Hepatic NADH reductive stress underlies common variation in metabolic traits.

The cellular NADH/NAD+ ratio is fundamental to biochemistry, but the extent to which it reflects versus drives metabolic physiology in vivo is poorly understood. Here we report the in vivo application of Lactobacillus brevis (Lb)NOX1, a bacterial water-forming NADH oxidase, to assess the metabolic consequences of directly lowering the hepatic cytosolic NADH/NAD+ ratio in mice. By combining this genetic tool with metabolomics, we identify circulating α-hydroxybutyrate levels as a robust marker of an elevated hepatic cytosolic NADH/NAD+ ratio, also known as reductive stress. In humans, elevations in circulating α-hydroxybutyrate levels have previously been associated with impaired glucose tolerance2, insulin resistance3 and mitochondrial disease4, and are associated with a common genetic variant in GCKR5, which has previously been associated with many seemingly disparate metabolic traits. Using LbNOX, we demonstrate that NADH reductive stress mediates the effects of GCKR variation on many metabolic traits, including circulating triglyceride levels, glucose tolerance and FGF21 levels. Our work identifies an elevated hepatic NADH/NAD+ ratio as a latent metabolic parameter that is shaped by human genetic variation and contributes causally to key metabolic traits and diseases. Moreover, it underscores the utility of genetic tools such as LbNOX to empower studies of 'causal metabolism'.

Disrupted glucose homeostasis and skeletal-muscle-specific glucose uptake in an exocyst knockout mouse model.

Skeletal muscle is responsible for the majority of glucose disposal following meals, and this is achieved by insulin-mediated trafficking of glucose transporter type 4 (GLUT4) to the cell membrane. The eight-protein exocyst trafficking complex facilitates targeted docking of membrane-bound vesicles, a process underlying the regulated delivery of fuel transporters. We previously demonstrated the role of exocyst subunit EXOC5 in insulin-stimulated GLUT4 exocytosis and glucose uptake in cultured rat skeletal myoblasts. However, the in vivo role of EXOC5 in skeletal muscle remains unclear. Using mice with inducible, skeletal-muscle-specific knockout of exocyst subunit EXOC5 (Exoc5-SMKO), we examined how muscle-specific disruption of the exocyst would affect glucose homeostasis in vivo. We found that both male and female Exoc5-SMKO mice displayed elevated fasting glucose levels. Additionally, male Exoc5-SMKO mice had impaired glucose tolerance and lower serum insulin levels. Using indirect calorimetry, we observed that male Exoc5-SMKO mice have a reduced respiratory exchange ratio during the light period and lower energy expenditure. Using the hyperinsulinemic-euglycemic clamp method, we further showed that insulin-stimulated skeletal muscle glucose uptake is reduced in Exoc5-SMKO males compared with wild-type controls. Overall, our findings indicate that EXOC5 and the exocyst are necessary for insulin-stimulated glucose uptake in skeletal muscle and regulate glucose homeostasis in vivo.

GABA-stimulated adipose-derived stem cells suppress subcutaneous adipose inflammation in obesity.

Accumulating evidence suggests that subcutaneous and visceral adipose tissues are differentially associated with metabolic disorders. In obesity, subcutaneous adipose tissue is beneficial for metabolic homeostasis because of repressed inflammation. However, the underlying mechanism remains unclear. Here, we demonstrate that γ-aminobutyric acid (GABA) sensitivity is crucial in determining fat depot-selective adipose tissue macrophage (ATM) infiltration in obesity. In diet-induced obesity, GABA reduced monocyte migration in subcutaneous inguinal adipose tissue (IAT), but not in visceral epididymal adipose tissue (EAT). Pharmacological modulation of the GABAB receptor affected the levels of ATM infiltration and adipose tissue inflammation in IAT, but not in EAT, and GABA administration ameliorated systemic insulin resistance and enhanced insulin-dependent glucose uptake in IAT, accompanied by lower inflammatory responses. Intriguingly, compared with adipose-derived stem cells (ADSCs) from EAT, IAT-ADSCs played key roles in mediating GABA responses that repressed ATM infiltration in high-fat diet-fed mice. These data suggest that selective GABA responses in IAT contribute to fat depot-selective suppression of inflammatory responses and protection from insulin resistance in obesity.

Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Obesity-mediated inflammation is a major cause of insulin resistance, and macrophages play an important role in this process. The 78-kDa glucose-regulated protein (GRP78) is a major endoplasmic reticulum chaperone that modulates unfolded protein response (UPR), and mice with GRP78 heterozygosity were resistant to diet-induced obesity. Here, we show that mice with macrophage-selective ablation of GRP78 (Lyz- GRP78-/-) are protected from skeletal muscle insulin resistance without changes in obesity compared with wild-type mice after 9 wk of high-fat diet. GRP78-deficient macrophages demonstrated adapted UPR with up-regulation of activating transcription factor (ATF)-4 and M2-polarization markers. Diet-induced adipose tissue inflammation was reduced, and bone marrow-derived macrophages from Lyz- GRP78-/- mice demonstrated a selective increase in IL-6 expression. Serum IL-13 levels were elevated by >4-fold in Lyz- GRP78-/- mice, and IL-6 stimulated the myocyte expression of IL-13 and IL-13 receptor. Lastly, recombinant IL-13 acutely increased glucose metabolism in Lyz- GRP78-/- mice. Taken together, our data indicate that GRP78 deficiency activates UPR by increasing ATF-4, and promotes M2-polarization of macrophages with a selective increase in IL-6 secretion. Macrophage-derived IL-6 stimulates the myocyte expression of IL-13 and regulates muscle glucose metabolism in a paracrine manner. Thus, our findings identify a novel crosstalk between macrophages and skeletal muscle in the modulation of obesity-mediated insulin resistance.-Kim, J. H., Lee, E., Friedline, R. H., Suk, S., Jung, D. Y., Dagdeviren, S., Hu, X., Inashima, K., Noh, H. L., Kwon, J. Y., Nambu, A., Huh, J. R., Han, M. S., Davis, R. J., Lee, A. S., Lee, K. W., Kim, J. K. Endoplasmic reticulum chaperone GRP78 regulates macrophage function and insulin resistance in diet-induced obesity.

Hirsutenone Directly Targets PI3K and ERK to Inhibit Adipogenesis in 3T3-L1 Preadipocytes.

Adipogenesis is a key driver of the expansion of adipose tissue mass that causes obesity. Hirsutenone (HST) is an active botanical diarylheptanoid present in Alnus species. In this study, we evaluated the effects of HST on adipogenesis, its mechanisms of action and the molecular targets involved. Using Oil Red O staining, we observed that HST dose-dependently suppresses lipid accumulation during adipogenesis in 3T3-L1 preadipocytes, concomitant with a decrease in peroxisome proliferator-activated receptor-γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα) and fatty acid synthase (FAS) protein expression. This inhibitory effect was largely limited to the early stage of adipogenesis, which includes mitotic clonal expansion (MCE), as evidenced by delayed cell cycle entry of preadipocytes from G1 to S phase. Furthermore, the regulation of MCE was accompanied by suppression of phosphatidylinositol 3-kinase (PI3K) and extracellular-regulated kinase (ERK) activity. HST was also shown to bind directly to PI3K and ERK1 in a non-ATP competitive manner. Our results suggest that HST attenuates adipogenesis by directly targeting PI3K and ERK during MCE in 3T3-L1 preadipocytes, underscoring the potential therapeutic application of HST in preventing obesity.

A fermented barley and soybean formula enhances skin hydration.

Skin hydration is one of the primary aims of beauty and anti-aging treatments. Barley (Hordeum vulgare) and soybean (Glycine max) are major food crops, but can also be used as ingredients for the maintenance of skin health. We developed a natural product-based skin treatment using a barley and soybean formula (BS) incorporating yeast fermentation, and evaluated its skin hydration effects as a dietary supplement in a clinical study. Participants ingested a placebo- (n = 33) or BS- (3 g/day) containing drink (n = 32) for 8 weeks. A significant increase in hydration in the BS group as compared to the placebo group was observed on the faces of subjects after 4 and 8 weeks, and on the forearm after 4 weeks. Decreases in stratum corneum (SC) thickness were also observed on the face and forearm. BS enhanced hyaluronan (HA) and skin barrier function in vitro and reduced Hyal2 expression in human dermal fibroblasts (HDF). BS also recovered ultraviolet (UV) B-induced downregulation of HA in HaCaT cells. These results suggest that BS has promising potential for development as a health functional food to enhance skin health.

IFNγ-IL12 axis regulates intercellular crosstalk in metabolic dysfunction-associated steatotic liver disease.

Obesity is a major cause of metabolic dysfunction-associated steatohepatitis (MASH) and is characterized by inflammation and insulin resistance. Interferon-γ (IFNγ) is a pro-inflammatory cytokine elevated in obesity and modulating macrophage functions. Here, we show that male mice with loss of IFNγ signaling in myeloid cells (Lyz-IFNγR2-/-) are protected from diet-induced insulin resistance despite fatty liver. Obesity-mediated liver inflammation is also attenuated with reduced interleukin (IL)-12, a cytokine primarily released by macrophages, and IL-12 treatment in vivo causes insulin resistance by impairing hepatic insulin signaling. Following MASH diets, Lyz-IFNγR2-/- mice are rescued from developing liver fibrosis, which is associated with reduced fibroblast growth factor (FGF) 21 levels. These results indicate critical roles for IFNγ signaling in macrophages and their release of IL-12 in modulating obesity-mediated insulin resistance and fatty liver progression to MASH. In this work, we identify the IFNγ-IL12 axis in regulating intercellular crosstalk in the liver and as potential therapeutic targets to treat MASH.

Beta-cell specific Insr deletion promotes insulin hypersecretion and improves glucose tolerance prior to global insulin resistance.

Insulin receptor (Insr) protein is present at higher levels in pancreatic ß-cells than in most other tissues, but the consequences of ß-cell insulin resistance remain enigmatic. Here, we use an Ins1cre knock-in allele to delete Insr specifically in ß-cells of both female and male mice. We compare experimental mice to Ins1cre-containing littermate controls at multiple ages and on multiple diets. RNA-seq of purified recombined ß-cells reveals transcriptomic consequences of Insr loss, which differ between female and male mice. Action potential and calcium oscillation frequencies are increased in Insr knockout ß-cells from female, but not male mice, whereas only male ßInsrKO islets have reduced ATP-coupled oxygen consumption rate and reduced expression of genes involved in ATP synthesis. Female ßInsrKO and ßInsrHET mice exhibit elevated insulin release in ex vivo perifusion experiments, during hyperglycemic clamps, and following i.p. glucose challenge. Deletion of Insr does not alter ß-cell area up to 9 months of age, nor does it impair hyperglycemia-induced proliferation. Based on our data, we adapt a mathematical model to include ß-cell insulin resistance, which predicts that ß-cell Insr knockout improves glucose tolerance depending on the degree of whole-body insulin resistance. Indeed, glucose tolerance is significantly improved in female ßInsrKO and ßInsrHET mice compared to controls at 9, 21 and 39 weeks, and also in insulin-sensitive 4-week old males. We observe no improved glucose tolerance in older male mice or in high fat diet-fed mice, corroborating the prediction that global insulin resistance obscures the effects of ß-cell specific insulin resistance. The propensity for hyperinsulinemia is associated with mildly reduced fasting glucose and increased body weight. We further validate our main in vivo findings using an Ins1-CreERT transgenic line and find that male mice have improved glucose tolerance 4 weeks after tamoxifen-mediated Insr deletion. Collectively, our data show that ß-cell insulin resistance in the form of reduced ß-cell Insr contributes to hyperinsulinemia in the context of glucose stimulation, thereby improving glucose homeostasis in otherwise insulin sensitive sex, dietary and age contexts.

Distinct Changes in Gut Microbiota Are Associated with Estradiol-Mediated Protection from Diet-Induced Obesity in Female Mice.

A decrease in ovarian estrogens in postmenopausal women increases the risk of weight gain, cardiovascular disease, type 2 diabetes, and chronic inflammation. While it is known that gut microbiota regulates energy homeostasis, it is unclear if gut microbiota is associated with estradiol regulation of metabolism. In this study, we tested if estradiol-mediated protection from high-fat diet (HFD)-induced obesity and metabolic changes are associated with longitudinal alterations in gut microbiota in female mice. Ovariectomized adult mice with vehicle or estradiol (E2) implants were fed chow for two weeks and HFD for four weeks. As reported previously, E2 increased energy expenditure, physical activity, insulin sensitivity, and whole-body glucose turnover. Interestingly, E2 decreased the tight junction protein occludin, suggesting E2 affects gut epithelial integrity. Moreover, E2 increased Akkermansia and decreased Erysipleotrichaceae and Streptococcaceae. Furthermore, Coprobacillus and Lactococcus were positively correlated, while Akkermansia was negatively correlated, with body weight and fat mass. These results suggest that changes in gut epithelial barrier and specific gut microbiota contribute to E2-mediated protection against diet-induced obesity and metabolic dysregulation. These findings provide support for the gut microbiota as a therapeutic target for treating estrogen-dependent metabolic disorders in women.

Maternal exposure to high-fat diet during pregnancy and lactation predisposes normal weight offspring mice to develop hepatic inflammation and insulin resistance.

Increasing evidence shows a potential link between the perinatal nutrient environment and metabolic outcome in offspring. Here, we investigated the effects of maternal feeding of a high-fat diet (HFD) during the perinatal period on hepatic metabolism and inflammation in male offspring mice at weaning and in early adulthood. Female C57BL/6 J mice were fed HFD or normal chow (NC) for 4 weeks before mating and during pregnancy and lactation. The male offspring mice were weaned onto an NC diet, and metabolic and molecular experiments were performed in early adulthood. At postnatal day 21, male offspring mice from HFD-fed dams (Off-HFD) showed significant increases in whole body fat mass and fasting levels of glucose, insulin, and cholesterol compared to male offspring mice from NC-fed dams (Off-NC). The RT-qPCR analysis showed two- to fivefold increases in hepatic inflammatory markers (MCP-1, IL-1ß, and F4/80) in Off-HFD mice. Hepatic expression of G6Pase and PEPCK was elevated by fivefold in the Off-HFD mice compared to the Off-NC mice. Hepatic expression of GLUT4, IRS-1, and PDK4, as well as lipid metabolic genes, CD36, SREBP1c, and SCD1 were increased in the Off-HFD mice compared to the Off-NC mice. In contrast, CPT1a mRNA levels were reduced by 60% in the Off-HFD mice. At postnatal day 70, despite comparable body weights to the Off-NC mice, Off-HFD mice developed hepatic inflammation with increased expression of MCP-1, CD68, F4/80, and CD36 compared to the Off-NC mice. Despite normal body weight, Off-HFD mice developed insulin resistance with defects in hepatic insulin action and insulin-stimulated glucose uptake in skeletal muscle and brown fat, and these metabolic effects were associated with hepatic inflammation in Off-HFD mice. Our findings indicate hidden, lasting effects of maternal exposure to HFD during pregnancy and lactation on metabolic homeostasis of normal weight offspring mice.

Muscle-Specific Insulin Receptor Overexpression Protects Mice From Diet-Induced Glucose Intolerance but Leads to Postreceptor Insulin Resistance.

Skeletal muscle insulin resistance is a prominent early feature in the pathogenesis of type 2 diabetes. In attempt to overcome this defect, we generated mice overexpressing insulin receptors (IR) specifically in skeletal muscle (IRMOE). On normal chow, IRMOE mice have body weight similar to that of controls but an increase in lean mass and glycolytic muscle fibers and reduced fat mass. IRMOE mice also show higher basal phosphorylation of IR, IRS-1, and Akt in muscle and improved glucose tolerance compared with controls. When challenged with high-fat diet (HFD), IRMOE mice are protected from diet-induced obesity. This is associated with reduced inflammation in fat and liver, improved glucose tolerance, and improved systemic insulin sensitivity. Surprisingly, however, in both chow and HFD-fed mice, insulin-stimulated Akt phosphorylation is significantly reduced in muscle of IRMOE mice, indicating postreceptor insulin resistance. RNA sequencing reveals downregulation of several postreceptor signaling proteins that contribute to this resistance. Thus, enhancing early insulin signaling in muscle by overexpression of the IR protects mice from diet-induced obesity and its effects on glucose metabolism. However, chronic overstimulation of this pathway leads to postreceptor desensitization, indicating the critical balance between normal signaling and hyperstimulation of the insulin signaling pathway.

Hyperinsulinemia drives hepatic insulin resistance in male mice with liver-specific Ceacam1 deletion independently of lipolysis.

BACKGROUND: CEACAM1 regulates insulin sensitivity by promoting insulin clearance. Accordingly, global C57BL/6J.Cc1-/- null mice display hyperinsulinemia due to impaired insulin clearance at 2 months of age, followed by insulin resistance, steatohepatitis, visceral obesity and leptin resistance at 6 months. The study aimed at investigating the primary role of hepatic CEACAM1 in insulin and lipid homeostasis independently of its metabolic effect in extra-hepatic tissues. METHODS: Liver-specific C57BL/6J.AlbCre+Cc1fl/fl mice were generated and their metabolic phenotype was characterized by comparison to that of their littermate controls at 2-9 months of age, using hyperinsulinemic-euglycemic clamp analysis and indirect calorimetry. The effect of hyperphagia on insulin resistance was assessed by pair-feeding experiments. RESULTS: Liver-specific AlbCre+Cc1fl/fl mutants exhibited impaired insulin clearance and hyperinsulinemia at 2 months, followed by hepatic insulin resistance (assessed by hyperinsulinemic-euglycemic clamp analysis) and steatohepatitis at ~ 7 months of age, at which point visceral obesity and hyperphagia developed, in parallel to hyperleptinemia and blunted hypothalamic STAT3 phosphorylation in response to an intraperitoneal injection of leptin. Hyperinsulinemia caused hypothalamic insulin resistance, followed by increased fatty acid synthase activity, which together with defective hypothalamic leptin signaling contributed to hyperphagia and reduced physical activity. Pair-feeding experiment showed that hyperphagia caused systemic insulin resistance, including blunted insulin signaling in white adipose tissue and lipolysis, at 8-9 months of age. CONCLUSION: AlbCre+Cc1fl/fl mutants provide an in vivo demonstration of the key role of impaired hepatic insulin clearance and hyperinsulinemia in the pathogenesis of secondary hepatic insulin resistance independently of lipolysis. They also reveal an important role for the liver-hypothalamic axis in the regulation of energy balance and subsequently, systemic insulin sensitivity.

Orobol, an Enzyme-Convertible Product of Genistein, exerts Anti-Obesity Effects by Targeting Casein Kinase 1 Epsilon.

Soy isoflavones, particularly genistein, have been shown to exhibit anti-obesity effects. When compared with the isoflavones genistin, daidzin, coumestrol, genistein, daidzein, 6-o-dihydroxyisoflavone, equol, 3'-o-dihydroxyisoflavone, and 8-o-dihydroxyisoflavone, a remarkably higher inhibitory effect on lipid accumulation was observed for orobol treatment during adipogenesis in 3T3-L1 cells. To identify the cellular target of orobol, its pharmacological effect on 395 human kinases was analyzed. Of the 395 kinases, orobol showed the lowest half maximal inhibitory concentration (IC50) for Casein Kinase 1 epsilon (CK1ε), and bound to this target in an ATP-competitive manner. A computer modeling study revealed that orobol may potentially dock with the ATP-binding site of CK1ε via several hydrogen bonds and van der Waals interactions. The phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, a substrate of CK1ε, was inhibited by orobol in isobutylmethylxanthine, dexamethasone and insulin (MDI)-induced 3T3-L1 cells. It was also found that orobol attenuates high fat diet-induced weight gain and lipid accumulation without affecting food intake in C57BL/6J mice. These findings underline orobol's potential for development as a novel agent for the prevention and treatment of obesity.

Mss51 deletion enhances muscle metabolism and glucose homeostasis in mice.

Myostatin is a negative regulator of muscle growth and metabolism and its inhibition in mice improves insulin sensitivity, increases glucose uptake into skeletal muscle, and decreases total body fat. A recently described mammalian protein called MSS51 is significantly downregulated with myostatin inhibition. In vitro disruption of Mss51 results in increased levels of ATP, ß-oxidation, glycolysis, and oxidative phosphorylation. To determine the in vivo biological function of Mss51 in mice, we disrupted the Mss51 gene by CRISPR/Cas9 and found that Mss51-KO mice have normal muscle weights and fiber-type distribution but reduced fat pads. Myofibers isolated from Mss51-KO mice showed an increased oxygen consumption rate compared with WT controls, indicating an accelerated rate of skeletal muscle metabolism. The expression of genes related to oxidative phosphorylation and fatty acid ß-oxidation were enhanced in skeletal muscle of Mss51-KO mice compared with that of WT mice. We found that mice lacking Mss51 and challenged with a high-fat diet were resistant to diet-induced weight gain, had increased whole-body glucose turnover and glycolysis rate, and increased systemic insulin sensitivity and fatty acid ß-oxidation. These findings demonstrate that MSS51 modulates skeletal muscle mitochondrial respiration and regulates whole-body glucose and fatty acid metabolism, making it a potential target for obesity and diabetes.

Salicornia Extract Ameliorates Salt-Induced Aggravation of Nonalcoholic Fatty Liver Disease in Obese Mice Fed a High-Fat Diet.

High-fat and high-salt intakes are among the major risks of chronic diseases including obesity, nonalcoholic fatty liver disease (NAFLD), and nonalcoholic steatohepatitis (NASH). Salicornia is a halophytic plant known to exert antioxidant, antidiabetic, and hypolipidemic effects, and Salicornia-extracted salt (SS) has been used as a salt substitute. In this study, the effects of SS and purified salt (PS) on the aggravation of NAFLD/NASH were compared. C57BL/6J male mice (8-wk-old) were fed a high-fat diet (HFD) for 6 mo and divided into 3 dietary groups, which were additionally fed HFD, HFD + SS, and HFD + PS for 13 wk. PS induced aggravation of NAFLD/NASH in HFD-fed mice. Although the actual salt intake was same between the PS and SS groups as 1% of the diet (extrapolated from the World Health Organization [WHO] guideline), SS induced less liver injury and hepatic steatosis compared to PS. The hepatic mRNA expressions of inflammatory cytokines and fibrosis marker were significantly lower in the SS group than the PS group. Oxidative stress is one of the major causes of inflammation in NAFLD/NASH. Results of the component analysis showed that the major polyphenols that exhibited antioxidant activity in the Salicornia water extract were ferulic acid, caffeic acid, and isorhamnetin. These results suggest that even the level of salt intake recommended by WHO can accelerate the progression of liver disease in obese individuals consuming HFD. It is proposed that SS can be a salt substitute for obese individuals who consume HFD.

Gingerenone A, a polyphenol present in ginger, suppresses obesity and adipose tissue inflammation in high-fat diet-fed mice.

SCOPE: Ginger exerts protective effects on obesity and its complications. Our objectives here are to identify bioactive compounds that inhibit adipogenesis and lipid accumulation in vitro, elucidate the anti-obesity effect of gingerenone A (GA) in diet-induced obesity (DIO), and investigate whether GA affects adipose tissue inflammation (ATI). METHODS AND RESULTS: Oil red O staining showed that GA had the most potent inhibitory effect on adipogenesis and lipid accumulation in 3T3-L1 cells among ginger components tested at a single concentration (40 µM). Consistent with in vitro data, GA attenuates DIO by reducing fat mass in mice. This was accompanied by a modulation of fatty acid metabolism via activation of AMP-activated protein kinase (AMPK) in vitro and in vivo. Additionally, GA suppressed ATI by inhibiting macrophage recruitment and downregulating pro-inflammatory cytokines. CONCLUSION: These results suggest that GA may be used as a potential therapeutic candidate for the treatment of obesity and its complications by suppressing adipose expansion and inflammation.