Cellular senescence in hepatocytes contributes to metabolic disturbances in NASH.

Cellular senescence is a state of irreversible cell cycle arrest and has been shown to play a key role in many diseases, including metabolic diseases. To investigate the potential contribution of hepatocyte cellular senescence to the metabolic derangements associated with non-alcoholic steatohepatitis (NASH), we treated human hepatocyte cell lines HepG2 and IHH with the senescence-inducing drugs nutlin-3a, doxorubicin and etoposide. The senescence-associated markers p16, p21, p53 and beta galactosidase were induced upon drug treatment, and this was associated with increased lipid storage, increased expression of lipid transporters and the development of hepatic steatosis. Drug-induced senescence also led to increased glycogen content, and increased VLDL secretion from hepatocytes. Senescence was also associated with an increase in glucose and fatty acid oxidation capacity, while de novo lipogenesis was decreased. Surprisingly, cellular senescence caused an overall increase in insulin signaling in hepatocytes, with increased insulin-stimulated phosphorylation of IR, Akt, and MAPK. Together, these data indicate that hepatic senescence plays a causal role in the development of NASH pathogenesis, by modulating glucose and lipid metabolism, favoring steatosis. Our findings contribute to a better understanding of the mechanisms linking cellular senescence and fatty liver disease and support the development of new therapies targeting senescent cells for the treatment of NASH.

BMP4 and Gremlin 1 regulate hepatic cell senescence during clinical progression of NAFLD/NASH.

The role of hepatic cell senescence in human non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) is not well understood. To examine this, we performed liver biopsies and extensive characterization of 58 individuals with or without NAFLD/NASH. Here, we show that hepatic cell senescence is strongly related to NAFLD/NASH severity, and machine learning analysis identified senescence markers, the BMP4 inhibitor Gremlin 1 in liver and visceral fat, and the amount of visceral adipose tissue as strong predictors. Studies in liver cell spheroids made from human stellate and hepatocyte cells show BMP4 to be anti-senescent, anti-steatotic, anti-inflammatory and anti-fibrotic, whereas Gremlin 1, which is particularly highly expressed in visceral fat in humans, is pro-senescent and antagonistic to BMP4. Both senescence and anti-senescence factors target the YAP/TAZ pathway, making this a likely regulator of senescence and its effects. We conclude that senescence is an important driver of human NAFLD/NASH and that BMP4 and Gremlin 1 are novel therapeutic targets.

Chronic hyperinsulinemia promotes human hepatocyte senescence.

OBJECTIVE: Cellular senescence, an irreversible proliferative cell arrest, is caused by excessive intracellular or extracellular stress/damage. Increased senescent cells have been identified in multiple tissues in different metabolic and other aging-related diseases. Recently, several human and mouse studies emphasized the involvement of senescence in development and progression of NAFLD. Hyperinsulinemia, seen in obesity, metabolic syndrome, and other conditions of insulin resistance, has been linked to senescence in adipocytes and neurons. Here, we investigate the possible direct role of chronic hyperinsulinemia in the development of senescence in human hepatocytes. METHODS: Using fluorescence microscopy, immunoblotting, and gene expression, we tested senescence markers in human hepatocytes subjected to chronic hyperinsulinemia in vitro and validated the data in vivo by using liver-specific insulin receptor knockout (LIRKO) mice. The consequences of hyperinsulinemia were also studied in senescent hepatocytes following doxorubicin as a model of stress-induced senescence. Furthermore, the effects of senolytic agents in insulin- and doxorubicin-treated cells were analyzed. RESULTS: Results showed that exposing the hepatocytes to prolonged hyperinsulinemia promotes the onset of senescence by increasing the expression of p53 and p21. It also further enhanced the senescent phenotype in already senescent hepatocytes. Addition of insulin signaling pathway inhibitors prevented the increase in cell senescence, supporting the direct contribution of insulin. Furthermore, LIRKO mice, in which insulin signaling in the liver is abolished due to deletion of the insulin receptor gene, showed no differences in senescence compared to their wild-type counterparts despite having marked hyperinsulinemia indicating these are receptor-mediated effects. In contrast, the persistent hyperinsulinemia in LIRKO mice enhanced senescence in white adipose tissue. In vitro, senolytic agents dasatinib and quercetin reduced the prosenescent effects of hyperinsulinemia in hepatocytes. CONCLUSION: Our findings demonstrate a direct link between chronic hyperinsulinemia and hepatocyte senescence. This effect can be blocked by reducing the levels of insulin receptors or administration of senolytic drugs, such as dasatinib and quercetin.

Emerging Role of Bone Morphogenetic Protein 4 in Metabolic Disorders.

Bone morphogenetic proteins (BMPs) are a group of signaling molecules that belong to the TGF-ß superfamily. Initially discovered for their ability to induce bone formation, BMPs are known to play a diverse and critical array of biological roles. We here focus on recent evidence showing that BMP4 is an important regulator of white/beige adipogenic differentiation with important consequences for thermogenesis, energy homeostasis, and development of obesity in vivo. BMP4 is highly expressed in, and released by, human adipose tissue, and serum levels are increased in obesity. Recent studies have now shown BMP4 to play an important role not only for white/beige/brown adipocyte differentiation and thermogenesis but also in regulating systemic glucose homeostasis and insulin sensitivity. It also has important suppressive effects on hepatic glucose production and lipid metabolism. Cellular BMP4 signaling/action is regulated by both ambient cell/systemic levels and several endogenous and systemic BMP antagonists. Reduced BMP4 signaling/action can contribute to the development of obesity, insulin resistance, and associated metabolic disorders. In this article, we summarize the pleiotropic functions of BMP4 in the pathophysiology of these diseases and also consider the therapeutic implications of targeting BMP4 in the prevention/treatment of obesity and its associated complications.

Involvement of Glucagon in Preventive Effect of Menthol Against High Fat Diet Induced Obesity in Mice.

Glucagon mediated mechanisms have been shown to play clinically significant role in energy expenditure. The present study was designed to understand whether pharmacological mimicking of cold using menthol (TRPM8 modulator) can induce glucagon-mediated energy expenditure to prevent weight gain and related complications. Acute oral and topical administration of TRPM8 agonists (menthol and icilin) increased serum glucagon concentration which was prevented by pre-treatment with AMTB, a TRPM8 blocker. Chronic administration of menthol (50 and 100 mg/kg/day for 12 weeks) to HFD fed animals prevented weight gain, insulin resistance, adipose tissue hypertrophy and triacylglycerol deposition in liver. These effects were not restricted to oral administration, but also observed upon the topical application of menthol (10% w/v). The metabolic alterations caused by menthol in liver and adipose tissue mirrored the known effects of glucagon, such as increased glycogenolysis and gluconeogenesis in the liver, and enhanced thermogenic activity of white and brown adipose tissue. Correlation analysis suggests a strong correlation between glucagon dependent changes and energy expenditure markers. Interestingly, in-vitro treatment of the serum of menthol treated mice increased energy expenditure markers in mature 3T3L1 adipocytes, which was prevented in the presence of non-competitive glucagon receptor antagonist, L-168,049, indicating that menthol-induced increase in serum glucagon is responsible for increase in energy expenditure phenotype. In conclusion, the present work provides evidence that glucagon plays an important role in the preventive effect of menthol against HFD-induced weight gain and related complications.

Dihydrocapsiate supplementation prevented high-fat diet-induced adiposity, hepatic steatosis, glucose intolerance, and gut morphological alterations in mice.

Despite the lipolytic and thermogenic properties of capsaicin, its putative use as a weight-lowering dietary supplement has been limited because of the burning sensation caused by capsaicin when it comes in contact with mucous membranes. A potential alternative to capsaicin are the capsinoids, nonpungent capsaicin analogs that exhibit effects similar to capsaicin. Whereas the antiobesity properties of capsinoids have been reported, the effectiveness of FDA-approved synthetic dihydrocapsiate has not yet been investigated. In the present study, we hypothesized that dihydrocapsiate might ameliorate high-fat diet (HFD)-induced metabolic disorders in a manner similar to capsaicin and therefore can be its nonpungent alternative. To test this hypothesis, HFD-fed mice were orally administered dihydrocapsiate (2 and 10mg/kg body weight) for 12weeks. Dihydrocapsiate modestly reduced the HFD-induced weight gain and significantly prevented the associated hyperglyceridemia and hyperinsulinemia while improving glucose tolerance. Histological and gene expression analysis showed that dihydrocapsiate significantly prevented the lipid accumulation in white adipose tissue and brown adipose tissue via targeting genes involved in energy expenditure and mitochondrial biogenesis, respectively. Dihydrocapsiate corrected hepatic triglyceride concentrations and normalized expression of genes regulating hepatic lipid and glucose metabolism. Moreover, dihydrocapsiate administration significantly improved gut morphology and altered gut microbial composition, resulting in reduced host energy availability. Collectively, these results indicate that dihydrocapsiate administration improved glucose tolerance, prevented adiposity and hepatic steatosis, as well as improved HFD-induced gut alterations, positing dihydrocapsiate as a potential food ingredient for the dietary management of HFD-induced metabolic alterations.

Kodo millet whole grain and bran supplementation prevents high-fat diet induced derangements in a lipid profile, inflammatory status and gut bacteria in mice.

The protective role of kodo millet whole grain and bran supplementation in diet induced obesity has not been investigated. Here we have studied the role of kodo millet supplementation in age matched Swiss albino mice that were randomly divided into groups and fed their respective diets for 16 weeks. A high fat diet increased weight gain, reduced glucose tolerance, increased serum lipids, altered hepatic and adipocyte gene expression and caused dysbiosis in the intestinal beneficial bacteria. Kodo millet supplementation did not affect weight gain but it improved glucose tolerance and prevented an increase in the serum cholesterol and lipid parameters (P ≤ 0.05), modulated adipogenesis related gene expression, decreased serum IL-6 and LPS levels (P ≤ 0.05), promoted selected beneficial gut bacterial abundances (Lactobacillus sp., Bifidobacteria, Akkermansia and Roseburia spp.) and improved the total short chain fatty acid production (P ≤ 0.05) and acetate levels (P ≤ 0.05) in cecal contents. This study provides evidence that kodo millet supplementation alleviates high-fat diet induced changes and hence can be incorporated as a functional ingredient for the management of obesity.

Cinnamaldehyde supplementation prevents fasting-induced hyperphagia, lipid accumulation, and inflammation in high-fat diet-fed mice.

Cinnamaldehyde, a bioactive component of cinnamon, is increasingly gaining interest for its preventive and therapeutic effects against metabolic complications like type-2 diabetes. This study is an attempt to understand the effect of cinnamaldehyde in high-fat diet (HFD)-associated increase in fasting-induced hyperphagia and related hormone levels, adipose tissue lipolysis and inflammation, and selected cecal microbial count in mice. Cinnamaldehyde, at 40 µM dose, prevented lipid accumulation and altered gene expression toward lipolytic phenotype in 3T3-L1 preadipocyte cell lines. In vivo, cinnamaldehyde coadministration prevented HFD-induced body weight gain, decreased fasting-induced hyperphagia, as well as circulating leptin and leptin/ghrelin ratio. In addition to that, cinnamaldehyde altered serum biochemical parameters related to lipolysis, that is, glycerol and free fatty acid levels. At transcriptional level, cinnamaldehyde increased anorectic gene expression in hypothalamus and lipolytic gene expression in visceral white adipose tissue. Furthermore, cinnamaldehyde also decreased serum IL-1ß and inflammatory gene expression in visceral white adipose tissue. However, cinnamaldehyde did not modulate the population of selected gut microbial (Lactobacillus, Bifidibaceria, and Roseburia) count in cecal content. In conclusion, cinnamaldehyde increased adipose tissue lipolysis, decreased fasting-induced hyperphagia, normalized circulating levels of leptin/ghrelin ratio, and reduced inflammation in HFD-fed mice, which augurs well for its antiobesity role.

Microarray based gene expression analysis of murine brown and subcutaneous adipose tissue: significance with human.

BACKGROUND: Two types of adipose tissues, white (WAT) and brown (BAT) are found in mammals. Increasingly novel strategies are being proposed for the treatment of obesity and its associated complications by altering amount and/or activity of BAT using mouse models. METHODOLOGY/PRINCIPLE FINDINGS: The present study was designed to: (a) investigate the differential expression of genes in LACA mice subcutaneous WAT (sWAT) and BAT using mouse DNA microarray, (b) to compare mouse differential gene expression with previously published human data; to understand any inter- species differences between the two and (c) to make a comparative assessment with C57BL/6 mouse strain. In mouse microarray studies, over 7003, 1176 and 401 probe sets showed more than two-fold, five-fold and ten-fold change respectively in differential expression between murine BAT and WAT. Microarray data was validated using quantitative RT-PCR of key genes showing high expression in BAT (Fabp3, Ucp1, Slc27a1) and sWAT (Ms4a1, H2-Ob, Bank1) or showing relatively low expression in BAT (Pgk1, Cox6b1) and sWAT (Slc20a1, Cd74). Multi-omic pathway analysis was employed to understand possible links between the organisms. When murine two fold data was compared with published human BAT and sWAT data, 90 genes showed parallel differential expression in both mouse and human. Out of these 90 genes, 46 showed same pattern of differential expression whereas the pattern was opposite for the remaining 44 genes. Based on our microarray results and its comparison with human data, we were able to identify genes (targets) (a) which can be studied in mouse model systems to extrapolate results to human (b) where caution should be exercised before extrapolation of murine data to human. CONCLUSION: Our study provides evidence for inter species (mouse vs human) differences in differential gene expression between sWAT and BAT. Critical understanding of this data may help in development of novel ways to engineer one form of adipose tissue to another using murine model with focus on human.

Finger millet bran supplementation alleviates obesity-induced oxidative stress, inflammation and gut microbial derangements in high-fat diet-fed mice.

Several epidemiological studies have shown that the consumption of finger millet (FM) alleviates diabetes-related complications. In the present study, the effect of finger millet whole grain (FM-WG) and bran (FM-BR) supplementation was evaluated in high-fat diet-fed LACA mice for 12 weeks. Mice were divided into four groups: control group fed a normal diet (10 % fat as energy); a group fed a high-fat diet; a group fed the same high-fat diet supplemented with FM-BR; a group fed the same high-fat diet supplemented with FM-WG. The inclusion of FM-BR at 10 % (w/w) in a high-fat diet had more beneficial effects than that of FM-WG. FM-BR supplementation prevented body weight gain, improved lipid profile and anti-inflammatory status, alleviated oxidative stress, regulated the expression levels of several obesity-related genes, increased the abundance of beneficial gut bacteria (Lactobacillus, Bifidobacteria and Roseburia) and suppressed the abundance of Enterobacter in caecal contents (P≤ 0·05). In conclusion, FM-BR supplementation could be an effective strategy for preventing high-fat diet-induced changes and developing FM-BR-enriched functional foods.

Capsaicin induces "brite" phenotype in differentiating 3T3-L1 preadipocytes.

OBJECTIVE: Targeting the energy storing white adipose tissue (WAT) by pharmacological and dietary means in order to promote its conversion to energy expending "brite" cell type holds promise as an anti-obesity approach. Present study was designed to investigate/revisit the effect of capsaicin on adipogenic differentiation with special reference to induction of "brite" phenotype during differentiation of 3T3-L1 preadipocytes. METHODS: Multiple techniques such as Ca2+ influx assay, Oil Red-O staining, nutrigenomic analysis in preadipocytes and matured adipocytes have been employed to understand the effect of capsaicin at different doses. In addition to in-vitro experiments, in-vivo studies were carried out in high-fat diet (HFD) fed rats treated with resiniferatoxin (RTX) (a TRPV1 agonist) and in mice administered capsaicin. RESULTS: TRPV1 channels are expressed in preadipocytes but not in adipocytes. In preadipocytes, both capsaicin and RTX stimulate Ca2+ influx in dose-dependent manner. This stimulation may be prevented by capsazepine, a TRPV1 antagonist. At lower doses, capsaicin inhibits lipid accumulation and stimulates TRPV1 gene expression, while at higher doses it enhances accumulation of lipids and suppresses expression of its receptor. In doses of 0.1-100 µM, capsaicin promotes expression of major pro-adipogenic factor PPARγ and some of its downstream targets. In concentrations of 1 µM, capsaicin up-regulates anti-adipogenic genes. Low-dose capsaicin treatment of 3T3-L1 preadipocytes differentiating into adipocytes results in increased expression of brown fat cell marker genes. In white adipose of mice, capsaicin administration leads to increase in browning-specific genes. Global TRPV1 ablation (i.p. by RTX administration) leads to increase in locomotor activity with no change in body weight. CONCLUSION: Our findings suggest the dual modulatory role of capsaicin in adipogenesis. Capsaicin inhibits adipogenesis in 3T3-L1 via TRPV1 activation and induces brown-like phenotype whereas higher doses.

Capsaicin-induced transcriptional changes in hypothalamus and alterations in gut microbial count in high fat diet fed mice.

Obesity is a global health problem and recently it has been seen as a growing concern for developing countries. Several bioactive dietary molecules have been associated with amelioration of obesity and associated complications and capsaicin is one among them. The present work is an attempt to understand and provide evidence for the novel mechanisms of anti-obesity activity of capsaicin in high fat diet (HFD)-fed mice. Swiss albino mice divided in three groups (n=8-10) i.e. control, HFD fed and capsaicin (2mg/kg, po)+HFD fed were administered respective treatment for 3months. After measuring phenotypic and serum related biochemical changes, effect of capsaicin on HFD-induced transcriptional changes in hypothalamus, white adipose tissue (WAT) (visceral and subcutaneous), brown adipose tissue (BAT) and gut microbial alterations was studied and quantified. Our results suggest that, in addition to its well-known effects, oral administration of capsaicin (a) modulates hypothalamic satiety associated genotype, (b) alters gut microbial composition, (c) induces "browning" genotype (BAT associated genes) in subcutaneous WAT and (d) increases expression of thermogenesis and mitochondrial biogenesis genes in BAT. The present study provides evidence for novel and interesting mechanisms to explain the anti-obesity effect of capsaicin.

Role of transient receptor potential channels in adipocyte biology.

The transient receptor potential (TRP) channel superfamily is a family of 28 nonselective cation channels expressed on the plasma membrane with a high permeability to calcium. Role of TRP channels, especially TRP vanilloid 1, TRP ankyrin 1 and TRP melastatin 8, is widely documented in nociception. During the last few years, there has been a consistent increase in reports indicating the presence and significance of these channels in different tissues including bladder, skin, respiratory system and brain. Based on calcium permeability of these channels and the much published role of calcium and its signaling in adipogenesis, there is a potential for importance of these ion channels in adipocyte biology. This review provides insight into the involvement of TRP channels in adipocyte differentiation, obesity and associated complications. Furthermore, the authors will focus to evaluate these channels as potential therapeutic targets for the management of obesity.