BMP, activin, membrane-bound inhibitor (BAMBI) acts as a pseudo-receptor for the transforming growth factor (TGF)-β type I receptor family and a negative modulator of TGF-β kinase signaling, and BAMBI(-/-) mice show mild endothelial dysfunction. Because diabetic glomerular disease is associated with TGF-β overexpression and microvascular alterations, we examined the effect of diabetes on glomerular BAMBI mRNA levels. In isolated glomeruli from biopsies of patients with diabetic nephropathy and in glomeruli from mice with type 2 diabetes, BAMBI was downregulated. We then examined the effects of BAMBI deletion on streptozotocin-induced diabetic glomerulopathy in mice. BAMBI(-/-) mice developed more albuminuria, with a widening of foot processes, than BAMBI(+/+) mice, along with increased activation of alternative TGF-β pathways such as extracellular signal-related kinase (ERK)1/2 and Smad1/5 in glomeruli and cortices of BAMBI(-/-) mice. Vegfr2 and Angpt1, genes controlling glomerular endothelial stability, were downmodulated in glomeruli from BAMBI(-/-) mice with diabetes. Incubation of glomeruli from nondiabetic BAMBI(+/+) or BAMBI(-/-) mice with TGF-β resulted in the downregulation of Vegfr2 and Angpt1, effects that were more pronounced in BAMBI(-/-) mice and were prevented by a MEK inhibitor. The downregulation of Vegfr2 in diabetes was localized to glomerular endothelial cells using a histone yellow reporter under the Vegfr2 promoter. Thus, BAMBI modulates the effects of diabetes on glomerular permselectivity in association with altered ERK1/2 and Smad1/5 signaling. Future therapeutic interventions with inhibitors of alternative TGF-β signaling may therefore be of interest in diabetic nephropathy.

Brain energy consumption induced by electrical stimulation increases systemic glucose tolerance in normal-weight men. In obesity, fundamental reductions in brain energy levels, gray matter density, and cortical metabolism, as well as chronically impaired glucose tolerance, suggest that disturbed neuroenergetic regulation may be involved in the development of overweight and obesity. Here, we induced neuronal excitation by anodal transcranial direct current stimulation versus sham, examined cerebral energy consumption with (31)P magnetic resonance spectroscopy, and determined systemic glucose uptake by euglycemic-hyperinsulinemic glucose clamp in 15 normal-weight and 15 obese participants. We demonstrate blunted brain energy consumption and impaired systemic glucose uptake in obese compared with normal-weight volunteers, indicating neuroenergetic dysregulation in obese humans. Broadening our understanding of reduced multifocal gray matter volumes in obesity, our findings show that reduced appetite- and taste-processing area morphometry is associated with decreased brain energy levels. Specifically, gray matter volumes of the insula relate to brain energy content in obese participants. Overall, our results imply that a diminished cerebral energy supply may underlie the decline in brain areas assigned to food intake regulation and therefore the development of obesity.

Members of the microRNA (miR)-30 family have been reported to promote adipogenesis and inhibit osteogenesis, yet their role in the regulation of thermogenesis remains unknown. In this study, we show that miR-30b/c concentrations are greatly increased during adipocyte differentiation and are stimulated by cold exposure or the β-adrenergic receptor activator. Overexpression and knockdown of miR-30b and -30c induced and suppressed, respectively, the expression of thermogenic genes such as UCP1 and Cidea in brown adipocytes. Forced expression of miR-30b/c also significantly increased thermogenic gene expression and mitochondrial respiration in primary adipocytes derived from subcutaneous white adipose tissue, demonstrating a promoting effect of miRNAs on the development of beige fat. In addition, knockdown of miR-30b/c repressed UCP1 expression in brown adipose tissue in vivo. miR-30b/c targets the 3'-untranslated region of the receptor-interacting protein 140 (RIP140), and overexpression of miR-30b/c significantly reduced RIP140 expression. Consistent with RIP140 as a target of miR-30b/c in regulating thermogenic gene expression, overexpression of RIP140 greatly suppressed the promoting effect of miR-30b/c on the expression of UCP1 and Cidea in brown adipocytes. Taken together, the data from our study identify miR-30b/c as a key regulator of thermogenesis and uncover a new mechanism underlying the regulation of brown adipose tissue function and the development of beige fat.

Insulin and exercise stimulate glucose uptake into skeletal muscle via different pathways. Both stimuli converge on the translocation of the glucose transporter GLUT4 from intracellular vesicles to the cell surface. Two Rab guanosine triphosphatases-activating proteins (GAPs) have been implicated in this process: AS160 for insulin stimulation and its homolog, TBC1D1, are suggested to regulate exercise-mediated glucose uptake into muscle. TBC1D1 has also been implicated in obesity in humans and mice. We investigated the role of TBC1D1 in glucose metabolism by generating TBC1D1(-/-) mice and analyzing body weight, insulin action, and exercise. TBC1D1(-/-) mice showed normal glucose and insulin tolerance, with no difference in body weight compared with wild-type littermates. GLUT4 protein levels were reduced by ∼40% in white TBC1D1(-/-) muscle, and TBC1D1(-/-) mice showed impaired exercise endurance together with impaired exercise-mediated 2-deoxyglucose uptake into white but not red muscles. These findings indicate that the RabGAP TBC1D1 plays a key role in regulating GLUT4 protein levels and in exercise-mediated glucose uptake in nonoxidative muscle fibers.

Monocyte-to-macrophage differentiation is a critical event that accentuates atherosclerosis by promoting an inflammatory environment within the vessel wall. In this study, we investigated the molecular mechanisms responsible for monocyte-to-macrophage differentiation and, subsequently, the effect of metformin in regressing angiotensin II (Ang-II)-mediated atheromatous plaque formation in ApoE(-/-) mice. AMPK activity was dose and time dependently downregulated during phorbol myristate acetate (PMA)-induced monocyte-to-macrophage differentiation, which was accompanied by an upregulation of proinflammatory cytokine production. Of note, AMPK activators metformin and AICAR significantly attenuated PMA-induced monocyte-to-macrophage differentiation and proinflammatory cytokine production. However, inhibition of AMPK activity alone by compound C was ineffective in promoting monocyte-to-macrophage differentiation in the absence of PMA. On the other hand, inhibition of c-Jun N-terminal kinase activity inhibited PMA-induced inflammation but not differentiation, suggesting that inflammation and differentiation are independent events. In contrast, inhibition of STAT3 activity inhibited both inflammation and monocyte-to-macrophage differentiation. By decreasing STAT3 phosphorylation, metformin and AICAR through increased AMPK activation caused inhibition of monocyte-to-macrophage differentiation. Metformin attenuated Ang-II-induced atheromatous plaque formation and aortic aneurysm in ApoE(-/-) mice partly by reducing monocyte infiltration. We conclude that the AMPK-STAT3 axis plays a pivotal role in regulating monocyte-to-macrophage differentiation and that by decreasing STAT3 phosphorylation through increased AMPK activity, AMPK activators inhibit monocyte-to-macrophage differentiation.

Long-term use of glucocorticoids (GCs) causes numerous adverse effects, including glucose/lipid abnormalities, osteoporosis, and muscle wasting. The pathogenic mechanism, however, is not completely understood. In this study, we used plasminogen activator inhibitor-1 (PAI-1)-deficient mice to explore the role of PAI-1 in GC-induced glucose/lipid abnormalities, osteoporosis, and muscle wasting. Corticosterone markedly increased the levels of circulating PAI-1 and the PAI-1 mRNA level in the white adipose tissue of wild-type mice. PAI-1 deficiency significantly reduced insulin resistance and glucose intolerance but not hyperlipidemia induced by GC. An in vitro experiment revealed that active PAI-1 treatment inhibits insulin-induced phosphorylation of Akt and glucose uptake in HepG2 hepatocytes. However, this was not observed in 3T3-L1 adipocytes and C2C12 myotubes, indicating that PAI-1 suppressed insulin signaling in hepatocytes. PAI-1 deficiency attenuated the GC-induced bone loss presumably via inhibition of apoptosis of osteoblasts. Moreover, the PAI-1 deficiency also protected from GC-induced muscle loss. In conclusion, the current study indicated that PAI-1 is involved in GC-induced glucose metabolism abnormality, osteopenia, and muscle wasting in mice. PAI-1 may be a novel therapeutic target to mitigate the adverse effects of GC.

Imeglimin is the first in a new class of oral glucose-lowering agents currently in phase 2b development. Although imeglimin improves insulin sensitivity in humans, the molecular mechanisms are unknown. This study used a model of 16-week high-fat, high-sucrose diet (HFHSD) mice to characterize its antidiabetic effects. Six-week imeglimin treatment significantly decreased glycemia, restored normal glucose tolerance, and improved insulin sensitivity without modifying organs, body weights, and food intake. This was associated with an increase in insulin-stimulated protein kinase B phosphorylation in the liver and muscle. In liver mitochondria, imeglimin redirects substrate flows in favor of complex II, as illustrated by increased respiration with succinate and by the restoration of respiration with glutamate/malate back to control levels. In addition, imeglimin inhibits complex I and restores complex III activities, suggesting an increase in fatty acid oxidation, which is supported by an increase in hepatic 3-hydroxyacetyl-CoA dehydrogenase activity and acylcarnitine profile and the reduction of liver steatosis. Imeglimin also reduces reactive oxygen species production and increases mitochondrial DNA. Finally, imeglimin effects on mitochondrial phospholipid composition could participate in the benefit of imeglimin on mitochondrial function. In conclusion, imeglimin normalizes glucose tolerance and insulin sensitivity by preserving mitochondrial function from oxidative stress and favoring lipid oxidation in liver of HFHSD mice.

An acute bout of exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise, insulin sensitivity to increased glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wild-type mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr(649) and Ser(711) in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK activation is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin.

Oxidative stress plays a critical role in the vascular complications of type 2 diabetes. We examined the effect of type 2 diabetes on NADPH oxidase in human vessels and explored the mechanisms of this interaction. Segments of internal mammary arteries (IMAs) with their perivascular adipose tissue (PVAT) and thoracic adipose tissue were obtained from 386 patients undergoing coronary bypass surgery (127 with type 2 diabetes). Type 2 diabetes was strongly correlated with hypoadiponectinemia and increased vascular NADPH oxidase-derived superoxide anions (O2˙(-)). The genetic variability of the ADIPOQ gene and circulating adiponectin (but not interleukin-6) were independent predictors of NADPH oxidase-derived O2˙(-). However, adiponectin expression in PVAT was positively correlated with vascular NADPH oxidase-derived O2˙(-). Recombinant adiponectin directly inhibited NADPH oxidase in human arteries ex vivo by preventing the activation/membrane translocation of Rac1 and downregulating p22(phox) through a phosphoinositide 3-kinase/Akt-mediated mechanism. In ex vivo coincubation models of IMA/PVAT, the activation of arterial NADPH oxidase triggered a peroxisome proliferator-activated receptor-γ-mediated upregulation of the adiponectin gene in the neighboring PVAT via the release of vascular oxidation products. We demonstrate for the first time in humans that reduced adiponectin levels in individuals with type 2 diabetes stimulates vascular NADPH oxidase, while PVAT "senses" the increased NADPH oxidase activity in the underlying vessel and responds by upregulating adiponectin gene expression. This PVAT-vessel interaction is identified as a novel therapeutic target for the prevention of vascular complications of type 2 diabetes.

A polymorphism located in the G6PC2 gene, which encodes an islet-specific glucose-6-phosphatase catalytic subunit, is the most important common determinant of variations in fasting blood glucose (FBG) levels in humans. Studies of G6pc2 knockout (KO) mice suggest that G6pc2 represents a negative regulator of basal glucose-stimulated insulin secretion (GSIS) that acts by hydrolyzing glucose-6-phosphate (G6P), thereby reducing glycolytic flux. However, this conclusion conflicts with the very low estimates for the rate of glucose cycling in pancreatic islets, as assessed using radioisotopes. We have reassessed the rate of glucose cycling in pancreatic islets using a novel stable isotope method. The data show much higher levels of glucose cycling than previously reported. In 5 mmol/L glucose, islets from C57BL/6J chow-fed mice cycled ∼16% of net glucose uptake. The cycling rate was further increased at 11 mmol/L glucose. Similar cycling rates were observed using islets from high fat-fed mice. Importantly, glucose cycling was abolished in G6pc2 KO mouse islets, confirming that G6pc2 opposes the action of the glucose sensor glucokinase by hydrolyzing G6P. The demonstration of high rates of glucose cycling in pancreatic islets explains why G6pc2 deletion enhances GSIS and why variants in G6PC2 affect FBG in humans.

Dysfunction in effector memory has been proposed to contribute to autoimmunity in type 1 diabetes (T1D). Using a unique cohort of age- and sex-matched T1D patients, nonaffected siblings, and unrelated control children, we undertook a detailed analysis of proliferation, activation, effector responses, and apoptosis in reactivated CD4(+)Tm cells during T-cell receptor stimulation. Across cohorts, there was no difference in the proliferation of reactivated CD4(+)Tm cells. In T1D patients and siblings, CD4(+)Tm cells easily acquired the activated CD25(+) phenotype and effectively transitioned from a central (CD62L(+)Tcm) to an effector memory (CD62L(-)Tem) phenotype with an elevated cytokine "signature" comprising interferon (IFN)-γ and interleukin-10 in T1D patients and IFN-γ in siblings. This amplified Tem phenotype also exhibited an exaggerated immune shutdown with heightened sensitivity to activation-induced cell death and Fas-independent apoptosis. Apoptosis resulted in the elimination of one-half of the effector memory in T1D patients and siblings compared with one-third of the effector memory in control subjects. These data suggest genetic/environment-driven immune alteration in T1D patients and siblings that manifests in an exaggerated CD4(+)Tem response and shutdown by apoptosis. Further immunological studies are required to understand how this exaggerated CD4(+)Tem response fits within the pathomechanisms of T1D and how the effector memory can be modulated for disease treatment and/or prevention.

The flavonoid luteolin has various pharmacological activities. However, few studies exist on the in vivo mechanism underlying the actions of luteolin in hepatic steatosis and obesity. The aim of the current study was to elucidate the action of luteolin on obesity and its comorbidity by analyzing its transcriptional and metabolic responses, in particular the luteolin-mediated cross-talk between liver and adipose tissue in diet-induced obese mice. C57BL/6J mice were fed a normal, high-fat, and high-fat + 0.005% (weight for weight) luteolin diet for 16 weeks. In high fat-fed mice, luteolin improved hepatic steatosis by suppressing hepatic lipogenesis and lipid absorption. In adipose tissue, luteolin increased PPARγ protein expression to attenuate hepatic lipotoxicity, which may be linked to the improvement in circulating fatty acid (FA) levels by enhancing FA uptake genes and lipogenic genes and proteins in adipose tissue. Interestingly, luteolin also upregulated the expression of genes controlling lipolysis and the tricarboxylic acid (TCA) cycle prior to lipid droplet formation, thereby reducing adiposity. Moreover, luteolin improved hepatic insulin sensitivity by suppressing SREBP1 expression that modulates Irs2 expression through its negative feedback and gluconeogenesis. Luteolin ameliorates the deleterious effects of diet-induced obesity and its comorbidity via the interplay between liver and adipose tissue.

Recent studies suggest improved outcomes and survival in obese heart failure patients (i.e., the obesity paradox), although obesity and heart failure unfavorably alter cardiac function and metabolism. We investigated the effects of weight loss on cardiac function and metabolism in obese heart failure mice. Obesity and heart failure were induced by feeding mice a high-fat (HF) diet (60% kcal from fat) for 4 weeks, following which an abdominal aortic constriction (AAC) was produced. Four weeks post-AAC, mice were switched to a low-fat (LF) diet (12% kcal from fat; HF AAC LF) or maintained on an HF (HF AAC HF) for a further 10 weeks. After 18 weeks, HF AAC LF mice weighed less than HF AAC HF mice. Diastolic function was improved in HF AAC LF mice, while cardiac hypertrophy was decreased and accompanied by decreased SIRT1 expression, increased FOXO1 acetylation, and increased atrogin-1 expression compared with HF AAC HF mice. Insulin-stimulated glucose oxidation was increased in hearts from HF AAC LF mice, compared with HF AAC HF mice. Thus lowering body weight by switching to LF diet in obese mice with heart failure is associated with decreased cardiac hypertrophy and improvements in both cardiac insulin sensitivity and diastolic function, suggesting that weight loss does not negatively impact heart function in the setting of obesity.

Insulin sensitivity, insulin secretion, insulin clearance, and glucose effectiveness exhibit strong genetic components, although few studies have examined their genetic architecture or influence on type 2 diabetes (T2D) risk. We hypothesized that loci affecting variation in these quantitative traits influence T2D. We completed a multicohort genome-wide association study to search for loci influencing T2D-related quantitative traits in 4,176 Mexican Americans. Quantitative traits were measured by the frequently sampled intravenous glucose tolerance test (four cohorts) or euglycemic clamp (three cohorts), and random-effects models were used to test the association between loci and quantitative traits, adjusting for age, sex, and admixture proportions (Discovery). Analysis revealed a significant (P < 5.00 × 10(-8)) association at 11q14.3 (MTNR1B) with acute insulin response. Loci with P < 0.0001 among the quantitative traits were examined for translation to T2D risk in 6,463 T2D case and 9,232 control subjects of Mexican ancestry (Translation). Nonparametric meta-analysis of the Discovery and Translation cohorts identified significant associations at 6p24 (SLC35B3/TFAP2A) with glucose effectiveness/T2D, 11p15 (KCNQ1) with disposition index/T2D, and 6p22 (CDKAL1) and 11q14 (MTNR1B) with acute insulin response/T2D. These results suggest that T2D and insulin secretion and sensitivity have both shared and distinct genetic factors, potentially delineating genomic components of these quantitative traits that drive the risk for T2D.

Although dogma predicts that under normal circumstances, potentially offensive autoreactive cells are silenced by mechanisms of immune tolerance, islet antigen-reactive B lymphocytes are known to play a crucial role in the development of autoimmunity in type 1 diabetes (T1D). Thus, participation of these cells in T1D may reflect escape from silencing mechanisms. Consistent with this concept, we found that in healthy subjects, high-affinity insulin-binding B cells occur exclusively in the anergic naive IgD(+), IgM(-) B-cell (BND) compartment. Antigen receptors expressed by these cells are polyreactive and have N-region additions, Vh usage, and charged complementarity-determining region 3 consistent with autoreactivity. Consistent with a potential early role in autoimmunity, these high-affinity insulin-binding B cells are absent from the anergic compartment of some first-degree relatives and all prediabetic and new-onset (<1 year) T1D patients tested, but return to normal levels in individuals diabetic for >1 year. Interestingly, these changes were correlated by transient loss of the entire BND compartment. These findings suggest that environmental events such as infection or injury may, by disrupting B-cell anergy, dispose individuals toward autoimmunity, the precise nature of which is specified by genetic risk factors, such as HLA alleles.

Circulating transthyretin (TTR) is a critical determinant of plasma retinol-binding protein 4 (RBP4) levels. Elevated RBP4 levels cause insulin resistance, and the lowering of RBP4 levels improves glucose homeostasis. Since lowering TTR levels increases renal clearance of RBP4, we determined whether decreasing TTR levels with antisense oligonucleotides (ASOs) improves glucose metabolism and insulin sensitivity in obesity. TTR-ASO treatment of mice with genetic or diet-induced obesity resulted in an 80-95% decrease in circulating levels of TTR and RBP4. Treatment with TTR-ASOs, but not control ASOs, decreased insulin levels by 30-60% and improved insulin sensitivity in ob/ob mice and high-fat diet-fed mice as early as after 2 weeks of treatment. The reduced insulin levels were sustained for up to 9 weeks of treatment and were associated with reduced adipose tissue inflammation. Body weight was not changed. TTR-ASO treatment decreased LDL cholesterol in high-fat diet-fed mice. The glucose infusion rate during a hyperinsulinemic-euglycemic clamp was increased by 50% in high-fat diet-fed mice treated with TTR-ASOs, demonstrating improved insulin sensitivity. This was also demonstrated by 20% greater inhibition of hepatic glucose production, a 45-60% increase of glucose uptake into skeletal and cardiac muscle, and a twofold increase in insulin signaling in muscle. These data show that decreasing circulating TTR levels or altering TTR-RBP4 binding could be a potential therapeutic approach for the treatment of type 2 diabetes.

The ability to maintain skeletal muscle mass appears to be impaired in insulin-resistant conditions, such as type 2 diabetes, that are characterized by muscle lipid accumulation. The current study investigated the effect of acutely increasing lipid availability on muscle protein synthesis. Seven healthy young male volunteers underwent a 7-h intravenous infusion of l-[ring-(2)H5]phenylalanine on two randomized occasions combined with 0.9% saline or 10% Intralipid at 100 mL/h. After a 4-h "basal" period, a 21-g bolus of amino acids was administered and a 3-h hyperinsulinemic-euglycemic clamp was commenced ("fed" period). Muscle biopsy specimens were obtained from the vastus lateralis at 1.5, 4, and 7 h. Lipid infusion reduced fed whole-body glucose disposal by 20%. Furthermore, whereas the mixed muscle fractional synthetic rate increased from the basal to the fed period during saline infusion by 2.2-fold, no change occurred during lipid infusion, despite similar circulating insulin and leucine concentrations. This "anabolic resistance" to insulin and amino acids with lipid infusion was associated with a complete suppression of muscle 4E-BP1 phosphorylation. We propose that increased muscle lipid availability may contribute to anabolic resistance in insulin-resistant conditions by impairing translation initiation.