Papers by Carsten Schmitz-Peiffer
Diabetologia, 2007
Aims/hypothesis Insulin resistance in skeletal muscle is strongly associated with lipid oversuppl... more Aims/hypothesis Insulin resistance in skeletal muscle is strongly associated with lipid oversupply, but the intracellular metabolites and underlying mechanisms are unclear. We therefore sought to identify the lipid intermediates through which the common unsaturated fatty acid linoleate causes defects in IRS-1 signalling in L6 myotubes and mouse skeletal muscle. Materials and methods Cells were pre-treated with 1 mmol/l linoleate for 24 h. Subsequent insulin-stimulated IRS-1 tyrosine phosphorylation and its association with the p85 subunit of phosphatidylinositol 3-kinase were determined by immunoblotting. Intracellular lipid species and protein kinase C activation were modulated by overexpression of diacylglycerol kinase ɛ, which preferentially converts unsaturated diacylglycerol into phosphatidic acid, or by inhibition of lysophosphatidic acid acyl transferase with lisofylline, which reduces phosphatidic acid synthesis. Phosphatidic acid species in linoleate-treated cells or muscle from insulin-resistant mice fed a safflower oil-based high-fat diet that was rich in linoleate were analysed by mass spectrometry. Results Linoleate pretreatment reduced IRS-1 tyrosine phosphorylation and p85 association. Overexpression of diacylglycerol kinase ɛ reversed the activation of protein kinase C isoforms by linoleate, but paradoxically further diminished IRS-1 tyrosine phosphorylation. Conversely, lisofylline treatment restored IRS-1 phosphorylation. Mass spectrometry indicated that the dilinoleoyl-phosphatidic acid content increased from undetectable levels to almost 20% of total phosphatidic acid in L6 cells and to 8% of total in the muscle of mice fed a high-fat diet. Micelles containing dilinoleoyl-phosphatidic acid specifically inhibited IRS-1 tyrosine phosphorylation and glycogen synthesis in L6 cells. Conclusions/interpretation These data indicate that linoleatederived phosphatidic acid is a novel lipid species that contributes independently of protein kinase C to IRS-1 signalling defects in muscle cells in response to lipid oversupply.
Aims/hypothesis We examined the time-dependent effects of deletion of the gene encoding protein k... more Aims/hypothesis We examined the time-dependent effects of deletion of the gene encoding protein kinase C epsilon (Prkce) on glucose homeostasis, insulin secretion and hepatic lipid metabolism in fat-fed mice. Methods Prkce −/− and wild-type (WT) mice were fed a high-fat diet for 1 to 16 weeks and subjected to i.p. glucose tolerance tests (ipGTT) and indirect calorimetry. We also investigated gene expression and protein levels by RT-PCR, quantitative protein profiling (isobaric tag for relative and absolute quantification; iTRAQ) and immunoblotting. Lipid levels, mitochondrial oxidative capacity and lipid metabolism were assessed in liver and primary hepatocytes. Results While fat-fed WT mice became glucose intolerant after 1 week, Prkce −/− mice exhibited normal glucose and insulin levels. iTRAQ suggested differences in lipid metabolism and oxidative phosphorylation between fat-fed WT and Prkce −/− animals. Liver triacylglycerols were increased in fat-fed Prkce −/− mice, resulting from altered lipid partitioning which promoted esterification of fatty acids in hepatocytes. In WT mice, fat feeding elevated oxygen consumption in vivo and in isolated liver mitochondria, but these increases were not seen in Prkce −/− mice. Prkce −/− hepatocytes also exhibited reduced production of reactive oxygen species (ROS) in the presence of palmitate. After 16 weeks of fat feeding, however, the improved glucose tolerance in fat-fed Prkce −/− mice was instead associated with increased insulin secretion during ipGTT, as we have previously reported. Conclusions/interpretation Prkce deletion ameliorates dietinduced glucose intolerance via two temporally distinct phenotypes. Protection against insulin resistance is associated with changes in hepatic lipid partitioning, which may reduce the acute inhibitory effects of fatty acid catabolism, such as ROS generation. In the longer term, enhancement of glucose-stimulated insulin secretion prevails. Keywords Diabetes. Insulin resistance. Lipid metabolism. Liver. PKCε. Prkce. Protein kinase C epsilon Abbreviations ipGTT Intraperitoneal glucose tolerance test IRS Insulin receptor substrate iTRAQ Isobaric tag for relative and absolute quantitation PKC Protein kinase C ROS Reactive oxygen species WT Wild-type Electronic supplementary material The online version of this article
Diabetes, 2009
OBJECTIVE-Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of -cell... more OBJECTIVE-Insufficient insulin secretion is a hallmark of type 2 diabetes, and exposure of -cells to elevated lipid levels (lipotoxicity) contributes to secretory dysfunction. Functional ablation of protein kinase C ε (PKCε) has been shown to improve glucose homeostasis in models of type 2 diabetes and, in particular, to enhance glucose-stimulated insulin secretion (GSIS) after lipid exposure. Therefore, we investigated the lipid-dependent mechanisms responsible for the enhanced GSIS after inactivation of PKCε. RESEARCH DESIGN AND METHODS-We cultured islets isolated from PKCε knockout (PKCεKO) mice in palmitate prior to measuring GSIS, Ca 2ϩ responses, palmitate esterification products, lipolysis, lipase activity, and gene expression. RESULTS-The enhanced GSIS could not be explained by increased expression of another PKC isoform or by alterations in glucose-stimulated Ca 2ϩ influx. Instead, an upregulation of the amplifying pathways of GSIS in lipid-cultured PKCεKO -cells was revealed under conditions in which functional ATP-sensitive K ϩ channels were bypassed. Furthermore, we showed increased esterification of palmitate into triglyceride pools and an enhanced rate of lipolysis and triglyceride lipase activity in PKCεKO islets. Acute treatment with the lipase inhibitor orlistat blocked the enhancement of GSIS in lipid-cultured PKCεKO islets, suggesting that a lipolytic product mediates the enhancement of glucose-amplified insulin secretion after PKCε deletion. CONCLUSIONS-Our findings demonstrate a mechanistic link between lipolysis and the amplifying pathways of GSIS in murine -cells, and they suggest an interaction between PKCε and lipolysis. These results further highlight the therapeutic potential of PKCε inhibition to enhance GSIS from the -cell under conditions of lipid excess.
Journal of Biological …, 1995
Neuropeptide Y (NPY) and norepinephrine, found co-localized in sympathetic neurons innervating bl... more Neuropeptide Y (NPY) and norepinephrine, found co-localized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have ...
Bioscience Reports
Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in Western countries, and is linked ... more Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in Western countries, and is linked to the development of liver cancer and Type 2 diabetes (T2D). It is strongly associated with obesity, but the dysregulation of liver lipid storage is not fully understood. Fatty acid oversupply to hepatocytes can establish a vicious cycle involving diminished protein folding, endoplasmic reticulum (ER) stress, insulin resistance and further lipogenesis. This commentary discusses the recent findings of Lai et al. published in Bioscience Reports, that implicate protein kinase C delta (PKCδ) activation by fatty acids in the inhibition of the SERCA Ca2+ pump, resulting in reduced ER Ca2+ loading and protein misfolding. PKCδ therefore represents a target for the treatment of both steatosis and insulin resistance, key to the prevention of NAFLD and T2D.
Obesity Research & Clinical Practice
Cell metabolism, Jan 8, 2018
Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action thr... more Protein kinase C epsilon (PKCɛ) activation in the liver is proposed to inhibit insulin action through phosphorylation of the insulin receptor. Here, however, we demonstrated that global, but not liver-specific, deletion of PKCɛ in mice protected against diet-induced glucose intolerance and insulin resistance. Furthermore, PKCɛ-dependent alterations in insulin receptor phosphorylation were not detected. Adipose-tissue-specific knockout mice did exhibit improved glucose tolerance, but phosphoproteomics revealed no PKCɛ-dependent effect on the activation of insulin signaling pathways. Altered phosphorylation of adipocyte proteins associated with cell junctions and endosomes was associated with changes in hepatic expression of several genes linked to glucose homeostasis and lipid metabolism. The primary effect of PKCɛ on glucose homeostasis is, therefore, not exerted directly in the liver as currently posited, and PKCɛ activation in this tissue should be interpreted with caution. Howeve...
Biochimica et biophysica acta, Nov 1, 2016
In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N... more In a recent study, we showed that in response to high fat feeding C57BL/6, 129X1, DBA/2 and FVB/N mice all developed glucose intolerance, while BALB/c mice displayed minimal deterioration in glucose tolerance and insulin action. Lipidomic analysis of livers across these five strains has revealed marked strain-specific differences in ceramide (Cer) and sphingomyelin (SM) species with high-fat feeding; with increases in C16-C22 (long-chain) and reductions in C>22 (very long-chain) Cer and SM species observed in the four strains that developed HFD-induced glucose intolerance. Intriguingly, the opposite pattern was observed in sphingolipid species in BALB/c mice. These strain-specific changes in sphingolipid acylation closely correlated with ceramide synthase 2 (CerS2) protein content and activity, with reduced CerS2 levels/activity observed in glucose intolerant strains and increased content in BALB/c mice. Overexpression of CerS2 in primary mouse hepatocytes induced a specific elev...
Molecular Endocrinology, 2016
Isoforms of flavin-containing monooxygenase (FMO) are involved in xenobiotic metabolism, but have... more Isoforms of flavin-containing monooxygenase (FMO) are involved in xenobiotic metabolism, but have also been implicated in the regulation of glucose and lipid homeostasis and in the development of atherosclerosis. However, we have recently shown that improved insulin action is associated with increased FMO expression in livers of protein kinase C (PKC)-deficient mice. Here we investigated whether FMO3 expression affected insulin signalling, glucose metabolism and ER stress in hepatocytes. HepG2 and IHH hepatocytes were transfected with FMO3 cDNA for overexpression, or small interfering RNA for knockdown. Cells were treated with palmitate to induce insulin resistance and insulin signalling, phosphoenolpyruvate carboxykinase (PEPCK) gene expression and ER stress markers were examined by immunoblotting and RT-PCR. Glycogen synthesis was measured using [(14)C]glucose. Palmitate treatment reduced insulin signalling at the level of Akt phosphorylation and glycogen synthesis, which were little affected by FMO3 overexpression. However, the fatty acid also increased the levels of several endoplasmic reticulum (ER) stress markers and activation of caspase 3, which were counteracted by FMO3 overexpression and exacerbated by FMO3 knockdown. While FMO3 expression did not reverse lipid effects on protein thiol redox in hepatocytes, it did prevent upregulation of the gluconeogenic enzyme PEPCK by pharmacological ER stress inducers or by palmitate. ER stress and PEPCK levels were also reduced in livers of fat-fed PKCδ-deficient mice. Our data indicate that FMO3 can contribute to the regulation of glucose metabolism in the liver by reducing lipid-induced ER stress and the expression of PEPCK, independently of insulin signal transduction.
Biochemical Journal, 1998
We have previously detected a number of protein kinase C (PKC) α-binding proteins in skeletal mus... more We have previously detected a number of protein kinase C (PKC) α-binding proteins in skeletal muscle cytosol by blot overlay assay, and now identify the major, 69 kDa binding protein as annexin VI by immunoblotting and overlay assay of hydroxyapatite chromatography fractions. Annexin VI was also detected in immunoprecipitates of PKC α. Annexin VI and PKC α are both calcium-dependent phospholipid-binding proteins, and detection of the interaction was dependent on the presence of calcium and phosphatidylserine (PS). The association probably involves specific protein-protein interactions rather than mere bridging by lipid molecules: firstly, detection of PKC α-annexin VI complexes by overlay assay was not diminished when PS concentrations were increased over a 10-fold range, while that of other PKC α-binding protein complexes was reduced or abolished; secondly, the presence in the overlay assay of a PKC pseudosubstrate peptide, analogous to a PKC sequence previously found to be involve...
American journal of physiology. Endocrinology and metabolism, 2000
Muscle insulin resistance in the chronic high-fat-fed rat is associated with increased membrane t... more Muscle insulin resistance in the chronic high-fat-fed rat is associated with increased membrane translocation and activation of the novel, lipid-responsive, protein kinase C (nPKC) isozymes PKC-theta and -epsilon. Surprisingly, fat-induced insulin resistance can be readily reversed by one high-glucose low-fat meal, but the underlying mechanism is unclear. Here, we have used this model to determine whether changes in the translocation of PKC-theta and -epsilon are associated with the acute reversal of insulin resistance. We measured cytosol and particulate PKC-alpha and nPKC-theta and -epsilon in muscle in control chow-fed Wistar rats (C) and 3-wk high-fat-fed rats with (HF-G) or without (HF-F) a single high-glucose meal. PKC-theta and -epsilon were translocated to the membrane in muscle of insulin-resistant HF-F rats. However, only membrane PKC-theta was reduced to the level of chow-fed controls when insulin resistance was reversed in HF-G rats [% PKC-theta at membrane, 23.0 +/- 4.4...
The American journal of physiology, 1999
Chronic glucose infusion results in hyperinsulinemia and causes lipid accumulation and insulin re... more Chronic glucose infusion results in hyperinsulinemia and causes lipid accumulation and insulin resistance in rat muscle. To examine possible mechanisms for the insulin resistance, alterations in malonyl-CoA and long-chain acyl-CoA (LCA-CoA) concentration and the distribution of protein kinase C (PKC) isozymes, putative links between muscle lipids and insulin resistance, were determined. Cannulated rats were infused with glucose (40 mg. kg(-1). min(-1)) for 1 or 4 days. This increased red quadriceps muscle LCA-CoA content (sum of 6 species) by 1.3-fold at 1 day and 1.4-fold at 4 days vs. saline-infused controls (both P < 0.001 vs. control). The concentration of malonyl-CoA was also increased (1.7-fold at 1 day, P < 0.01, and 2.2-fold at 4 days, P < 0.001 vs. control), suggesting an even greater increase in cytosolic LCA-CoA. The ratio of membrane to cytosolic PKC-epsilon was increased twofold in the red gastrocnemius after both 1 and 4 days, suggesting chronic activation. No...
The Biochemical journal, Jan 15, 1996
We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the prese... more We have investigated protein kinase C (PKC) in skeletal muscle cytosol and demonstrated the presence of two major activities. These did not correspond to different PKC isoenzymes but seemed to represent two species of PKC alpha as deduced by: elution during hydroxyapatite chromatography at KH2PO4 concentrations expected of PKC alpha; detection of the two species by three specific but unrelated anti-(PKC alpha) antibodies; immunodepletion of both activities with anti-(PKC alpha) antibody; and demonstration of identical requirements of both Ca2+ ions and lipid for activation. These species, termed PKC alpha 1 and PKC alpha 2, phosphorylated the modified conventional PKC pseudosubstrate peptide (19-31, Ser-25) equally well. Importantly, however, the activities differed in that PKC alpha 1 phosphorylated histone IIIS, and also peptides derived from the EGF receptor and glycogen synthase, to a much greater extent than did PKC alpha 2. Similarly, incubation of crude muscle extracts with e...
Advances in experimental medicine and biology, 1998
Malonyl CoA is a regulator of carnitine palmitoyl transferase 1 (CPT1), the enzyme that controls ... more Malonyl CoA is a regulator of carnitine palmitoyl transferase 1 (CPT1), the enzyme that controls the transfer of long chain fatty acyl CoA into mitochondria where it is oxidized. Recent studies indicate that in skeletal muscle the concentration of malonyl CoA is acutely (minutes) regulated by changes in its fuel supply and energy expenditure. In response to changes in fuel supply, regulation appears to be due to alterations in the cytosolic concentration of citrate, which is both an allosteric activator of acetyl CoA carboxylase (ACC), the enzyme that catalyzes malonyl CoA synthesis and a source of its precursor, cytosolic acetyl CoA. During exercise and immediately thereafter regulation by citrate appears to be lost and malonyl CoA levels diminish as the result of a decrease in ACC activity secondary to phosphorylation. Sustained increases in the concentration of malonyl CoA have been observed in muscle of a number of insulin-resistant rodents including the Zucker (fa/fa) and GK ra...
The American journal of physiology, 1997
We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is a... more We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is associated with the translocation of the novel protein kinase C epsilon (nPKC epsilon) from cytosolic to particulate fractions in red skeletal muscle and also the downregulation of cytosolic nPKC theta. Here we have further investigated the link between insulin resistance and PKC by assessing the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC isoenzymes in cytosolic fractions of red muscle from fat-fed rats, reducing their apparent activation and/or downregulation, whereas PKC in control rats was unaffected. Because BRL-49653 also improves insulin-stimulated glucose uptake in fat-fed rats and reduces muscle lipid storage, especially diglyceride content, these results strengthen the association between lipid availability, nPKC activation, and skeletal muscle insulin resistance and support the hypothesis th...
PROTEOMICS, 2014
Insulin resistance contributes to the development of Type 2 diabetes, and is associated with lipi... more Insulin resistance contributes to the development of Type 2 diabetes, and is associated with lipid oversupply. Deletion of isoforms of the lipid-activated protein kinase C (PKC) family, PKCδ or PKCε, improves insulin action in fat-fed mice, but differentially affects hepatic lipid metabolism. To investigate the mechanisms involved, we employed an in vivo adaptation of SILAC to examine the effects of a fat diet together with deletion of PKCδ or PKCε on the expression of liver proteins. We identified a total of 3359 and 3488 proteins from the PKCδ and PKCε knockout study groups, respectively, and showed that several enzymes of lipid metabolism were affected by the fat diet. In fat-fed mice, 23 proteins showed changes upon PKCδ deletion while 19 proteins were affected by PKCε deletion. Enzymes of retinol metabolism were affected by the absence of either PKC. Pathway analysis indicated that monosaccharide metabolism was affected only upon PKCδ deletion, while isoprenoid biosynthesis was affected in a PKCε-specific manner. Certain proteins were regulated inversely, including HIV-1 tat interactive protein 2 (Htatip2). Overexpression or knockdown of Htatip2 in hepatocytes affected fatty acid storage and oxidation, consistent with a novel role in mediating the differential effects of PKC isoforms on lipid metabolism. All MS data have been deposited in the ProteomeXchange with identifier PXD000971 (http://proteomecentral.proteomexchange.org/dataset/PXD000971).
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Papers by Carsten Schmitz-Peiffer