Lipid Intermediates Research Articles

MECHANISMS IN ENDOCRINOLOGY: Skeletal muscle lipotoxicity in insulin resistance and type 2 diabetes: a causal mechanism or an innocent bystander?

Dysfunctional adipose tissue is associated with an increased risk of developing type 2 diabetes (T2D). One characteristic of a dysfunctional adipose tissue is the reduced expandability of the subcutaneous adipose tissue leading to ectopic storage of fat in organs and/or tissues involved in the pathogenesis of T2D that can cause lipotoxicity. Accumulation of lipids in the skeletal muscle is associated with insulin resistance, but the majority of previous studies do not prove any causality. Most studies agree that it is not the intramuscular lipids per se that causes insulin resistance, but rather lipid intermediates such as diacylglycerols, fatty acyl-CoAs and ceramides and that it is the localization, composition and turnover of these intermediates that play an important role in the development of insulin resistance and T2D. Adipose tissue is a more active tissue than previously thought, and future research should thus aim at examining the exact role of lipid composition, cellular localization and the dynamics of lipid turnover on the development of insulin resistance. In addition, ectopic storage of fat has differential impact on various organs in different phenotypes at risk of developing T2D; thus, understanding how adipogenesis is regulated, the interference with metabolic outcomes and what determines the capacity of adipose tissue expandability in distinct population groups is necessary. This study is a review of the current literature on the adipose tissue expandability hypothesis and how the following ectopic lipid accumulation as a consequence of a limited adipose tissue expandability may be associated with insulin resistance in muscle and liver.

Physiological Suppression of Lipotoxic Liver Damage by Complementary Actions of HDAC3 and SCAP/SREBP

Liver fat accumulation precedes non-alcoholic steatohepatitis, an increasing cause of end-stage liver disease. Histone deacetylase 3 (HDAC3) is required for hepatic triglyceride homeostasis, and sterol regulatory element binding protein (SREBP) regulates the lipogenic response to feeding, but the crosstalk between these pathways is unknown. Here we show that inactivation of SREBP by hepatic deletion of SREBP cleavage activating protein (SCAP) abrogates the increase in lipogenesis caused by loss of HDAC3, but fatty acid oxidation remains defective. This combination leads to accumulation of lipid intermediates and to an energy drain that collectively cause oxidative stress, inflammation, liver damage, and, ultimately, synthetic lethality. Remarkably, this phenotype is prevented by ectopic expression of nuclear SREBP1c, revealing a surprising benefit of de novo lipogenesis and triglyceride synthesis in preventing lipotoxicity. These results demonstrate that HDAC3 and SCAP control symbiotic pathways of liver lipid metabolism that are critical for suppression of lipotoxicity.

Lipid modulation of skeletal muscle mass and function.

Loss of skeletal muscle mass is a characteristic feature of various pathologies including cancer, diabetes, and obesity, as well as being a general feature of ageing. However, the processes underlying its pathogenesis are not fully understood and may involve multiple factors. Importantly, there is growing evidence which supports a role for fatty acids and their derived lipid intermediates in the regulation of skeletal muscle mass and function. In this review, we discuss evidence pertaining to those pathways which are involved in the reduction, increase and/or preservation of skeletal muscle mass by such lipids under various pathological conditions, and highlight studies investigating how these processes may be influenced by dietary supplementation as well as genetic and/or pharmacological intervention.

Lipid-Induced Insulin Resistance in Skeletal Muscle: The Chase for the Culprit Goes from Total Intramuscular Fat to Lipid Intermediates, and Finally to Species of Lipid Intermediates.

The skeletal muscle is the largest organ in the body. It plays a particularly pivotal role in glucose homeostasis, as it can account for up to 40% of the body and for up to 80%–90% of insulin-stimulated glucose disposal. Hence, insulin resistance (IR) in skeletal muscle has been a focus of much research and review. The fact that skeletal muscle IR precedes β-cell dysfunction makes it an ideal target for countering the diabetes epidemic. It is generally accepted that the accumulation of lipids in the skeletal muscle, due to dietary lipid oversupply, is closely linked with IR. Our understanding of this link between intramyocellular lipids (IMCL) and glycemic control has changed over the years. Initially, skeletal muscle IR was related to total IMCL. The inconsistencies in this explanation led to the discovery that particular lipid intermediates are more important than total IMCL. The two most commonly cited lipid intermediates for causing skeletal muscle IR are ceramides and diacylglycerol (DAG) in IMCL. Still, not all cases of IR and dysfunction in glycemic control have shown an increase in either or both of these lipids. In this review, we will summarise the latest research results that, using the lipidomics approach, have elucidated DAG and ceramide species that are involved in skeletal muscle IR in animal models and human subjects.

Duodenal-jejunal bypass surgery induces hepatic lipidomic alterations associated with ameliorated hepatic steatosis in mice.

Bariatric surgery induces weight loss and improvement of insulin resistance; one aspect of both is an amelioration of hepatic steatosis. This study was undertaken to assess the changes in the hepatic lipidome after duodenal-jejunal bypass (DJB) surgery. A DJB surgical model was developed and characterized in diet-induced obese mice. In comparison with sham-operated mice, an unbiased lipidomic profiling of hepatic lipids was performed together with measurements of gene expression within key pathways of hepatic lipid metabolism. In the liver of DJB mice, a dramatic reduction (by 77%) in hepatic triacylglycerols was observed. Global lipidomic profiling identified marked decreases of triacylglycerols comprised of medium length fatty acids and with low double bond content. Specific diacylglycerol species were also among the most dramatic decreases in hepatic lipids, whereas lysophosphatidic acids and phosphatidic acids were increased. Expression of fatty acid transporter and lipogenic genes was down-regulated. From in-depth analysis of hepatic lipid composition, specific lipid intermediates were identified that are preferentially changed following DJB surgery. These changes were most likely due to DJB-induced weight loss, and only further studies will be able to distinguish weight loss-dependent from weight loss-independent changes.

Determining the Turnover of Glycosphingolipid Species by Stable-Isotope Tracer Lipidomics

In this study, we used water-soluble stable mass isotope precursors to measure the turnover of endogenous ceramide (Cer) and glycosphingolipids (GSLs) in HEp-2 cells. Cells incubated in the presence of [13C-U]glucose showed rapid incorporation of hexose residues with an increased mass of 6Da into GSLs. Different turnover rates of GSL classes and their molecular species were observed. Approximately 30% of the glucosylceramide, 50% of the lactosylceramide, and 50% of the globotriaosylceramide species showed a much slower turnover than the rest. This demonstrates the existence of different lipid pools, where a certain fraction of species survived for a long time in the cells. The species with the shortest N-amidated fatty acyl groups (C16:0 and C18:0) showed a more rapid turnover than those with the longest N-amidated fatty acids (C24:0 and C24:1). Experiments with addition of [13C-U]serine were performed to study de novo synthesis of Cer from serine and palmitoyl-CoA. These experiments revealed that de novo synthesis contributes to a minor extent to the total synthesis of new sphingolipids and showed that there is a more rapid formation of the longest Cer species (C24:0 and C24:1) than of the shortest species (C16:0), that is, the opposite as observed for the GSLs in the experiments with [13C-U]glucose. In conclusion, this FLUX lipidomics experimental approach with the addition of [13C-U]glucose to cells allows us to not only study the total turnover but also permit observations of lipid intermediates and metabolic flow of endogenous GSL species at the molecular lipid level.

Features of the Phosphatidylinositol Cycle and its Role in Signal Transduction.

The phosphatidylinositol cycle (PI-cycle) has a central role in cell signaling. It is the major pathway for the synthesis of phosphatidylinositol and its phosphorylated forms. In addition, some lipid intermediates of the PI-cycle, including diacylglycerol and phosphatidic acid, are also important lipid signaling agents. The PI-cycle has some features that are important for the understanding of its role in the cell. As a cycle, the intermediates will be regenerated. The PI-cycle requires a large amount of metabolic energy. There are different steps of the cycle that occur in two different membranes, the plasma membrane and the endoplasmic reticulum. In order to complete the PI-cycle lipid must be transferred between the two membranes. The role of the Nir proteins in the process has recently been elucidated. The lipid intermediates of the PI-cycle are normally highly enriched with 1-stearoyl-2-arachidonoyl molecular species in mammals. This enrichment will be retained as long as the intermediates are segregated from other lipids of the cell. However, there is a significant fraction (>15%) of lipids in the PI-cycle of normal cells that have other acyl chains. Phosphatidylinositol largely devoid of arachidonoyl chains are found in cancer cells. Phosphatidylinositol species with less unsaturation will not be as readily converted to phosphatidylinositol-3,4,5-trisphosphate, the lipid required for the activation of Akt with resulting effects on cell proliferation. Thus, the cyclical nature of the PI-cycle, its dependence on acyl chain composition and its requirement for lipid transfer between two membranes, explain many of the biological properties of this cycle.

Genetic and Pharmacological Inhibition of Malonyl CoA Decarboxylase Does Not Exacerbate Age-Related Insulin Resistance in Mice.

Aging is associated with the development of chronic diseases such as insulin resistance and type 2 diabetes. A reduction in mitochondrial fat oxidation is postulated to be a key factor contributing to the progression of these diseases. Our aim was to investigate the contribution of impaired mitochondrial fat oxidation toward age-related disease. Mice deficient for malonyl CoA decarboxylase (MCD(-/-)), a mouse model of reduced fat oxidation, were allowed to age while life span and a number of physiological parameters (glucose tolerance, insulin tolerance, indirect calorimetry) were assessed. Decreased fat oxidation in MCD(-/-) mice resulted in the accumulation of lipid intermediates in peripheral tissues, but this was not associated with a worsening of age-associated insulin resistance and, conversely, improved longevity. This improvement was associated with reduced oxidative stress and reduced acetylation of the antioxidant enzyme superoxide dismutase 2 in muscle but not the liver of MCD(-/-) mice. These findings were recapitulated in aged mice treated with an MCD inhibitor (CBM-3001106), and these mice also demonstrated improvements in glucose and insulin tolerance. Therefore, our results demonstrate that in addition to decreasing fat oxidation, MCD inhibition also has novel effects on protein acetylation. These combined effects protect against age-related metabolic dysfunction, demonstrating that MCD inhibitors may have utility in the battle against chronic disease in the elderly.

Analysis of diacylglycerols by ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry: Double bond location and isomers separation

Diacylglycerols (DAGs) are important lipid intermediates and have been implicated in human diseases. Isomerism complicates their mass spectrometric analysis; in particular, it is difficult to identify fatty acid substituents and locate the double bond positions in unsaturated DAGs. We have developed an analytical strategy using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UPLC/Q-TOF MS) in conjunction with dimethyl disulfide (DMDS) derivatization and collision cross-section (CCS) measurement to characterize DAGs in biological samples. The method employs non-aqueous reversed-phase chromatographic separation and profile collision energy (CE) mode for MSE and MS/MS analyses. Three types of fragment ions were produced simultaneously. Hydrocarbon ions (m/z 50–200) obtained at high CE helped to distinguish unsaturated and saturated DAGs rapidly. Neutral loss ions and acylium ions (m/z 300–400) produced at low CE were used to identify fatty acid substituents. Informative methyl thioalkane fragment ions were used to locate the double bonds of unsaturated DAGs. Mono-methylthio derivatives were formed mainly by the reaction of DAGs with DMDS, where methyl thiol underwent addition to the first double bond farthest from the ester terminus of unsaturated fatty acid chains. The addition of CCS values maximized the separation of isomeric DAG species and improved the confidence of DAG identification. Fourteen DAGs were identified in mouse myotube cells based on accurate masses, characteristic fragment ions, DMDS derivatization, and CCS values.

A Model for the Function of COQ9: the Isoprenoid‐Precursor Presenter

Coenzyme Q (CoQ) is a vital part of the electron transport chain that is synthesized de novo in mitochondria by the combined action of at least 10 proteins (COQ1–10). Defects in CoQ biosynthesis lead to primary CoQ deficiency, which has serious human health implications ranging from encephalomyopathy to cerebellar ataxia. Coenzyme Q biosynthesis protein 9 (COQ9) is a crucial part of this pathway, but its exact role remains unknown. A recently‐published manuscript from our lab revealed that COQ9 contains a small‐molecule binding domain, which co‐crystallized with a phospholipid, and demonstrated that COQ9 associates functionally and physically with COQ7. COQ7 catalyzes the hydroxylation of the CoQ intermediate, demethoxy‐CoQ (DMQ), in one of the last steps of CoQ biosynthesis. CoQ and its isoprenylated intermediates are highly hydrophobic, with mature CoQ residing predominantly within the inner mitochondrial membrane, while the CoQ biosynthetic enzymes are peripheral. This apparent lack of accessibility produces a biosynthetic barrier that we envision may be overcome by a protein capable of chaperoning and presenting lipid intermediates to the CoQ biosynthetic enzymes. Due to its ability to bind lipids, its physical association with COQ7, and the accumulation of DMQ in both yeast and mice harboring mutant versions of COQ9, we hypothesize that COQ9 fills this role. To test this hypothesis, I have enriched DMQ from bacteria using organic lipid extraction and column chromatography, and have optimized assays using liposomes to test the isoprenoid transport abilities of COQ9. In this presentation, I discuss the results of these experiments and the insights they have provided toward elucidating the biochemical role of COQ9 in CoQ biosynthesis, understanding CoQ biosynthesis as a whole, and ultimately treating CoQ deficiency.

Novel Citronellyl-Based Photoprobes Designed to Identify ER Proteins Interacting with Dolichyl Phosphate in Yeast and Mammalian Cells.

Dolichyl phosphate-linked mono- and oligosaccharides (DLO) are essential intermediates in protein N-glycosylation, C- and O-mannosylation and GPI anchor biosynthesis. While many membrane proteins in the endoplasmic reticulum (ER) involved in the assembly of DLOs are known, essential proteins believed to be required for the transbilayer movement (flip-flopping) and proteins potentially involved in the regulation of DLO synthesis remain to be identified. The synthesis of a series of Dol-P derivatives composed of citronellyl-based photoprobes with benzophenone groups equipped with alkyne moieties for Huisgen "click" chemistry is now described to utilize as tools for identifying ER proteins involved in regulating the biosynthesis and transbilayer movement of lipid intermediates. In vitro enzymatic assays were used to establish that the photoprobes contain the critical structural features recognized by pertinent enzymes in the dolichol pathway. ER proteins that photoreacted with the novel probes were identified by MS. The potential of the newly designed photoprobes, m-PAL-Cit-P and p-PAL-Cit-P, for identifying previously unidentified Dol-P-interacting proteins is supported by the observation that they are enzymatically mannosylated by Man-P-Dol synthase (MPDS) from Chinese Hamster Ovary (CHO) cells at an enzymatic rate similar to that for Dol-P. MS analyses reveal that DPM1, ALG14 and several other yeast ER proteins involved in DLO biosynthesis and lipid-mediated protein O-mannosylation photoreacted with the novel probes. The newly-designed photoprobes described in this paper provide promising new tools for the identification of yet to be identified Dol-P interacting ER proteins in yeast and mammalian cells, including the Dol-P flippase required for the "re-cycling" of the glycosyl carrier lipid from the lumenal monolayer of the ER to the cytoplasmic leaflet for new rounds of DLO synthesis.

Evidence for Intramyocardial Disruption of Lipid Metabolism and Increased Myocardial Ketone Utilization in Advanced Human Heart Failure.

The failing human heart is characterized by metabolic abnormalities, but these defects remains incompletely understood. In animal models of heart failure there is a switch from a predominance of fatty acid utilization to the more oxygen-sparing carbohydrate metabolism. Recent studies have reported decreases in myocardial lipid content, but the inclusion of diabetic and nondiabetic patients obscures the distinction of adaptations to metabolic derangements from adaptations to heart failure per se. We performed both unbiased and targeted myocardial lipid surveys using liquid chromatography-mass spectroscopy in nondiabetic, lean, predominantly nonischemic, advanced heart failure patients at the time of heart transplantation or left ventricular assist device implantation. We identified significantly decreased concentrations of the majority of myocardial lipid intermediates, including long-chain acylcarnitines, the primary subset of energetic lipid substrate for mitochondrial fatty acid oxidation. We report for the first time significantly reduced levels of intermediate and anaplerotic acyl-coenzyme A (CoA) species incorporated into the Krebs cycle, whereas the myocardial concentration of acetyl-CoA was significantly increased in end-stage heart failure. In contrast, we observed an increased abundance of ketogenic β-hydroxybutyryl-CoA, in association with increased myocardial utilization of β-hydroxybutyrate. We observed a significant increase in the expression of the gene encoding succinyl-CoA:3-oxoacid-CoA transferase, the rate-limiting enzyme for myocardial oxidation of β-hydroxybutyrate and acetoacetate. These findings indicate increased ketone utilization in the severely failing human heart independent of diabetes mellitus, and they support the role of ketone bodies as an alternative fuel and myocardial ketone oxidation as a key metabolic adaptation in the failing human heart.

Ceramide Induces Human Hepcidin Gene Transcription through JAK/STAT3 Pathway.

Changes in lipid metabolism and iron content are observed in the livers of patients with fatty liver disease. The expression of hepcidin, an iron-regulatory and acute phase protein synthesized by the liver, is also modulated. The potential interaction of lipid and iron metabolism is largely unknown. We investigated the role of lipid intermediate, ceramide in the regulation of human hepcidin gene, HAMP. Human hepatoma HepG2 cells were treated with cell-permeable ceramide analogs. Ceramide induced significant up-regulation of HAMP mRNA expression in HepG2 cells. The effect of ceramide on HAMP expression was mediated through transcriptional mechanisms because it was completely blocked with actinomycin D treatment. Reporter assays also confirmed the activation of 0.6 kb HAMP promoter by ceramide. HepG2 cells treated with ceramide displayed increased phosphorylation of STAT3, JNK, and NF-κB proteins. However, ceramide induced the binding of STAT3, but not NF-κB or c-Jun, to HAMP promoter, as shown by the chromatin immunoprecipitation assays. The mutation of STAT3 response element within 0.6 kb HAMP promoter region significantly inhibited the stimulatory effect of ceramide on HAMP promoter activity. Similarly, the inhibition of STAT3 with a pan-JAK kinase inhibitor and STAT3 siRNA pool also diminished the induction of both HAMP promoter activity and mRNA expression by ceramide. In conclusion, we have shown a direct role for ceramide in the activation of hepatic HAMP transcription via STAT3. Our findings suggest a crosstalk between lipid and iron metabolism in the liver, which may contribute to the pathogenesis of obesity-related fatty liver disease.

Mechanisms of intrahepatic triglyceride accumulation.

Hepatic steatosis defined as lipid accumulation in hepatocytes is very frequently found in adults and obese adolescents in the Western World. Etiologically, obesity and associated insulin resistance or excess alcohol intake are the most frequent causes of hepatic steatosis. However, steatosis also often occurs with chronic hepatitis C virus (HCV) infection and is also found in rare but potentially life-threatening liver diseases of pregnancy. Clinical significance and outcome of hepatic triglyceride accumulation are highly dependent on etiology and histological pattern of steatosis. This review summarizes current concepts of pathophysiology of common causes of hepatic steatosis, including non-alcoholic fatty liver disease (NAFLD), alcoholic fatty liver disease, chronic HCV infections, drug-induced forms of hepatic steatosis, and acute fatty liver of pregnancy. Regarding the pathophysiology of NAFLD, this work focuses on the close correlation between insulin resistance and hepatic triglyceride accumulation, highlighting the potential harmful effects of systemic insulin resistance on hepatic metabolism of fatty acids on the one side and the role of lipid intermediates on insulin signalling on the other side. Current studies on lipid droplet morphogenesis have identified novel candidate proteins and enzymes in NAFLD.

ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana.

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature.

Carnitine supplementation in high-fat diet-fed rats does not ameliorate lipid-induced skeletal muscle mitochondrial dysfunction in vivo.

Muscle lipid overload and the associated accumulation of lipid intermediates play an important role in the development of insulin resistance. Carnitine insufficiency is a common feature of insulin-resistant states and might lead to incomplete fatty acid oxidation and impaired export of lipid intermediates out of the mitochondria. The aim of the present study was to test the hypothesis that carnitine supplementation reduces high-fat diet-induced lipotoxicity, improves muscle mitochondrial function, and ameliorates insulin resistance. Wistar rats were fed either normal chow or a high-fat diet for 15 wk. One group of high-fat diet-fed rats was supplemented with 300 mg·kg(-1)·day(-1) L-carnitine during the last 8 wk. Muscle mitochondrial function was measured in vivo by (31)P magnetic resonance spectroscopy (MRS) and ex vivo by high-resolution respirometry. Muscle lipid status was determined by (1)H MRS (intramyocellular lipids) and tandem mass spectrometry (acylcarnitines). High-fat diet feeding induced insulin resistance and was associated with decreases in muscle and blood free carnitine, elevated levels of muscle lipids and acylcarnitines, and an increased number of muscle mitochondria that showed an improved capacity to oxidize fat-derived substrates when tested ex vivo. This was, however, not accompanied by an increase in muscle oxidative capacity in vivo, indicating that in vivo mitochondrial function was compromised. Despite partial normalization of muscle and blood free carnitine content, carnitine supplementation did not induce improvements in muscle lipid status, in vivo mitochondrial function, or insulin sensitivity. Carnitine insufficiency, therefore, does not play a major role in high-fat diet-induced muscle mitochondrial dysfunction in vivo.

Lipid-mediated muscle insulin resistance: different fat, different pathways?

Increased dietary fat intake and lipolysis result in excessive lipid availability, which relates to impaired insulin sensitivity. Over the last years, several mechanisms possibly underlying lipid-mediated insulin resistance evolved. Lipid intermediates such as diacylglycerols (DAG) associate with changes in insulin sensitivity in many models. DAG activate novel protein kinase C (PKC) isoforms followed by inhibitory serine phosphorylation of insulin receptor substrate 1 (IRS1). Activation of Toll-like receptor 4 (TLR4) raises another lipid class, ceramides (CER), which induce pro-inflammatory pathways and lead to inhibition of Akt phosphorylation. Inhibition of glucosylceramide and ganglioside synthesis results in improved insulin sensitivity and increased activatory tyrosine phosphorylation of IRS1 in the muscle. Incomplete fat oxidation can increase acylcarnitines (ACC), which in turn stimulate pro-inflammatory pathways. This review analyzed the effects of lipid metabolites on insulin action in skeletal muscle of humans and rodents. Despite the evidence for the association of both DAG and CER with insulin resistance, its causal relevance may differ depending on the subcellular localization and the tested cohorts, e.g., athletes. Nevertheless, recent data indicate that individual lipid species and their degree of fatty acid saturation, particularly membrane and cytosolic C18:2 DAG, specifically activate PKCθ and induce both acute lipid-induced and chronic insulin resistance in humans.

Diaminopimelic Acid Amidation in Corynebacteriales: NEW INSIGHTS INTO THE ROLE OF LtsA IN PEPTIDOGLYCAN MODIFICATION

A gene named ltsA was earlier identified in Rhodococcus and Corynebacterium species while screening for mutations leading to increased cell susceptibility to lysozyme. The encoded protein belonged to a huge family of glutamine amidotransferases whose members catalyze amide nitrogen transfer from glutamine to various specific acceptor substrates. We here describe detailed physiological and biochemical investigations demonstrating the specific role of LtsA protein from Corynebacterium glutamicum (LtsACg) in the modification by amidation of cell wall peptidoglycan diaminopimelic acid (DAP) residues. A morphologically altered but viable ΔltsA mutant was generated, which displays a high susceptibility to lysozyme and β-lactam antibiotics. Analysis of its peptidoglycan structure revealed a total loss of DAP amidation, a modification that was found in 80% of DAP residues in the wild-type polymer. The cell peptidoglycan content and cross-linking were otherwise not modified in the mutant. Heterologous expression of LtsACg in Escherichia coli yielded a massive and toxic incorporation of amidated DAP into the peptidoglycan that ultimately led to cell lysis. In vitro assays confirmed the amidotransferase activity of LtsACg and showed that this enzyme used the peptidoglycan lipid intermediates I and II but not, or only marginally, the UDP-MurNAc pentapeptide nucleotide precursor as acceptor substrates. As is generally the case for glutamine amidotransferases, either glutamine or NH4(+) could serve as the donor substrate for LtsACg. The enzyme did not amidate tripeptide- and tetrapeptide-truncated versions of lipid I, indicating a strict specificity for a pentapeptide chain length.

Sphingosine 1-phosphate counteracts insulin signaling in pancreatic β-cells via the sphingosine 1-phosphate receptor subtype 2.

Glucolipotoxic stress has been identified as a key player in the progression of pancreatic β-cell dysfunction contributing to insulin resistance and the development of type 2 diabetes mellitus (T2D). It has been suggested that bioactive lipid intermediates, formed under lipotoxic conditions, are involved in these processes. Here, we show that sphingosine 1-phosphate (S1P) levels are not only increased in palmitate-stimulated pancreatic β-cells but also regulate β-cell homeostasis in a divergent manner. Although S1P possesses a prosurvival effect in β-cells, an enhanced level of the sphingolipid antagonizes insulin-mediated cell growth and survival via the sphingosine 1-phosphate receptor subtype 2 (S1P2) followed by an inhibition of Akt-signaling. In an attempt to investigate the role of the S1P/S1P2 axis in vivo, the New Zealand obese (NZO) diabetic mouse model, characterized by β-cell loss under high-fat diet (HFD) conditions, was used. The occurrence of T2D was accompanied by an increase of plasma S1P levels. To examine whether S1P contributes to the morphologic changes of islets via S1P2, the receptor antagonist JTE-013 was administered. Most interestingly, JTE-013 rescued β-cell damage clearly indicating an important role of the S1P2 in β-cell homeostasis. Therefore, the present study provides a new therapeutic strategy to diminish β-cell dysfunction and the development of T2D.

The Lipin Protein Family, Cellular Lipid Storage, and Disease

Several years ago we identified the Lipin gene family in a mutant inbred mouse strain with lipodystrophy and insulin resistance. The lipin proteins are phosphatidate phosphatase enzymes, which dephosphorylate phosphatidic acid to diacylglycerol, the penultimate step in triacylglycerol biosynthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in their ability to associate with multiple cellular compartments that include the cytosol, endoplasmic reticulum membrane, and nucleus. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins—lipin‐1, lipin‐2, and lipin‐3. In mice and humans, the levels of lipin activity are a determinant of triacylglycerol storage in several tissues, and humans with deficiency in lipin‐1 or lipin‐2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of “lipinopathies” in mouse models and in humans has revealed that lipin activity regulates lipid intermediates that influence fundamental cellular processes including adipocyte and chondrocyte differentiation, cerebellar function, intestinal fat absorption, and autophagy. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.