Lysophosphatidylinositols Are Upregulated After Human ß-Cell Loss and Potentiate Insulin Release.

In this study, we identified new lipid species associated with the loss of pancreatic ß-cells triggering diabetes. We performed lipidomics measurements on serum from prediabetic mice lacking ß-cell prohibitin-2 (a model of monogenic diabetes) patients without previous history of diabetes but scheduled for pancreaticoduodenectomy resulting in the acute reduction of their ß-cell mass (∼50%), and patients with type 2 diabetes (T2D). We found lysophosphatidylinositols (lysoPIs) were the main circulating lipid species altered in prediabetic mice. The changes were confirmed in the patients with acute reduction of their ß-cell mass and in those with T2D. Increased lysoPIs significantly correlated with HbA1c (reflecting glycemic control), fasting glycemia, and disposition index, and did not correlate with insulin resistance or obesity in human patients with T2D. INS-1E ß-cells as well as pancreatic islets isolated from nondiabetic mice and human donors exposed to exogenous lysoPIs showed potentiated glucose-stimulated and basal insulin secretion. Finally, addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. Overall, lysoPIs appear to be lipid species upregulated in the prediabetic stage associated with the loss of ß-cells and that support the secretory function of the remaining ß-cells. ARTICLE HIGHLIGHTS: Circulating lysophosphatidylinositols (lysoPIs) are increased in situations associated with ß-cell loss in mice and humans such as (pre-)diabetes, and hemipancreatectomy. Pancreatic islets isolated from nondiabetic mice and human donors, as well as INS-1E ß-cells, exposed to exogenous lysoPIs exhibited potentiated glucose-stimulated and basal insulin secretion. Addition of exogenous lysoPIs partially rescued impaired glucose-stimulated insulin secretion in islets from mice and humans in the diabetic state. LysoPIs appear as lipid species being upregulated already in the prediabetic stage associated with the loss of ß-cells and supporting the function of the remaining ß-cells.

Type 2 diabetes disrupts circadian orchestration of lipid metabolism and membrane fluidity in human pancreatic islets.

Recent evidence suggests that circadian clocks ensure temporal orchestration of lipid homeostasis and play a role in pathophysiology of metabolic diseases in humans, including type 2 diabetes (T2D). Nevertheless, circadian regulation of lipid metabolism in human pancreatic islets has not been explored. Employing lipidomic analyses, we conducted temporal profiling in human pancreatic islets derived from 10 nondiabetic (ND) and 6 T2D donors. Among 329 detected lipid species across 8 major lipid classes, 5% exhibited circadian rhythmicity in ND human islets synchronized in vitro. Two-time point-based lipidomic analyses in T2D human islets revealed global and temporal alterations in phospho- and sphingolipids. Key enzymes regulating turnover of sphingolipids were rhythmically expressed in ND islets and exhibited altered levels in ND islets bearing disrupted clocks and in T2D islets. Strikingly, cellular membrane fluidity, measured by a Nile Red derivative NR12S, was reduced in plasma membrane of T2D diabetic human islets, in ND donors' islets with disrupted circadian clockwork, or treated with sphingolipid pathway modulators. Moreover, inhibiting the glycosphingolipid biosynthesis led to strong reduction of insulin secretion triggered by glucose or KCl, whereas inhibiting earlier steps of de novo ceramide synthesis resulted in milder inhibitory effect on insulin secretion by ND islets. Our data suggest that circadian clocks operative in human pancreatic islets are required for temporal orchestration of lipid homeostasis, and that perturbation of temporal regulation of the islet lipid metabolism upon T2D leads to altered insulin secretion and membrane fluidity. These phenotypes were recapitulated in ND islets bearing disrupted clocks.

The GLP-1R agonist liraglutide limits hepatic lipotoxicity and inflammatory response in mice fed a methionine-choline deficient diet.

Nonalcoholic fatty liver disease (NAFLD) is the most common hepatic disorder related to type 2 diabetes (T2D). The disease can evolve toward nonalcoholic steatohepatitis (NASH), a state of hepatic inflammation and fibrosis. There is presently no drug that effectively improves and/or prevents NAFLD/NASH/fibrosis. GLP-1 receptor agonists (GLP-1Ra) are effective in treating T2D. As with the endogenous gut incretins, GLP-1Ra potentiate glucose-induced insulin secretion. In addition, GLP-1Ra limit food intake and weight gain, additional beneficial properties in the context of obesity/insulin-resistance. Nevertheless, these pleiotropic effects of GLP-1Ra complicate the elucidation of their direct action on the liver. In the present study, we used the classical methionine-choline deficient (MCD) dietary model to investigate the potential direct hepatic actions of the GLP-1Ra liraglutide. A 4-week infusion of liraglutide (570 µg/kg/day) did not impact body weight, fat accretion or glycemic control in MCD-diet fed mice, confirming the suitability of this model for avoiding confounding factors. Liraglutide treatment did not prevent lipid deposition in the liver of MCD-fed mice but limited the accumulation of C16 and C24-ceramide/sphingomyelin species. In addition, liraglutide treatment alleviated hepatic inflammation (in particular accumulation of M1 pro-inflammatory macrophages) and initiation of fibrosis. Liraglutide also influenced the composition of gut microbiota induced by the MCD-diet. This included recovery of a normal Bacteroides proportion and, among the Erysipelotrichaceae family, a shift between Allobaculum and Turicibacter genera. In conclusion, liraglutide prevents accumulation of C16 and C24-ceramides/sphingomyelins species, inflammation and initiation of fibrosis in MCD-diet-fed mice liver, suggesting beneficial hepatic actions independent of weight loss and global hepatic steatosis.

Ether lipids, sphingolipids and toxic 1-deoxyceramides as hallmarks for lean and obese type 2 diabetic patients.

AIM: The worldwide increase in obesity and type 2 diabetes (T2D) represents a major health challenge. Chronically altered lipids induced by obesity further promote the development of T2D, and the accumulation of toxic lipid metabolites in serum and peripheral organs may contribute to the diabetic phenotype. METHODS: To better understand the complex metabolic pattern of lean and obese T2D and non-T2D individuals, we analysed the lipid profile of human serum, skeletal muscle and visceral adipose tissue of two cohorts by systematic mass spectrometry-based lipid analysis. RESULTS: Lipid homeostasis was strongly altered in a disease- and tissue-specific manner, allowing us to define T2D signatures associated with obesity from those that were obesity independent. Lipid changes encompassed lyso-, diacyl- and ether-phospholipids. Moreover, strong changes in sphingolipids included cytotoxic 1-deoxyceramide accumulation in a disease-specific manner in serum and visceral adipose tissue. The high amounts of non-canonical 1-deoxyceramide present in human adipose tissue most likely come from cell-autonomous synthesis because 1-deoxyceramide production increased upon differentiation to adipocytes in mouse cell culture experiments. CONCLUSION: Taken together, the observed lipidome changes in obesity and T2D will facilitate the identification of T2D patient subgroups and represent an important step towards personalized medicine in diabetes.

Circadian Lipidomics: Analysis of Lipid Metabolites Around the Clock.

Lipidomics has been defined as the large-scale analysis of lipids in organelles, cells, tissues, or whole organisms. Including the temporal aspects of lipid metabolic changes into this analysis allows to access yet another important aspect of lipid regulation. The resulting methodology, circadian lipidomics, has thus emerged as a novel tool to address the enormous complexity, which is present among cellular lipids. Here, we describe how mass spectrometry-based circadian lipidomics can be applied to study the impact of peripheral clocks on lipid metabolism in human primary cells and tissues, exemplified by studies in human pancreatic islets and skeletal myotubes.

Multi-technique comparison of atherogenic and MCD NASH models highlights changes in sphingolipid metabolism.

Lipotoxicity is a key player in the pathogenesis of nonalcoholic steatohepatitis (NASH), a progressive subtype of nonalcoholic fatty liver disease (NAFLD). In the present study, we combine histological, transcriptional and lipidomic approaches to dissociate common and specific alterations induced by two classical dietary NASH models (atherogenic (ATH) and methionine/choline deficient (MCD) diet) in C57BL/6J male mice. Despite a similar degree of steatosis, MCD-fed mice showed more pronounced liver damage and a worsened pro-inflammatory and pro-fibrogenic environment than ATH-fed mice. Regarding lipid metabolism, the ATH diet triggered hepatic counter regulatory mechanisms, while the MCD diet worsened liver lipid accumulation by a concomitant increase in lipid import and reduction in lipid export. Liver lipidomics revealed sphingolipid enrichment in both NASH models that was accompanied by an upregulation of the ceramide biosynthesis pathway and a significant rise in dihydroceramide levels. In contrast, the phospholipid composition was not substantially altered by the ATH diet, whereas the livers of MCD-fed mice presented a reduced phosphatidylcholine to phosphatidylethanolamine (PC/PE) ratio and a strong depletion in phospholipids containing the sum of 34-36 carbons in their fatty acid chains. Therefore, the assessment of liver damage at the histological and transcriptional level combined with a lipidomic analysis reveals sphingolipids as shared mediators in liver lipotoxicity and pathogenesis of NASH.

Transcriptomic analyses reveal rhythmic and CLOCK-driven pathways in human skeletal muscle.

Circadian regulation of transcriptional processes has a broad impact on cell metabolism. Here, we compared the diurnal transcriptome of human skeletal muscle conducted on serial muscle biopsies in vivo with profiles of human skeletal myotubes synchronized in vitro. More extensive rhythmic transcription was observed in human skeletal muscle compared to in vitro cell culture as a large part of the in vivo mRNA rhythmicity was lost in vitro. siRNA-mediated clock disruption in primary myotubes significantly affected the expression of ~8% of all genes, with impact on glucose homeostasis and lipid metabolism. Genes involved in GLUT4 expression, translocation and recycling were negatively affected, whereas lipid metabolic genes were altered to promote activation of lipid utilization. Moreover, basal and insulin-stimulated glucose uptake were significantly reduced upon CLOCK depletion. Our findings suggest an essential role for the circadian coordination of skeletal muscle glucose homeostasis and lipid metabolism in humans.

Mitochondrial disruption in peroxisome deficient cells is hepatocyte selective but is not mediated by common hepatic peroxisomal metabolites.

The structural disruption of the mitochondrial inner membrane in hepatocytes lacking functional peroxisomes along with selective impairment of respiratory complexes and depletion of mitochondrial DNA was previously reported. In search for the molecular origin of these mitochondrial alterations, we here show that these are tissue selective as they do neither occur in peroxisome deficient brain nor in peroxisome deficient striated muscle. Given the hepatocyte selectivity, we investigated the potential involvement of metabolites that are primarily handled by hepatic peroxisomes. Levels of these metabolites were manipulated in L-Pex5 knockout mice and/or compared with levels in different mouse models with a peroxisomal ß-oxidation deficiency. We show that neither the deficiency of docosahexaenoic acid nor the accumulation of branched chain fatty acids, dicarboxylic acids or C27 bile acid intermediates are solely responsible for the mitochondrial anomalies. In conclusion, we demonstrate that peroxisomal inactivity differentially impacts mitochondria depending on the cell type but the cause of the mitochondrial destruction needs to be further explored.

Lipidomics reveals diurnal lipid oscillations in human skeletal muscle persisting in cellular myotubes cultured in vitro.

Circadian clocks play an important role in lipid homeostasis, with impact on various metabolic diseases. Due to the central role of skeletal muscle in whole-body metabolism, we aimed at studying muscle lipid profiles in a temporal manner. Moreover, it has not been shown whether lipid oscillations in peripheral tissues are driven by diurnal cycles of rest-activity and food intake or are able to persist in vitro in a cell-autonomous manner. To address this, we investigated lipid profiles over 24 h in human skeletal muscle in vivo and in primary human myotubes cultured in vitro. Glycerolipids, glycerophospholipids, and sphingolipids exhibited diurnal oscillations, suggesting a widespread circadian impact on muscle lipid metabolism. Notably, peak levels of lipid accumulation were in phase coherence with core clock gene expression in vivo and in vitro. The percentage of oscillating lipid metabolites was comparable between muscle tissue and cultured myotubes, and temporal lipid profiles correlated with transcript profiles of genes implicated in their biosynthesis. Lipids enriched in the outer leaflet of the plasma membrane oscillated in a highly coordinated manner in vivo and in vitro. Lipid metabolite oscillations were strongly attenuated upon siRNA-mediated clock disruption in human primary myotubes. Taken together, our data suggest an essential role for endogenous cell-autonomous human skeletal muscle oscillators in regulating lipid metabolism independent of external synchronizers, such as physical activity or food intake.

Glucose Homeostasis: Regulation by Peripheral Circadian Clocks in Rodents and Humans.

Most organisms, including humans, have developed an intrinsic system of circadian oscillators, allowing the anticipation of events related to the rotation of Earth around its own axis. The mammalian circadian timing system orchestrates nearly all aspects of physiology and behavior. Together with systemic signals, emanating from the central clock that resides in the hypothalamus, peripheral oscillators orchestrate tissue-specific fluctuations in gene expression, protein synthesis, and posttranslational modifications, driving overt rhythms in physiology and behavior. There is increasing evidence on the essential roles of the peripheral oscillators, operative in metabolically active organs in the regulation of body glucose homeostasis. Here, we review some recent findings on the molecular and cellular makeup of the circadian timing system and its implications in the temporal coordination of metabolism in health and disease.

Paraoxonase 1 (PON1) and pomegranate influence circadian gene expression and period length.

The circadian timing system regulates key aspects of mammalian physiology. Here, we analyzed the effect of the endogenous antioxidant paraoxonase 1 (PON1), a high-density lipoprotein-associated lipolactonase that hydrolyses lipid peroxides and attenuates atherogenesis, on circadian gene expression in C57BL/6J and PON1KO mice fed a normal chow diet or a high-fat diet (HFD). Expression levels of core-clock transcripts Nr1d1, Per2, Cry2 and Bmal1 were altered in skeletal muscle in PON1-deficient mice in response to HFD. These findings were supported by circadian bioluminescence reporter assessments in mouse C2C12 and human primary myotubes, synchronized in vitro, where administration of PON1 or pomegranate juice modulated circadian period length.

Human skeletal myotubes display a cell-autonomous circadian clock implicated in basal myokine secretion.

OBJECTIVE: Circadian clocks are functional in all light-sensitive organisms, allowing an adaptation to the external world in anticipation of daily environmental changes. In view of the potential role of the skeletal muscle clock in the regulation of glucose metabolism, we aimed to characterize circadian rhythms in primary human skeletal myotubes and investigate their roles in myokine secretion. METHODS: We established a system for long-term bioluminescence recording in differentiated human myotubes, employing lentivector gene delivery of the Bmal1-luciferase and Per2-luciferase core clock reporters. Furthermore, we disrupted the circadian clock in skeletal muscle cells by transfecting siRNA targeting CLOCK. Next, we assessed the basal secretion of a large panel of myokines in a circadian manner in the presence or absence of a functional clock. RESULTS: Bioluminescence reporter assays revealed that human skeletal myotubes, synchronized in vitro, exhibit a self-sustained circadian rhythm, which was further confirmed by endogenous core clock transcript expression. Moreover, we demonstrate that the basal secretion of IL-6, IL-8 and MCP-1 by synchronized skeletal myotubes has a circadian profile. Importantly, the secretion of IL-6 and several additional myokines was strongly downregulated upon siClock-mediated clock disruption. CONCLUSIONS: Our study provides for the first time evidence that primary human skeletal myotubes possess a high-amplitude cell-autonomous circadian clock, which could be attenuated. Furthermore, this oscillator plays an important role in the regulation of basal myokine secretion by skeletal myotubes.

LAPTM4B facilitates late endosomal ceramide export to control cell death pathways.

Lysosome-associated protein transmembrane-4b (LAPTM4B) associates with poor prognosis in several cancers, but its physiological function is not well understood. Here we use novel ceramide probes to provide evidence that LAPTM4B interacts with ceramide and facilitates its removal from late endosomal organelles (LEs). This lowers LE ceramide in parallel with and independent of acid ceramidase-dependent catabolism. In LAPTM4B-silenced cells, LE sphingolipid accumulation is accompanied by lysosomal membrane destabilization. However, these cells resist ceramide-driven caspase-3 activation and apoptosis induced by chemotherapeutic agents or gene silencing. Conversely, LAPTM4B overexpression reduces LE ceramide and stabilizes lysosomes but sensitizes to drug-induced caspase-3 activation. Together, these data uncover a cellular ceramide export route from LEs and identify LAPTM4B as its regulator. By compartmentalizing ceramide, LAPTM4B controls key sphingolipid-mediated cell death mechanisms and emerges as a candidate for sphingolipid-targeting cancer therapies.

HCV 3a core protein increases lipid droplet cholesteryl ester content via a mechanism dependent on sphingolipid biosynthesis.

Hepatitis C virus (HCV) infected patients often develop steatosis and the HCV core protein alone can induce this phenomenon. To gain new insights into the pathways leading to steatosis, we performed lipidomic profiling of HCV core protein expressing-Huh-7 cells and also assessed the lipid profile of purified lipid droplets isolated from HCV 3a core expressing cells. Cholesteryl esters, ceramides and glycosylceramides, but not triglycerides, increased specifically in cells expressing the steatogenic HCV 3a core protein. Accordingly, inhibitors of cholesteryl ester biosynthesis such as statins and acyl-CoA cholesterol acyl transferase inhibitors prevented the increase of cholesteryl ester production and the formation of large lipid droplets in HCV core 3a-expressing cells. Furthermore, inhibition of de novo sphingolipid biosynthesis by myriocin - but not of glycosphingolipid biosynthesis by miglustat - affected both lipid droplet size and cholesteryl ester level. The lipid profile of purified lipid droplets, isolated from HCV 3a core-expressing cells, confirmed the particular increase of cholesteryl ester. Thus, both sphingolipid and cholesteryl ester biosynthesis are affected by the steatogenic core protein of HCV genotype 3a. These results may explain the peculiar lipid profile of HCV-infected patients with steatosis.

The peroxisomal enzyme L-PBE is required to prevent the dietary toxicity of medium-chain fatty acids.

Specific metabolic pathways are activated by different nutrients to adapt the organism to available resources. Although essential, these mechanisms are incompletely defined. Here, we report that medium-chain fatty acids contained in coconut oil, a major source of dietary fat, induce the liver ω-oxidation genes Cyp4a10 and Cyp4a14 to increase the production of dicarboxylic fatty acids. Furthermore, these activate all ω- and ß-oxidation pathways through peroxisome proliferator activated receptor (PPAR) α and PPARγ, an activation loop normally kept under control by dicarboxylic fatty acid degradation by the peroxisomal enzyme L-PBE. Indeed, L-pbe(-/-) mice fed coconut oil overaccumulate dicarboxylic fatty acids, which activate all fatty acid oxidation pathways and lead to liver inflammation, fibrosis, and death. Thus, the correct homeostasis of dicarboxylic fatty acids is a means to regulate the efficient utilization of ingested medium-chain fatty acids, and its deregulation exemplifies the intricate relationship between impaired metabolism and inflammation.

On the future of mass-spectrometry-based lipidomics.

Lipids have highly diverse functions that go beyond cellular membrane structure and energy storage. One of the great challenges in lipid research will be to understand how the enormous complexity of lipid homeostasis is maintained. Genetic approaches combined with mass spectrometry-based lipidomics will help to elucidate how cells create and maintain their nonrandom lipid distribution within tissues, cells, organelles and lipid bilayers. Lipid homeostasis is crucial for many cellular processes and we are currently only beginning to understand the specific functions of lipids and the local environment that they create.

The yeast p5 type ATPase, spf1, regulates manganese transport into the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a large, multifunctional and essential organelle. Despite intense research, the function of more than a third of ER proteins remains unknown even in the well-studied model organism Saccharomyces cerevisiae. One such protein is Spf1, which is a highly conserved, ER localized, putative P-type ATPase. Deletion of SPF1 causes a wide variety of phenotypes including severe ER stress suggesting that this protein is essential for the normal function of the ER. The closest homologue of Spf1 is the vacuolar P-type ATPase Ypk9 that influences Mn(2+) homeostasis. However in vitro reconstitution assays with Spf1 have not yielded insight into its transport specificity. Here we took an in vivo approach to detect the direct and indirect effects of deleting SPF1. We found a specific reduction in the luminal concentration of Mn(2+) in ∆spf1 cells and an increase following it's overexpression. In agreement with the observed loss of luminal Mn(2+) we could observe concurrent reduction in many Mn(2+)-related process in the ER lumen. Conversely, cytosolic Mn(2+)-dependent processes were increased. Together, these data support a role for Spf1p in Mn(2+) transport in the cell. We also demonstrate that the human sequence homologue, ATP13A1, is a functionally conserved orthologue. Since ATP13A1 is highly expressed in developing neuronal tissues and in the brain, this should help in the study of Mn(2+)-dependent neurological disorders.

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels.

Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules.