Deletion of protein kinase Cε in mice has limited effects on liver metabolite levels but alters fasting ketogenesis and gluconeogenesis.

AIMS/HYPOTHESIS: Protein kinase Cε (PKCε) is emerging as a key mediator of lipid-induced insulin resistance in liver and hepatic lipid metabolism itself. We investigated whether PKCε plays a role in other metabolic processes, to further examine its suitability as a therapeutic target. METHODS: We measured amino acid, organic acid and sugar levels by liquid and gas chromatography-mass spectrometry of liver extracts from chow and fat-fed wild-type (WT) and PKCε-deficient (Prkce(-/-)) mice. Fed and fasting glucose, ketone and fatty acid levels were measured in blood. Triacylglycerol levels and gluconeogenic and ketogenic enzyme expression were measured in liver. The effect of fasting on epididymal fat pad mass was also determined. RESULTS: Metabolomic analysis indicated that the short-term high-fat diet affected over 20 compounds, including a 50% reduction in the glucogenic amino acid alanine. Prkce deletion resulted only in a reduction of 4-hydroxyproline and aspartate and an increase in glutamate. However, upon fasting, Prkce(-/-) mice were better able to maintain blood glucose levels and also exhibited lower levels of the ketone ß-hydroxybutyrate compared with WT mice. Upon fasting, Prkce deletion also resulted in lower liver and plasma lipids and a smaller reduction in fat pad mass. CONCLUSIONS/INTERPRETATION: Metabolomic analysis provided new insights into the effects of a high-fat diet on liver metabolite levels. Glucose homeostasis under fasting conditions is improved in Prkce(-/-) mice, which, in turn, may reduce the mobilisation of lipid from adipose tissue, reducing the availability of ketogenic substrate in the liver. Together with the protection against fat-diet-induced glucose intolerance previously observed in the fed state, these findings indicate PKCε as a unique therapeutic target for the improvement of glucose homeostasis.

Time-dependent effects of Prkce deletion on glucose homeostasis and hepatic lipid metabolism on dietary lipid oversupply in mice.

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 diet-induced 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.

Saturated- and n-6 polyunsaturated-fat diets each induce ceramide accumulation in mouse skeletal muscle: reversal and improvement of glucose tolerance by lipid metabolism inhibitors.

Lipid-induced insulin resistance is associated with intracellular accumulation of inhibitory intermediates depending on the prevalent fatty acid (FA) species. In cultured myotubes, ceramide and phosphatidic acid (PA) mediate the effects of the saturated FA palmitate and the unsaturated FA linoleate, respectively. We hypothesized that myriocin (MYR), an inhibitor of de novo ceramide synthesis, would protect against glucose intolerance in saturated fat-fed mice, while lisofylline (LSF), a functional inhibitor of PA synthesis, would protect unsaturated fat-fed mice. Mice were fed diets enriched in saturated fat, n-6 polyunsaturated fat, or chow for 6 wk. Saline, LSF (25 mg/kg x d), or MYR (0.3 mg/kg x d) were administered by mini-pumps in the final 4 wk. Glucose homeostasis was examined by glucose tolerance test. Muscle ceramide and PA were analyzed by mass spectrometry. Expression of LASS isoforms (ceramide synthases) was evaluated by immunoblotting. Both saturated and polyunsaturated fat diets increased muscle ceramide and induced glucose intolerance. MYR and LSF reduced ceramide levels in saturated and unsaturated fat-fed mice. Both inhibitors also improved glucose tolerance in unsaturated fat-fed mice, but only LSF was effective in saturated fat-fed mice. The discrepancy between ceramide and glucose tolerance suggests these improvements may not be related directly to changes in muscle ceramide and may involve other insulin-responsive tissues. Changes in the expression of LASS1 were, however, inversely correlated with alterations in glucose tolerance. The demonstration that LSF can ameliorate glucose intolerance in vivo independent of the dietary FA type indicates it may be a novel intervention for the treatment of insulin resistance.

Diverse roles for protein kinase C delta and protein kinase C epsilon in the generation of high-fat-diet-induced glucose intolerance in mice: regulation of lipogenesis by protein kinase C delta.

AIMS/HYPOTHESIS: This study aimed to determine whether protein kinase C (PKC) delta plays a role in the glucose intolerance caused by a high-fat diet, and whether it could compensate for loss of PKCepsilon in the generation of insulin resistance in skeletal muscle. METHODS: Prkcd (-/-), Prkce (-/-) and wild-type mice were fed high-fat diets and subjected to glucose tolerance tests. Blood glucose levels and insulin responses were determined during the tests. Insulin signalling in liver and muscle was assessed after acute in vivo insulin stimulation by immunoblotting with phospho-specific antibodies. Activation of PKC isoforms in muscle from Prkce (-/-) mice was assessed by determining intracellular distribution. Tissues and plasma were assayed for triacylglycerol accumulation, and hepatic production of lipogenic enzymes was determined by immunoblotting. RESULTS: Both Prkcd (-/-) and Prkce (-/-) mice were protected against high-fat-diet-induced glucose intolerance. In Prkce (-/-) mice this was mediated through enhanced insulin availability, while in Prkcd (-/-) mice the reversal occurred in the absence of elevated insulin. Neither the high-fat diet nor Prkcd deletion affected maximal insulin signalling. The activation of PKCdelta in muscle from fat-fed mice was enhanced by Prkce deletion. PKCdelta-deficient mice exhibited reduced liver triacylglycerol accumulation and diminished production of lipogenic enzymes. CONCLUSIONS/INTERPRETATION: Deletion of genes encoding isoforms of PKC can improve glucose intolerance, either by enhancing insulin availability in the case of Prkce, or by reducing lipid accumulation in the case of Prkcd. The absence of PKCepsilon in muscle may be compensated by increased activation of PKCdelta in fat-fed mice, suggesting that an additional role for PKCepsilon in this tissue is masked.