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To determine the relative time courses of changes in peripheral and hepatic insulin action and skeletal muscle GLUT4 protein levels after a streptozotocin (STZ) injection in rats, we performed hyperinsulinemic (14–18 nM), euglycemic (7.5 mM) clamps in control (n = 8) and diabetic rats at 1 (n = 7), 3 (n = 8), 7 (n = 8), and 14 (n = 6) days after intraperitoneal STZ (65 mg/kg). Basal plasma glucose concentrations increased from 8.1 ± 0.2 mM in control rats to 23.5 ± 1.2 mM 1 day after STZ (P < 0.01) and remained constant thereafter. Basal plasma insulin levels were ∼ 35% of control levels in all STZ groups (P < 0.01). Insulin-stimulated whole-body glucose uptake decreased significantly as early as one day after STZ injection (P < 0.01), resulting predominantly from a decrease in whole-body glycolysis. Insulin action to suppress hepatic glucose output was normal on day 1 after STZ but impaired markedly on day 3 and thereafter (P < 0.01). Insulin-stimulated glucose uptake in individual skeletal muscles was not altered until day 7 after STZ, and the magnitudes of decreases in skeletal muscle insulin action on days 7 and 14 were not fully accounted for by the decreases in GLUT4 protein level measured from the same muscles. Our data indicate that there is a temporal hierarchy in the development of insulin resistance in STZ-induced diabetes. Insulin resistance is manifested initially (within one day) as a reduction in insulin-mediated glucose uptake and glycolysis in tissues other than skeletal muscle; available data suggest that the liver is a potential site of the initial reduction in insulin-mediated glucose uptake. Resistance of hepatic glucose output to suppression by insulin appears next, by day 3. Insulin resistance in skeletal muscle appears last (between days 3 and 7 after STZ administration), and the resistance in muscle cannot be fully accounted for by reduced GLUT4 expression.

Dysfunction of hepatic insulin receptor tyrosine kinase (IRTK) causes the development of type 2 diabetes. However, the molecular mechanism regulating IRTK activity in the liver remains poorly understood. Here, we show that phosphoinositide 3-kinase enhancer A (PIKE-A) is a new insulin-dependent enhancer of hepatic IRTK. Liver-specific Pike-knockout (LPKO) mice display glucose intolerance with impaired hepatic insulin sensitivity. Specifically, insulin-provoked phosphoinositide 3-kinase/Akt signalling is diminished in the liver of LPKO mice, leading to the failure of insulin-suppressed gluconeogenesis and hyperglycaemia. Thus, hepatic PIKE-A has a key role in mediating insulin signal transduction and regulating glucose homeostasis in the liver.

Insulin resistance is a major characteristic of obesity and type 2 diabetes, and develops in multiple organs, including the heart. Compared with its role in other organs, the physiological role of insulin resistance in the heart is not well understood. The heart uses lipid as a primary fuel, but glucose becomes an important source of energy in ischemia. The impaired ability to utilize glucose might contribute to cell death and abnormal function in the diabetic heart. Recent discoveries regarding the role of inflammation, mitochondrial dysfunction and endoplasmic reticulum (ER) stress in obesity have advanced our understanding of how insulin resistance develops in peripheral organs. In this review, we examine these findings in relation to the diabetic heart to provide new insights into the mechanism of cardiac insulin resistance.

OBJECTIVE To determine the mechanisms by which blockade of adenosine A2B receptors (A2BRs) reduces insulin resistance. RESEARCH DESIGN AND METHODS We investigated the effects of deleting or blocking the A2BR on insulin sensitivity using glucose tolerance tests (GTTs) and hyperinsulinemic-euglycemic clamps in mouse models of type 2 diabetes. The effects of diabetes on A2BR transcription and signaling were measured in human and mouse macrophages and mouse endothelial cells. In addition, tag single nucleotide polymorphisms (SNPs) in ∼42 kb encompassing the A2BR gene, ADORA2B, were evaluated for associations with markers of diabetes and inflammation. RESULTS Treatment of mice with the nonselective adenosine receptor agonist 5′-N-ethylcarboxamidoadensoine (NECA) increased fasting blood glucose and slowed glucose disposal during GTTs. These responses were inhibited by A2BR deletion or blockade and minimally affected by deletion of A1Rs or A2ARs. During hyperinsulinemic-euglycemic clamp of diabetic KKAY mice, A2BR antagonism increased glucose infusion rate, reduced hepatic glucose production, and increased glucose uptake into skeletal muscle and brown adipose tissue. Diabetes caused a four- to sixfold increase in A2BR mRNA in endothelial cells and macrophages and resulted in enhanced interleukin (IL)-6 production in response to NECA due to activation of protein kinases A and C. Five consecutive tag SNPs in ADORA2B were highly correlated with IL-6 and C-reactive protein (CRP). Diabetes had a highly significant independent effect on variation in inflammatory markers. The strength of associations between several ADORA2B SNPs and inflammatory markers was increased when accounting for diabetes status. CONCLUSIONS Diabetes affects the production of adenosine and the expression of A2BRs that stimulate IL-6 and CRP production, insulin resistance, and the association between ADORA2B SNPs and inflammatory markers. We hypothesize that increased A2BR signaling in diabetes increases insulin resistance in part by elevating proinflammatory mediators. Selective A2BR blockers may be useful to treat insulin resistance.

OBJECTIVE Phosphoinositide 3-kinase enhancer A (PIKE-A) is a proto-oncogene that promotes tumor growth and transformation by enhancing Akt activity. However, the physiological functions of PIKE-A in peripheral tissues are unknown. Here, we describe the effect of PIKE deletion in mice and explore the role of PIKE-A in obesity development. RESEARCH DESIGN AND METHODS Whole-body PIKE knockout mice were generated and subjected to high-fat–diet feeding for 20 weeks. The glucose tolerance, tissue-specific insulin sensitivity, adipocyte differentiation, and lipid oxidation status were determined. The molecular mechanism of PIKE in the insulin signaling pathway was also studied. RESULTS We show that PIKE-A regulates obesity development by modulating AMP-activated protein kinase (AMPK) phosphorylation. PIKE-A is important for insulin to suppress AMPK phosphorylation. The expression of PIKE-A is markedly increased in adipose tissue of obese mice, whereas depletion of PIKE-A inhibits adipocyte differentiation. PIKE knockout mice exhibit a prominent phenotype of lipoatrophy and are resistant to high-fat diet–induced obesity, liver steatosis, and diabetes. PIKE knockout mice also have augmented lipid oxidation, which is accompanied by enhanced AMPK phosphorylation in both muscle and adipose tissue. Moreover, insulin sensitivity is improved in PIKE-A–deficient muscle and fat, thus protecting the animals from diet-induced diabetes. CONCLUSIONS Our results suggest that PIKE-A is implicated in obesity and associated diabetes development by negatively regulating AMPK activity.

Our work focuses on genetic and molecular mechanisms for the reciprocal regulation of bone and energy metabolism orchestrated by leptin and osteocalcin. In the context of this reciprocal regulation, the finding that leptin inhibits insulin secretion by beta cells while osteocalcin favors it is surprising. In exploring the molecular bases of this paradox we found that leptin, as is the case for most of its functions, uses a neuronal relay to inhibit insulin secretion. Cell-specific gene-deletion experiments revealed that a component of this neuronal regulation is the sympathetic innervation to osteoblasts. Under the control of leptin the sympathetic tone favors expression in osteoblasts of Esp, which inhibits the metabolic activity of osteocalcin. We further identify ATF4 as a transcription factor that regulates Esp expression and thereby insulin secretion and sensitivity. Taken together these data illustrate the tight connections between bone remodeling and energy metabolism and add further credence to the notion that the osteoblast is a bona fide endocrine cell type.

Post-transplant malignancy of donor origin is a rare complication of organ transplantation, most likely transmitted as micrometastases within the parenchyma of the donor organ or from circulating tumor cells contained within the organ. Patient survival is dependent upon early diagnoses, and differentiation of the malignancy as of donor or recipient derivation is important in developing a treatment modality. The utilization of fluorescent in situ hybridization chromosome analysis and DNA sequence analysis of the tumor cells can assist in this determination. This case report describes the management of donor transmitted malignant melanoma in a liver graft recipient and a review of the current literature.

The recent demonstration that osteoblasts have a role in controlling energy metabolism suggests that they express cell-specific regulatory genes involved in this process. Activating transcription factor 4 (ATF4) is a transcription factor that accumulates predominantly in osteoblasts, where it regulates virtually all functions linked to the maintenance of bone mass. Since Atf4-/- mice have smaller fat pads than littermate controls, we investigated whether ATF4 also influences energy metabolism. Here, we have shown, through analysis of Atf4-/- mice, that ATF4 inhibits insulin secretion and decreases insulin sensitivity in liver, fat, and muscle. Several lines of evidence indicated that this function of ATF4 occurred through its osteoblastic expression. First, insulin sensitivity is enhanced in the liver of Atf4-/- mice, but not in cultured hepatocytes from these mice. Second, mice overexpressing ATF4 in osteoblasts only [termed here alpha1(I)Collagen-Atf4 mice] displayed a decrease in insulin secretion and were insulin insensitive. Third, the alpha1(I)Collagen-Atf4 transgene corrected the energy metabolism phenotype of Atf4-/- mice. Fourth, and more definitely, mice lacking ATF4 only in osteoblasts presented the same metabolic abnormalities as Atf4-/- mice. Molecularly, ATF4 favored expression in osteoblasts of Esp, which encodes a product that decreases the bioactivity of osteocalcin, an osteoblast-specific secreted molecule that enhances secretion of and sensitivity to insulin. These results provide a transcriptional basis to the observation that osteoblasts fulfill endocrine functions and identify ATF4 as a regulator of most functions of osteoblasts.

Insulin resistance, the impaired ability of insulin to stimulate glucose utilization, is a major characteristic of type 2 diabetes. Insulin sensitivity can be measured using a variety of techniques that are commonly employed in diabetes research and care. Of these, hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity. The euglycemic clamp is widely used in clinics and laboratories to measure insulin action on glucose utilization in humans and animals for clinical and basic science research. Incorporation of radioactive-labeled glucose during euglycemic clamps makes it possible to measure glucose metabolism in individual organs. In recent years, euglycemic clamps have been actively performed in transgenic animal models of obesity, diabetes, and its complications, and have significantly advanced our understanding on the etiology and pathogenesis of type 2 diabetes. This chapter describes our standardized methods of the euglycemic clamp and associated surgical and biochemical procedures to measure insulin sensitivity in conscious rodents.

Insulin resistance, the impaired ability of insulin to stimulate glucose utilization, is a major characteristic of type 2 diabetes. Insulin sensitivity can be measured using a variety of techniques that are commonly employed in diabetes research and care. Of these, hyperinsulinemic-euglycemic clamp is the gold-standard method to assess insulin sensitivity. The euglycemic clamp is widely used in clinics and laboratories to measure insulin action on glucose utilization in humans and animals for clinical and basic science research. Incorporation of radioactive-labeled glucose during euglycemic clamps makes it possible to measure glucose metabolism in individual organs. In recent years, euglycemic clamps have been actively performed in transgenic animal models of obesity, diabetes, and its complications, and have significantly advanced our understanding on the etiology and pathogenesis of type 2 diabetes. This chapter describes our standardized methods of the euglycemic clamp and associated surgical and biochemical procedures to measure insulin sensitivity in conscious rodents.

A high-fat diet causes activation of the regulatory protein c-Jun NH 2 -terminal kinase 1 (JNK1) and triggers development of insulin resistance. JNK1 is therefore a potential target for therapeutic treatment of metabolic syndrome. We explored the mechanism of JNK1 signaling by engineering mice in which the Jnk1 gene was ablated selectively in adipose tissue. JNK1 deficiency in adipose tissue suppressed high-fat diet–induced insulin resistance in the liver. JNK1-dependent secretion of the inflammatory cytokine interleukin-6 by adipose tissue caused increased expression of liver SOCS3, a protein that induces hepatic insulin resistance. Thus, JNK1 activation in adipose tissue can cause insulin resistance in the liver.

Increasing evidence implicates the role of inflammation in diabetes and complications. Macrophages are shown to infiltrate adipose tissue in obesity, and inflammatory cytokines alter glucose metabolism in peripheral organs. Male C57BL/6 mice were fed high-fat diet (HFD; 55% fat by calories) or chow diet for 6 weeks, and heart samples were taken for analysis (n = 5~7). Chronic HFD increased whole body fat mass, measured by 1 H-MRS, by 3-fold, and elevated plasma IL-6 and TNF-α levels by 40%. Diet-induced obesity caused inflammation in heart and increased macrophage-specific CD68 levels by 5-fold (Fig. 1) (* P < 0.05 vs Chow). Diet-induced cardiac inflammation was associated with significant increases in toll-like receptor 4 (TLR4) and MyD88 levels in heart (Fig. 2). HFD also increased cardiomyocyte SOCS3 levels by more than 3-fold (Fig. 3). Myocardial glucose metabolism was measured using intravenous injection of 2-[ 14 C]deoxyglucose in awake mice (n = 6). Chronic HFD reduced myocardial glucose uptake by 50%, and this was associated with significant reductions in total GLUT4 and GLUT1 protein levels. Further, Thr 172 phosphorylation of AMPK, a critical regulator of energy balance, was markedly reduced in heart following HFD (Fig. 4). These results demonstrate that diet-induced obesity causes macrophage infiltration and inflammation in heart by increasing TLR4 signaling in cardiomyocytes. Similar to the effects of inflammation on peripheral glucose metabolism, diet-induced cardiac inflammation reduced myocardial glucose metabolism by downregulating AMPK and GLUT protein levels. Thus, our findings underscore an important role of inflammation in diabetic heart.

OBJECTIVE—Liver-specific inactivation of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) by a dominant-negative transgene (l-SACC1 mice) impaired insulin clearance, caused insulin resistance, and increased hepatic lipogenesis. To discern whether this phenotype reflects a physiological function of CEACAM1 rather than the effect of the dominant-negative transgene, we characterized the metabolic phenotype of mice with null mutation of the Ceacam1 gene (Cc1−/−). RESEARCH DESIGN AND METHODS—Mice were originally generated on a mixed C57BL/6x129sv genetic background and then backcrossed 12 times onto the C57BL/6 background. More than 70 male mice of each of the Cc1−/− and wild-type Cc1+/+ groups were subjected to metabolic analyses, including insulin tolerance, hyperinsulinemic-euglycemic clamp studies, insulin secretion in response to glucose, and determination of fasting serum insulin, C-peptide, triglyceride, and free fatty acid levels. RESULTS—Like l-SACC1, Cc1−/− mice exhibited impairment of insulin clearance and hyperinsulinemia, which caused insulin resistance beginning at 2 months of age, when the mutation was maintained on a mixed C57BL/6x129sv background, but not until 5–6 months of age on a homogeneous inbred C57BL/6 genetic background. Hyperinsulinemic-euglycemic clamp studies revealed that the inbred Cc1−/− mice developed insulin resistance primarily in liver. Despite substantial expression of CEACAM1 in pancreatic β-cells, insulin secretion in response to glucose in vivo and in isolated islets was normal in Cc1−/− mice (inbred and outbred strains). CONCLUSIONS—Intact insulin secretion in response to glucose and impairment of insulin clearance in l-SACC1 and Cc1−/− mice suggest that the principal role of CEACAM1 in insulin action is to mediate insulin clearance in liver.

Long-term postplacement costs increase the global cost of endovascular aneurysm repair (EVAR) by 44%. Secondary procedures and endoleaks significantly increase long-term expense. This study evaluates device-specific long-term postplacement costs using two different endografts. AneuRx and Zenith endografts were used to treat 250 patients with abdominal aortic aneurysms between December 1998 and June 2006 at a single institution. A relative value unit-based hospital cost accounting system was used to calculate both direct and indirect hospital departmental costs. Institutional overhead expenses, costs of professional services, and outpatient visits were also included in cost determinations. All costs were valued in 2006 dollars. To examine long-term costs, patients with

Increasing evidence implicates the role of inflammation in the pathogenesis of diabetes and complications. Inflammatory cytokines (IL-6, TNF-α) are elevated in obese diabetic subjects, and are shown to modulate glucose metabolism in peripheral organs. In this report, we examined the effects of IL-6 on cardiac metabolism and insulin action in vivo. Male C57BL/6 mice were intravenously treated with IL-6 (16 ng/hr) or saline (control) for 2 hrs, and [ 14 C]2-deoxyglucose was intravenously injected in awake mice to measure myocardial glucose metabolism (n=9~10). Hyperinsulinemic-euglycemic clamps (2.5 mU/kg/min insulin infusion) were also performed in IL-6 or saline-treated mice (n=4~5) to measure cardiac insulin action. Acute treatment with IL-6 caused a 25% increase in myocardial STAT3 activity and significantly reduced basal myocardial glucose metabolism (Fig. 1 ; * P< 0.05). IL-6 treatment also reduced insulin-stimulated glucose uptake in heart, and these effects were associated with marked decreases in AMPK activity (Thr-phosphorylation of AMPK; Fig. 2 ) and IRS-1 tyrosine phosphorylation (Fig. 3 ). Acute IL-6 treatment increased myocardial expression of PKC-𝛉, which has been shown to mediate insulin resistance in peripheral organs (Fig. 4 ). These results indicate that IL-6 is a potent negative regulator of myocardial glucose metabolism and insulin action, and the underlying mechanism may involve IL-6 mediated activation of PKC-𝛉 and defects in AMPK and insulin signaling activity. Thus, our findings suggest a potential role of IL-6 in the pathogenesis of diabetic heart failure.

Although insulin resistance has been traditionally associated with type 2 diabetes, recent evidence in humans and animal models indicates that insulin resistance may also develop in type 1 diabetes. A point mutation of insulin 2 gene in Ins2Akitamice leads to pancreatic β-cell apoptosis and hyperglycemia, and these mice are commonly used to investigate type 1 diabetes and complications. Since insulin resistance plays an important role in diabetic complications, we performed hyperinsulinemic-euglycemic clamps in awake Ins2Akitaand wild-type mice to measure insulin action and glucose metabolism in vivo. Nonobese Ins2Akitamice developed insulin resistance, as indicated by an ∼80% reduction in glucose infusion rate during clamps. Insulin resistance was due to ∼50% decreases in glucose uptake in skeletal muscle and brown adipose tissue as well as hepatic insulin action. Skeletal muscle insulin resistance was associated with a 40% reduction in total GLUT4 and a threefold increase in PKCε levels in Ins2Akitamice. Chronic phloridzin treatment lowered systemic glucose levels and normalized muscle insulin action, GLUT4 and PKCε levels in Ins2Akitamice, indicating that hyperglycemia plays a role in insulin resistance. Echocardiography showed significant cardiac remodeling with ventricular hypertrophy that was ameliorated following chronic phloridzin treatment in Ins2Akitamice. Overall, we report for the first time that nonobese, insulin-deficient Ins2Akitamice develop type 2 diabetes phenotypes including peripheral and hepatic insulin resistance and cardiac remodeling. Our findings provide important insights into the pathogenesis of metabolic abnormalities and complications affecting type 1 diabetes and lean type 2 diabetes subjects.

As a new mouse model of obesity-induced diabetes generated by combining quantitative trait loci from New Zealand Obese (NZO/HlLt) and Nonobese Nondiabetic (NON/LtJ) mice, NONcNZO10/LtJ (RCS10) male mice developed type 2 diabetes characterized by maturity onset obesity, hyperglycemia, and insulin resistance. To metabolically profile the progression to diabetes in preobese and obese states, a 2-h hyperinsulinemic euglycemic clamp was performed and organ-specific changes in insulin action were assessed in awake RCS10 and NON/LtJ (control) males at 8 and 13 wk of age. Prior to development of obesity and attendant increases in hepatic lipid content, 8-wk-old RCS10 mice developed insulin resistance in liver and skeletal muscle due to significant decreases in insulin-stimulated glucose uptake and GLUT4 expression in muscle. Transition to an obese and hyperglycemic state by 13 wk of age exacerbated insulin resistance in skeletal muscle, liver, and heart associated with organ-specific increases in lipid content. Thus, this polygenic mouse model of type 2 diabetes, wherein plasma insulin is only modestly elevated and obesity develops with maturity yet insulin action and glucose metabolism in skeletal muscle and liver are reduced at an early prediabetic age, should provide new insights into the etiology of type 2 diabetes.

In the postabsorptive state, certain tissues, including the brain, require glucose as the sole source of energy. After an overnight fast, hepatic glycogen stores are depleted, and gluconeogenesis becomes essential for preventing life-threatening hypoglycemia. Mice with a targeted deletion of KLF15, a member of the Kruppel-like family of transcription factors, display severe hypoglycemia after an overnight (18 hr) fast. We provide evidence that defective amino acid catabolism promotes the development of fasting hypoglycemia in KLF15-/- mice by limiting gluconeogenic substrate availability. KLF15-/- liver and skeletal muscle show markedly reduced mRNA expression of amino acid-degrading enzymes. Furthermore, the enzymatic activity of alanine aminotransferase (ALT), which converts the critical gluconeogenic amino acid alanine into pyruvate, is decreased (approximately 50%) in KLF15-/- hepatocytes. Consistent with this observation, intraperitoneal injection of pyruvate, but not alanine, rescues fasting hypoglycemia in KLF15-/- mice. We conclude that KLF15 plays an important role in the regulation of gluconeogenesis.

Obesity is a major factor central to the development of insulin resistance and type 2 diabetes. The identification and characterization of genes involved in regulation of adiposity, insulin sensitivity, and glucose uptake are key to the design and development of new drug therapies for this disease. In this study, we show that the polarity kinase Par-1b/MARK2 is required for regulating glucose metabolism in vivo . Mice null for Par-1b were lean, insulin hypersensitive, resistant to high-fat diet-induced weight gain, and hypermetabolic. 18 F-FDG microPET and hyperinsulinemic–euglycemic clamp analyses demonstrated increased glucose uptake into white and brown adipose tissue, but not into skeletal muscle of Par-1b null mice relative to wild-type controls. Taken together, these data indicate that Par-1b is a regulator of glucose metabolism and adiposity in the whole animal and may be a valuable drug target for the treatment of both type 2 diabetes and obesity.

Fatty acid–binding proteins (FABPs) are cytosolic fatty acid chaperones that play a critical role in systemic regulation of lipid and glucose metabolism. In animals lacking the adipocyte/macrophage FABP isoforms aP2 and mal1, there is strong protection against diet-induced obesity, insulin resistance, type 2 diabetes, fatty liver disease, and hypercholesterolemic atherosclerosis. On high-fat diet, FABP-deficient mice also exhibit enhanced muscle AMP-activated kinase (AMPK) and reduced liver stearoyl-CoA desaturase-1 (SCD-1) activities. Here, we performed a cross between aP2−/−, mal1−/−, and leptin-deficient (ob/ob) mice to elucidate the role of leptin action on the metabolic phenotype of aP2-mal1 deficiency. The extent of obesity in the ob/ob-aP2-mal1−/− mice was comparable with ob/ob mice. However, despite severe obesity, ob/ob-aP2-mal1−/− mice remained euglycemic and demonstrated improved peripheral insulin sensitivity. There was also a striking protection from liver fatty infiltration in the ob/ob-aP2-mal1−/− mice with strong suppression of SCD-1 activity. On the other hand, the enhanced muscle AMPK activity in aP2-mal1−/− mice was lost in the ob/ob background. These results indicated that both decreased body weight and enhanced muscle AMPK activity in aP2-mal1−/− mice are potentially leptin dependent but improved systemic insulin sensitivity and protection from liver fatty infiltration are largely unrelated to leptin action and that insulin-sensitizing effects of FABP deficiency are, at least in part, independent of its effects on total-body adiposity.

The protein tyrosine phosphatase SHP-1 is a well-known inhibitor of activation-promoting signaling cascades in hematopoietic cells but its potential role in insulin target tissues is unknown. Here we show that Ptpn6(me-v/me-v) (also known as viable motheaten) mice bearing a functionally deficient SHP-1 protein are markedly glucose tolerant and insulin sensitive as compared to wild-type littermates, as a result of enhanced insulin receptor signaling to IRS-PI3K-Akt in liver and muscle. Downregulation of SHP-1 activity in liver of normal mice by adenoviral expression of a catalytically inert mutant of SHP-1, or after small hairpin RNA-mediated SHP-1 silencing, further confirmed this phenotype. Tyrosine phosphorylation of CEACAM1, a modulator of hepatic insulin clearance, and clearance of serum [125I]-insulin were markedly increased in SHP-1-deficient mice or SHP-1-deficient hepatic cells in vitro. These findings show a novel role for SHP-1 in the regulation of glucose homeostasis through modulation of insulin signaling in liver and muscle as well as hepatic insulin clearance.

Acute activation of the serine-threonine kinase Akt is cardioprotective and increases glucose uptake, at least in part, through enhanced expression of GLUT4 on the sarcolemma. The effects of chronic Akt activation on glucose uptake in the heart remain unclear. To address this issue, we examined the effects of chronic Akt activation on glucose uptake, glycogen storage, and relevant glucose transporters in the hearts of transgenic mice. We found that chronic cardiac activation of Akt led to a substantial increase in the rate of basal glucose uptake ( P < 0.05) but blunted the response to insulin (1.9 vs. 18.1-fold increase compared with baseline) using NMR in ex vivo perfused heart. Basal glucose uptake was also increased in Akt transgenic mice in vivo ( P < 0.005). These changes were associated with an increase on glycogen deposition, examined with histochemical staining, biochemical (>6-fold, P < 0.001) and in vivo radioactive (5-fold, P < 0.01) assays. Studies in chimeric hearts of female X-linked transgenic Akt mice suggested that increased glycogen deposition occurred as a cell autonomous effect of transgene expression. Interestingly, although sarcolemmal GLUT1 was not significantly altered, chronic Akt activation actually decreased plasma membrane GLUT4. Moreover, intracellular pools of GLUT1 were modestly reduced, whereas intracellular GLUT4 was substantially reduced. It seems likely that neither GLUT1 nor GLUT4 explains the increase in basal glucose uptake but that these reductions contribute to the loss of insulin responsiveness that we observed. These data demonstrate that chronic Akt activation increases basal glucose uptake and glycogen deposition while inhibiting the response to insulin.

Type 2 diabetes is a heterogeneous disease characterized by insulin resistance and altered glucose and lipid metabolism in multiple organs. To understand the complex series of events that occur during the development of obesity-associated diabetes, we examined the temporal pattern of changes in insulin action and glucose metabolism in individual organs during chronic high-fat feeding in C57BL/6 mice. Insulin-stimulated cardiac glucose metabolism was significantly reduced after 1.5 weeks of high-fat feeding, and cardiac insulin resistance was associated with blunted Akt-mediated insulin signaling and GLUT4 levels. Insulin resistance in skeletal muscle, adipose tissue, and liver developed in parallel after 3 weeks of high-fat feeding. Diet-induced whole-body insulin resistance was associated with increased circulating levels of resistin and leptin but unaltered adiponectin levels. High-fat feeding caused insulin resistance in skeletal muscle that was associated with significantly elevated intramuscular fat content. In contrast, diet-induced hepatic insulin resistance developed before a marked increase in intrahepatic triglyceride levels. Cardiac function gradually declined over the course of high-fat feeding, and after 20 weeks of high-fat diet, cardiac dysfunction was associated with mild hyperglycemia, hyperleptinemia, and reduced circulating adiponectin levels. Our findings demonstrate that cardiac insulin resistance is an early adaptive event in response to obesity and develops before changes in whole-body glucose homeostasis. This suggests that obesity-associated defects in cardiac function may not be due to insulin resistance per se but may be attributable to chronic alteration in cardiac glucose and lipid metabolism and circulating adipokines.

Caveolin-3 (Cav-3) is expressed predominantly in skeletal muscle fibers, where it drives caveolae formation at the muscle cell's plasma membrane. In vitro studies have suggested that Cav-3 may play a positive role in insulin signaling and energy metabolism. We directly address the in vivo metabolic consequences of genetic ablation of Cav-3 in mice as it relates to insulin action, glucose metabolism, and lipid homeostasis. At age 2 mo, Cav-3 null mice are significantly larger than wild-type mice, and display significant postprandial hyperinsulinemia, whole body insulin resistance, and whole body glucose intolerance. Studies using hyperinsulinemic-euglycemic clamps revealed that Cav-3 null mice exhibited 20% and 40% decreases in insulin-stimulated whole body glucose uptake and whole body glycogen synthesis, respectively. Whole body insulin resistance was mostly attributed to 20% and 40% decreases in insulin-stimulated glucose uptake and glucose metabolic flux in the skeletal muscle of Cav-3 null mice. In addition, insulin-mediated suppression of hepatic glucose production was significantly reduced in Cav-3 null mice, indicating hepatic insulin resistance. Insulin-stimulated glucose uptake in white adipose tissue, which does not express Cav-3, was decreased by ∼70% in Cav-3 null mice, suggestive of an insulin-resistant state for this tissue. During fasting, Cav-3 null mice possess normal insulin receptor protein levels in their skeletal muscle. However, after 15 min of acute insulin stimulation, Cav-3 null mice show dramatically reduced levels of the insulin receptor protein, compared with wild-type mice treated identically. These results suggest that Cav-3 normally functions to increase the stability of the insulin receptor at the plasma membrane, preventing its rapid degradation, i.e., by blocking or slowing ligand-induced receptor downregulation. Thus our results demonstrate the importance of Cav-3 in regulating whole body glucose homeostasis in vivo and its possible role in the development of insulin resistance. These findings may have clinical implications for the early diagnosis and treatment of caveolinopathies.

Insulin-stimulated translocation of GLUT4 vesicles from an intracellular compartment to the plasma membrane in 3T3L1 adipocytes is mediated through a syntaxin 4 (Syn4)- and Munc18c-dependent mechanism. To investigate the impact of increasing Syn4 protein abundance on glucose homeostasis in vivo, we engineered tetracycline-repressible transgenic mice to overexpress Syn4 by fivefold in skeletal muscle and pancreas and threefold in adipose tissue. Increases in Syn4 caused increases in Munc18c protein, indicating that Syn4 regulates Munc18c expression in vivo. An important finding was that female Syn4 transgenic mice exhibited an increased rate of glucose clearance during glucose tolerance tests that was repressible by the administration of tetracycline. Insulin-stimulated glucose uptake in skeletal muscle was increased by twofold in Syn4 transgenic mice compared with wild-type mice as assessed by hyperinsulinemic-euglycemic clamp analysis, consistent with a twofold increase in insulin-stimulated GLUT4 translocation in skeletal muscle. Hepatic insulin action was unaffected. Moreover, insulin content and glucose-stimulated insulin secretion by islets isolated from Syn4 transgenic mice did not differ from that of wild-type mice. In sum, these data suggest that increasing the number of Syn4-Munc18c “fusion sites” at the plasma membrane of skeletal muscle increases the amount of GLUT4 available to increase the overall rate of insulin-mediated glucose uptake in vivo.

Heart-type fatty acid-binding protein (H-FABP) is required for high rates of skeletal muscle long-chain fatty acid (LCFA) oxidation and esterification. Here we assessed whether H-FABP affects soleus muscle glucose uptake when measured in vitro in the absence of LCFA. Wild-type and H-FABP null mice were fed a standard chow or high-fat diet before muscle isolation. With the chow, the mutation increased insulin-dependent deoxyglucose uptake by 141% ( P < 0.01) at 0.02 mU/ml of insulin but did not cause a significant effect at 2 mU/ml of insulin; skeletal muscle triglyceride and long-chain acyl-CoA (LCA-CoA) levels remained normal. With the high-fat diet, the mutation increased insulin-dependent deoxyglucose uptake by 190% ( P < 0.01) at 2 mU/ml of insulin, thus partially preventing insulin resistance, and it completely prevented the threefold ( P < 0.001) diet-induced increase of muscle triglyceride levels; however, muscle LCA-CoA levels showed little or no reduction. With both diets, the mutation reduced the basal (insulin-independent) soleus muscle deoxyglucose uptake by 28% ( P < 0.05). These results establish a close relation between FABP-dependent lipid pools and insulin sensitivity and indicate the existence of a nonacute, antagonistic, and H-FABP-dependent fatty acid regulation of basal and insulin-dependent muscle glucose uptake.

The circulating level of the inflammatory cytokine interleukin (IL)-6 is elevated in various insulin-resistant states including type 2 diabetes, obesity, cancer, and HIV-associated lipodystrophy. To determine the role of IL-6 in the development of insulin resistance, we examined the effects of IL-6 treatment on whole-body insulin action and glucose metabolism in vivo during hyperinsulinemic-euglycemic clamps in awake mice. Pretreatment of IL-6 blunted insulin’s ability to suppress hepatic glucose production and insulin-stimulated insulin receptor substrate (IRS)-2–associated phosphatidylinositol (PI) 3-kinase activity in liver. Acute IL-6 treatment also reduced insulin-stimulated glucose uptake in skeletal muscle, and this was associated with defects in insulin-stimulated IRS-1–associated PI 3-kinase activity and increases in fatty acyl-CoA levels in skeletal muscle. In contrast, we found that co-treatment of IL-10, a predominantly anti-inflammatory cytokine, prevented IL-6–induced defects in hepatic insulin action and signaling activity. Additionally, IL-10 co-treatment protected skeletal muscle from IL-6 and lipid-induced defects in insulin action and signaling activity, and these effects were associated with decreases in intramuscular fatty acyl-CoA levels. This is the first study to demonstrate that inflammatory cytokines IL-6 and IL-10 alter hepatic and skeletal muscle insulin action in vivo, and the mechanism may involve cytokine-induced alteration in intracellular fat contents. These findings implicate an important role of inflammatory cytokines in the pathogenesis of insulin resistance.

Three mouse lines with altered growth hormone (GH) signaling were used to study GH's role in adiposity. Dwarf GH receptor knockout mice (GHR -/-) and bovine GH antagonist expressing mice (GHA) had an increased percent body fat with most of the excess fat mass accumulating in the subcutaneous region. Giant bovine GH expressing mice (bGH) had a reduced percent body fat. Only GHA mice consumed significantly more food per body weight. Serum leptin levels were significantly increased in GHA mice and decreased in bGH mice but unchanged in the GHR -/- mice. Interestingly, serum adiponectin levels were significantly increased in the GHR -/- and GHA lines but decreased in bGH mice. These data suggest that suppression or absence of GH action and enhanced GH action indeed have opposite metabolic effects in terms of adiposity. Interestingly, adiponectin levels were positively correlated with previously reported insulin sensitivity of these mice, but also positively correlated with adiposity, which is contrary to findings in other mouse models. Thus, adiponectin levels were negatively correlated with GH function suggesting a role for adiponectin in GH-induced insulin resistance.

To determine the role of adipocytes and the tissue-specific nature in the insulin sensitizing action of rosiglitazone, we examined the effects of 3 weeks of rosiglitazone treatment on insulin signaling and action during hyperinsulinemic-euglycemic clamps in awake A-ZIP/F-1 (fatless), fat-transplanted fatless, and wild-type littermate mice. We found that 53 and 66% decreases in insulin-stimulated glucose uptake and insulin receptor substrate (IRS)-1–associated phosphatidylinositol (PI) 3-kinase activity in skeletal muscle of fatless mice were normalized after rosiglitazone treatment. These effects of rosiglitazone treatment were associated with 50% decreases in triglyceride and fatty acyl-CoA contents in the skeletal muscle of rosiglitazone-treated fatless mice. In contrast, rosiglitazone treatment exacerbated hepatic insulin resistance in the fatless mice and did not affect already reduced IRS-2–associated PI 3-kinase activity in liver. The worsening of insulin action in liver was associated with 30% increases in triglyceride and fatty acyl-CoA contents in the liver of rosiglitazone-treated fatless mice. In conclusion, these data support the hypothesis that rosiglitazone treatment enhanced insulin action in skeletal muscle mostly by its ability to repartition fat away from skeletal muscle.

The virtually fatless A-ZIP/F-1 mouse is profoundly insulin resistant, diabetic, and a good model for humans with severe generalized lipoatrophy. Like a number of other mouse models of diabetes, the A-ZIP/F-1 mouse has elevated serum corticosterone levels. Leptin infusion lowers the corticosterone levels, suggesting that leptin deficiency contributes to the hypercorticosteronemic state. To test the hypothesis that the increased glucocorticoids contribute to the diabetes and insulin resistance, we examined the effect of adrenalectomy on A-ZIP/F-1 mice. Adrenalectomy significantly decreased the blood glucose, serum insulin, and glycated hemoglobin levels. Hyperinsulinemic-euglycemic clamps were performed to characterize the changes in whole-body and tissue insulin sensitivity. The adrenalectomized A-ZIP/F-1 mice displayed a marked improvement in insulin-induced suppression of endogenous glucose production, indicating increased hepatic insulin sensitivity. Adrenalectomy also increased muscle glucose uptake and glycogen synthesis. These results suggest that the chronically increased serum corticosterone levels contribute to the diabetes of the A-ZIP/F-1 mice and that removal of the glucocorticoid excess improves the insulin sensitivity in both muscle and liver.

Both classical brown adipocytes and brown-like beige adipocytes are considered as promising therapeutic targets for obesity; however, their development, relative importance and functional coordination are not well understood. Here we show that a modest expression of miR-378/378* in adipose tissue specifically increases classical brown fat (BAT) mass, but not white fat (WAT) mass. Remarkably, BAT expansion, rather than miR-378 per se, suppresses formation of beige adipocytes in subcutaneous WAT. Despite this negative feedback, the expanded BAT depot is sufficient to prevent both genetic and high-fat diet-induced obesity. At the molecular level, we find that miR-378 targets phosphodiesterase Pde1b in BAT but not in WAT. Indeed, miR-378 and Pde1b inversely regulate brown adipogenesis in vitro in the absence of phosphodiesterase inhibitor isobutylmethylxanthine. Our work identifies miR-378 as a key regulatory component underlying classical BAT-specific expansion and obesity resistance, and adds novel insights into the physiological crosstalk between BAT and WAT.

9 páginas, 5 figuras, 3 tablas -- PAGS nros. 1926-1934, Peripheral insulin resistance and impaired insulin action are the primary characteristics of type 2 diabetes. The first observable defect in this major disorder occurs in muscle, where glucose disposal in response to insulin is impaired. We have developed a transgenic mouse with a dominant-negative insulin-like growth factor-I receptor (KR–IGF-IR) specifically targeted to the skeletal muscle. Expression of KR–IGF-IR resulted in the formation of hybrid receptors between the mutant and the endogenous IGF-I and insulin receptors, thereby abrogating the normal function of these receptors and leading to insulin resistance. Pancreatic β-cell dysfunction developed at a relative early age, resulting in diabetes. These mice provide an excellent model to study the molecular mechanisms underlying the development of human type 2 diabetes, The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Insulin resistance is a major factor in the pathogenesis of type 2 diabetes and is strongly associated with obesity. Increased concentrations of intracellular fatty acid metabolites have been postulated to interfere with insulin signaling by activation of a serine kinase cascade involving PKCtheta in skeletal muscle. Uncoupling protein 3 (UCP3) has been postulated to dissipate the mitochondrial proton gradient and cause metabolic inefficiency. We therefore hypothesized that overexpression of UCP3 in skeletal muscle might protect against fat-induced insulin resistance in muscle by conversion of intramyocellular fat into thermal energy. Wild-type mice fed a high-fat diet were markedly insulin resistant, a result of defects in insulin-stimulated glucose uptake in skeletal muscle and hepatic insulin resistance. Insulin resistance in these tissues was associated with reduced insulin-stimulated insulin receptor substrate 1- (IRS-1-) and IRS-2-associated PI3K activity in muscle and liver, respectively. In contrast, UCP3-overexpressing mice were completely protected against fat-induced defects in insulin signaling and action in these tissues. Furthermore, these changes were associated with a lower membrane-to-cytosolic ratio of diacylglycerol and reduced PKCtheta activity in whole-body fat-matched UCP3 transgenic mice. These results suggest that increasing mitochondrial uncoupling in skeletal muscle may be an excellent therapeutic target for type 2 diabetes mellitus.

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

The earliest defect in developing type 2 diabetes is insulin resistance, characterized by decreased glucose transport and metabolism in muscle and adipocytes. The glucose transporter GLUT4 mediates insulin-stimulated glucose uptake in adipocytes and muscle by rapidly moving from intracellular storage sites to the plasma membrane. In insulin-resistant states such as obesity and type 2 diabetes, GLUT4 expression is decreased in adipose tissue but preserved in muscle. Because skeletal muscle is the main site of insulin-stimulated glucose uptake, the role of adipose tissue GLUT4 downregulation in the pathogenesis of insulin resistance and diabetes is unclear. To determine the role of adipose GLUT4 in glucose homeostasis, we used Cre/loxP DNA recombination to generate mice with adipose-selective reduction of GLUT4 (G4A-/-). Here we show that these mice have normal growth and adipose mass despite markedly impaired insulin-stimulated glucose uptake in adipocytes. Although GLUT4 expression is preserved in muscle, these mice develop insulin resistance in muscle and liver, manifested by decreased biological responses and impaired activation of phosphoinositide-3-OH kinase. G4A-/- mice develop glucose intolerance and hyperinsulinaemia. Thus, downregulation of GLUT4 and glucose transport selectively in adipose tissue can cause insulin resistance and thereby increase the risk of developing diabetes.

Studies from several laboratories suggest that oxidized LDL may play an important role in atherogenesis. Our group previously showed that treatment of aortic endothelial cells with low levels of MM-LDL caused increased expression of MCP-1, M-CSF, tissue factor, and a monocyte-binding protein. In these studies MM-LDL was produced by storage of native LDL. We now show that cocultures of endothelial and smooth muscle cells can also produce MM-LDL from native LDL. This production of MM-LDL by cells is prevented by preincubating the LDL with probucol or vitamin E. However, addition of antioxidants to MM-LDL did not block its action. In past studies we also showed that endothelial cells exhibit differential sensitivity to the effects of MM-LDL. We report herein that in resistant cells there is no elevation of catalase, glutathione peroxidase, or copper-zinc-dependent SOD. However, maganese-dependent SOD is elevated in resistant cells. Ways in which MM-LDL production may be elevated in poorly controlled diabetics subjects are discussed.

Scanning tunneling microscopy (STM) and point-probe electrical conductivity measurements of electrochemically protonated films of the emeraldine-base form of the conducting polymer, polyaniline are reported. The conductivity varies spatially, dependent on the size (L(M) \m=~\ 200 to 300 angstroms) of granular metallic regions which relate directly to the inhomogeneous micromorphology of the electrodeposited films. The normalized conductivity at zero bias is observed to increase with doping, indicating an increase in states at the Fermi level. The STM electronic measurements also show regions of negative differential resistance. Negative differential resistance is observed for all samples, although more frequently on less oxidized samples.

Obesity and insulin resistance in skeletal muscle are two major factors in the pathogenesis of type 2 diabetes. Mice with muscle-specific inactivation of the insulin receptor gene (MIRKO) are normoglycemic but have increased fat mass. To identify the potential mechanism for this important association, we examined insulin action in specific tissues of MIRKO and control mice under hyperinsulinemic-euglycemic conditions. We found that insulin-stimulated muscle glucose transport and glycogen synthesis were decreased by about 80% in MIRKO mice, whereas insulin-stimulated fat glucose transport was increased threefold in MIRKO mice. These data demonstrate that selective insulin resistance in muscle promotes redistribution of substrates to adipose tissue thereby contributing to increased adiposity and development of the prediabetic syndrome.

To examine whether growth hormone (GH) induces peripheral insulin resistance by altering plasma free fatty acid (FFA) or insulin levels, the effects of GH infusion on insulin-stimulated glucose fluxes were studied in conscious rats under two protocols. In study 1, either saline ( n = 7) or human recombinant GH (21 μg ⋅ kg−1⋅ h−1; n = 8) was infused for 300 min, and insulin-stimulated glucose fluxes were estimated during the final 150-min period of hyperinsulinemic euglycemic clamps. In study 2, hyperinsulinemic euglycemic clamps were first conducted for 150 min (to raise plasma insulin and suppress FFA levels), and saline or GH ( n = 7 for each) was subsequently infused for the following 300-min clamp period. In study 1, GH infusion in the basal state did not significantly alter plasma FFA or insulin levels. In contrast, GH infusion decreased insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis by 32, 27, and 40%, respectively ( P < 0.05). In study 2, GH infusion during hyperinsulinemic euglycemic clamps did not alter plasma FFA or insulin levels ( P > 0.05). GH infusion had no effect on insulin-stimulated glucose uptake during the initial 150 min but eventually decreased insulin-stimulated glucose uptake by 37% ( P < 0.05), similar to the results in study 1. These data indicate that GH induces peripheral insulin resistance independent of plasma FFA and insulin levels. The induction of insulin resistance was preceded by suppression of glycogen synthesis, consistent with the hypothesis that metabolic impairment precedes and causes development of peripheral insulin resistance.

The relationships between postabsorptive glucose concentration and hepatic glucose output (HGO) and glucose clearance were studied in rats one day after treatment with various doses of streptozotocin (STZ; 0, 15, 30, 40, 50, or 75 mg/kg; n = 6 per dose; study 1). Glucose fluxes were estimated using a prolonged (6-h) infusion of [3-3H]glucose to ensure complete tracer equilibration at hyperglycemia. Postabsorptive glucose was significantly increased at the high doses of STZ (50 and 75 mg/kg; P < 0.01) and was strongly correlated with glucose clearance across all doses ( r = −0.85, P < 0.001) but less strongly with HGO ( r = 0.46, P < 0.01). In the group treated with 50 mg/kg STZ, postabsorptive glucose was increased twofold compared with the control (i.e., zero dose) group, with no change in HGO and a 45% decrease in glucose clearance, indicating that the hyperglycemia was due to a decrease in glucose clearance. To understand the cellular mechanisms of decreased glucose clearance in STZ diabetic rats, skeletal muscle glucose clearance and intracellular glucose and glucose 6-phosphate (G-6- P) concentrations were determined in normal and STZ (50 mg/kg) diabetic rats at their postabsorptive glucose levels as well as at matched hyperglycemia (12 mM; study 2). Glucose clearance was significantly decreased in soleus ( P< 0.05) muscles of the diabetic rats, and this was associated with significantly decreased intracellular glucose and G-6- P levels at matched hyperglycemia ( P < 0.05), suggestive of decreased glucose transport. In conclusion, postabsorptive hyperglycemia in STZ diabetic rats was largely due to decreased glucose clearance, although increased HGO may also have been a contributing factor at the highest STZ dose. The decrease in postabsorptive glucose clearance in STZ diabetic rats appeared to be associated with an impairment of glucose transport in soleus (type I) muscles.

Metabolic syndrome describes a set of obesity-related disorders that increase diabetes, cardiovascular, and mortality risk. Studies of liver-specific protein-tyrosine phosphatase 1b (PTP1b) deletion mice (L-PTP1b[superscript −/−]) suggest that hepatic PTP1b inhibition would mitigate metabolic-syndrome through amelioration of hepatic insulin resistance, endoplasmic-reticulum stress, and whole-body lipid metabolism. However, the altered molecular-network states underlying these phenotypes are poorly understood. We used mass spectrometry to quantify protein-phosphotyrosine network changes in L-PTP1b[superscript −/−] mouse livers relative to control mice on normal and high-fat diets. We applied a phosphosite-set-enrichment analysis to identify known and novel pathways exhibiting PTP1b- and diet-dependent phosphotyrosine regulation. Detection of a PTP1b-dependent, but functionally uncharacterized, set of phosphosites on lipid-metabolic proteins motivated global lipidomic analyses that revealed altered polyunsaturated-fatty-acid (PUFA) and triglyceride metabolism in L-PTP1b[superscript −/−] mice. To connect phosphosites and lipid measurements in a unified model, we developed a multivariate-regression framework, which accounts for measurement noise and systematically missing proteomics data. This analysis resulted in quantitative models that predict roles for phosphoproteins involved in oxidation–reduction in altered PUFA and triglyceride metabolism., Pfizer Inc. (grant), National Institutes of Health (U.S.) (grant 5R24DK090963), National Institutes of Health (U.S.) (grant U54-CA112967), National Institutes of Health (U.S.) (grant CA49152 R37), National Institutes of Health (U.S.) (grant R01-DK080756), National Mouse Metabolic Phenotyping Center at UMASS (Grant (U24-DK093000)), National Science Foundation (U.S.) (Graduate Research Fellowship)

Recent studies have suggested that the insulin receptor tyrosine kinase inhibitor, membrane glycoprotein PC-1, may play a role in certain insulin resistant states. In the present study, we examined whether either insulin receptor function or PC-1 activity was altered during the development of insulin resistance that occurs with high fat feeding in normal rats. Over the course of 14 days of high fat feeding, both maximal and submaximal (physiological) insulin-stimulated skeletal muscle glucose uptake decreased gradually; after 14 days of high fat feeding, submaximal and maximal insulin-stimulated glucose uptake decreased by approximately 40 and approximately 50%, respectively. In contrast, in the same muscles (tibialis anterior) of these animals, neither insulin receptor content nor insulin-stimulated insulin receptor autophosphorylation was altered after 14 days of high fat feeding. PC-1 has both nucleotide pyrophosphatase (EC 3.6.1.9) and alkaline phosphodiesterase I (EC 3.1.4.1) enzyme activities. These enzyme activities showed no changes during the course of 14 days of high fat feeding. Individual data revealed that there was no significant correlation between insulin-stimulated glucose uptake and alkaline phosphodiesterase or nucleotide pyrophosphatase activity (P0.05). Together, these data indicate that neither defects in insulin receptor function nor elevated PC-1 activities are involved in the development of insulin resistance in rats with high fat feeding, and the insulin resistance induced with high fat feeding is likely due to postreceptor defects in skeletal muscle.

Postabsorptive hepatic glucose output (HGO) was estimated in normal (n = 9) and streptozotocin (STZ) diabetic rats after a 6-h [3-3H]glucose infusion. In diabetic rats, HGO was estimated at ambient (n = 12) or normal (achieved via phlorizin infusion; n = 9) glucose concentrations. HGO was not statistically different between normal and diabetic rats (63 +/- 3 vs. 77 +/- 10 mumol.kg-1.min-1; P> 0.05). HGO was also normal in diabetic rats even when plasma glucose was normalized with phlorizin infusion (71 +/- 5 vs. 63 +/- 3 mumol.kg-1.min-1; P> 0.05). In contrast, peripheral glucose uptake, when estimated at matched euglycemia, was lower by approximately 25% in diabetic than in normal rate (46 +/- 6 vs. 62 +/- 3 mumol.kg-1.min-1; P < 0.01). In addition, acute changes in plasma glucose concentrations did not have significant effects on HGO or peripheral glucose uptake in diabetic rats (P> 0.05), resulting in markedly decreased glucose clearance at ambient hyperglycemia (P < 0.001). In conclusion, postabsorptive HGO was not elevated in a majority (17 of 21) of STZ diabetic rats with severe hyperglycemia and therefore was not responsible for postabsorptive hyperglycemia. Our data suggest that an impairment in the ability of glucose to regulate peripheral glucose uptake or HGO develops in STZ diabetes and contributes to postabsorptive hyperglycemia.

To examine whether impairment of intracellular glucose metabolism precedes insulin resistance, we determined the time courses of changes in insulin-stimulated glucose uptake, glycolysis, and glycogen synthesis during high-fat feeding in rats. Animals were fed with a high-fat (66.5%) diet ad libitum for 0, 2, 4, 7, or 14 days (n = 10–11 in each group) after 5 days of a low-fat (12.5%) diet. Submaximal and maximal insulin-stimulated glucose fluxes were estimated in whole body and individual skeletal muscles using the glucose clamp technique combined with D-[3-3H]glucose infusion and 2-[1-14C]deoxyglucose injection. Both submaximal and maximal insulin-stimulated glucose uptake in whole body decreased gradually with high-fat feeding. However, the decreases were minimal and not statistically significant during the initial few days (i.e., 2 and 4 days) of high-fat feeding (P > 0.05). In contrast, insulin-stimulated whole-body glycolysis (both maximal and submaximal) significantly decreased by ∼30% with 2 days of high-fat feeding and remained suppressed thereafter (P < 0.05). Similar patterns of changes in insulin-stimulated glucose uptake and glycolysis were also observed in skeletal muscle. Insulin-stimulated glycogen synthesis and glucose-6-phosphate (G-6-P) concentrations in skeletal muscle increased significantly during the initial few days of high-fat feeding and gradually returned to control levels by day 14, suggesting that increased G-6-P concentrations were responsible for increased glycogen synthesis. Thus, suppression of insulin-stimulated glycolysis and a compensatory increase in glycogen synthesis (presumably arising from the glucose-fatty acid cycle) preceded decreases in insulin-stimulated glucose uptake in skeletal muscle during high-fat feeding. These findings suggest that the insulin resistance may develop as a secondary response to impaired intracellular glucose metabolism.

The effects of elevated plasma free fatty acid (FFA) levels on insulin-stimulated whole-body and skeletal muscle glucose transport, glucose uptake, glycolysis, and glycogen synthesis were studied in conscious rats during hyperinsulinemic-euglycemic clamps with (n = 26) or without (n = 23) Intralipid and heparin infusion. Whole-body and skeletal muscle glucose uptake, glycolysis, and glycogen synthesis were estimated using d-[3-3H]glucose and 2-[14C]deoxyglucose (study 1), and glucose transport activity was assessed by analyzing plasma kinetics of l-[14C]glucose and 3-O-[3H]-methylglucose (study 2). Plasma FFA levels decreased during the clamps without intralipid but increased above basal during the clamps with Intralipid infusion (P < 0.01 for both). Elevated plasma FFA levels decreased insulin-stimulated whole-body glucose uptake by ∼ 15% and ∼ 20% during physiological and maximal insulin clamps, respectively (P < 0.01). Similarly, insulin-stimulated glucose uptake was also decreased in individual skeletal muscles with Intralipid infusion (P < 0.05). The most profound effect of elevated plasma FFA levels was a 30–50% suppression of insulin-stimulated glycolysis in whole body and individual skeletal muscles in both clamps. In contrast, physiological insulin-stimulated glycogen synthesis was increased with elevated plasma FFA levels in whole body and individual skeletal muscles (P < 0.05). Glucose-6-phosphate (G-6-P) levels were increased in soleus and extensor digitorum longus (EDL) muscles with Intralipid infusion in both clamps (P < 0.05). Intralipid infusion did not alter the time profiles of plasma l-glucose and 3-O-methylglucose after an intravenous injection during maximal insulin clamps, and compartmental analysis indicated no significant effect of elevated FFA levels on glucose transport activity in insulin-sensitive tissues (P > 0.05). Thus, elevated plasma FFA decreased insulin-stimulated glucose uptake in skeletal muscle by suppressing glycolysis and increasing G-6-P levels. These findings suggest that the classic glucose-fatty acid cycle was the predominant mechanism underlying the inhibitory effect of FFA on skeletal muscle glucose uptake.