Restriction of essential amino acids dictates the systemic metabolic response to dietary protein dilution.

Dietary protein dilution (DPD) promotes metabolic-remodelling and -health but the precise nutritional components driving this response remain elusive. Here, by mimicking amino acid (AA) supply from a casein-based diet, we demonstrate that restriction of dietary essential AA (EAA), but not non-EAA, drives the systemic metabolic response to total AA deprivation; independent from dietary carbohydrate supply. Furthermore, systemic deprivation of threonine and tryptophan, independent of total AA supply, are both adequate and necessary to confer the systemic metabolic response to both diet, and genetic AA-transport loss, driven AA restriction. Dietary threonine restriction (DTR) retards the development of obesity-associated metabolic dysfunction. Liver-derived fibroblast growth factor 21 is required for the metabolic remodelling with DTR. Strikingly, hepatocyte-selective establishment of threonine biosynthetic capacity reverses the systemic metabolic response to DTR. Taken together, our studies of mice demonstrate that the restriction of EAA are sufficient and necessary to confer the systemic metabolic effects of DPD.

The androgen receptor in bone marrow progenitor cells negatively regulates fat mass.

It is well established that testosterone negatively regulates fat mass in humans and mice; however, the mechanism by which testosterone exerts these effects is poorly understood. We and others have shown that deletion of the androgen receptor (AR) in male mice results in a phenotype that mimics the three key clinical aspects of hypogonadism in human males; increased fat mass and decreased bone and muscle mass. We now show that replacement of the Ar gene specifically in mesenchymal progenitor cells (PCs) residing in the bone marrow of Global-ARKO mice, in the absence of the AR in all other tissues (PC-AR Gene Replacements), completely attenuates their increased fat accumulation. Inguinal subcutaneous white adipose tissue and intra-abdominal retroperitoneal visceral adipose tissue depots in PC-AR Gene Replacement mice were 50-80% lower than wild-type (WT) and 75-90% lower than Global-ARKO controls at 12 weeks of age. The marked decrease in subcutaneous and visceral fat mass in PC-AR Gene Replacements was associated with an increase in the number of small adipocytes and a healthier metabolic profile compared to WT controls, characterised by normal serum leptin and elevated serum adiponectin levels. Euglycaemic/hyperinsulinaemic clamp studies reveal that the PC-AR Gene Replacement mice have improved whole-body insulin sensitivity with higher glucose infusion rates compared to WT mice and increased glucose uptake into subcutaneous and intra-abdominal fat. In conclusion, these data provide the first evidence for an action of androgens via the AR in mesenchymal bone marrow PCs to negatively regulate fat mass and improve metabolic function.

Muscle-specific androgen receptor deletion shows limited actions in myoblasts but not in myofibers in different muscles in vivo.

The aim of this study was to investigate the direct muscle cell-mediated actions of androgens by comparing two different mouse lines. The cre-loxP system was used to delete the DNA-binding activity of the androgen receptor (AR) in mature myofibers (MCK mAR(ΔZF2)) in one model and the DNA-binding activity of the AR in both proliferating myoblasts and myofibers (α-actin mAR(ΔZF2)) in another model. We found that hind-limb muscle mass was normal in MCK mAR(ΔZF2) mice and that relative mass of only some hind-limb muscles was reduced in α-actin mAR(ΔZF2) mice. This suggests that myoblasts and myofibers are not the major cellular targets mediating the anabolic actions of androgens on male muscle during growth and development. Levator ani muscle mass was decreased in both mouse lines, demonstrating that there is a myofiber-specific effect in this unique androgen-dependent muscle. We found that the pattern of expression of genes including c-myc, Fzd4 and Igf2 is associated with androgen-dependent changes in muscle mass; therefore, these genes are likely to be mediators of anabolic actions of androgens. Further research is required to identify the major targets of androgen actions in muscle, which are likely to include indirect actions via other tissues.

Identification of ABCC8 as a contributory gene to impaired early-phase insulin secretion in NZO mice.

Type 2 diabetes (T2D) is associated with defective insulin secretion, which in turn contributes to worsening glycaemic control and disease progression. The genetic cause(s) associated with impaired insulin secretion in T2D are not well elucidated. Here we used the polygenic New Zealand Obese (NZO) mouse model, which displays all the cardinal features of T2D including hyperglycaemia to identify genes associated with ß-cell dysfunction. A genome-wide scan identified a major quantitative trait locus (QTL) on chromosome 7 associated with defective glucose-mediated insulin secretion. Using congenic strains, the locus was narrowed to two candidate genes encoding the components of the KATP channel: Abcc8 (SUR1) and Kcnj11 (Kir6.2). The NZO Abcc8 allele was associated with a ∼211 bp deletion in its transcript and reduced expression of SUR1. Transgenic NZO mice were generated that expressed the WT Abcc8/Kcnj11 genes and displayed significant improvements in early-phase glucose-mediated insulin secretion and glucose tolerance, confirming Abcc8 as a causative gene. Importantly, we showed that despite improving ß-cell function in the NZO transgenic mice, there was no enhancement of insulin sensitivity or body weight. This study provides evidence for a role of Abcc8 in early-phase glucose-mediated insulin secretion and validates this gene as a contributor to ß-cell dysfunction in T2D.

Contribution of the hypothalamus and gut to weight gain susceptibility and resistance in mice.

Obesity susceptibility in humans and in rodent strains varies in response to the consumption of high-energy density (HED) diets. However, the exact mechanism(s) involved in this susceptibility remain(s) unresolved. The aim of the present study was to gain greater insight into this susceptibility by using C57BL/6J (B6) mice that were separated into obesity-prone (diet-induced obese (DIO)) and obesity-resistant (diet-induced resistant (DR)) groups following an HED diet for 6 weeks. Physiological, biochemical and gene expression assessments of energy balance were performed in the DIO and DR mice on an HED diet and chow-fed mice. The increased weight gain of the DIO mice as compared to the DR mice was associated with increased energy intake and higher plasma leptin and adiponectin levels but not with reduced physical activity or resting energy expenditure. Hypothalamic Pomc gene expression was elevated, but there were no changes in Npy or Agrp expression. Adipose tissue leptin and adiponectin gene expression were significantly reduced in the DIO group as compared to the DR group. Interestingly, ileum expression of G protein-coupled receptor (Gpr) 40 (Gpr40) was significantly increased, whereas Gpr120, Gpr119, Gpr41, and glucagon-like peptide 1 (Glp1) were reduced. Contrastingly, the lower weight gain of the DR group was associated with elevated adipose tissue leptin and adiponectin gene expression, but there were no differences in plasma hormone or hypothalamic gene expression levels as compared to chow-fed mice. Therefore, the present data demonstrate that susceptibility and resistance to diet-induced weight gain in B6 mice appears to be predominantly driven by peripheral rather than hypothalamic modifications, and changes in gut-specific receptors are a potentially important contributor to this variation.

Targeting VEGF-B as a novel treatment for insulin resistance and type 2 diabetes.

The prevalence of type 2 diabetes is rapidly increasing, with severe socioeconomic impacts. Excess lipid deposition in peripheral tissues impairs insulin sensitivity and glucose uptake, and has been proposed to contribute to the pathology of type 2 diabetes. However, few treatment options exist that directly target ectopic lipid accumulation. Recently it was found that vascular endothelial growth factor B (VEGF-B) controls endothelial uptake and transport of fatty acids in heart and skeletal muscle. Here we show that decreased VEGF-B signalling in rodent models of type 2 diabetes restores insulin sensitivity and improves glucose tolerance. Genetic deletion of Vegfb in diabetic db/db mice prevented ectopic lipid deposition, increased muscle glucose uptake and maintained normoglycaemia. Pharmacological inhibition of VEGF-B signalling by antibody administration to db/db mice enhanced glucose tolerance, preserved pancreatic islet architecture, improved ß-cell function and ameliorated dyslipidaemia, key elements of type 2 diabetes and the metabolic syndrome. The potential use of VEGF-B neutralization in type 2 diabetes was further elucidated in rats fed a high-fat diet, in which it normalized insulin sensitivity and increased glucose uptake in skeletal muscle and heart. Our results demonstrate that the vascular endothelium can function as an efficient barrier to excess muscle lipid uptake even under conditions of severe obesity and type 2 diabetes, and that this barrier can be maintained by inhibition of VEGF-B signalling. We propose VEGF-B antagonism as a novel pharmacological approach for type 2 diabetes, targeting the lipid-transport properties of the endothelium to improve muscle insulin sensitivity and glucose disposal.

Normal muscle glucose uptake in mice deficient in muscle GLUT4.

Skeletal muscle insulin resistance is a major characteristic underpinning type 2 diabetes. Impairments in the insulin responsiveness of the glucose transporter, Glut4 (Slc2a4), have been suggested to be a contributing factor to this disturbance. We have produced muscle-specific Glut4 knockout (KO) mice using Cre/LoxP technology on a C57BL6/J background and shown undetectable levels of GLUT4 in both skeletal muscle and heart. Our aim was to determine whether complete deletion of muscle GLUT4 does in fact lead to perturbations in glucose homoeostasis. Glucose tolerance, glucose turnover and 2-deoxyglucose uptake into muscle and fat under basal and insulin-stimulated conditions were assessed in 12-week-old KO and control mice using the oral glucose tolerance test (OGTT) and hyperinsulinaemic/euglycaemic clamp respectively. KO mice weighed ~17% less and had significantly heavier hearts compared with control mice. Basally, plasma glucose and plasma insulin were significantly lower in the KO compared with control mice, which conferred normal glucose tolerance. Despite the lack of GLUT4 in the KO mouse muscle, glucose uptake was not impaired in skeletal muscle but was reduced in heart under insulin-stimulated conditions. Neither GLUT1 nor GLUT12 protein levels were altered in the skeletal muscle or heart tissue of our KO mice. High-fat feeding did not alter glucose tolerance in the KO mice but led to elevated plasma insulin levels during the glucose tolerance test. Our study demonstrates that deletion of muscle GLUT4 does not adversely affect glucose disposal and glucose tolerance and that compensation from other transporters may contribute to this unaltered homoeostasis of glucose.

The role of liver fructose-1,6-bisphosphatase in regulating appetite and adiposity.

Liver fructose-1,6-bisphosphatase (FBPase) is a regulatory enzyme in gluconeogenesis that is elevated by obesity and dietary fat intake. Whether FBPase functions only to regulate glucose or has other metabolic consequences is not clear; therefore, the aim of this study was to determine the importance of liver FBPase in body weight regulation. To this end we performed comprehensive physiologic and biochemical assessments of energy balance in liver-specific transgenic FBPase mice and negative control littermates of both sexes. In addition, hepatic branch vagotomies and pharmacologic inhibition studies were performed to confirm the role of FBPase. Compared with negative littermates, liver-specific FBPase transgenic mice had 50% less adiposity and ate 15% less food but did not have altered energy expenditure. The reduced food consumption was associated with increased circulating leptin and cholecystokinin, elevated fatty acid oxidation, and 3-ß-hydroxybutyrate ketone levels, and reduced appetite-stimulating neuropeptides, neuropeptide Y and Agouti-related peptide. Hepatic branch vagotomy and direct pharmacologic inhibition of FBPase in transgenic mice both returned food intake and body weight to the negative littermates. This is the first study to identify liver FBPase as a previously unknown regulator of appetite and adiposity and describes a novel process by which the liver participates in body weight regulation.

The liver: Key in regulating appetite and body weight.

Liver fructose-1,6-bisphosphatase (FBPase) is a regulatory enzyme in gluconeogenesis that is elevated by obesity and dietary fat intake. Whether FBPase functions only in glucose metabolism or has other metabolic roles is currently unclear. In our recently published study, we examined the importance of liver FBPase in body weight regulation by performing a series of comprehensive physiological and biochemical assessments of energy balance and specific intervention studies in our transgenic mouse line that overexpresses FBPase specifically in the liver. Compared with negative littermates, these FBPase transgenic mice weighed 10% less, had 50% less adiposity, ate 15% less food but did not have altered energy expenditure. Increased circulating leptin and cholecystokinin levels, elevated fatty acid oxidation and reduced appetite stimulating neuropeptides, neuropeptide Y (NPY) and agouti-related peptide (AGRP), underpinned this phenotype. Blocking the action of FBPase returned food intake and body weight to those of the negative littermates. Our study is the first to identify liver FBPase as a previously unknown regulator of appetite and adiposity. Importantly, this work recognizes the liver as an important organ in appetite and body weight regulation. This commentary will provide further insight and expand on this novel concept that the liver does in fact play an important role in adiposity.

Deficiency in interferon-gamma results in reduced body weight and better glucose tolerance in mice.

Obesity is a chronic low-grade inflammatory disease caused by increased energy intake and reduced energy expenditure. Studies using animal models with deletion of inflammatory cytokines have produced conflicting results with some showing increased weight gain and others showing no effect or even reduced body weights. Clearly, more work is necessary to understand the role of cytokines on body weight control. The aim of this study was to determine the effect of interferon-γ deletion (IFNγ(-/-)) on body weight regulation and glucose metabolism. Male IFNγ(-/-) and wild-type C57BL/6 mice were fed a low-fat chow diet, and body weight, food intake, and energy expenditure were monitored over 20 wk. At the end of the study, ip glucose tolerance test, insulin tolerance test, basal glucose turnover, and hyperinsulinemic/euglycemic clamps were performed. Expression levels of arcuate nucleus neuropeptide Y, Agouti-related peptide, and proopiomelanocortin mRNA as well as circulating leptin levels were also determined. IFNγ(-/-) mice had improved glucose tolerance with reduced rate of glucose appearance and increased insulin sensitivity due to greater suppression of endogenous glucose output, which was associated with decreased hepatic glucose-6-phosphatase activity. In addition, we also observed reduced body weight associated with decreased food intake and increased physical activity. Neuropeptide Y and Agouti-related peptide mRNA expression was reduced, whereas proopiomelanocortin mRNA expression was increased, as were plasma leptin levels. Global deletion of IFNγ in mice resulted in reduced body weight associated with negative energy balance, improved glucose tolerance, and hepatic insulin sensitivity. Our findings demonstrate that IFNγ plays a critical role in the regulation of body weight and glucose metabolism.

A primary defect in glucose production alone cannot induce glucose intolerance without defects in insulin secretion.

Increased glucose production is associated with fasting hyperglycaemia in type 2 diabetes but whether or not it causes glucose intolerance is unclear. This study sought to determine whether a primary defect in gluconeogenesis (GNG) resulting in elevated glucose production is sufficient to induce glucose intolerance in the absence of insulin resistance and impaired insulin secretion. Progression of glucose intolerance was assessed in phosphoenolpyruvate carboxykinase (PEPCK) transgenic rats, a genetic model with a primary increase in GNG. Young (4-5 weeks of age) and adult (12-14 weeks of age) PEPCK transgenic and Piebald Virol Glaxo (PVG/c) control rats were studied. GNG, insulin sensitivity, insulin secretion and glucose tolerance were assessed by intraperitoneal and intravascular substrate tolerance tests and hyperinsulinaemic/euglycaemic clamps. Despite elevated GNG and increased glucose appearance, PEPCK transgenic rats displayed normal glucose tolerance due to adequate glucose disposal and robust glucose-mediated insulin secretion. Glucose intolerance only became apparent in the PEPCK transgenic rats following the development of insulin resistance (both hepatic and peripheral) and defective glucose-mediated insulin secretion. Taken together, a single genetic defect in GNG leading to increased glucose production does not adversely affect glucose tolerance. Insulin resistance and impaired glucose-mediated insulin secretion are required to precipitate glucose intolerance in a setting of chronic glucose oversupply.

Increased adiposity in DNA binding-dependent androgen receptor knockout male mice associated with decreased voluntary activity and not insulin resistance.

In men, as testosterone levels decrease, fat mass increases and muscle mass decreases. Increased fat mass in men, in particular central obesity, is a major risk factor for type 2 diabetes, cardiovascular disease, and all-cause mortality. Testosterone treatment has been shown to decrease fat mass and increase fat-free mass. We hypothesize that androgens act directly via the DNA binding-dependent actions of the androgen receptor (AR) to regulate genes controlling fat mass and metabolism. The aim of this study was to determine the effect of a global DNA binding-dependent (DBD) AR knockout (DBD-ARKO) on the metabolic phenotype in male mice by measuring body mass, fat mass, food intake, voluntary physical activity, resting energy expenditure, substrate oxidation rates, serum glucose, insulin, lipid, and hormone levels, and metabolic gene expression levels and second messenger protein levels. DBD-ARKO males have increased adiposity despite a decreased total body mass compared with wild-type (WT) males. DBD-ARKO males showed reduced voluntary activity, decreased food intake, increased serum leptin and adiponectin levels, an altered lipid metabolism gene profile, and increased phosphorylated CREB levels compared with WT males. This study demonstrates that androgens acting via the DNA binding-dependent actions of the AR regulate fat mass and metabolism in males and that the increased adiposity in DBD-ARKO male mice is associated with decreased voluntary activity, hyperleptinemia and hyperadiponectinemia and not with insulin resistance, increased food intake, or decreased resting energy expenditure.

High-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and cholecystokinin: implications for circulatory control.

Gastric leptin and cholecystokinin (CCK) act on vagal afferents to induce cardiovascular effects and reflex inhibition of splanchnic sympathetic nerve discharge (SSND) and may act cooperatively in these responses. We sought to determine whether these effects are altered in animals that developed obesity in response to a medium high-fat diet (MHFD). Male Sprague-Dawley rats were placed on a low-fat diet (LFD; n = 8) or a MHFD (n = 24) for 13 wk, after which the animals were anesthetized and artificially ventilated. Arterial pressure was monitored and blood was collected for the determination of plasma leptin and CCK. SSND responses to leptin (15 µg/kg) and CCK (2 µg/kg) administered close to the coeliac artery were evaluated. Collectively, MHFD animals had significantly higher plasma leptin but lower plasma CCK levels than LFD rats (P < 0.05), and this corresponded to attenuated or reversed SSND responses to CCK (LFD, -21 ± 2%; and MHFD, -12 ± 2%; P < 0.05) and leptin (LFD, -6 ± 2%; and MHFD, 4 ± 1%; P < 0.001). Alternatively, animals on the MHFD were stratified into obesity-prone (OP; n = 8) or obesity-resistant (OR; n = 8) groups according to their weight gain falling within the upper or lower tertile, respectively. OP rats had significantly higher resting arterial pressure, adiposity, and plasma leptin but lower plasma CCK compared with LFD rats (P < 0.05). The SSND responses to CCK or leptin were not significantly different between OP and OR animals. These results demonstrate that a high-fat diet is associated with blunted splanchnic sympathoinhibitory responses to gastric leptin and CCK and may impact on sympathetic vasomotor mechanisms involved in circulatory control.

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.

Increased glucose production in mice overexpressing human fructose-1,6-bisphosphatase in the liver.

Increased endogenous glucose production (EGP) predominantly from the liver is a characteristic feature of type 2 diabetes, which positively correlates with fasting hyperglycemia. Gluconeogenesis is the biochemical pathway shown to significantly contribute to increased EGP in diabetes. Fructose-1,6-bisphosphatase (FBPase) is a regulated enzyme in gluconeogenesis that is increased in animal models of obesity and insulin resistance. However, whether a specific increase in liver FBPase can result in increased EGP has not been shown. The objective of this study was to determine the role of upregulated liver FBPase in glucose homeostasis. To achieve this goal, we generated human liver FBPase transgenic mice under the control of the transthyretin promoter, using insulator sequences to flank the transgene and protect it from site-of-integration effects. This resulted in a liver-specific model, as transgene expression was not detected in other tissues. Mice were studied under the following conditions: 1) at two ages (24 wk and 1 yr old), 2) after a 60% high-fat diet, and 3) when bred to homozygosity. Hemizygous transgenic mice had an approximately threefold increase in total liver FBPase mRNA with concomitant increases in FBPase protein and enzyme activity levels. After high-fat feeding, hemizygous transgenics were glucose intolerant compared with negative littermates (P < 0.02). Furthermore, when bred to homozygosity, chow-fed transgenic mice showed a 5.5-fold increase in liver FBPase levels and were glucose intolerant compared with negative littermates, with a significantly higher rate of EGP (P < 0.006). This is the first study to show that FBPase regulates EGP and whole body glucose homeostasis in a liver-specific transgenic model. Our homozygous transgenic model may be useful for testing human FBPase inhibitor compounds with the potential to treat patients with type 2 diabetes.

Evaluating the glucose tolerance test in mice.

The objective of this study was to determine the optimal conditions under which to assess glucose tolerance in chow- and high-fat-fed C57BL/6J mice. Mice were fed either chow or high-fat diet for 8 wk. Variables tested were fasting duration (0-, 3-, 6-, and 24-h and overnight fasting), route of administration (intraperitoneal vs. oral) load of glucose given (2, 1, or 0.5 g/kg and fixed 50-mg dose), and state of consciousness. Basal glucose concentrations were increased in high-fat- compared with chow-fed mice following 6 h of fasting (9.1 +/- 0.3 vs. 7.9 +/- 0.4 mmol/l P = 0.01). Glucose tolerance was most different and therefore significant (P = 0.001) in high-fat-fed mice after 6 h of fasting (1,973 +/- 96 vs. 1,248 +/- 83 mmol.l(-1).120 min(-1)). The difference in glucose tolerance was greater following an OGTT (142%), in contrast to an IPGTT, with a 127% difference between high fat and chow. We also found that administering 2 g/kg of glucose resulted in a greater level of significance (P = 0.0008) in glucose intolerance in high-fat- compared with chow-fed mice. A fixed dose of 50 mg glucose regardless of body weight was enough to show glucose intolerance in high-fat- vs. chow-fed mice. Finally, high-fat-fed mice showed glucose intolerance compared with their chow-fed counterparts whether they were tested under conscious or anesthetized conditions. We conclude that 2 g/kg glucose administered orally following 6 h of fasting is best to assess glucose tolerance in mice under these conditions.

Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function.

OBJECTIVE: Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic beta-cell lines exposed to high fat. However, whether specific beta-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet beta-cell FBPase can result in reduced glucose-mediated insulin secretion. RESEARCH DESIGN AND METHODS: To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet beta-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic beta-cell lines (MIN6) stably overexpressing huFBPase. RESULTS: FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels. CONCLUSIONS: Taken together, these results suggest that upregulation of FBPase in pancreatic islet beta-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.

Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.

Elevated levels of tumor necrosis factor (TNFalpha) are implicated in the development of insulin resistance, but the mechanisms mediating these chronic effects are not completely understood. We demonstrate that TNFalpha signaling through TNF receptor (TNFR) 1 suppresses AMPK activity via transcriptional upregulation of protein phosphatase 2C (PP2C). This in turn reduces ACC phosphorylation, suppressing fatty-acid oxidation, increasing intramuscular diacylglycerol accumulation, and causing insulin resistance in skeletal muscle, effects observed both in vitro and in vivo. Importantly even at pathologically elevated levels of TNFalpha observed in obesity, the suppressive effects of TNFalpha on AMPK signaling are reversed in mice null for both TNFR1 and 2 or following treatment with a TNFalpha neutralizing antibody. Our data demonstrate that AMPK is an important TNFalpha signaling target and is a contributing factor to the suppression of fatty-acid oxidation and the development of lipid-induced insulin resistance in obesity.

Ciliary neurotrophic factor suppresses hypothalamic AMP-kinase signaling in leptin-resistant obese mice.

We examined the actions of a second-generation ciliary neurotrophic factor analog (CNTF(Ax15)) on AMP-activated protein kinase (AMPK), a known regulator of food intake. Unlike leptin CNTF(Ax15) has been shown to reduce food intake in obese rodents and humans. Intraperitoneal injection of CNTF(Ax15) acutely (45 min) reduced hypothalamic AMPKalpha2 activity, AMPKalpha2Thr172 phosphorylation, and acetyl-coenzyme A carboxylase phosphorylation, effects not observed 2 or 6 h after injection. Intracerebroventricular CNTF(Ax15) reduced food intake, increased arcuate nucleus (ARC) signal transducer and activator of transcription 3 phosphorylation, and reduced AMPK signaling but not in the paraventricular nucleus (PVN), posterior hypothalamus, or cortex. To compare the effects of leptin and CNTF(Ax15) in a diet-induced model of obesity, mice were fed a control carbohydrate or high-fat diet (HFD) for 12 wk. Leptin treatment ip reduced food intake in control mice but not in mice fed a HFD. In contrast, ip CNTF markedly reduced food intake in both control and HFD animals. Both leptin and CNTF reduced AMPK activity and acetyl-coenzyme A carboxylase phosphorylation in the ARC and PVN of control-fed mice. A HFD blunted leptin but not CNTF effects on AMPK signaling in the ARC and PVN. In summary, these data demonstrate that CNTF(Ax15) bypasses diet-induced leptin resistance to reduce hypothalamic AMPK activity.

Expression of human fructose-1,6-bisphosphatase in the liver of transgenic mice results in increased glycerol gluconeogenesis.

In type 2 diabetes, increased endogenous glucose production (EGP) as a result of elevated gluconeogenesis contributes to hyperglycemia. An increase in glycerol gluconeogenesis has led to the suggestion that, in obese human subjects with type 2 diabetes, there may be an increase in the activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase). The aim of this study was to generate transgenic mice that overexpress human liver FBPase in the liver and assess the consequences to whole-body and hepatic glucose metabolism. FBPase transgenic mice had significantly higher levels of transgene expression in the liver and, as a result, had increased FBPase protein and enzyme activity levels in the liver. This resulted in an increase in the rate of glycerol conversion to glucose but not in EGP. The increased expression of FBPase in the liver did not result in any significant differences compared with littermate control mice in insulin or glucose tolerance. Therefore, it appears that, on its own, an increase in FBPase does not lead to impaired regulation of EGP and hence does not affect whole-body glucose metabolism. This suggests that, for EGP to be increased, other factors associated with obesity are also required.