Mice lacking MAP kinase phosphatase-1 have enhanced MAP kinase activity and resistance to diet-induced obesity

The mitogen-activated protein kinases (MAPK) play critical roles in the pathogenesis of diabetes and obesity. The MAPKs are inactivated by MAPK phosphatases (MKPs) either in the cytosol or nucleus. Here we show that mice lacking the nuclear-localized MKP, MKP-1 (mkp-1(-/-)), have enhanced Erk, p38 MAPK and c-Jun NH(2)-terminal kinase (JNK) activities in insulin-responsive tissues as compared with wild-type mice. Although JNK promotes insulin resistance, mkp-1(-/-) mice exhibited unimpaired insulin-mediated signaling and glucose homeostasis. We reconciled these results by demonstrating that in mkp-1(-/-) mice, JNK activity was increased in the nucleus, but not the cytosol. Significantly, mkp-1(-/-) mice are resistant to diet-induced obesity due to enhanced energy expenditure, but succumb to glucose intolerance on a high fat diet. These results suggest that nuclear regulation of the MAPKs by MKP-1 is essential for the management of metabolic homeostasis in a manner that is spatially uncoupled from the cytosolic actions of the MAPKs.     

Maintenance of metabolic homeostasis by sestrin2 and sestrin3

Chronic activation of mammalian target of rapamycin?complex 1 (mTORC1) and p70 S6 kinase (S6K) in?response to hypernutrition contributes to obesity-associated metabolic pathologies, including hepatosteatosis and insulin resistance. Sestrins are?stress-inducible proteins that activate AMP-activated protein kinase (AMPK) and suppress mTORC1-S6K activity, but their role in mammalian physiology and metabolism has not been investigated. We show that Sestrin2-encoded by the Sesn2 locus, whose expression is induced upon hypernutrition-maintains metabolic homeostasis in liver of obese mice. Sesn2 ablation exacerbates obesity-induced mTORC1-S6K activation, glucose intolerance, insulin resistance, and hepatosteatosis, all of which are reversed by AMPK activation. Furthermore, concomitant ablation of Sesn2 and Sesn3 provokes hepatic mTORC1-S6K activation and insulin resistance even in the absence of nutritional overload and obesity. These results demonstrate an important homeostatic function for the stress-inducible Sestrin protein family in the control of mammalian lipid and glucose metabolism.     

Chromium attenuates high-fat diet-induced nonalcoholic fatty liver disease in KK/HlJ mice

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease associated with insulin resistance, oxidative stress, and inflammation. Evidence indicates that chromium has a role in the regulation of glucose and lipid metabolism and may improve insulin sensitivity. In this study, we report that chromium supplementation has a beneficial effect against NAFLD. We found that KK/HlJ mice developed obesity and progressed to NAFLD after feeding with high-fat diet for 8 weeks. High-fat-fed KK/HlJ mice showed hepatocyte injury and hepatic triglyceride accumulation, which was accompanied by insulin resistance, oxidative stress, and inflammation. Chromium supplementation prevented progression of NAFLD and the beneficial effects were accompanied by reduction of hepatic triglyceride accumulation, elevation of hepatic lipid catabolic enzyme, improvement of glucose and lipid metabolism, suppression of inflammation as well as resolution of oxidative stress, probably through enhancement of insulin signaling. Our findings suggest that chromium could serve as a hepatoprotective agent against NAFLD.     

Erythropoietin Inhibits Gluconeogenesis and Inflammation in the Liver and Improves Glucose Intolerance in High-Fat Diet-Fed Mice

Erythropoietin (EPO) has multiple biological functions, including the modulation of glucose metabolism. However, the mechanisms underlying the action of EPO are still obscure. This study is aimed at investigating the potential mechanisms by which EPO improves glucose tolerance in an animal model of type 2 diabetes. Male C57BL/6 mice were fed with high-fat diet (HFD) for 12 weeks and then treated with EPO (HFD-EPO) or vehicle saline (HFD-Con) for two week. The levels of fasting blood glucose, serum insulin and glucose tolerance were measured and the relative levels of insulin-related phosphatidylinositol 3-kinase (PI3K)/Akt, insulin receptor (IR) and IR substrate 1 (IRS1) phosphorylation were determined. The levels of phosphoenolpyruvate carboxykinase (PEPCK), glucose-6- phosphatase (G6Pase), toll like receptor 4 (TLR4), tumor necrosis factor (TNF)-α and IL-6 expression and nuclear factor-κB (NF-κB) and c-Jun N-terminal kinase (JNK), extracellular-signal-regulated kinase (ERK) and p38 MAPK activation in the liver were examined. EPO treatment significantly reduced the body weights and the levels of fasting blood glucose and serum insulin and improved the HFD-induced glucose intolerance in mice. EPO treatment significantly enhanced the levels of Akt, but not IR and IRS1, phosphorylation, accompanied by inhibiting the PEPCK and G6Pase expression in the liver. Furthermore, EPO treatment mitigated the HFD-induced inflammatory TNF-α and IL-6 production, TLR4 expression, NF-κB and JNK, but not ERK and p38 MAPK, phosphorylation in the liver. Therefore, our data indicated that EPO treatment improved glucose intolerance by inhibiting gluconeogenesis and inflammation in the livers of HFD-fed mice.     

Probiotic culture supernatant improves metabolic function through FGF21-adiponectin pathway in mice

High-fat/high-fructose diet plus intermittent hypoxia exposure (HFDIH) causes metabolic disorders such as insulin resistance, obesity, nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. The purpose of this study is to examine the effects and understand the mechanism of action of Lactobacillus rhamnosus GG culture supernatant (LGGs) on HFDIH-induced metabolic dysfunction. Mice were fed high-fat:high-fructose diet for 15 weeks. After 3 weeks of feeding, the mice were exposed to chronic intermittent hypoxia for the next 12 weeks (HFDIH), and LGGs was supplemented over the entire experiment. HFDIH exposure significantly led to metabolic disorders. LGGs treatment showed significant improvements in indices of metabolic disorders including fat mass, energy expenditure, glucose intolerance, insulin resistance, increased hepatic steatosis and liver injury. HFDIH mice markedly increased adipose inflammation and adipocyte size, and reduced circulating adiponectin, which was restored by LGGs treatment. LGGs treatment increased hepatic FGF21 mRNA expression and circulating FGF21 protein levels, which were associated with increased hepatic PPARα expression and fecal butyrate concentration. In addition, HFDIH-induced hepatic fat accumulation and apoptosis were significantly reduced by LGGs supplementation. In summary, LGGs treatment increased energy expenditure and insulin sensitivity and prevented metabolic abnormalities in HFDIH mice, and this is associated with the FGF21-adiponectin signaling pathway. LGGs may be a potential prevention/treatment strategy in subjects with the metabolic syndrome.     

Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: Cause or consequence?

Abstract

Non-alcoholic fatty liver disease (NAFLD) is considered the hepatic manifestation of the metabolic syndrome and refers to a spectrum of disorders ranging from steatosis to steatohepatitis, a disease stage characterized by inflammation, fibrosis, cell death and insulin resistance (IR). Due to its association with obesity and IR the impact of NAFLD is growing worldwide. Consistent with the role of mitochondria in fatty acid (FA) metabolism, impaired mitochondrial function is thought to contribute to NAFLD and IR. Indeed, mitochondrial dysfunction and impaired mitochondrial respiratory chain have been described in patients with non-alcoholic steatohepatitis and skeletal muscle of obese patients. However, recent data have provided evidence that pharmacological and genetic models of mitochondrial impairment with reduced electron transport stimulate insulin sensitivity and protect against diet-induced obesity, hepatosteatosis and IR. These beneficial metabolic effects of impaired mitochondrial oxidative phosphorylation may be related not only to the reduction of reactive oxygen species production that regulate insulin signaling but also to decreased mitochondrial FA overload that generate specific metabolites derived from incomplete FA oxidation (FAO) in the TCA cycle. In line with the Randle cycle, reduced mitochondrial FAO rates may alleviate the repression on glucose metabolism in obesity. In addition, the redox paradox in insulin signaling and the delicate mitochondrial antioxidant balance in steatohepatitis add another level of complexity to the role of mitochondria in NAFLD and IR. Thus, better understanding the role of mitochondria in FA metabolism and glucose homeostasis may provide novel strategies for the treatment of NAFLD and IR.     

Long-term adjustment of hepatic lipid metabolism after chronic stress and the role of FGF21

Chronic stress leads to post-traumatic stress disorder (PTSD) and metabolic disorders including fatty liver. We hypothesized that stress-induced molecular mechanisms alter energy metabolism, thereby promoting hepatic lipid accumulation even after a stress-free recovery period. In this context, we investigated fibroblast growth factor-21 (FGF21) as protective for energy and glucose homeostasis. FGF21 knockout mice (B6.129S6(SJL)-Fgf21tm1.2Djm; FGF21KO) and control mice (C57BL6; WT) were subjected to chronic variable stress. Mice were examined directly after acute intervention (Cvs) and long-term after 3 months of recovery (3mCvs). In WT, Cvs reduced insulin sensitivity and hepatic lipid accumulation, whilst fatty acid uptake increased. FGF21KO mice responded to Cvs with improved glucose tolerance, insulin resistance but liver triglycerides and plasma lipids were unaltered. Hepatic gene expression was specifically altered by genotype and stress e.g. by PPARa and SREBP-1 regulated genes. The stress-induced alteration of hepatic metabolism persisted after stress recovery. In hepatocytes at 3mCvs, differential gene regulation and secreted proteins indicated a genotype specific progression of liver dysfunction. Overall, at 3mCvs FGF21 was involved in maintaining mitochondrial activity, attenuating de novo lipogenesis, increased fatty acid uptake and histone acetyltransferase activity. Glucocorticoid release and binding to the FGF21 promoter may contribute to prolonged FGF21 release and protection against hepatic lipid accumulation. In conclusion, we showed that stress favors fatty liver disease and FGF21 protected against hepatic lipid accumulation after previous chronic stress loading by i) restored physiological function, ii) modulated gene expression via DNA-modifying enzymes, and iii) maintained energy metabolism.     

Hepatic fibroblast growth factor 21 is regulated by PPARalpha and is a key mediator of hepatic lipid metabolism in ketotic states

Mice fed a high-fat, low-carbohydrate ketogenic diet (KD) exhibit marked changes in hepatic metabolism and energy homeostasis. Here, we identify liver-derived fibroblast growth factor 21 (FGF21) as an endocrine regulator of the ketotic state. Hepatic expression and circulating levels of FGF21 are induced by both KD and fasting, are rapidly suppressed by refeeding, and are in large part downstream of PPARα. Importantly, adenoviral knockdown of hepatic FGF21 in KD-fed mice causes fatty liver, lipemia, and reduced serum ketones, due at least in part to altered expression of key genes governing lipid and ketone metabolism. Hence, induction of FGF21 in liver is required for the normal activation of hepatic lipid oxidation, triglyceride clearance, and ketogenesis induced by KD. These findings identify hepatic FGF21 as a critical regulator of lipid homeostasis and identify a physiological role for this hepatic hormone.     

Postnatal overfeeding promotes early onset and exaggeration of high-fat diet-induced nonalcoholic fatty liver disease through disordered hepatic lipid metabolism in rats

Exposure to overnutrition in critical or sensitive developmental periods may increase the risk of developing obesity and metabolic syndrome in adults. Nonalcoholic fatty liver disease (NAFLD) is the hepatic manifestation of the metabolic syndrome, but the relationship among postnatal nutrition, lipid metabolism, and NAFLD progression during development remains poorly understood. Here we investigated in a rat model whether postnatal overfeeding increases susceptibility to NAFLD in response to a high-fat diet. Litters from Sprague-Dawley dams were culled to three (small litters) or ten (normal litters) pups and then weaned onto a standard or high-fat diet at postnatal day 21 to generate normal-litter, small-litter, normal-litter/high-fat, and small-litter/high-fat groups. At age 16 weeks, the small-litter and both high-fat groups showed obesity, dyslipidemia, and insulin resistance. Hepatic disorders appeared earlier in the small-litter/high-fat rats with greater liver mass gain and higher hepatic triglycerides and steatosis score versus normal-litter/high-fat rats. Hepatic acetyl-CoA carboxylase activity and mRNA expression were increased in small-litter rats and aggravated in small-litter/high-fat rats but not in normal-litter/high-fat rats. The high expression in small-litter/high-fat rats coincided with high sterol regulatory element-binding protein-1c mRNA and protein expression. However, mRNA expression of enzymes involved in hepatic fatty acid oxidation (carnitine palmitoyltransferase 1) and output (microsomal triglyceride transfer protein) was decreased under a high-fat diet regardless of litter size. In conclusion, overfeeding related to small-litter rearing during lactation contributes to the NAFLD phenotype when combined with a high-fat diet, possibly through up-regulated hepatic lipogenesis.     

Mitogen-activated protein kinase (MAPK) phosphatase-1 and -4 attenuate p38 MAPK during dexamethasone-induced insulin resistance in 3T3-L1 adipocytes

Prolonged use of glucocorticoids induces pronounced insulin resistance in vivo. In vitro, treatment of 3T3-L1 adipocytes with dexamethasone for 48 h reduces the maximal level of insulin- and stress (arsenite)-induced glucose uptake by approximately 50%. Although phosphatidylinositol 3-kinase signaling was slightly attenuated, phosphorylation of its downstream effectors such as protein kinase B and protein kinase C-lambda remained intact. Nor was any effect of dexamethasone treatment observed on insulin- or arsenite-induced translocation of glucose transporter 4 (GLUT4) toward the plasma membrane. However, for a maximal response to either arsenite- or insulin-induced glucose uptake in these cells, functional p38 MAPK signaling is required. Dexamethasone treatment markedly attenuated p38 MAPK phosphorylation coincident with an up-regulation of the MAPK phosphatases MKP-1 and MKP-4. Employing lentivirus-mediated ectopic expression in fully differentiated 3T3-L1 adipocytes demonstrated a differential effect of these phosphatases: whereas MKP-1 was a more potent inhibitor of insulin-induced glucose uptake, MKP-4 more efficiently inhibited arsenite-induced glucose uptake. This coincided with the effects of these phosphatases on p38 MAPK phosphorylation, i.e. MKP-1 and MKP-4 attenuated p38 MAPK phosphorylation by insulin and arsenite, respectively. Taken together, these data provide evidence that in 3T3-L1 adipocytes dexamethasone inhibits the activation of the GLUT4 in the plasma membrane by a p38 MAPK-dependent process, rather than in a defect in GLUT4 translocation per se.     

Dynamics of insulin signalling in liver during hyperinsulinemic euglycaemic clamp conditions in vivo and the effects of high-fat feeding in male mice

Insulin is an important regulator of hepatic carbohydrate, lipid, and protein metabolism, and the regulation of these processes by insulin is disturbed under conditions of insulin resistance and type 2 diabetes. Despite these alterations, the impact of insulin resistance on insulin signalling in the liver is not well defined. Variations in time and dose of insulin stimulation as well as plasma glucose levels may underlie this. The present study aimed at determining the dynamics of activation of hepatic insulin signalling in vivo at insulin concentrations resembling those achieved after a meal, and addressing the effects of high-fat feeding. An unexpected finding of this study was the biphasic activation pattern of the IRS-PI3K-PKB/Akt pathway. Our findings indicate that the first burst of activation contributes to regulation of glucose metabolism. The physiological function of the second peak is still unknown, but may involve regulation of protein synthesis. Finally, high-fat feeding caused hepatic insulin resistance, as illustrated by a reduced suppression of hepatic glucose production. A sustained increased phosphorylation of the serine/threonine kinases p70S6kinase and Jun N-terminal kinase in the absence of insulin may underlie the abrogated phosphorylation of the IRS proteins and their downstream targets.     

Circulating fibroblast growth factor 21 levels are closely associated with hepatic fat content: a cross-sectional study

Background and Aims

Fibroblasts growth factor 21 (FGF21), a liver-secreted endocrine factor involved in regulating glucose and lipid metabolism, has been shown to be elevated in patients with non-alcoholic fatty liver disease (NAFLD). This study aimed to evaluate the quantitative correlation between serum FGF21 level and hepatic fat content.

Methods

A total of 138 subjects (72 male and 66 female) aged from 18 to 65 years with abnormal glucose metabolism and B-ultrasonography diagnosed fatty liver were enrolled in the study. Serum FGF21 levels were determined by an in-house chemiluminescence immunoassay and hepatic fat contents were measured by proton magnetic resonance spectroscopy.

Results

Serum FGF21 increased progressively with the increase of hepatic fat content, but when hepatic fat content increased to the fourth quartile, FGF21 tended to decline. Serum FGF21 concentrations were positively correlated with hepatic fat content especially in subjects with mild/moderate hepatic steatosis (r = 0.276, p = 0.009). Within the range of hepatic steatosis from the first to third quartile, FGF21 was superior to any other traditional clinical markers including ALT to reflect hepatic fat content. When the patients with severe hepatic steatosis (the fourth quartile) were included, the quantitative correlation between FGF21 and hepatic fat content was weakened.

Conclusions

Serum FGF21 was a potential biomarker to reflect the hepatic fat content in patients with mild or moderate NAFLD. In severe NAFLD patients, FGF21 concentration might decrease due to liver inflammation or injury.     

Restoration of impaired p38 activation by insulin in insulin resistant skeletal muscle cells treated with thiazolidinediones

We have previously reported that thiazolidinediones (TZDs) are able to restore the tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1, activation of phosphatidyl inositol 3-kinase and glucose uptake in insulin resistant skeletal muscle cells [21]. In this study, we investigated the effects of insulin stimulation and TZDs on the role of mitogen-activated protein kinase (MAPK) in insulin resistant skeletal muscle cells. All the three MAPKs [extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAPK] were activated by insulin in the sensitive skeletal muscle cells. In contrast, activation of p38 MAPK was impaired in insulin resistant cells, where as ERK and JNK were activated by insulin. Treatment with TZDs resulted in the restoration of p38 MAPK activity in insulin resistant cells. The treatment of cells with p38 MAPK inhibitor, SB203580, blocked the insulin stimulated glucose uptake in sensitive as well as resistant cells and it also prevented the activation of p38 by insulin. These results suggest the potential involvement of p38 as well as the mechanistic role of TZDs in insulin resistance.     

Interleukin-38 alleviates hepatic steatosis through AMPK/autophagy-mediated suppression of endoplasmic reticulum stress in obesity models

Interleukin-38 (IL-38), recently recognized as a cytokine with anti-inflammatory properties that mitigate type 2 diabetes, has been associated with indicators of insulin resistance and nonalcoholic fatty liver disease (NAFLD). This study investigated the impact of IL-38 on hepatic lipid metabolism and endoplasmic reticulum (ER) stress. We assessed protein expression levels using Western blot analysis, while monodansylcadaverine staining was employed to detect autophagosomes in hepatocytes. Oil red O staining was utilized to examine lipid deposition. The study revealed elevated serum IL-38 levels in high-fat diet (HFD)-fed mice and IL-38 secretion from mouse keratinocytes. IL-38 treatment attenuated lipogenic lipid accumulation and ER stress markers in hepatocytes exposed to palmitate. Furthermore, IL-38 treatment increased AMP-activated protein kinase (AMPK) phosphorylation and autophagy. The effects of IL-38 on lipogenic lipid deposition and ER stress were nullified in cultured hepatocytes by suppressing AMPK through small interfering (si) RNA or 3-methyladenine (3MA). In animal studies, IL-38 administration mitigated hepatic steatosis by suppressing the expression of lipogenic proteins and ER stress markers while reversing AMPK phosphorylation and autophagy markers in the livers of HFD-fed mice. Additionally, AMPK siRNA, but not 3MA, mitigated IL-38-enhanced fatty acid oxidation in hepatocytes. In summary, IL-38 alleviates hepatic steatosis through AMPK/autophagy signaling-dependent attenuation of ER stress and enhancement of fatty acid oxidation via the AMPK pathway, suggesting a therapeutic strategy for treating NAFLD.     

Methionine restriction restores a younger metabolic phenotype in adult mice with alterations in fibroblast growth factor 21

Methionine restriction (MR) decreases body weight and adiposity and improves glucose homeostasis in rodents. Similar to caloric restriction, MR extends lifespan, but is accompanied by increased food intake and energy expenditure. Most studies have examined MR in young animals; therefore, the aim of this study was to investigate the ability of MR to reverse age‐induced obesity and insulin resistance in adult animals. Male C57BL/6J mice aged 2 and 12 months old were fed MR (0.172% methionine) or control diet (0.86% methionine) for 8 weeks or 48 h. Food intake and whole‐body physiology were assessed and serum/tissues analyzed biochemically. Methionine restriction in 12‐month‐old mice completely reversed age‐induced alterations in body weight, adiposity, physical activity, and glucose tolerance to the levels measured in healthy 2‐month‐old control‐fed mice. This was despite a significant increase in food intake in 12‐month‐old MR‐fed mice. Methionine restriction decreased hepatic lipogenic gene expression and caused a remodeling of lipid metabolism in white adipose tissue, alongside increased insulin‐induced phosphorylation of the insulin receptor (IR) and Akt in peripheral tissues. Mice restricted of methionine exhibited increased circulating and hepatic gene expression levels of FGF21, phosphorylation of eIF2a, and expression of ATF4, with a concomitant decrease in IRE1α phosphorylation. Short‐term 48‐h MR treatment increased hepatic FGF21 expression/secretion and insulin signaling and improved whole‐body glucose homeostasis without affecting body weight. Our findings suggest that MR feeding can reverse the negative effects of aging on body mass, adiposity, and insulin resistance through an FGF21 mechanism. These findings implicate MR dietary intervention as a viable therapy for age‐induced metabolic syndrome in adult humans.