Development of muscle insulin resistance after liver insulin resistance in high-fat-fed rats

Muscle and hepatic insulin resistance are two major defects of non-insulin-dependent diabetes mellitus. Dietary factors may be important in the etiology of insulin resistance. We studied progressive changes in the development of high-fat-diet-induced insulin resistance in tissues of the adult male Wistar rat. In vivo insulin action was compared 3 days and 3 wk after isocaloric synthetic high-fat or high-starch feeding (59 and 10% cal as fat, respectively). Basal and insulin-stimulated glucose metabolism were assessed in the conscious 5- to 7-h fasted state with the euglycemic clamp (600 pM insulin) with a [3-3H]-glucose infusion. Fat feeding significantly reduced suppressibility of hepatic glucose output by insulin after both 3 days and 3 wk of diet (P less than 0.01). However, a significant impairment of insulin-mediated peripheral glucose disposal was only present after 3 wk of diet. Further in vivo [3H]-2-deoxyglucose uptake studies supported this finding and demonstrated adipose but not muscle insulin resistance after 3 days of high-fat feeding. Muscle triglyceride accumulation due to fat feeding was not significant at 3 days but had doubled by 3 wk in red muscle (P less than 0.001) compared with starch-fed controls. By 3 wk, high-fat-fed animals had developed significant glucose intolerance. We conclude that fat feeding induces insulin resistance in liver and adipose tissue before skeletal muscle with early metabolic changes favoring an oversupply of energy substrate to skeletal muscle relative to metabolic needs. This may generate later muscle insulin resistance.     

Local effect of burn on skeletal muscle insulin responsiveness

Basal and insulin-stimulated glucose metabolism by skeletal muscle were studied 3 days after a 3-sec scald of one hindlimb of the rat. Soleus muscles from the burned and unburned limb of the burned rats, as well as from timed controls, were incubated with [U-¹⁴C]glucose and 0, 0.1, 1.0, or 10.0 mU insulin/ml. Basal glucose uptake by soleus muscle from the burned limb was 144% (P < 0.001) greater than that of the controls. The glucose uptake by muscle from the unburned limb did not differ from that of controls. Insulin increased glucose uptake in control and unburned muscles but had no effect in burned muscles. Basal lactic acid release by soleus muscle from the burned limb and the contralateral unburned limb of burned rats was 123% (P < 0.001) and 24% (P < 0.01) higher, respectively, than that of soleus muscle from control rats. Of the lactic acid released by muscles from control rats or the unburned limb of burned rats, 35% was derived from exogenous glucose. In contrast, 50–55% of the lactic acid released by muscles from the burned limb was derived from exogenous glucose reflecting a predominant conversion of glucose to lactic acid. Insulin had no effect on the rate of lactic acid release and did not change the proportion of glucose converted to lactic acid by any of the three muscle groups. The basal rate of glucose conversion to CO₂ by muscle from the burned limb was elevated 133% (P < 0.01) and that by muscle from the unburned limb was not altered as compared to controls. Insulin did not stimulate the conversion of glucose to CO₂ by any of the three muscle groups, and in the presence of insulin the release of CO₂ by burned, unburned, and control muscle did not differ. The basal rate of glucose incorporation into glycogen was the same in all three muscle groups. Insulin stimulated glucose incorporation into glycogen to a similar degree in muscles from control rats and the unburned limb of burned rats. However, the stimulatory effect of insulin was completely absent in soleus muscle from the burned limb. It is concluded that thermal injury suppresses the insulin-induced augmentation of glucose uptake and glucose incorporation into glycogen in skeletal muscles in the region of the burn but does not alter insulin sensitivity of skeletal muscles in the unburned region.     

Effect of insulin on amino acid uptake and protein turnover in skeletal muscle from septic rats. Evidence for insulin resistance of protein breakdown

We investigated the effect of different concentrations of insulin (0, 10, 1 X 10(2), 1 X 10(3), 1 X 10(4), and 1 X 10(5) mU/L [0, 70, 7 X 10(2), 7 X 10(3), 7 X 10(4), and 7 X 10(5) pmol/L]) on amino acid (alpha-aminoisobutyric acid) uptake and protein synthesis and breakdown in incubated extensor digitorum longus (EDL) and soleus muscles of rats. We studied three groups: untreated, fed rats; sham-operated rats; and septic rats. Sepsis was induced by cecal ligation and puncture. The alpha-aminoisobutyric acid uptake was increased by insulin in all three groups. Protein synthesis was maximally stimulated by 30% to 40% by 1 X 10(2) mU/L (7 X 10(2) pmol/L) of insulin in all three groups. Protein degradation in soleus muscle was not affected by insulin. In EDL muscles from untreated and sham-operated rats, protein breakdown was reduced by 15% to 20% by 1 X 10(2) mU/L (7 X 10(2) pmol/L) of insulin. In contrast, protein breakdown was not inhibited by insulin in septic EDL muscle until the concentration of the hormone was increased to 1 X 10(4) mU/L (7 X 10(4) pmol/L), at which concentration the hormonal effect was less than half that in nonseptic muscle. The results suggest a postreceptor insulin resistance of protein breakdown in septic muscle, while the response to the hormone of amino acid transport and protein synthesis was not altered in sepsis.     

Mechanism of amino acid-induced skeletal muscle insulin resistance in humans

Plasma concentrations of amino acids are frequently elevated in insulin-resistant states, and a protein-enriched diet can impair glucose metabolism. This study examined effects of short-term plasma amino acid (AA) elevation on whole-body glucose disposal and cellular insulin action in skeletal muscle. Seven healthy men were studied for 5.5 h during euglycemic (5.5 mmol/l), hyperinsulinemic (430 pmol/l), fasting glucagon (65 ng/l), and growth hormone (0.4 microg/l) somatostatin clamp tests in the presence of low (approximately 1.6 mmol/l) and increased (approximately 4.6 mmol/l) plasma AA concentrations. Glucose turnover was measured with D-[6,6-(2)H(2)]glucose. Intramuscular concentrations of glycogen and glucose-6-phosphate (G6P) were monitored using (13)C and (31)P nuclear magnetic resonance spectroscopy, respectively. A approximately 2.1-fold elevation of plasma AAs reduced whole-body glucose disposal by 25% (P < 0.01). Rates of muscle glycogen synthesis decreased by 64% (180--315 min, 24 plus minus 3; control, 67 plus minus 10 micromol center dot l(-1) center dot min(-1); P < 0.01), which was accompanied by a reduction in G6P starting at 130 min (DeltaG6P(260--300 min), 18 plus minus 19; control, 103 plus minus 33 micromol/l; P < 0.05). In conclusion, plasma amino acid elevation induces skeletal muscle insulin resistance in humans by inhibition of glucose transport/phosphorylation, resulting in marked reduction of glycogen synthesis.     

Mechanisms of hyperglycemia-induced insulin resistance in whole body and skeletal muscle of type I diabetic patients.

To examine the mechanisms of hyperglycemia-induced insulin resistance, eight insulin-dependent (type I) diabetic men were studied twice, after 24 h of hyperglycemia (mean blood glucose 20.0 +/- 0.3 mM, i.v. glucose) and after 24 h of normoglycemia (7.1 +/- 0.4 mM, saline) while receiving identical diets and insulin doses. Whole-body and forearm glucose uptake were determined during a 300-min insulin infusion (serum free insulin 359 +/- 22 and 373 +/- 29 pM, after hyper- and normoglycemia, respectively). Muscle biopsies were taken before and at the end of the 300-min insulin infusion. Plasma glucose levels were maintained constant during the 300-min period by keeping glucose for 150 min at 16.7 +/- 0.1 mM after 24-h hyperglycemia and increasing it to 16.5 +/- 0.1 mM after normoglycemia and by allowing it thereafter to decrease in both studies to normoglycemia. During the normoglycemic period (240-300 min), total glucose uptake (25.0 +/- 2.8 vs. 33.8 +/- 3.9 mumol.kg-1 body wt.min-1, P less than 0.05) was 26% lower, forearm glucose uptake (11 +/- 4 vs. 18 +/- 3 mumol.kg-1 forearm.min-1, P less than 0.05) was 35% lower, and nonoxidative glucose disposal (8.9 +/- 2.2 vs. 19.4 +/- 3.3 mumol.kg-1 body wt-1min-1, P less than 0.01) was 54% lower after 24 h of hyper- and normoglycemia, respectively. Glucose oxidation rates were similar. Basal muscle glycogen content was similar after 24 h of hyperglycemia (234 +/- 23 mmol/kg dry muscle) and normoglycemia (238 +/- 22 mmol/kg dry muscle). Insulin increased muscle glycogen to 273 +/- 22 mmol/kg dry muscle after 24 h of hyperglycemia and to 296 +/- 33 mmol/kg dry muscle after normoglycemia (P less than 0.05 vs. 0 min for both). Muscle ATP, free glucose, glucose-6-phosphate, and fructose-6-phosphate concentrations were similar after both 24-h treatment periods and did not change in response to insulin. We conclude that a marked decrease in whole-body, muscle, and nonoxidative glucose disposal can be induced by hyperglycemia alone.     

IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas transplant recipients

Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied by altered signal transduction, skeletal muscle biopsies were obtained from pancreas-kidney transplant recipients (n = 4), nondiabetic kidney transplant recipients (receiving the same immunosuppressive drugs; n = 5), and healthy subjects (n = 6) before and during a euglycemic-hyperinsulinemic clamp. Basal insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal IRS-1 Ser (312) and Ser (616) phosphorylation was also increased in nondiabetic kidney transplant recipients. Insulin increased phosphorylation of IRS-1 at Ser (312) but not Ser (616) in healthy subjects, with impairments noted in nondiabetic kidney and pancreas-kidney transplant recipients. Insulin action on ERK-1/2 and Akt phosphorylation was impaired in pancreas-kidney transplant recipients and was preserved in nondiabetic kidney transplant recipients. Importantly, insulin stimulation of the Akt substrate AS160 was impaired in nondiabetic kidney and pancreas-kidney transplant recipients. In conclusion, peripheral insulin resistance in pancreas-kidney transplant recipients may arise from a negative feedback regulation of the canonical insulin-signaling cascade from excessive serine phosphorylation of IRS-1, possibly as a consequence of immunosuppressive therapy and hyperinsulinemia.     

Effect of laser therapy on skeletal muscle repair process in diabetic rats

Skeletal muscle myopathy is a common source of disability in diabetic patients. This study evaluated whether low-level laser therapy (LLLT) influences the healing morphology of injured skeletal muscle. Sixty-five male Wistar rats were divided as follows: (1) sham; (2) control; (3) diabetic; (4) diabetic sham; (5) nondiabetic cryoinjured submitted to LLLT (LLLT); (6) diabetic cryoinjured submitted to LLLT (D-LLLT); and (7) diabetic cryoinjured non-treated (D). Diabetes was induced with streptozotocin. Anterior tibialis muscle was cryoinjured and received LLLT daily (780 nm, 5 J/cm², 10 s per point; 0.2 J; total treatment, 1.6 J). Euthanasia occurred on day 1 in groups 1, 2, 3, and 4 and on days 1, 7, and 14 in groups 5, 6, and 7. Muscle samples were processed for H&E and Picrosirius Red and photographed. Leukocytes, myonecrosis, fibrosis, and immature fibers were manually quantified using the ImageJ software. On day 1, all cryoinjured groups were in the inflammatory phase. The D group exhibited more myonecrosis than LLLT group (p?<?0.05). On day 14, the LLLT group was in the remodeling phase; the D group was still in the proliferative phase, with fibrosis, chronic inflammation, and granulation tissue; and the D-LLLT group was in an intermediary state in relation to the two previous groups. Under polarized light, on day 14, the LLLT and D-LLLT groups had organized collagen bundles in the perimysium, whereas the diabetic groups exhibited fibrosis. LLLT can have a positive effect on the morphology of skeletal muscle during the tissue repair process by enhancing the reorganization of myofibers and the perimysium, reducing fibrosis.     

Suppressor of cytokine signaling 3 expression and insulin resistance in skeletal muscle of obese and type 2 diabetic patients

Interleukin-6 (IL-6) could be a possible mediator of insulin resistance. We investigated whether IL-6 could inhibit insulin signaling in human skeletal myotubes and whether suppressor of cytokine signaling 3 (SOCS-3) could be related to insulin resistance in vivo in humans. IL-6 inhibited insulin signaling and induced SOCS-3 expression in differentiated myotubes. SOCS-3 mRNA levels were significantly increased in the skeletal muscle of type 2 diabetic patients compared with control subjects and correlated with reduced insulin-stimulated glucose uptake. In contrast, SOCS-3 mRNA levels were reduced in muscle of obese nondiabetic subjects compared with type 2 diabetic patients, despite similar circulating concentrations of IL-6. Increased SOCS-3 mRNA levels in diabetes were not attributable to hyperglycemia, as type 1 diabetic patients had normal SOCS-3 mRNA expression in muscle. However, the combination of high glucose and IL-6 levels in type 2 diabetic patients may induce SOCS-3 expression, as has been seen in human muscle cells. In subcutaneous adipose tissue, SOCS-3 mRNA levels were increased in obese individuals and strongly correlated with IL-6 expression, supporting a paracrine effect of IL-6 on SOCS-3 expression in fat. Taken together, our results showed that SOCS-3 expression in human skeletal muscle in vivo is not related to insulin resistance in the presence of elevated IL-6 concentrations and suggest that cytokine action could differ in type 2 diabetic patients and nondiabetic obese subjects.     

Fuel selection in human skeletal muscle in insulin resistance: a reexamination

For many years, the Randle glucose fatty acid cycle has been invoked to explain insulin resistance in skeletal muscle of patients with type 2 diabetes or obesity. Increased fat oxidation was hypothesized to reduce glucose metabolism. The results of a number of investigations have shown that artificially increasing fat oxidation by provision of excess lipid does decrease glucose oxidation in the whole body. However, results obtained with rodent or human systems that more directly examined muscle fuel selection have found that skeletal muscle in insulin resistance is accompanied by increased, rather than decreased, muscle glucose oxidation under basal conditions and decreased glucose oxidation under insulin-stimulated circumstances, producing a state of "metabolic inflexibility." Such a situation could contribute to the accumulation of triglyceride within the myocyte, as has been observed in insulin resistance. Recent knowledge of insulin receptor signaling indicates that the accumulation of lipid products in muscle can interfere with insulin signaling and produce insulin resistance. Therefore, although the Randle cycle is a valid physiological principle, it may not explain insulin resistance in skeletal muscle.     

The effect of burn plasma on skeletal muscle proteolysis in rats

We studied the effects of plasma from burned rats on skeletal muscle proteolysis. Major burn injury (40% total body surface area (TBSA)) was produced in male Sprague-Dawley rats. Fluid resuscitation was given with intraperitoneal Ringer's solution (4cm(3)/(kg%) TBSA). Plasma was harvested daily for 5 days after burn injury from the tail blood vessel. This plasma was added in vitro to incubated soleus muscles from healthy animals. The incubation medium was assayed for amino acids by HPLC. Glutamine, glutamate, leucine and alanine were tested to monitor the amino acid profile in the medium. Results showed there was no significant change during the initial 4 days after injury, except that glutamine and alanine increased significantly on the first day. However, all of them had a tendency to increase on the fifth day after injury. Present results suggest that the humoral effect on muscle proteolysis did not exist during the initial days after burning. The humoral effect on skeletal muscle proteolysis may have been present 5 days post-burn.     

Lactate production and pyruvate dehydrogenase activity in fat and skeletal muscle from diabetic rats.

This study was initiated to explore the possibility that an increase in the supply of gluconeogenic precursors contributes to the overproduction of glucose by the liver in NIDDM patients. To address this issue, a form of experimental NIDDM was produced in rats by injecting a low dose (38 mg/kg) of STZ and comparing lactate and alanine production and PDH activity in skeletal muscle and isolated adipocytes from normal and diabetic rats. Skeletal muscle lactate production was measured by using a hindlimb perfusion technique and was significantly greater (P < 0.01) in the diabetic rats compared with two groups of control rats: one perfused at normal glucose levels and the other perfused at glucose concentrations comparable with those observed in diabetic rats. Alanine production by hindlimb from diabetic rats was 46% greater than hindlimbs from control rats perfused at normal glucose levels (P < 0.01) but was not significantly greater than control rats perfused at diabetic glucose levels. The percentage of glucose converted to lactate by muscle from both control groups was 4-5%, significantly lower than the 18% conversion rate observed in diabetic animals (P < 0.001). An increase in the ratio of lactate produced/glucose transport by isolated adipocytes from diabetic rats also was observed when measured in both the basal state (0.65 +/- 0.12 vs. 0.15 +/- 0.03, P < 0.01) and in the presence of maximal amounts of insulin (0.15 +/- 0.02 vs. 0.04 +/- 0.01, P < 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Involvement of protein kinase C in human skeletal muscle insulin resistance and obesity

This study was conducted to investigate the possible involvement of protein kinase C (PKC) and serine/threonine phosphorylation of the insulin receptor in insulin resistance and/or obesity. Insulin receptor tyrosine kinase activity was depressed in muscle from obese insulin-resistant patients compared with lean insulin-responsive control subjects. Alkaline phosphatase treatment resulted in a significant 48% increase in in vitro insulin-stimulated receptor tyrosine kinase activity in obese but not lean muscle. To investigate the involvement of PKC in skeletal muscle insulin resistance and/or obesity, membrane-associated PKC activity and the protein content of various PKC isoforms were measured in human skeletal muscle from lean, insulin-responsive, and obese insulin-resistant patients. Membrane-associated PKC activity was not changed; however, PKC-beta protein content, assayed by Western blot analysis, was significantly higher, whereas PKC-theta, -eta, and -mu were significantly lower in muscle from obese patients compared with muscle from lean control subjects. Incubation of muscle strips with insulin significantly increased membrane-associated PKC activity in muscle from obese but not lean subjects. PKC-delta, -beta, and -theta were translocated from the cytosol to the membrane fraction in response to insulin treatment. These results suggest that in skeletal muscle from insulin-resistant obese patients, insulin receptor tyrosine kinase activity was reduced because of hyperphosphorylation on serine/threonine residues. Membrane-associated PKC-beta protein was elevated under basal conditions, and membrane-associated total PKC activity was increased under insulin-stimulated conditions in muscle from obese insulin-resistant patients. Thus, we postulate that the decreased tyrosine kinase activity of the insulin receptor may be caused by serine/threonine phosphorylation by PKC.     

Phosphorylation of insulin receptors solubilized from rat skeletal muscle

A method has been developed to solubilize insulin receptors from skeletal muscles. Rat hindlimb muscles were rapidly frozen in liquid nitrogen, powdered, extracted with buffered Triton X-100, and partially purified by differential centrifugation followed by wheat germ agglutinin affinity chromatography. The solubilized receptors exhibit typical curvilinear Scatchard plots in insulin binding assays: rapid, Mn2+-dependent autophosphorylation of the beta-subunit on exposure to insulin as well as insulin-stimulated kinase activity toward histone H2B. Furthermore, when intact soleus muscles were incubated in phosphate-depleted medium containing Na2H[32P]PO4, addition of insulin stimulated the in situ phosphorylation of the beta-subunit of the insulin receptor. The ability to rapidly and efficiently isolate insulin receptors from skeletal muscle may permit investigation of factors that modulate insulin action in this tissue.     

Adenovirus-mediated adiponectin expression augments skeletal muscle insulin sensitivity in male Wistar rats

In this study, we investigated the chronic in vivo effect of adiponectin on insulin sensitivity and glucose metabolism by overexpressing the adiponectin protein in male Wistar rats using intravenous administration of an adenovirus (Adv-Adipo). Virally infected liver secreted adiponectin as high and low molecular weight complexes. After 7 days of physiological or supraphysiological hyperadiponectinemia, the animals displayed enhanced insulin sensitivity during the glucose tolerance and insulin tolerance tests. Glucose clamp studies performed at submaximal and maximal insulin infusion rates (4 and 25 mU x kg(-1) x min(-1), respectively) also demonstrated increased insulin sensitivity in Adv-Adipo animals, with the insulin-stimulated glucose disposal rate being increased by 20-67%. In contrast, insulin's effect on the suppression of hepatic glucose output and plasma free fatty acid levels was not enhanced in Adv-Adipo rats compared with controls, suggesting that high levels of adiponectin expression in the liver may lead to a local desensitization. Consistent with the clamp data, the activation of AMP-activated protein kinase was significantly enhanced in skeletal muscle (by 50%) but not in liver. One interesting finding was that in male Wistar rats, both AdipoR1 and AdipoR2 expression levels were higher in skeletal muscle than in liver, as it is the case in humans. These results indicate that chronic adiponectin treatment enhances insulin sensitivity and could serve as a therapy for human insulin resistance.     

Overexpressing human lipoprotein lipase in mouse skeletal muscle is associated with insulin resistance

Lipoprotein lipase (LPL) plays a rate-limiting role in triglyceride-rich lipoprotein metabolism and is expressed in most tissues. Overexpression of LPL in skeletal muscle has been linked with higher plasma glucose levels suggesting insulin resistance (Jensen et al., Am J Physiol 273:R683-R689, 1997). The aim of our study was to ascertain whether the overexpression of human LPL in skeletal muscle leads to insulin resistance and to investigate the mechanism. Respiratory quotient measurements in both transgenic (MCKhLPL) and nontransgenic mice on a high-carbohydrate diet were conducted and showed a shift in fuel usage in transgenic mice when fasting but not when actively feeding. An increase in citrate and glucose 6-phosphate levels in fasted MCKhLPL mice further supports this preferential use of lipids. When challenged with an intraperitoneal injection of glucose (1 g/kg), MCKhLPL mice had a higher plasma glycemic excursion than nontransgenic mice. No differences in insulin response were observed between the two groups. Further investigation using hyperinsulinemic-euglycemic clamps revealed insulin resistance in MCKhLPL mice. Despite signs of insulin resistance, there was no associated increase in free fatty acids, hypertriglyceridemia, or hyperinsulinemia in MCKhLPL mice. In conclusion, MCKhLPL mice are insulin resistant, presumably due to increased delivery of lipoprotein-derived fatty acids to muscle.     

Altered skeletal muscle lipase expression and activity contribute to insulin resistance in humans

OBJECTIVE

Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance.

RESEARCH DESIGN AND METHODS

We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling.

RESULTS

Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = −0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (−30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance.

CONCLUSIONS

Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes.Skeletal muscle insulin resistance is a strong risk factor of type 2 diabetes and cardiovascular diseases (1,2). Dysfunctional adipose tissue can lead to lipid oversupply and increased flux of free fatty acids (FFAs) into skeletal muscle and is associated with the accumulation of intramyocellular triacylglycerols (IMTGs) (3–5). This chronic lipid overload in tissues can evolve to a state of lipotoxicity leading to cell dysfunction (6). The term “lipotoxicity” defines more generally in skeletal muscle a state of lipid overload (increased concentrations of long-chain acyl-CoA, diacylglycerols [DAGs], and ceramide) causing insulin resistance (7–12). DAGs have been shown to activate novel protein kinase C (PKC) isoforms, such as the novel PKCθ leading to insulin receptor substrate-1 (IRS-1) serine phosphorylation and impaired downstream insulin signaling (10–13). DAGs can be formed through multiple pathways but are also formed as intermediates during triacylglycerol (TAG) synthesis and hydrolysis (14). The control of lipolysis in skeletal muscle has mainly been attributed to hormone-sensitive lipase (HSL), which exhibits a 10-fold higher specific activity for DAG than TAG (15). HSL null mice display normal TAG hydrolase activity after an overnight fast and accumulate large amounts of DAG (16). Recently it was shown that adipose triglyceride lipase (ATGL) plays a major role in the regulation of cellular TAG stores in various tissues of the body, including heart and skeletal muscle (17,18). ATGL specifically drives the hydrolysis of TAG into DAG (18). ATGL-deficient mice are more insulin sensitive and glucose tolerant despite a threefold increase in TAG content in their skeletal muscle (17). The molecular mechanism underlying this phenotype remains unclear. In the current study, we hypothesized that an imbalance of ATGL relative to HSL could increase intracellular DAG concentrations and promote insulin resistance. To test this hypothesis, we first examined the relationship between muscle ATGL expression and whole-body insulin sensitivity in a wide range of subjects. We next manipulated the expression/activity of ATGL and HSL in vitro in cultured human primary skeletal muscle cells and evaluated its impact on lipid pools and insulin signaling.     

A possible link between skeletal muscle mitochondrial efficiency and age-induced insulin resistance

The transition from young to adult age is associated with decreased insulin sensitivity. To investigate whether changes in skeletal muscle mitochondrial function could be involved in the development of insulin resistance, we measured the oxidative capacity and energetic efficiency of subsarcolemmal and intermyofibrillar mitochondria isolated from the skeletal muscle of 60- and 180-day-old rats. Mitochondrial efficiency was tested by measuring the degree of thermodynamic coupling and optimal thermodynamic efficiency, as well as mitochondrial proton leak, which was determined in both the absence (basal) and the presence (fatty acid induced) of palmitate. Serum glucose, insulin, and HOMA index were also measured. The results show that in adult rats, concomitant with increased HOMA index, skeletal muscle mitochondria display higher respiratory capacity and energy efficiency. In fact, thermodynamic coupling and optimal thermodynamic efficiency significantly increased and fatty acid-induced proton leak was significantly lower in the skeletal muscle mitochondria from adult than in younger rats. A deleterious consequence of increased mitochondrial efficiency would be a reduced utilization of energy substrates, especially fatty acids, leading to intracellular triglyceride accumulation and lipotoxicity, thus contributing to the onset of skeletal muscle insulin resistance.     

Muscle-specific IRS-1 Ser->Ala transgenic mice are protected from fat-induced insulin resistance in skeletal muscle

OBJECTIVE—Insulin resistance in skeletal muscle plays a critical role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance are poorly understood. In this study, we examine the role of serine phosphorylation of insulin receptor substrate (IRS)-1 in mediating fat-induced insulin resistance in skeletal muscle in vivo.RESEARCH DESIGN AND METHODS—To directly assess the role of serine phosphorylation in mediating fat-induced insulin resistance in skeletal muscle, we generated muscle-specific IRS-1 Ser³⁰², Ser³⁰⁷, and Ser⁶¹² mutated to alanine (Tg IRS-1 Ser→Ala) and IRS-1 wild-type (Tg IRS-1 WT) transgenic mice and examined insulin signaling and insulin action in skeletal muscle in vivo.RESULTS—Tg IRS-1 Ser→Ala mice were protected from fat-induced insulin resistance, as reflected by lower plasma glucose concentrations during a glucose tolerance test and increased insulin-stimulated muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. In contrast, Tg IRS-1 WT mice exhibited no improvement in glucose tolerance after high-fat feeding. Furthermore, Tg IRS-1 Ser→Ala mice displayed a significant increase in insulin-stimulated IRS-1–associated phosphatidylinositol 3-kinase activity and Akt phosphorylation in skeletal muscle in vivo compared with WT control littermates.CONCLUSIONS—These data demonstrate that serine phosphorylation of IRS-1 plays an important role in mediating fat-induced insulin resistance in skeletal muscle in vivo.Insulin resistance in skeletal muscle plays a major role in the pathogenesis of type 2 diabetes, yet the cellular mechanisms responsible for insulin resistance in skeletal muscle are poorly understood (1). Reduced insulin-stimulated glucose transport activity exists with reduced insulin receptor substrate (IRS)-1–associated phosphatidylinositol 3-kinase (PI3-kinase) activity in patients with type 2 diabetes and the offspring of type 2 diabetic parents (2–5). Increased serine phosphorylation of IRS-1 has been suggested to be responsible for this phenomenon (6), and, consistent with this hypothesis, recent studies have demonstrated hyperserine phosphorylation of IRS-1 on Ser³⁰², Ser³⁰⁷, Ser⁶¹², and Ser⁶³⁶ in several insulin-resistant rodent models (7–10), as well as in lean insulin-resistant offspring of type 2 diabetic parents (11). Circulating factors that are increased in obese and inflammatory states, such as tumor necrosis factor-α, activate Ser/Thr kinases (12,13). Also, recent studies (14–18) have demonstrated a strong relationship between intramyocelullar lipid accumulation and insulin resistance in muscle independent of alterations in circulating adipocytokines. Intramyocellular fatty acid metabolites, such as diacylglycerol, have been postulated to activate a serine kinase cascade leading to increased serine phosphorylation of IRS-1. Furthermore, high-fat diet–induced insulin resistance has been abrogated in rodent models in which certain Ser/Thr kinases (c-Jun N-terminal kinase, inhibitor of nuclear factor κB kinase β subunit, S6 kinase-1, and protein kinase C-θ) were either knocked down or pharmacologically inhibited (8,9,19–21). However, it remains unknown whether increased IRS-1 serine phosphorylation plays a causative role in the pathogenesis of fat-induced insulin resistance in skeletal muscle or whether it is merely an associated phenomenon. To address this question, we generated IRS-1 Ser³⁰², Ser³⁰⁷, and Ser⁶¹² to Ala mutant–overexpression (Tg IRS-1 Ser→Ala) mice using a muscle-specific myosin light-chain-2 promoter and assessed insulin responsiveness in vivo by intraperitoneal glucose tolerance tests and hyperinsulinemic-euglycemic clamp studies.     

Counteraction of type 1 diabetic alterations by engineering skeletal muscle to produce insulin: insights from transgenic mice

Insulin replacement therapy in type 1 diabetes is imperfect because proper glycemic control is not always achieved. Most patients develop microvascular, macrovascular, and neurological complications, which increase with the degree of hyperglycemia. Engineered muscle cells continuously secreting basal levels of insulin might be used to improve the efficacy of insulin treatment. Here we examined the control of glucose homeostasis in healthy and diabetic transgenic mice constitutively expressing mature human insulin in skeletal muscle. Fed transgenic mice were normoglycemic and normoinsulinemic and, after an intraperitoneal glucose tolerance test, showed increased glucose disposal. When treated with streptozotocin (STZ), transgenic mice showed increased insulinemia and reduced hyperglycemia when fed and normoglycemia and normoinsulinemia when fasted. Injection of low doses of soluble insulin restored normoglycemia in fed STZ-treated transgenic mice, while STZ-treated controls remained highly hyperglycemic, indicating that diabetic transgenic mice were more sensitive to the hypoglycemic effects of insulin. Furthermore, STZ-treated transgenic mice presented normalization of both skeletal muscle and liver glucose metabolism. These results indicate that skeletal muscle may be a key target tissue for insulin production and suggest that muscle cells secreting basal levels of insulin, in conjunction with insulin therapy, may permit tight regulation of glycemia.