizing the cell (e.g., protein and lipid synthesis). Much less studied is the notion that a decrease in AMPK below baseline values may also be a physiologically or pathophysiologically relevant event. In keeping with such a possibility, decreased AMPK activity has been observed in tissues of many obese insulin-resistant rodents (3) and in liver (4,5) and adipose tissue (6) of rats starved for 48 h when they are refed. One consequence of decreased AMPK activity could be increases in mammalian target of rapamycin (mTOR)/p70S6 kinase (p70S6K) signaling and protein synthesis because both are decreased by AMPK activation (7).In the present study, we assessed whether fuel-induced increases in protein synthesis, mTOR/p70S6K signaling, and insulin resistance in skeletal muscle are mediated by decreases in AMPK activity. Toward this end, rat extensor digitorum longus (EDL) muscles were incubated for different time periods with various concentrations of leucine or glucose and the above parameters were assessed. The results indicate that elevated concentrations of leucine and glucose decrease AMPK activity, increase protein synthesis and mTOR/p70S6 phosphorylation, and cause insulin resistance and that activation of AMPK by pharmacological agents prevents these events from occurring. Finally, the data suggest that the decrease in AMPK activity caused by both leucine and glucose is not mediated by changes in the AMP-to-ATP ratio but is associated with an increase in the lactate/pyruvate ratio.     

Diet-Induced Obesity Prevents Interstitial Dispersion of Insulin in Skeletal Muscle

OBJECTIVE

Obesity causes insulin resistance, which has been interpreted as reduced downstream insulin signaling. However, changes in access of insulin to sensitive tissues such as skeletal muscle may also play a role. Insulin injected directly into skeletal muscle diffuses rapidly through the interstitial space to cause glucose uptake. When insulin resistance is induced by exogenous lipid infusion, this interstitial diffusion process is curtailed. Thus, the possibility exists that hyperlipidemia, such as that seen during obesity, may inhibit insulin action to muscle cells and exacerbate insulin resistance. Here we asked whether interstitial insulin diffusion is reduced in physiological obesity induced by a high-fat diet (HFD).

RESEARCH DESIGN AND METHODS

Dogs were fed a regular diet (lean) or one supplemented with bacon grease for 9–12 weeks (HFD). Basal insulin (0.2 mU · min⁻¹ · kg⁻¹) euglycemic clamps were performed on fat-fed animals (n = 6). During clamps performed under anesthesia, five sequential doses of insulin were injected into the vastus medialis of one hind limb (INJ); the contralateral limb (NINJ) served as a control.

RESULTS

INJ lymph insulin showed an increase above NINJ in lean animals, but no change in HFD-fed animals. Muscle glucose uptake observed in lean animals did not occur in HFD-fed animals.

CONCLUSIONS

Insulin resistance induced by HFD caused a failure of intramuscularly injected insulin to diffuse through the interstitial space and failure to cause glucose uptake, compared with normal animals. High-fat feeding prevents the appearance of injected insulin in the interstitial space, thus reducing binding to skeletal muscle cells and glucose uptake.Insulin resistance is associated with a number of diseases, including cardiovascular disease, hypertension (1), cancer (2), and obesity (1,3), as well as type 2 diabetes. Much research into insulin resistance has focused on insulin signaling, suggesting that target cell (e.g., skeletal muscle) response to insulin is the primary defect. Most recently, mitochondrial dysfunction (4,5) and intracellular fat accumulation (6) have been proposed as primary causes of insulin resistance. Despite these efforts, there is no definitive conclusion as to the primary cause of cellular insulin resistance.There are a number of loci where insulin action can be altered, including the direct effect of inflammatory cytokines to attenuate distribution of blood flow and glucose uptake (7), and direct effects of plasma free fatty acids (FFAs) to impede insulin signaling in the cell (4) and/or reduce capillary recruitment (8). To act, insulin must cross the capillary endothelium, traverse the interstitial fluid compartment, and thence access and bind to insulin receptors. A reduction in insulin''s ability to diffuse through the interstitial space in lipid-induced insulin resistance (9) suggests that access to sensitive tissues (such as skeletal muscle) may be important in addition to cellular insulin resistance. In insulin-resistant situations, it is therefore important to examine whether insulin can access cells prior to stimulating cell signaling.A high-fat diet (HFD), which induces obesity and leads to deposition of fat in tissues (10,11), does not always increase basal FFAs in humans or canines (12,13); but large elevations of FFAs can be observed at night in canines (13) and humans (14). Given the uncertainty regarding the effect of HFDs on FFA levels in the circulation, it is not yet known whether the effects of lipid infusion on insulin action can be extrapolated to explain how an HFD per se would affect insulin action and the ability of insulin to diffuse through skeletal muscle.Previous studies in our laboratory have shown that insulin injected directly into healthy muscle is disseminated throughout the interstitial space, where the hormone accesses myocytes and stimulates glucose uptake (15). In a model of insulin resistance where plasma FFAs were raised by lipid infusion, injected insulin was surprisingly not detected in the interstitial space (lymph) of skeletal muscle, supporting absence of elevated interstitial insulin. Under this condition there was little stimulation of glucose disposal (9). This latter result suggests that high plasma FFAs may prevent insulin''s access to skeletal muscle, resulting in insulin resistance. It cannot be concluded whether this is a direct effect of elevated plasma FFAs, or of acute insulin resistance induced by lipid infusion. Therefore, it was important to examine whether lipid elevated physiologically by an HFD would similarly limit insulin''s access to muscle cells. For example, in dogs fed a control diet, insulin stimulates the extravascular distribution of insulin without effects on vascular distribution volume (16), suggesting that insulin can increase its own access to insulin-sensitive tissues. This effect was lost after induction of insulin resistance by an HFD (17), possibly due to an alteration in the diffusion of insulin through the interstitial space. In the present report, we examined whether insulin resistance induced by an HFD would simulate lipid infusion studies in which injected insulin was not detected in lymph. If so, this would implicate insulin access to muscle cells as an important factor in dietary fat–induced insulin resistance.