Ceramides Increase Fatty Acid Utilization in Intestinal Progenitors to Enhance Stemness and Increase Tumor Risk.

BACKGROUND & AIMS: Cancers of the alimentary tract, including esophageal adenocarcinomas, colorectal cancers, and cancers of the gastric cardia, are common comorbidities of obesity. Prolonged, excessive delivery of macronutrients to the cells lining the gut can increase one's risk for these cancers by inducing imbalances in the rate of intestinal stem cell proliferation vs differentiation, which can produce polyps and other aberrant growths. We investigated whether ceramides, which are sphingolipids that serve as a signal of nutritional excess, alter stem cell behaviors to influence cancer risk. METHODS: We profiled sphingolipids and sphingolipid-synthesizing enzymes in human adenomas and tumors. Thereafter, we manipulated expression of sphingolipid-producing enzymes, including serine palmitoyltransferase (SPT), in intestinal progenitors of mice, cultured organoids, and Drosophila to discern whether sphingolipids altered stem cell proliferation and metabolism. RESULTS: SPT, which diverts dietary fatty acids and amino acids into the biosynthetic pathway that produces ceramides and other sphingolipids, is a critical modulator of intestinal stem cell homeostasis. SPT and other enzymes in the sphingolipid biosynthesis pathway are up-regulated in human intestinal adenomas. They produce ceramides, which serve as prostemness signals that stimulate peroxisome-proliferator activated receptor-α and induce fatty acid binding protein-1. These actions lead to increased lipid utilization and enhanced proliferation of intestinal progenitors. CONCLUSIONS: Ceramides serve as critical links between dietary macronutrients, epithelial regeneration, and cancer risk.

Targeting a ceramide double bond improves insulin resistance and hepatic steatosis.

Ceramides contribute to the lipotoxicity that underlies diabetes, hepatic steatosis, and heart disease. By genetically engineering mice, we deleted the enzyme dihydroceramide desaturase 1 (DES1), which normally inserts a conserved double bond into the backbone of ceramides and other predominant sphingolipids. Ablation of DES1 from whole animals or tissue-specific deletion in the liver and/or adipose tissue resolved hepatic steatosis and insulin resistance in mice caused by leptin deficiency or obesogenic diets. Mechanistic studies revealed ceramide actions that promoted lipid uptake and storage and impaired glucose utilization, none of which could be recapitulated by (dihydro)ceramides that lacked the critical double bond. These studies suggest that inhibition of DES1 may provide a means of treating hepatic steatosis and metabolic disorders.

Short chain fatty acids induce UCP2-mediated autophagy in hepatic cells.

Short-chain fatty acids (SCFAs) are gut microbial fermentation products derived from dietary fiber sources. Although depletion of gut microflora has been linked to the development of liver disease, the direct effects of SCFAs on intracellular hepatic processes are not well understood. In this study, we demonstrated that the SCFAs, propionate and butyrate, regulated autophagic flux in hepatic cells in a cell-autonomous manner. Induction of autophagy by SCFAs required PPARγ stimulation of Uncoupling Protein 2 (UCP2) expression that was associated with reduced intracellular ATP levels and activation of PRKAA1/AMPK (protein kinase, AMP-activated, alpha 1 catalytic subunit). In addition, elimination of gut flora by chronic antibiotic treatment diminished basal hepatic autophagy in mice suggesting that gut microbiota can regulate hepatic autophagy. These findings provide novel insights into the interplay between diet, gut microbiota, short chain fatty acids, and hepatic autophagic signaling.

Adipocyte Ceramides Regulate Subcutaneous Adipose Browning, Inflammation, and Metabolism.

Adipocytes package incoming fatty acids into triglycerides and other glycerolipids, with only a fraction spilling into a parallel biosynthetic pathway that produces sphingolipids. Herein, we demonstrate that subcutaneous adipose tissue of type 2 diabetics contains considerably more sphingolipids than non-diabetic, BMI-matched counterparts. Whole-body and adipose tissue-specific inhibition/deletion of serine palmitoyltransferase (Sptlc), the first enzyme in the sphingolipid biosynthesis cascade, in mice markedly altered adipose morphology and metabolism, particularly in subcutaneous adipose tissue. The reduction in adipose sphingolipids increased brown and beige/brite adipocyte numbers, mitochondrial activity, and insulin sensitivity. The manipulation also increased numbers of anti-inflammatory M2 macrophages in the adipose bed and induced secretion of insulin-sensitizing adipokines. By comparison, deletion of serine palmitoyltransferase from macrophages had no discernible effects on metabolic homeostasis or adipose function. These data indicate that newly synthesized adipocyte sphingolipids are nutrient signals that drive changes in the adipose phenotype to influence whole-body energy expenditure and nutrient metabolism.

A Role for Ceramides, but Not Sphingomyelins, as Antagonists of Insulin Signaling and Mitochondrial Metabolism in C2C12 Myotubes.

The accumulation of sphingolipids in obesity leads to impairments in insulin sensitivity and mitochondrial metabolism, but the precise species driving these defects is unclear. We have modeled these obesity-induced effects in cultured C2C12 myotubes, using BSA-conjugated palmitate to increase synthesis of endogenous sphingolipids and to inhibit insulin signaling and oxidative phosphorylation. Palmitate (a) induced the accumulation of sphingomyelin (SM) precursors such as sphinganine, dihydroceramide, and ceramide; (b) inhibited insulin stimulation of a central modulator of anabolic metabolism, Akt/PKB; (c) inhibited insulin-stimulated glycogen synthesis; and (d) decreased oxygen consumption and ATP synthesis. Under these conditions, palmitate failed to alter levels of SMs, which are the most abundant sphingolipids, suggesting that they are not the primary intermediates accounting for the deleterious palmitate effects. Treating cells with a pharmacological inhibitor of SM synthase or using CRISPR to knock out the Sms2 gene recapitulated the palmitate effects by inducing the accumulation of SM precursors and impairing insulin signaling and mitochondrial metabolism. To profile the sphingolipids that accumulate in obesity, we performed lipidomics on quadriceps muscles from obese mice with impaired glucose tolerance. Like the cultured myotubes, these tissues accumulated ceramides but not SMs. Collectively, these data suggest that SM precursors such as ceramides, rather than SMs, are likely nutritional antagonists of metabolic function in skeletal muscle.

Dihydroceramides: From Bit Players to Lead Actors.

Sphingolipid synthesis involves a highly conserved biosynthetic pathway that produces fundamental precursors of complex sphingolipids. The final reaction involves the insertion of a double bond into dihydroceramides to generate the more abundant ceramides, which are converted to sphingomyelins and glucosylceramides/gangliosides by the addition of polar head groups. Although ceramides have long been known to mediate cellular stress responses, the dihydroceramides that are transiently produced during de novo sphingolipid synthesis were deemed inert. Evidence published in the last few years suggests that these dihydroceramides accumulate to a far greater extent in tissues than previously thought. Moreover, they have biological functions that are distinct and non-overlapping with those of the more prevalent ceramides. Roles are being uncovered in autophagy, hypoxia, and cellular proliferation, and the lipids are now implicated in the etiology, treatment, and/or diagnosis of diabetes, cancer, ischemia/reperfusion injury, and neurodegenerative diseases. This minireview summarizes recent findings on this emerging class of bioactive lipids.

Role of seipin in lipid droplet morphology and hepatitis C virus life cycle.

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

PTEN protein phosphatase activity regulates hepatitis C virus secretion through modulation of cholesterol metabolism.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection is dependent on lipid metabolism. Hepatocyte steatosis occurs frequently in HCV infection, but the relationship between steatosis and HCV life cycle is unclear. We showed that HCV induces steatosis via the downregulation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN). We here investigated how PTEN may affect HCV production. METHODS: The effect of overexpression or silencing of PTEN on HCV secretion was assessed in genomic-length Jc1 infected HuH7 cells. The role of PTEN protein and lipid phosphatase activities on lipid metabolism and infectious viral particle secretion was investigated using dominant-negative PTEN mutants. The importance of cholesterol metabolism for PTEN-dependent lipid droplet biogenesis and viral particle secretion was examined using statins. RESULTS: PTEN silencing in Jc1 infected HuH7 cells stimulated HCV particle secretion, while PTEN overexpression decreased virus egress. Viral secretion was also increased by overexpression of protein phosphatase-deleted (PTENY138L), but not lipid phosphatase-deleted (PTENG129E), PTEN mutant, thus indicating that the protein phosphatase activity of PTEN controls viral secretion. Similarly, PTENY138L, but not PTENG129E mutant induced the formation of large lipid droplets. PTENY138L mutant did not affect biosynthesis of triglycerides, but promoted the biosynthesis of cholesterol esters. Consistently, statins prevented the increased cholesterol ester production, large lipid droplet formation, and viral secretion in cells expressing the PTENY138L mutant. CONCLUSIONS: Downregulation of PTEN protein phosphatase activity by HCV affects cholesterol metabolism, thereby inducing the appearance of large lipid droplets and increasing virion egress.

Current understanding of insulin resistance in hepatitis C.

Important breakthroughs have been made in recent years into understanding the close interaction between hepatitis C virus (HCV) infection and glucose homeostasis. Both cross-sectional and longitudinal studies have demonstrated that infection with HCV is associated with an increased risk of developing insulin resistance and Type 2 diabetes. A direct effect of HCV on hepatocyte insulin signaling has been shown in experimental models. Some preliminary observations seem to suggest that indirect mechanisms involving extrahepatic organs might also play a role. The interaction between HCV and glucose metabolism has significant clinical consequences. Insulin resistance and Type 2 diabetes not only accelerate the histological and clinical progression of chronic hepatitis C, but also reduce the virological response to IFN-α-based therapy. Thus, understanding the mechanisms underlying HCV-associated glucose metabolism derangements is of paramount interest in order to improve the clinical management of chronic hepatitis C. This article will focus on the studies that consistently argue in favor of an interrelation between HCV and insulin resistance and will highlight the latest discoveries in this field.

Viral genotype-specific role of PNPLA3, PPARG, MTTP, and IL28B in hepatitis C virus-associated steatosis.

BACKGROUND & AIMS: Steatosis is a prominent feature of hepatitis C, especially in patients infected with genotype 3. The analysis of genetic polymorphisms influencing steatosis in chronic hepatitis C has been limited by the studies' small sample size, and important single nucleotide polymorphisms (SNPs), such as those in the patatin-like phospholipase family 3 protein (PNPLA3), were never evaluated. METHODS: We analyzed the role of SNPs, from 19 systematically selected candidate genes, on steatosis in 626 Caucasian hepatitis C virus (HCV) infected patients. SNPs were extracted from a genome-wide association-generated dataset. Associations of alleles with the presence and/or different severity of steatosis were evaluated by univariate and multivariate logistic regression, accounting for all relevant covariates. RESULTS: The risk of steatosis was increased by carriage of I148M in PNPLA3, but only in patients with HCV genotypes non-3 (odds ratio [OR]=1.9, 95% confidence interval [CI]=1.6-2.3, p<0.001) and similar, albeit weaker associations were found for SNPs in peroxisome proliferator-activated receptor-γ (PPARG) and interleukin-28B (IL28B). Carriage of a SNP in the microsomal triglyceride transfer protein (MTTP) increased the risk of steatosis, but only in patients with HCV genotype 3 (rs1800803, OR=3.4, 95% CI=2.4-4.9, p=0.001). CONCLUSIONS: The rs738409 SNP in PNPLA3 is associated with an increased risk of steatosis in patients infected with HCV genotypes non-3. Host genes affect steatosis depending on the infecting HCV genotype, suggesting their interaction with viral factors.

Rab4b is a small GTPase involved in the control of the glucose transporter GLUT4 localization in adipocyte.

BACKGROUND: Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport. METHODOLOGY/PRINCIPAL FINDINGS: We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment. CONCLUSION/SIGNIFICANCE: Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes.

The Rab4 effector Rabip4 plays a role in the endocytotic trafficking of Glut 4 in 3T3-L1 adipocytes.

Insulin regulates glucose uptake in the adipocytes by modulating Glut 4 localization, a traffic pathway involving the endocytic small GTPases Rab4, Rab5, and RabThe expression of the Rab4 effector Rabip4 leads to a 30% increase in glucose uptake and Glut 4 translocation in the presence of insulin, without modifications in the basal condition. This effect was not due to modifications of Glut 4 expression or insulin signaling, suggesting that Rabip4 controls Glut 4 trafficking. We present evidence that Rabip4 defines a subdomain of early endosomes and that Rabip4 is redistributed to the plasma membrane by insulin. Rabip4 is mostly absent from structures positive for early endosome antigen 1, Rab11 or transferrin receptors and from Glut 4 sequestration compartments. However, Rabip4 vesicles can be reached by internalized transferrin and Glut 4. Thus, Rabip4 probably defines an endocytic sorting platform for Glut 4 towards its sequestration pool. The expression of a form of Rabip4 unable to bind Rab4 does not modify basal and insulin-induced glucose transport. However, it induces an increase in the amount of Glut 4 at the plasma membrane and perturbs Glut 4 traffic from endosomes towards its sequestration compartments. These observations suggest that the uncoupling between Rabip4 and Rab4 induces the insertion of Glut 4 molecules that are unable to transport glucose into the plasma membrane.

Helicobacter pylori VacA cytotoxin: a probe for a clathrin-independent and Cdc42-dependent pinocytic pathway routed to late endosomes.

The vacuolating cytotoxin VacA is a major virulence factor of Helicobacter pylori, a bacterium responsible for gastroduodenal ulcers and cancer. VacA associates with lipid rafts, is endocytosed, and reaches the late endocytic compartment where it induces vacuolation. We have investigated the endocytic and intracellular trafficking pathways used by VacA, in HeLa and gastric AGS cells. We report here that VacA was first bound to plasma-membrane domains localized above F-actin structures that were controlled by the Rac1 GTPase. VacA was subsequently pinocytosed by a clathrin-independent mechanism into cell peripheral early endocytic compartments lacking caveolin 1, the Rab5 effector early endosomes antigen-1 (EEA1) and transferrin. These compartments took up fluid-phase (as evidenced by the accumulation of fluorescent dextran) and glycosylphosphatidylinositol-anchored proteins (GPI-APs). VacA pinocytosis was controlled by Cdc42 and did not require cellular tyrosine kinases, dynamin 2, ADP-ribosylating factor 6, or RhoA GTPase activities. VacA was subsequently routed to EEA1-sorting endosomes and then sorted to late endosomes. During all these different endocytic steps, VacA was continuously associated with detergent resistant membrane domains. From these results we propose that VacA might be a valuable probe to study raft-associated molecules, pinocytosed by a clathrin-independent mechanism, and routed to the degradative compartment.

CD2AP, Rabip4, and Rabip4': analysis of interaction with Rab4a and regulation of endosomes morphology.

In this chapter, we describe various approaches that allow us to study interactions between the small GTPase Rab4a and its two effectors, Rabip4 and CD2AP. Two complementary approaches, one using the yeast two-hybrid system and the other using a GST pull-down assay, are described. We document the studies of the localization of these proteins by cellular fractionation. Finally, we develop cellular imaging techniques to study the morphology of vesicular structures containing Rab4a. We show that the coexpression of Rab4a with its effectors affects Rab4a-containing structures, giving a clear indication of their interaction in the mammalian cellular context.