Sexual dimorphism in the glucose homeostasis phenotype of the Aromatase Knockout (ArKO) mice.

We investigated the effects of estrogens on glucose homeostasis using the Aromatase Knockout (ArKO) mouse, which is unable to convert androgens into estrogens. The ArKO mouse is a model of total estrogen ablation which develops symptoms of metabolic syndrome. To determine the development and progression of whole body state of insulin resistance of ArKO mice, comprehensive whole body tolerance tests were performed on WT, ArKO and estrogen administrated mice at 3 and 12 months of age. The absence of estrogens in the male ArKO mice leads to hepatic insulin resistance, glucose and pyruvate intolerance from 3 to 12 months with consistent improvement upon estrogen treatment. Estrogen absence in the female ArKO mice leads to glucose intolerance without pyruvate intolerance or insulin resistance. The replacement of estrogens in the female WT and ArKO mice exhibited both insulin sensitizing and resistance effects depending on age and dosage. In conclusion, this study presents information on the sexually dimorphic roles of estrogens on glucose homeostasis regulation.

Effects of Estrogens on Adipokines and Glucose Homeostasis in Female Aromatase Knockout Mice.

The maintenance of glucose homeostasis within the body is crucial for constant and precise performance of energy balance and is sustained by a number of peripheral organs. Estrogens are known to play a role in the maintenance of glucose homeostasis. Aromatase knockout (ArKO) mice are estrogen-deficient and display symptoms of dysregulated glucose metabolism. We aim to investigate the effects of estrogen ablation and exogenous estrogen administration on glucose homeostasis regulation. Six month-old female wildtype, ArKO, and 17ß-estradiol (E2) treated ArKO mice were subjected to whole body tolerance tests, serum examination of estrogen, glucose and insulin, ex-vivo muscle glucose uptake, and insulin signaling pathway analyses. Female ArKO mice display increased body weight, gonadal (omental) adiposity, hyperinsulinemia, and liver triglycerides, which were ameliorated upon estrogen treatment. Tolerance tests revealed that estrogen-deficient ArKO mice were pyruvate intolerant hence reflecting dysregulated hepatic gluconeogenesis. Analyses of skeletal muscle, liver, and adipose tissues supported a hepatic-based glucose dysregulation, with a down-regulation of Akt phosphorylation (a key insulin signaling pathway molecule) in the ArKO liver, which was improved with E2 treatment. Concurrently, estrogen treatment lowered ArKO serum leptin and adiponectin levels and increased inflammatory adipokines such as tumour necrosis factor alpha (TNFα) and interleukin 6 (IL6). Furthermore, estrogen deficiency resulted in the infiltration of CD45 macrophages into gonadal adipose tissues, which cannot be reversed by E2 treatment. This study describes the effects of estrogens on glucose homeostasis in female ArKO mice and highlights a primary phenotype of hepatic glucose dysregulation and a parallel estrogen modified adipokine profile.

Hepatic glucose intolerance precedes hepatic steatosis in the male aromatase knockout (ArKO) mouse.

Estrogens are known to play a role in modulating metabolic processes within the body. The Aromatase knockout (ArKO) mice have been shown to harbor factors of Metabolic syndrome with central adiposity, hyperinsulinemia and male-specific hepatic steatosis. To determine the effects of estrogen ablation and subsequent replacement in males on whole body glucose metabolism, three- and six-month-old male ArKO mice were subjected to whole body glucose, insulin and pyruvate tolerance tests and analyzed for ensuing metabolic changes in liver, adipose tissue, and skeletal muscle. Estrogen-deficient male ArKO mice showed increased gonadal adiposity which was significantly reduced upon 17ß-estradiol (E2) treatment. Concurrently, elevated ArKO serum leptin levels were significantly reduced upon E2 treatment and lowered serum adiponectin levels were restored to wild type levels. Three-month-old male ArKO mice were hyperglycemic, and both glucose and pyruvate intolerant. These phenotypes continued through to 6 months of age, highlighting a loss of glycemic control. ArKO livers displayed changes in gluconeogenic enzyme expression, and in insulin signaling pathways upon E2 treatment. Liver triglycerides were increased in the ArKO males only after 6 months of age, which could be reversed by E2 treatment. No differences were observed in insulin-stimulated ex vivo muscle glucose uptake nor changes in ArKO adipose tissue and muscle insulin signaling pathways. Therefore, we conclude that male ArKO mice develop hepatic glucose intolerance by the age of 3 months which precedes the sex-specific development of hepatic steatosis. This can be reversed upon the administration of exogenous E2.

Deletion of skeletal muscle SOCS3 prevents insulin resistance in obesity.

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

Hematopoietic AMPK ß1 reduces mouse adipose tissue macrophage inflammation and insulin resistance in obesity.

Individuals who are obese are frequently insulin resistant, putting them at increased risk of developing type 2 diabetes and its associated adverse health conditions. The accumulation in adipose tissue of macrophages in an inflammatory state is a hallmark of obesity-induced insulin resistance. Here, we reveal a role for AMPK ß1 in protecting macrophages from inflammation under high lipid exposure. Genetic deletion of the AMPK ß1 subunit in mice (referred to herein as ß1(-/-) mice) reduced macrophage AMPK activity, acetyl-CoA carboxylase phosphorylation, and mitochondrial content, resulting in reduced rates of fatty acid oxidation. ß1(-/-) macrophages displayed increased levels of diacylglycerol and markers of inflammation, effects that were reproduced in WT macrophages by inhibiting fatty acid oxidation and, conversely, prevented by pharmacological activation of AMPK ß1-containing complexes. The effect of AMPK ß1 loss in macrophages was tested in vivo by transplantation of bone marrow from WT or ß1(-/-) mice into WT recipients. When challenged with a high-fat diet, mice that received ß1(-/-) bone marrow displayed enhanced adipose tissue macrophage inflammation and liver insulin resistance compared with animals that received WT bone marrow. Thus, activation of AMPK ß1 and increasing fatty acid oxidation in macrophages may represent a new therapeutic approach for the treatment of insulin resistance.

Macrophage deletion of SOCS1 increases sensitivity to LPS and palmitic acid and results in systemic inflammation and hepatic insulin resistance.

OBJECTIVE: Macrophage secretion of proinflammatory cytokines contributes to the pathogenesis of obesity-related insulin resistance. An important regulator of inflammation is the suppressor of cytokine signaling-1 (SOCS1), which inhibits the JAK-STAT and toll-like receptor-4 (TLR4) pathways. Despite the reported role of SOCS1 in inhibiting insulin signaling, it is surprising that a SOCS1 polymorphism that increases SOCS1 promoter activity is associated with enhanced insulin sensitivity despite obesity. In the current study, we investigated the physiological role of myeloid and lymphoid cell SOCS1 in regulating inflammation and insulin sensitivity. RESEARCH DESIGN AND METHODS: We used mice generated by crossing SOCS1 floxed mice with mice expressing Cre recombinase under the control of the LysM-Cre promoter (SOCS1 LysM-Cre). These mice have deletion of SOCS1 in macrophages and lymphocytes. We assessed macrophage inflammation using flow cytometry and serum cytokine levels using Bioplex assays. We then measured insulin sensitivity using glucose tolerance tests and the euglycemic-hyperinsulinemic clamp. Using bone marrow-derived macrophages, we tested the effects of SOCS1 deletion in regulating responses to the TLR4 ligands: lipopolysaccharide (LPS) and palmitic acid. RESULTS: SOCS1 LysM-Cre mice had increased macrophage expression of CD11c, enhanced sensitivity to LPS, and palmitic acid and increased serum concentrations of tumor necrosis factor-α, interleukin-6, and monocyte chemoattractant protein. Increased inflammation was associated with impaired glucose tolerance and hyperinsulinemia as a result of reduced hepatic but not skeletal muscle insulin sensitivity. CONCLUSIONS: The expression of SOCS1 in hematopoietic cells protects mice against systemic inflammation and hepatic insulin resistance potentially by inhibiting LPS and palmitate-induced TLR4 signaling in macrophages.

Antibody therapeutics - the evolving patent landscape.

The antibody patent landscape has evolved dramatically over the past 30 years, particularly in areas of technology relating to antibody modification to reduce immunogenicity in humans or improve antibody function. In some cases antibody techniques that were developed in the 1980s are still the subject of patent protection in the United States or Canada.

Whole body deletion of AMP-activated protein kinase {beta}2 reduces muscle AMPK activity and exercise capacity.

AMP-activated protein kinase (AMPK) ß subunits (ß1 and ß2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK ß2 (ß2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that ß2 KO mice are viable and breed normally. ß2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the ß1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in ß2 KO mice. During treadmill running ß2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of ß2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet ß2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK ß2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.

Liver-specific suppressor of cytokine signaling-3 deletion in mice enhances hepatic insulin sensitivity and lipogenesis resulting in fatty liver and obesity.

UNLABELLED: Obesity is associated with chronic inflammation and contributes to the development of insulin resistance and nonalcoholic fatty liver disease. The suppressor of cytokine signaling-3 (SOCS3) protein is increased in inflammation and is thought to contribute to the pathogenesis of insulin resistance by inhibiting insulin and leptin signaling. Therefore, we studied the metabolic effects of liver-specific SOCS3 deletion in vivo. We fed wild-type (WT) and liver-specific SOCS3 knockout (SOCS3 LKO) mice either a control diet or a high-fat diet (HFD) for 6 weeks and examined their metabolic phenotype. We isolated hepatocytes from WT and SOCS3 LKO mice and examined the effects of tumor necrosis factor α and insulin on Akt phosphorylation and fatty acid metabolism and lipogenic gene expression. Hepatocytes from control-fed SOCS3 LKO mice were protected from developing tumor necrosis factor α-induced insulin resistance but also had increased lipogenesis and expression of sterol response element-binding protein-1c target genes. Lean SOCS3 LKO mice fed a control diet had enhanced hepatic insulin sensitivity; however, when fed an HFD, SOCS3 LKO mice had increased liver fat, inflammation, and whole-body insulin resistance. SOCS3 LKO mice fed an HFD also had elevated hypothalamic SOCS3 and fatty acid synthase expression and developed greater obesity due to increased food intake and reduced energy expenditure. CONCLUSION: Deletion of SOCS3 in the liver increases liver insulin sensitivity in mice fed a control diet but paradoxically promotes lipogenesis, leading to the development of nonalcoholic fatty liver disease, inflammation, and obesity.

AMPK beta1 deletion reduces appetite, preventing obesity and hepatic insulin resistance.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that regulates appetite and fuel metabolism. We have generated AMPK beta1(-/-) mice on a C57Bl/6 background that are viable, fertile, survived greater than 2 years, and display no visible brain developmental defects. These mice have a 90% reduction in hepatic AMPK activity due to loss of the catalytic alpha subunits, with modest reductions of activity detected in the hypothalamus and white adipose tissue and no change in skeletal muscle or heart. On a low fat or an obesity-inducing high fat diet, beta1(-/-) mice had reduced food intake, reduced adiposity, and reduced total body mass. Metabolic rate, physical activity, adipose tissue lipolysis, and lipogenesis were similar to wild type littermates. The reduced appetite and body mass of beta1(-/-) mice were associated with protection from high fat diet-induced hyperinsulinemia, hepatic steatosis, and insulin resistance. We demonstrate that the loss of beta1 reduces food intake and protects against the deleterious effects of an obesity-inducing diet.

Oligomeric resistin impairs insulin and AICAR-stimulated glucose uptake in mouse skeletal muscle by inhibiting GLUT4 translocation.

The hormone resistin is elevated in obesity and impairs glucose homeostasis. Here, we examined the effect of oligomerized human resistin on insulin signaling and glucose metabolism in skeletal muscle and myotubes. This was investigated by incubating mouse extensor digitorum longus (EDL) and soleus muscles and L6 myotubes with physiological concentrations of resistin and assessing insulin-stimulated glucose uptake, cellular signaling, suppressor of cytokine signaling 3 (SOCS-3) mRNA, and GLUT4 translocation. We found that resistin at a concentration of 30 ng/ml decreased insulin-stimulated glucose uptake by 30-40% in soleus muscle and myotubes, whereas in EDL muscle insulin-stimulated glucose uptake was impaired at a resistin concentration of 100 ng/ml. Impaired insulin-stimulated glucose uptake was not associated with reduced Akt phosphorylation or IRS-1 protein or increased SOCS-3 mRNA expression. To further investigate the site(s) at which resistin impairs glucose uptake we treated myotubes and skeletal muscle with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) and found that, although resistin did not impair AMPK activation, it reduced AICAR-stimulated glucose uptake. These data suggested that resistin impairs glucose uptake at a point common to insulin and AMPK signaling pathways, and we thus measured AS160/TBC1D4 Thr(642) phosphorylation and GLUT4 translocation in myotubes. Resistin did not impair TBC1D4 phosphorylation but did reduce both insulin and AICAR-stimulated GLUT4 plasma membrane translocation. We conclude that resistin impairs insulin-stimulated glucose uptake by mechanisms involving reduced plasma membrane GLUT4 translocation but independent of the proximal insulin-signaling cascade, AMPK, and SOCS-3.

Thienopyridone drugs are selective activators of AMP-activated protein kinase beta1-containing complexes.

The AMP-activated protein kinase (AMPK) is an alphabetagamma heterotrimer that plays a pivotal role in regulating cellular and whole-body metabolism. Activation of AMPK reverses many of the metabolic defects associated with obesity and type 2 diabetes, and therefore AMPK is considered a promising target for drugs to treat these diseases. Recently, the thienopyridone A769662 has been reported to directly activate AMPK by an unexpected mechanism. Here we show that A769662 activates AMPK by a mechanism involving the beta subunit carbohydrate-binding module and residues from the gamma subunit but not the AMP-binding sites. Furthermore, A769662 exclusively activates AMPK heterotrimers containing the beta1 subunit. Our findings highlight the regulatory role played by the beta subunit in modulating AMPK activity and the possibility of developing isoform specific therapeutic activators of this important metabolic regulator.