Ceramide analog C2-cer induces a loss in insulin sensitivity in muscle cells through the salvage/recycling pathway.

Ceramides have been shown to play a major role in the onset of skeletal muscle insulin resistance and therefore in the prevalence of type 2 diabetes. However, many of the studies involved in the discovery of deleterious ceramide actions used a nonphysiological, cell-permeable, short-chain ceramide analog, the C2-ceramide (C2-cer). In the present study, we determined how C2-cer promotes insulin resistance in muscle cells. We demonstrate that C2-cer enters the salvage/recycling pathway and becomes deacylated, yielding sphingosine, re-acylation of which depends on the availability of long chain fatty acids provided by the lipogenesis pathway in muscle cells. Importantly, we show these salvaged ceramides are actually responsible for the inhibition of insulin signaling induced by C2-cer. Interestingly, we also show that the exogenous and endogenous monounsaturated fatty acid oleate prevents C2-cer to be recycled into endogenous ceramide species in a diacylglycerol O-acyltransferase 1-dependent mechanism, which forces free fatty acid metabolism towards triacylglyceride production. Altogether, the study highlights for the first time that C2-cer induces a loss in insulin sensitivity through the salvage/recycling pathway in muscle cells. This study also validates C2-cer as a convenient tool to decipher mechanisms by which long-chain ceramides mediate insulin resistance in muscle cells and suggests that in addition to the de novo ceramide synthesis, recycling of ceramide could contribute to muscle insulin resistance observed in obesity and type 2 diabetes.

SREBP-1c and lipogenesis in the liver: an update1.

Sterol Regulatory Element Binding Protein-1c is a transcription factor that controls the synthesis of lipids from glucose in the liver, a process which is of utmost importance for the storage of energy. Discovered in the early nineties by B. Spiegelman and by M. Brown and J. Goldstein, it has generated more than 5000 studies in order to elucidate its mechanism of activation and its role in physiology and pathology. Synthetized as a precursor found in the membranes of the endoplasmic reticulum, it has to be exported to the Golgi and cleaved by a mechanism called regulated intramembrane proteolysis. We reviewed in 2002 its main characteristics, its activation process and its role in the regulation of hepatic glycolytic and lipogenic genes. We particularly emphasized that Sterol Regulatory Element Binding Protein-1c is the mediator of insulin effects on these genes. In the present review, we would like to update these informations and focus on the response to insulin and to another actor in Sterol Regulatory Element Binding Protein-1c activation, the endoplasmic reticulum stress.

Roles of Ceramides in Non-Alcoholic Fatty Liver Disease.

Non-alcoholic fatty liver disease is one of the most common chronic liver diseases, ranging from simple steatosis to steatohepatitis, fibrosis, and cirrhosis. Its prevalence is rapidly increasing and presently affects around 25% of the general population of Western countries, due to the obesity epidemic. Liver fat accumulation induces the synthesis of specific lipid species and particularly ceramides, a sphingolipid. In turn, ceramides have deleterious effects on hepatic metabolism, a phenomenon called lipotoxicity. We review here the evidence showing the role of ceramides in non-alcoholic fatty liver disease and the mechanisms underlying their effects.

Dihydroceramides: their emerging physiological roles and functions in cancer and metabolic diseases.

Dihydroceramides (DhCers) are a type of sphingolipids that for a long time were regarded as biologically inactive. They are metabolic intermediates of the de novo sphingolipid synthesis pathway, and are converted into ceramides (Cers) with the addition of a double bond. Ceramides are abundant in tissues and have well-established biological functions. On the contrary, dihydroceramides are less prevalent, and despite their hitherto characterization as inert lipids, studies of the past decade began to unravel their implication in various biological processes distinct from those involving ceramides. These processes include cellular stress responses and autophagy, cell growth, pro-death or pro-survival pathways, hypoxia, and immune responses. In addition, their plasma concentration has been related to metabolic diseases and shown as a long-term predictor of type 2 diabetes onset. They are thus important players and potential biomarkers in pathologies ranging from diabetes to cancer and neurodegenerative diseases. The purpose of this mini-review is to highlight the emergence of dihydroceramides as a new class of bioactive sphingolipids by reporting recent advances on their biological characterization and pathological implications, focusing on cancer and metabolic diseases.

Dihydroceramides in Triglyceride-Enriched VLDL Are Associated with Nonalcoholic Fatty Liver Disease Severity in Type 2 Diabetes.

Plasma dihydroceramides are predictors of type 2 diabetes and related to metabolic dysfunctions, but the underlying mechanisms are not characterized. We compare the relationships between plasma dihydroceramides and biochemical and hepatic parameters in two cohorts of diabetic patients. Hepatic steatosis, steatohepatitis, and fibrosis are assessed by their plasma biomarkers. Plasma lipoprotein sphingolipids are studied in a sub-group of diabetic patients. Liver biopsies from subjects with suspected non-alcoholic fatty liver disease are analyzed for sphingolipid synthesis enzyme expression. Dihydroceramides, contained in triglyceride-rich very-low-density lipoprotein (VLDL), are associated with steatosis and steatohepatitis. Expression of sphingolipid synthesis enzymes is correlated with histological steatosis and inflammation grades. In conclusion, association of plasma dihydroceramides with nonalcoholic fatty liver might explain their predictive character for type 2 diabetes. Our results suggest a relationship between hepatic sphingolipid metabolism and steatohepatitis and an involvement of dihydroceramides in the synthesis/secretion of triglyceride-rich VLDL, a hallmark of NAFLD and type 2 diabetes dyslipidemia.

[ER stress and NAFLD]. / Stress du réticulum endoplasmique et stéatopathies métaboliques.

Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent pathology associated with obesity. It encompasses a spectrum of hepatic disorders ranging from steatosis to non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and hepatocellular carcinoma (HCC). Endoplasmic reticulum (ER) stress has been widely involved to drive in NAFLD progression through the activation of the unfolded protein response (UPR). While transient UPR activation can boost hepatic ER functions, its continuous activation upon a chronic ER stress contributes to lipid accumulation, inflammation and hepatocyte death, which are determinant factors for the progression to more severe stages. The aim of this review is to describe the mechanisms through which the UPR can take part in the transition from a healthy to a diseased liver and to report on possible ways of pharmacological manipulation against these pathological mechanisms.

TITLE: Stress du réticulum endoplasmique et stéatopathies métaboliques. ABSTRACT: Les stéatopathies métaboliques sont des pathologies en pleine expansion car très associées à l'obésité. Elles englobent un éventail de troubles hépatiques allant de la stéatose à la stéatohépatite non alcoolique (NASH) pouvant conduire à la cirrhose et au carcinome hépatocellulaire (CHC). Le stress du réticulum endoplasmique (RE), à travers l'activation de la voie UPR (Unfolded Protein Response), a été largement impliqué dans le développement et la progression de ces maladies métaboliques hépatiques. Alors que l'activation transitoire de la voie UPR fait partie intégrante de la physiologie hépatique, son activation chronique contribue à la stimulation de voies métaboliques et cellulaires (synthèse des lipides, inflammation, apoptose) qui sont déterminantes dans la progression vers des stades sévères. Le but de cette revue est de décrire comment la voie UPR participe au passage d'un foie sain à un foie malade au cours de l'obésité et d'analyser les perspectives thérapeutiques liées à la manipulation pharmacologique de cette voie.

Ceramide Transporter CERT Is Involved in Muscle Insulin Signaling Defects Under Lipotoxic Conditions.

One main mechanism of insulin resistance (IR), a key feature of type 2 diabetes, is the accumulation of saturated fatty acids (FAs) in the muscles of obese patients with type 2 diabetes. Understanding the mechanism that underlies lipid-induced IR is an important challenge. Saturated FAs are metabolized into lipid derivatives called ceramides, and their accumulation plays a central role in the development of muscle IR. Ceramides are produced in the endoplasmic reticulum (ER) and transported to the Golgi apparatus through a transporter called CERT, where they are converted into various sphingolipid species. We show that CERT protein expression is reduced in all IR models studied because of a caspase-dependent cleavage. Inhibiting CERT activity in vitro potentiates the deleterious action of lipotoxicity on insulin signaling, whereas overexpression of CERT in vitro or in vivo decreases muscle ceramide content and improves insulin signaling. In addition, inhibition of caspase activity prevents ceramide-induced insulin signaling defects in C2C12 muscle cells. Altogether, these results demonstrate the importance of physiological ER-to-Golgi ceramide traffic to preserve muscle cell insulin signaling and identify CERT as a major actor in this process.

Activation of AMPK-Regulated CRH Neurons in the PVH is Sufficient and Necessary to Induce Dietary Preference for Carbohydrate over Fat.

Food selection is essential for metabolic homeostasis and is influenced by nutritional state, food palatability, and social factors such as stress. However, the mechanism responsible for selection between a high-carbohydrate diet (HCD) and a high-fat diet (HFD) remains unknown. Here, we show that activation of a subset of corticotropin-releasing hormone (CRH)-positive neurons in the rostral region of the paraventricular hypothalamus (PVH) induces selection of an HCD over an HFD in mice during refeeding after fasting, resulting in a rapid recovery from the change in ketone metabolism. These neurons manifest activation of AMP-activated protein kinase (AMPK) during food deprivation, and this activation is necessary and sufficient for selection of an HCD over an HFD. Furthermore, this effect is mediated by carnitine palmitoyltransferase 1c (CPT1c). Thus, our results identify the specific neurons and intracellular signaling pathway responsible for regulation of the complex behavior of selection between an HCD and an HFD. VIDEO ABSTRACT.

Lipid environment induces ER stress, TXNIP expression and inflammation in immune cells of individuals with type 2 diabetes.

AIMS/HYPOTHESIS: Obesity and type 2 diabetes are concomitant with low-grade inflammation affecting insulin sensitivity and insulin secretion. Recently, the thioredoxin interacting protein (TXNIP) has been implicated in the activation process of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. In this study, we aim to determine whether the expression of TXNIP is altered in the circulating immune cells of individuals with type 2 vs type 1 diabetes and whether this can be related to specific causes and consequences of inflammation. METHODS: The expression of TXNIP, inflammatory markers, markers of the unfolded protein response (UPR) to endoplasmic reticulum (ER) stress and enzymes involved in sphingolipid metabolism was quantified by quantitative reverse transcription real-time PCR (qRT-PCR) in peripheral blood mononuclear cells (PBMCs) of 13 non-diabetic individuals, 23 individuals with type 1 diabetes and 81 with type 2 diabetes. A lipidomic analysis on the plasma of 13 non-diabetic individuals, 35 individuals with type 1 diabetes and 94 with type 2 diabetes was performed. The effects of ER stress or of specific lipids on TXNIP and inflammatory marker expression were analysed in human monocyte-derived macrophages (HMDMs) and THP-1 cells. RESULTS: The expression of TXNIP and inflammatory and UPR markers was increased in the PBMCs of individuals with type 2 diabetes when compared with non-diabetic individuals or individuals with type 1 diabetes. TXNIP expression was significantly correlated with plasma fasting glucose, plasma triacylglycerol concentrations and specific UPR markers. Induction of ER stress in THP-1 cells or cultured HMDMs led to increased expression of UPR markers, TXNIP, NLRP3 and IL-1ß. Conversely, a chemical chaperone reduced the expression of UPR markers and TXNIP in PBMCs of individuals with type 2 diabetes. The lipidomic plasma analysis revealed an increased concentration of saturated dihydroceramide and sphingomyelin in individuals with type 2 diabetes when compared with non-diabetic individuals and individuals with type 1 diabetes. In addition, the expression of specific enzymes of sphingolipid metabolism, dihydroceramide desaturase 1 and sphingomyelin synthase 1, was increased in the PBMCs of individuals with type 2 diabetes. Palmitate or C2 ceramide induced ER stress in macrophages as well as increased expression of TXNIP, NLRP3 and IL-1ß. CONCLUSIONS/INTERPRETATION: In individuals with type 2 diabetes, circulating immune cells display an inflammatory phenotype that can be linked to ER stress and TXNIP expression. Immune cell ER stress can in turn be linked to the specific exogenous and endogenous lipid environment found in type 2 diabetes.

Steatosis and NASH in type 2 diabetes.

Non Alcoholic Fatty Liver Disease (NAFLD) is currently the most common chronic liver disease in the world, encompassing various conditions ranging from simple steatosis, steatohepatitis, to fibrosis and cirrhosis. The association between NAFLD and Type 2 Diabetes (T2D) is strong and complex, given that the prevalence of NAFLD is particularly high in individuals with Type 2 Diabetes. In fact, insulin resistance occurring in this metabolic disease can promote NAFLD development, and vice versa, NAFLD can enhance insulin resistance. In this review, we focus on the mechanisms linking NAFLD and T2D, including fatty acid accumulation, inflammation, oxidative stress etc. We also discuss about situations showing a dissociation between steatosis and insulin resistance, in order to provide new insights for NAFLD therapeutic targets.

High carbohydrate diet induces nonalcoholic steato-hepatitis (NASH) in a desert gerbil.

A high intake of sugars has been linked to diet-induced health problems. The aim of this study was to assess whether the long-term consumption of a high-carbohydrate diet (HCD) would cause the hepatic histopathological and metabolic abnormalities that characterize nonalcoholic steatohepatitis (NASH) in a desert gerbil, Gerbillus gerbillus. Compared to natural diet, HCD leads to several metabolic disorders including adiposity, dyslipidemia, insulin resistance, ectopic fat deposition in the liver, which were associated with higher levels of transcripts of genes involved with fat synthesis, endoplasmic reticulum (ER) stress, and fibrosis. In the same way, the experimented animals showed enhanced oxidative stress. Taken together, these results demonstrate that HCD consumption in gerbils induces metabolic disorders and damaged liver, which are key contributors to NASH development. These results suggest that this rodent represents a valuable natural model for human diet-induced metabolic disorders and nonalcoholic fatty liver disease (NAFLD).

Short Term Palmitate Supply Impairs Intestinal Insulin Signaling via Ceramide Production.

The worldwide prevalence of metabolic diseases is increasing, and there are global recommendations to limit consumption of certain nutrients, especially saturated lipids. Insulin resistance, a common trait occurring in obesity and type 2 diabetes, is associated with intestinal lipoprotein overproduction. However, the mechanisms by which the intestine develops insulin resistance in response to lipid overload remain unknown. Here, we show that insulin inhibits triglyceride secretion and intestinal microsomal triglyceride transfer protein expression in vivo in healthy mice force-fed monounsaturated fatty acid-rich olive oil but not in mice force-fed saturated fatty acid-rich palm oil. Moreover, when mouse intestine and human Caco-2/TC7 enterocytes were treated with the saturated fatty acid, palmitic acid, the insulin-signaling pathway was impaired. We show that palmitic acid or palm oil increases ceramide production in intestinal cells and that treatment with a ceramide analogue partially reproduces the effects of palmitic acid on insulin signaling. In Caco-2/TC7 enterocytes, ceramide effects on insulin-dependent AKT phosphorylation are mediated by protein kinase C but not by protein phosphatase 2A. Finally, inhibiting de novo ceramide synthesis improves the response of palmitic acid-treated Caco-2/TC7 enterocytes to insulin. These results demonstrate that a palmitic acid-ceramide pathway accounts for impaired intestinal insulin sensitivity, which occurs within several hours following initial lipid exposure.

Correction: Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

[This corrects the article DOI: 10.1371/journal.pone.0149343.].

Glucocorticoids Inhibit Basal and Hormone-Induced Serotonin Synthesis in Pancreatic Beta Cells.

Diabetes is a major complication of chronic Glucocorticoids (GCs) treatment. GCs induce insulin resistance and also inhibit insulin secretion from pancreatic beta cells. Yet, a full understanding of this negative regulation remains to be deciphered. In the present study, we investigated whether GCs could inhibit serotonin synthesis in beta cell since this neurotransmitter has been shown to be involved in the regulation of insulin secretion. To this aim, serotonin synthesis was evaluated in vitro after treatment with GCs of either islets from CD1 mice or MIN6 cells, a beta-cell line. We also explored the effect of GCs on the stimulation of serotonin synthesis by several hormones such as prolactin and GLP 1. We finally studied this regulation in islet in two in vivo models: mice treated with GCs and with liraglutide, a GLP1 analog, and mice deleted for the glucocorticoid receptor in the pancreas. We showed in isolated islets and MIN6 cells that GCs decreased expression and activity of the two key enzymes of serotonin synthesis, Tryptophan Hydroxylase 1 (Tph1) and 2 (Tph2), leading to reduced serotonin contents. GCs also blocked the induction of serotonin synthesis by prolactin or by a previously unknown serotonin activator, the GLP-1 analog exendin-4. In vivo, activation of the Glucagon-like-Peptide-1 receptor with liraglutide during 4 weeks increased islet serotonin contents and GCs treatment prevented this increase. Finally, islets from mice deleted for the GR in the pancreas displayed an increased expression of Tph1 and Tph2 and a strong increased serotonin content per islet. In conclusion, our results demonstrate an original inhibition of serotonin synthesis by GCs, both in basal condition and after stimulation by prolactin or activators of the GLP-1 receptor. This regulation may contribute to the deleterious effects of GCs on beta cells.

Sustained Action of Ceramide on the Insulin Signaling Pathway in Muscle Cells: IMPLICATION OF THE DOUBLE-STRANDED RNA-ACTIVATED PROTEIN KINASE.

In vivo, ectopic accumulation of fatty acids in muscles leads to alterations in insulin signaling at both the IRS1 and Akt steps. However, in vitro treatments with saturated fatty acids or their derivative ceramide demonstrate an effect only at the Akt step. In this study, we adapted our experimental procedures to mimic the in vivo situation and show that the double-stranded RNA-dependent protein kinase (PKR) is involved in the long-term effects of saturated fatty acids on IRS1. C2C12 or human muscle cells were incubated with palmitate or directly with ceramide for short or long periods, and insulin signaling pathway activity was evaluated. PKR involvement was assessed through pharmacological and genetic studies. Short-term treatments of myotubes with palmitate, a ceramide precursor, or directly with ceramide induce an inhibition of Akt, whereas prolonged periods of treatment show an additive inhibition of insulin signaling through increased IRS1 serine 307 phosphorylation. PKR mRNA, protein, and phosphorylation are increased in insulin-resistant muscles. When PKR activity is reduced (siRNA or a pharmacological inhibitor), serine phosphorylation of IRS1 is reduced, and insulin-induced phosphorylation of Akt is improved. Finally, we show that JNK mediates ceramide-activated PKR inhibitory action on IRS1. Together, in the long term, our results show that ceramide acts at two distinct levels of the insulin signaling pathway (IRS1 and Akt). PKR, which is induced by both inflammation signals and ceramide, could play a major role in the development of insulin resistance in muscle cells.

Kidney Dysfunction in Adult Offspring Exposed In Utero to Type 1 Diabetes Is Associated with Alterations in Genome-Wide DNA Methylation.

BACKGROUND: Fetal exposure to hyperglycemia impacts negatively kidney development and function. OBJECTIVE: Our objective was to determine whether fetal exposure to moderate hyperglycemia is associated with epigenetic alterations in DNA methylation in peripheral blood cells and whether those alterations are related to impaired kidney function in adult offspring. DESIGN: Twenty nine adult, non-diabetic offspring of mothers with type 1 diabetes (T1D) (case group) were matched with 28 offspring of T1D fathers (control group) for the study of their leukocyte genome-wide DNA methylation profile (27,578 CpG sites, Human Methylation 27 BeadChip, Illumina Infinium). In a subset of 19 cases and 18 controls, we assessed renal vascular development by measuring Glomerular Filtration Rate (GFR) and Effective Renal Plasma Flow (ERPF) at baseline and during vasodilatation produced by amino acid infusion. RESULTS: Globally, DNA was under-methylated in cases vs. controls. Among the 87 CpG sites differently methylated, 74 sites were less methylated and 13 sites more methylated in cases vs. controls. None of these CpG sites were located on a gene known to be directly involved in kidney development and/or function. However, the gene encoding DNA methyltransferase 1 (DNMT1)--a key enzyme involved in gene expression during early development--was under-methylated in cases. The average methylation of the 74 under-methylated sites differently correlated with GFR in cases and controls. CONCLUSION: Alterations in methylation profile imprinted by the hyperglycemic milieu of T1D mothers during fetal development may impact kidney function in adult offspring. The involved pathways seem to be a nonspecific imprinting process rather than specific to kidney development or function.

Characterising the inhibitory actions of ceramide upon insulin signaling in different skeletal muscle cell models: a mechanistic insight.

Ceramides are known to promote insulin resistance in a number of metabolically important tissues including skeletal muscle, the predominant site of insulin-stimulated glucose disposal. Depending on cell type, these lipid intermediates have been shown to inhibit protein kinase B (PKB/Akt), a key mediator of the metabolic actions of insulin, via two distinct pathways: one involving the action of atypical protein kinase C (aPKC) isoforms, and the second dependent on protein phosphatase-2A (PP2A). The main aim of this study was to explore the mechanisms by which ceramide inhibits PKB/Akt in three different skeletal muscle-derived cell culture models; rat L6 myotubes, mouse C2C12 myotubes and primary human skeletal muscle cells. Our findings indicate that the mechanism by which ceramide acts to repress PKB/Akt is related to the myocellular abundance of caveolin-enriched domains (CEM) present at the plasma membrane. Here, we show that ceramide-enriched-CEMs are markedly more abundant in L6 myotubes compared to C2C12 myotubes, consistent with their previously reported role in coordinating aPKC-directed repression of PKB/Akt in L6 muscle cells. In contrast, a PP2A-dependent pathway predominantly mediates ceramide-induced inhibition of PKB/Akt in C2C12 myotubes. In addition, we demonstrate for the first time that ceramide engages an aPKC-dependent pathway to suppress insulin-induced PKB/Akt activation in palmitate-treated cultured human muscle cells as well as in muscle cells from diabetic patients. Collectively, this work identifies key mechanistic differences, which may be linked to variations in plasma membrane composition, underlying the insulin-desensitising effects of ceramide in different skeletal muscle cell models that are extensively used in signal transduction and metabolic studies.

Liver X receptor: from metabolism to cancer.

Cholesterol plays an indispensable role in regulating the properties of cell membranes in mammalian cells. Accumulation of cholesterol and its intermediates, such as oxysterols, lead to activation of the nuclear receptors LXRs (liver X receptors). LXR is an important regulator of cholesterol homoeostasis by controlling its transport and its neo-synthesis. Accumulating evidence indicates that the endogenous ligands of LXRs, oxysterols, play an active and important role in regulating the fate and function of immune cells. Indeed, LXRs are negative regu-lators of innate immunity by interfering with macrophage activation. Recent advances have highlighted a controversial role for LXR in cancer. In this issue of the Biochemical Journal, Wang et al. propose that LXR agonist directly controls IFN-γ (interferon-γ) expression, which limits tumour growth. This protective effect mediated by LXR appears to be dependent on IFN-γ. Thus, despite accumulation of endogenous ligand of LXR in cancer, activation of LXR seems protective. This novel evidence provides a new perspective for targeting LXR in cancer, although controversial studies can be also found in the literature. In order to avoid side effects associated with LXR agonists, molecular and cellular studies are required to decipher this unexpected action of LXRs.

Fetal PGC-1α overexpression programs adult pancreatic ß-cell dysfunction.

Adult ß-cell dysfunction, a hallmark of type 2 diabetes, can be programmed by adverse fetal environment. We have shown that fetal glucocorticoids (GCs) participate in this programming through inhibition of ß-cell development. Here we have investigated the molecular mechanisms underlying this regulation. We showed that GCs stimulate the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a coregulator of the GCs receptor (GR), and that the overexpression of PGC-1α represses genes important for ß-cell development and function. More precisely, PGC-1α inhibited the expression of the key ß-cell transcription factor pancreatic duodenal homeobox 1 (Pdx1). This repression required the GR and was mediated through binding of a GR/PGC-1α complex to the Pdx1 promoter. To explore PGC-1α function, we generated mice with inducible ß-cell PGC-1α overexpression. Mice overexpressing PGC-1α exhibited at adult age impaired glucose tolerance associated with reduced insulin secretion, decreased ß-cell mass, and ß-cell hypotrophy. Interestingly, PGC-1α expression in fetal life only was sufficient to impair adult ß-cell function whereas ß-cell PGC-1α overexpression from adult age had no consequence on ß-cell function. Altogether, our results demonstrate that the GR and PGC-1α participate in the fetal programming of adult ß-cell function through inhibition of Pdx1 expression.