Thioredoxin interacting protein (Txnip), a regulator of cellular oxidative stress, is induced by hyperglycemia and inhibits glucose uptake into fat and muscle, suggesting a role for Txnip in type 2 diabetes pathogenesis. Here, we tested the hypothesis that Txnip-null (knockout) mice are protected from insulin resistance induced by a high-fat diet.

Skeletal muscle-specific LPL knockout mouse (SMLPL(-/-)) were created to study the systemic impact of reduced lipoprotein lipid delivery in skeletal muscle on insulin sensitivity, body weight, and composition.

Type 2 diabetes (T2D) has reached an epidemic level globally. Most current treatments ameliorate the hyperglycemic symptom of the disease but are not effective in correcting its underlying cause. One important causal factor of T2D is ectopic accumulation of lipids in metabolically sensitive organs such as liver and muscle. Mitochondrial uncoupling, which reduces cellular energy efficiency and increases lipid oxidation, is an appealing therapeutic strategy. The challenge, however, is to discover safe mitochondrial uncouplers for practical use. Niclosamide is an anthelmintic drug approved by the US Food and Drug Administration that uncouples the mitochondria of parasitic worms. Here we show that niclosamide ethanolamine salt (NEN) uncouples mammalian mitochondria at upper nanomolar concentrations. Oral NEN increases energy expenditure and lipid metabolism in mice. It is also efficacious in preventing and treating hepatic steatosis and insulin resistance induced by a high-fat diet. Moreover, it improves glycemic control and delays disease progression in db/db mice. Given the well-documented safety profile of NEN, our study provides a potentially new and practical pharmacological approach for treating T2D.

Insulin constitutes a principal evolutionarily conserved hormonal axis for maintaining glucose homeostasis; dysregulation of this axis causes diabetes. PGC-1α (peroxisome-proliferator-activated receptor-γ coactivator-1α) links insulin signalling to the expression of glucose and lipid metabolic genes. The histone acetyltransferase GCN5 (general control non-repressed protein 5) acetylates PGC-1α and suppresses its transcriptional activity, whereas sirtuin 1 deacetylates and activates PGC-1α. Although insulin is a mitogenic signal in proliferative cells, whether components of the cell cycle machinery contribute to its metabolic action is poorly understood. Here we report that in mice insulin activates cyclin D1-cyclin-dependent kinase 4 (Cdk4), which, in turn, increases GCN5 acetyltransferase activity and suppresses hepatic glucose production independently of cell cycle progression. Through a cell-based high-throughput chemical screen, we identify a Cdk4 inhibitor that potently decreases PGC-1α acetylation. Insulin/GSK-3β (glycogen synthase kinase 3-beta) signalling induces cyclin D1 protein stability by sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 messenger RNA transcripts. Activated cyclin D1-Cdk4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1α activity on gluconeogenic genes. Loss of hepatic cyclin D1 results in increased gluconeogenesis and hyperglycaemia. In diabetic models, cyclin D1-Cdk4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycaemia. Our findings show that insulin uses components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell division.

Macrophage recruitment to adipose tissue is a reproducible feature of obesity. However, the events that result in chemokine production and macrophage recruitment to adipose tissue during states of energetic excess are not clear. Sirtuin 1 (SirT1) is an essential nutrient-sensing histone deacetylase, which is increased by caloric restriction and reduced by overfeeding. We discovered that SirT1 depletion causes anorexia by stimulating production of inflammatory factors in white adipose tissue and thus posit that decreases in SirT1 link overnutrition and adipose tissue inflammation.

Reducing the expression of the Indy (I'm Not Dead Yet) gene in lower organisms extends life span by mechanisms resembling caloric restriction. Similarly, deletion of the mammalian homolog, mIndy (Slc13a5), encoding for a plasma membrane tricarboxylate transporter, protects from aging- and diet-induced adiposity and insulin resistance in mice. The organ specific contribution to this phenotype is unknown. We examined the impact of selective inducible hepatic knockdown of mIndy on whole body lipid and glucose metabolism using 2'-O-methoxyethyl chimeric anti-sense oligonucleotides (ASOs) in high-fat fed rats. 4-week treatment with 2'-O-methoxyethyl chimeric ASO reduced mIndy mRNA expression by 91% (P=0.001) compared to control ASO. Besides similar body weights between both groups, mIndy-ASO treatment lead to a 74% reduction in fasting plasma insulin concentrations as well as a 35% reduction in plasma triglycerides. Moreover, hepatic triglyceride content was significantly reduced by the knockdown of mIndy, likely mediating a trend to decreased basal rates of endogenous glucose production as well as an increased suppression of hepatic glucose production by 25% during a hyperinsulinemic-euglycemic clamp. Together, these data suggest that inducible liver-selective reduction of mIndy in rats is able to ameliorate hepatic steatosis and insulin resistance, conditions occurring with high calorie diets and during aging.

Thyroid hormone mimetics are alluring potential therapies for diseases like dyslipidemia, nonalcoholic fatty liver disease (NAFLD), and insulin resistance. Though diiodothyronines are thought inactive, pharmacologic treatment with 3,5- Diiodo-L-Thyronine (T2) reportedly reduces hepatic lipid content and improves glucose tolerance in fat-fed male rats. To test this, male Sprague Dawley rats fed a safflower-oil based high-fat diet were treated with T2 (0.25 mg/kg-d) or vehicle. Neither 10 nor 30 days of T2 treatment had an effect on weight, adiposity, plasma fatty acids, or hepatic steatosis. Insulin action was quantified in vivo by a hyperinsulinemic-euglycemic clamp. T2 did not alter fasting plasma glucose or insulin concentration. Basal endogenous glucose production (EGP) rate was unchanged. During the clamp, there was no difference in insulin stimulated whole body glucose disposal. Insulin suppressed EGP by 60% ± 10 in T2-treated rats as compared with 47% ± 4 suppression in the vehicle group (p = 0.32). This was associated with an improvement in hepatic insulin signaling; insulin stimulated Akt phosphorylation was ~2.5 fold greater in the T2-treated group as compared with the vehicle-treated group (p = 0.003). There was no change in expression of genes thought to mediate the effect of T2 on hepatic metabolism, including genes that regulate hepatic lipid oxidation (ppara, carnitine palmitoyltransferase 1a), genes that regulate hepatic fatty acid synthesis (srebp1c, acetyl coa carboxylase, fatty acid synthase), and genes involved in glycolysis and gluconeogenesis (L-pyruvate kinase, glucose 6 phosphatase). Therefore, in contrast with previous reports, in Sprague Dawley rats fed an unsaturated fat diet, T2 administration failed to improve NAFLD or whole body insulin sensitivity. Though there was a modest improvement in hepatic insulin signaling, this was not associated with significant differences in hepatic insulin action. Further study will be necessary before diiodothyronines can be considered an effective treatment for NAFLD and dyslipidemia.

Mononuclear phagocytes (MNPs) are a highly heterogeneous group of cells that play important roles in maintaining the body's homeostasis. Here, we found CD301b (also known as MGL2), a lectin commonly used as a marker for alternatively activated macrophages, was selectively expressed by a subset of CD11b(+)CD11c(+)MHCII(+) MNPs in multiple organs including adipose tissues. Depleting CD301b(+) MNPs in vivo led to a significant weight loss with increased insulin sensitivity and a marked reduction in serum Resistin-like molecule (RELM) α, a multifunctional cytokine produced by MNPs. Reconstituting RELMα in CD301b(+) MNP-depleted animals restored body weight and normoglycemia. Thus, CD301b(+) MNPs play crucial roles in maintaining glucose metabolism and net energy balance.

Recent studies reveal a strong relationship between reduced mitochondrial content and insulin resistance in human skeletal muscle, although the underlying factors responsible for this association remain unknown. To address this question, we analyzed muscle biopsy samples from young, lean, insulin resistant (IR) offspring of parents with type 2 diabetes and control subjects by microarray analyses and found significant differences in expression of ~512 probe pairs. We then screened these genes for their potential involvement in the regulation of mitochondrial biogenesis using RNA interference and found that mRNA and protein expression of lipoprotein lipase (LPL) in skeletal muscle was significantly decreased in the IR offspring and was associated with decreased mitochondrial density. Furthermore, we show that LPL knockdown in muscle cells decreased mitochondrial content by effectively decreasing fatty acid delivery and subsequent activation of peroxisome proliferator-activated receptor (PPAR)-δ. Taken together, these data suggest that decreased mitochondrial content in muscle of IR offspring may be due in part to reductions in LPL expression in skeletal muscle resulting in decreased PPAR-δ activation.

Insulin resistance associated with altered fat partitioning in liver and adipose tissues is a prediabetic condition in obese adolescents. We investigated interactions between glucose tolerance, insulin sensitivity, and the expression of lipogenic genes in abdominal subcutaneous adipose and liver tissue in 53 obese adolescents. Based on their 2-h glucose tests they were stratified in the following groups: group 1, 2-h glucose level <120 mg/dL; group 2, 2-h glucose level between 120 and 140 mg/dL; and group 3, 2-h glucose level >140 mg/dL. Liver and adipose tissue insulin sensitivity were greater in group 1 than in group 2 and group 3, and muscle insulin sensitivity progressively decreased from group 1 to group 3. The expression of the carbohydrate-responsive element-binding protein (ChREBP) was decreased in adipose tissue but increased in the liver (eight subjects) in adolescents with impaired glucose tolerance or type 2 diabetes. The expression of adipose ChREBPα and ChREBPβ was inversely related to 2-h glucose level and positively correlated to insulin sensitivity. Improvement of glucose tolerance in four subjects was associated with an increase of ChREBP/GLUT4 expression in the adipose tissue. In conclusion, early in the development of prediabetes/type 2 diabetes in youth, ChREBPβ expression in adipose tissue predicts insulin resistance and, therefore, might play a role in the regulation of glucose tolerance.

Stored glycogen is an important source of energy for skeletal muscle. Human genetic disorders primarily affecting skeletal muscle glycogen turnover are well-recognised, but rare. We previously reported that a frameshift/premature stop mutation in PPP1R3A, the gene encoding RGL, a key regulator of muscle glycogen metabolism, was present in 1.36% of participants from a population of white individuals in the UK. However, the functional implications of the mutation were not known. The objective of this study was to characterise the molecular and physiological consequences of this genetic variant.

Metformin, the universal first-line treatment for type 2 diabetes, exerts its therapeutic glucose-lowering effects by inhibiting hepatic gluconeogenesis. However, the primary molecular mechanism of this biguanide remains unclear, though it has been suggested to act, at least partially, by mitochondrial complex I inhibition. Here we show that clinically relevant concentrations of plasma metformin achieved by acute intravenous, acute intraportal or chronic oral administration in awake normal and diabetic rats inhibit gluconeogenesis from lactate and glycerol but not from pyruvate and alanine, implicating an increased cytosolic redox state in mediating metformin's antihyperglycemic effect. All of these effects occurred independently of complex I inhibition, evidenced by unaltered hepatic energy charge and citrate synthase flux. Normalizing the cytosolic redox state by infusion of methylene blue or substrates that contribute to gluconeogenesis independently of the cytosolic redox state abrogated metformin-mediated inhibition of gluconeogenesis in vivo. Additionally, in mice expressing constitutively active acetyl-CoA carboxylase, metformin acutely decreased hepatic glucose production and increased the hepatic cytosolic redox state without altering hepatic triglyceride content or gluconeogenic enzyme expression. These studies demonstrate that metformin, at clinically relevant plasma concentrations, inhibits hepatic gluconeogenesis in a redox-dependent manner independently of reductions in citrate synthase flux, hepatic nucleotide concentrations, acetyl-CoA carboxylase activity, or gluconeogenic enzyme protein expression.

Park2 is an E3 ubiquitin ligase known for its role in mitochondrial quality control via the mitophagy pathway. Park2 KO mice are protected from diet-induced obesity and hepatic insulin sensitivity is improved in high-fat diet (HFD)-fed Park2 KO mice even under body weight-matched conditions. In order to better understand the cellular mechanism by which Park2 KO mice are protected from diet-induced hepatic insulin resistance, we determined changes in multiple pathways commonly associated with the pathogenesis of insulin resistance, namely levels of bioactive lipid species, activation of the endoplasmic reticulum (ER) stress response and changes in cytokine levels and signaling. We report for the first time that whole-body insulin sensitivity is unchanged in regular chow (RC)-fed Park2 KO mice, and that liver diacylglycerol levels are reduced and very-long-chain ceramides are increased in Park2 KO mice fed HFD for 1 week. Hepatic transcriptional markers of the ER stress response were reduced and plasma tumor necrosis factor-α (TNFα), interleukin-6 and -10 (IL6, IL10) were significantly increased in HFD-fed Park2 KO mice; however, there were no detectable differences in hepatic inflammatory signaling pathways between groups. Interestingly, hepatic adenylate charge was reduced in HFD-fed Park2 KO liver and was associated increased activation of AMPK. These data suggest that negative energy balance that contributed to protection from obesity during chronic HFD manifested at the level of the hepatocyte during short-term HFD feeding and contributed to the improved hepatic insulin sensitivity.

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp-/-) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp-/- mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp-/- mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKCε activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp-/- mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKCε activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp-/- mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKCε activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp-/- mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp-/- mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp-/- mice.

Hypothyroidism, a metabolic disease characterized by low thyroid hormone (TH) and high thyroid-stimulating hormone (TSH) levels in the serum, is strongly associated with nonalcoholic fatty liver disease (NAFLD). Hypothyroidism-induced NAFLD has generally been attributed to reduced TH signaling in the liver with a consequent decrease in lipid utilization. Here, we found that mildly hypothyroid mice develop NAFLD without down-regulation of hepatic TH signaling or decreased hepatic lipid utilization. NAFLD was induced by impaired suppression of adipose tissue lipolysis due to decreased insulin secretion and to a reduced response of adipose tissue itself to insulin. This condition leads to increased shuttling of fatty acids (FAs) to the liver, where they are esterified and accumulated as triglycerides. Lipid accumulation in the liver induces hepatic insulin resistance, which leads to impaired suppression of endogenous glucose production after feeding. Hepatic insulin resistance, synergistically with lowered insulin secretion, increases serum glucose levels, which stimulates de novo lipogenesis (DNL) in the liver. Up-regulation of DNL also contributes to NAFLD. In contrast, severely hypothyroid mice show down-regulation of TH signaling in their livers and profound suppression of adipose tissue lipolysis, which decreases delivery of FAs to the liver. The resulting lack of substrates for triglyceride esterification protects severely hypothyroid mice against NAFLD. Our findings demonstrate that NAFLD occurs when TH levels are mildly reduced, but, paradoxically, not when they are severely reduced. Our results show that the pathogenesis of hypothyroidism-induced NAFLD is both intra- and extrahepatic; they also reveal key metabolic differences between mild and severe hypothyroidism.

Multiple insulin-regulated enzymes participate in hepatic glycogen synthesis, and the rate-controlling step responsible for insulin stimulation of glycogen synthesis is unknown. We demonstrate that glucokinase (GCK)-mediated glucose phosphorylation is the rate-controlling step in insulin-stimulated hepatic glycogen synthesis in vivo, by use of the somatostatin pancreatic clamp technique using [13C6]glucose with metabolic control analysis (MCA) in three rat models: 1) regular chow (RC)-fed male rats (control), 2) high fat diet (HFD)-fed rats, and 3) RC-fed rats with portal vein glucose delivery at a glucose infusion rate matched to the control. During hyperinsulinemia, hyperglycemia dose-dependently increased hepatic glycogen synthesis. At similar levels of hyperinsulinemia and hyperglycemia, HFD-fed rats exhibited a decrease and portal delivery rats exhibited an increase in hepatic glycogen synthesis via the direct pathway compared with controls. However, the strong correlation between liver glucose-6-phosphate concentration and net hepatic glycogen synthetic rate was nearly identical in these three groups, suggesting that the main difference between models is the activation of GCK. MCA yielded a high control coefficient for GCK in all three groups. We confirmed these findings in studies of hepatic GCK knockdown using an antisense oligonucleotide. Reduced liver glycogen synthesis in lipid-induced hepatic insulin resistance and increased glycogen synthesis during portal glucose infusion were explained by concordant changes in translocation of GCK. Taken together, these data indicate that the rate of insulin-stimulated hepatic glycogen synthesis is controlled chiefly through GCK translocation.

Although it is well-established that reductions in the ratio of insulin to glucagon in the portal vein have a major role in the dysregulation of hepatic glucose metabolism in type-2 diabetes1-3, the mechanisms by which glucagon affects hepatic glucose production and mitochondrial oxidation are poorly understood. Here we show that glucagon stimulates hepatic gluconeogenesis by increasing the activity of hepatic adipose triglyceride lipase, intrahepatic lipolysis, hepatic acetyl-CoA content and pyruvate carboxylase flux, while also increasing mitochondrial fat oxidation-all of which are mediated by stimulation of the inositol triphosphate receptor 1 (INSP3R1). In rats and mice, chronic physiological increases in plasma glucagon concentrations increased mitochondrial oxidation of fat in the liver and reversed diet-induced hepatic steatosis and insulin resistance. However, these effects of chronic glucagon treatment-reversing hepatic steatosis and glucose intolerance-were abrogated in Insp3r1 (also known as Itpr1)-knockout mice. These results provide insights into glucagon biology and suggest that INSP3R1 may represent a target for therapies that aim to reverse nonalcoholic fatty liver disease and type-2 diabetes.

Acute psychological stress has long been known to decrease host fitness to inflammation in a wide variety of diseases, but how this occurs is incompletely understood. Using mouse models, we show that interleukin-6 (IL-6) is the dominant cytokine inducible upon acute stress alone. Stress-inducible IL-6 is produced from brown adipocytes in a beta-3-adrenergic-receptor-dependent fashion. During stress, endocrine IL-6 is the required instructive signal for mediating hyperglycemia through hepatic gluconeogenesis, which is necessary for anticipating and fueling "fight or flight" responses. This adaptation comes at the cost of enhancing mortality to a subsequent inflammatory challenge. These findings provide a mechanistic understanding of the ontogeny and adaptive purpose of IL-6 as a bona fide stress hormone coordinating systemic immunometabolic reprogramming. This brain-brown fat-liver axis might provide new insights into brown adipose tissue as a stress-responsive endocrine organ and mechanistic insight into targeting this axis in the treatment of inflammatory and neuropsychiatric diseases.

Intricate regulatory networks govern the net balance of cholesterol biosynthesis, uptake and efflux; however, the mechanisms surrounding cholesterol homeostasis remain incompletely understood. Here, we develop an integrative genomic strategy to detect regulators of LDLR activity and identify 250 genes whose knockdown affects LDL-cholesterol uptake and whose expression is modulated by intracellular cholesterol levels in human hepatic cells. From these hits, we focus on MMAB, an enzyme which catalyzes the conversion of vitamin B12 to adenosylcobalamin, and whose expression has previously been linked with altered levels of circulating cholesterol in humans. We demonstrate that hepatic levels of MMAB are modulated by dietary and cellular cholesterol levels through SREBP2, the master transcriptional regulator of cholesterol homeostasis. Knockdown of MMAB decreases intracellular cholesterol levels and augments SREBP2-mediated gene expression and LDL-cholesterol uptake in human and mouse hepatic cell lines. Reductions in total sterol content were attributed to increased intracellular levels of propionic and methylmalonic acid and subsequent inhibition of HMGCR activity and cholesterol biosynthesis. Moreover, mice treated with antisense inhibitors of MMAB display a significant reduction in hepatic HMGCR activity, hepatic sterol content and increased expression of SREBP2-mediated genes. Collectively, these findings reveal an unexpected role for the adenosylcobalamin pathway in regulating LDLR expression and identify MMAB as an additional control point by which cholesterol biosynthesis is regulated by its end product.

Maternal obesity increases the incidence of excess adiposity in newborns, resulting in lifelong diabetes risk. Elevated intrauterine fetal adiposity has been attributed to maternal hyperglycemia; however, this hypothesis does not account for the increased adiposity seen in newborns of mothers with obesity who have euglycemia. We aimed to explore the placental response to maternal hyperinsulinemia and the effect of insulin-like growth factor 2 (IGF-2) in promoting fetal adiposity by increasing storage and availability of nutrients to the fetus.