Lipid rather than glucose metabolism is implicated in altered insulin secretion caused by oleate in INS-1 cells.

A comprehensive metabolic study was carried out to understand how chronic exposure of pancreatic beta-cells to fatty acids causes high basal secretion and impairs glucose-induced insulin release. INS-1 beta-cells were exposed to 0.4 mM oleate for 3 days and subsequently incubated at 5 or 25 mM glucose, after which various parameters were measured. Chronic oleate promoted triglyceride deposition, increased fatty acid oxidation and esterification, and reduced malonyl-CoA at low glucose in association with elevated basal O(2) consumption and redox state. Oleate caused a modest (25%) reduction in glucose oxidation but did not affect glucose usage, the glucose 6-phosphate and citrate contents, and the activity of pyruvate dehydrogenase of INS-1 cells. Thus changes in glucose metabolism and a Randle-glucose/fatty acid cycle do not explain the altered secretory properties of beta-cells exposed to fatty acids. The main response of INS-1 cells to chronic oleate, which is to increase the oxidation and esterification of fatty acids, may contribute to cause high basal insulin secretion via increased production of reducing equivalents and/or the generation of complex lipid messenger molecule(s).

Atypical expression of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase in subcutaneous adipose tissue of male rats.

The mRNAs encoding mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (mtHMG-CoA synthase), the rate limiting enzyme in ketone body production, are highly expressed in subcutaneous (SC) and, to a lesser extent, in peri-epididymal (PE) rat adipose tissues. This atypical mtHMG-CoA synthase gene expression is dependent on the age (from 9 weeks of age) and sex (higher in male than in female) of the rats. In contrast, the expression of mtHMG-CoA synthase in SC adipose deposit is independent of the nutritional state (fed versus starved) or of the thermic environment (24 degrees C versus 4 degrees C). The expression of mtHMG-CoA synthase is suppressed in SC fat pads of castrated male rats whereas treatment of castrated rats with testosterone restores a normal level of expression. Moreover, testosterone injection induces the expression mtHMG-CoA synthase in SC adipose tissue of age-matched females. The presence of the mtHMG-CoA synthase immunoreactive protein confers to mitochondria isolated from SC adipose deposits, the capacity to produce ketone bodies at a rate similar to that found in liver mitochondria (SC = 13.7 +/- 0.7, liver = 16.4 +/- 1.4 nmol/min/mg prot). mtHMG-CoA synthase is expressed in the stromal vascular fraction (SVF) whatever the adipose deposit considered. While acetyl-CoA carboxylase (ACC) is only expressed in mature adipocytes, the other lipogenic enzymes, fatty acid synthase (FAS) and citrate cleavage enzyme (CCE), are expressed both in SVF cells and mature adipocytes. The expression of lipogenic enzyme genes is markedly reduced in adipocytes but not in SVF cells isolated from 48-h starved male rats. When SVF is subfractionated, mtHMG-CoA synthase mRNAs are mainly recovered in two fractions containing poorly digested structures such as microcapillaries whereas the lowest expression is found in the pre-adipocyte fraction. Interestingly, FAS and CCE mRNAs co-segregate with mtHMG-CoA synthase mRNA. The possible physiological relevance of such atypical expression of mtHMG-CoA synthase is discussed.

Long-term exposure of beta-INS cells to high glucose concentrations increases anaplerosis, lipogenesis, and lipogenic gene expression.

Chronic exposure of pancreatic beta-cells to high glucose has pleiotropic action on beta-cell function. In particular, it induces key glycolytic genes, promotes glycogen deposition, and causes beta-cell proliferation and altered insulin secretion characterized by sensitization to low glucose. Postglycolytic events, in particular, anaplerosis and lipid signaling, are thought to be implicated in beta-cell activation by glucose. To understand the biochemical nature of the beta-cell adaptive process to hyperglycemia, we studied the regulation by glucose of lipogenic genes in the beta-cell line INS-1. A 3-day exposure of cells to elevated glucose (5-25 mmol/l) increased the enzymatic activities of fatty acid synthase 3-fold, acetyl-CoA carboxylase 30-fold, and malic enzyme 1.3-fold. Pyruvate carboxylase and citrate lyase expression remained constant. Similar observations were made at the protein and mRNA levels except for malic enzyme mRNA, which did not vary. Metabolic gene expression changes were associated with chronically elevated levels of citrate, malate, malonyl-CoA, and conversion of glucose carbon into lipids, even in cells that were subsequently exposed to low glucose. Similarly, fatty acid oxidation was suppressed and phospholipid and triglyceride synthesis was enhanced independently of the external glucose concentration in cells preexposed to high glucose. The results suggest that a coordinated induction of glycolytic and lipogenic genes in conjunction with glycogen and triglyceride deposition, as well as increased anaplerosis and altered lipid partitioning, contribute to the adaptive process to hyperglycemia and glucose sensitization of the beta-cell.

Fatty acids rapidly induce the carnitine palmitoyltransferase I gene in the pancreatic beta-cell line INS-1.

Fatty acids are important metabolic substrates for the pancreatic beta-cell, and long term exposure of pancreatic islets to elevated concentrations of fatty acids results in an alteration of glucose-induced insulin secretion. Previous work suggested that exaggerated fatty acid oxidation may be implicated in this process by a mechanism requiring changes in metabolic enzyme expression. We have therefore studied the regulation of carnitine palmitoyltransferase I (CPT I) gene expression by fatty acids in the pancreatic beta-cell line INS-1 since this enzyme catalyzes the limiting step of fatty acid oxidation in various tissues. Palmitate, oleate, and linoleate (0.35 mM) elicited a 4-6-fold increase in CPT I mRNA. The effect was dose-dependent and was similar for saturated and unsaturated fatty acids. It was detectable after 1 h and reached a maximum after 3 h. The induction of CPT I mRNA by fatty acids did not require their oxidation, and 2-bromopalmitate, a nonoxidizable fatty acid, increased CPT I mRNA to the same extent as palmitate. The induction was not prevented by cycloheximide treatment of cells indicating that it was mediated by pre-existing transcription factors. Neither glucose nor pyruvate and various secretagogues had a significant effect except glutamine (7 mM) which slightly induced CPT I mRNA. The half-life of the CPT I transcript was unchanged by fatty acids, and nuclear run-on analysis showed a rapid (less than 45 min) and pronounced transcriptional activation of the CPT I gene by fatty acids. The increase in CPT I mRNA was followed by a 2-3-fold increase in CPT I enzymatic activity measured in isolated mitochondria. The increase in activity was time-dependent, detectable after 4 h, and close to maximal after 24 h. Fatty acid oxidation by INS-1 cells, measured at low glucose, was also 2-3-fold higher in cells cultured with fatty acid in comparison with control cells. Long term exposure of INS-1 cells to fatty acid was associated with elevated secretion of insulin at a low (5 mM) concentration of glucose and a decreased effect of higher glucose concentrations. It also resulted in a decreased oxidation of glucose. The results indicate that the CPT I gene is an early response gene induced by fatty acids at the transcriptional level in beta- (INS-1) cells. It is suggested that exaggerated fatty acid oxidation caused by CPT-1 induction is implicated in the process whereby fatty acids alter glucose-induced insulin secretion.

Expression of liver carnitine palmitoyltransferase I and II genes during development in the rat.

The enzyme activity and the expression (protein and mRNA concentrations) of genes encoding for hepatic carnitine palmitoyl-transferases (CPT) I and II were studied during neonatal development, in response to nutritional state at weaning and during the fed-starved transition in adult rats. The activity, the protein concentration and the level of mRNA encoding CPT I are low in foetal-rat liver and increase 5-fold during the first day of extra-uterine life. The activity and gene expression of CPT I are high during the entire suckling period, in the liver of 30-day-old rats weaned at 20 days on to a high-fat diet and in the liver of 48 h-starved adult rats. The activity and CPT I gene expression are markedly decreased in the liver of rats weaned on to a high-carbohydrate diet. By contrast, the activity, the protein concentration and the level of mRNA encoding CPT II are already high in the liver of term foetuses and remain at this level throughout the suckling period, irrespective of the nutritional state of the animals either at weaning or in the adult.

Developmental changes in mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase gene expression in rat liver, intestine and kidney.

The tissue-specific expression of the mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase gene was studied in 15-day-old suckling rats. The mRNA and protein were present in liver, intestine and kidney, but were absent from brain, heart, skeletal muscles, brown and white adipose tissues. Kidney-cortex mitochondria from suckling rats were able to produce low amounts of ketone bodies from oleate. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels increased slowly during foetal life and markedly after birth. The postnatal increase in liver HMG-CoA synthase mRNA could be due to the increase in plasma glucagon levels, since it rapidly induced the accumulation of HMG-CoA synthase mRNA in cultured foetal hepatocytes. Hepatic, intestinal and renal HMG-CoA synthase mRNA levels remained elevated throughout the suckling period or in rats weaned on to a high-fat carbohydrate-free diet (HF), but decreased by 50% in the liver and totally disappeared from the intestine and the kidney of rats weaned on to a high-carbohydrate low-fat diet (HC). When HC-weaned rats were fed on a HF-diet for a week, HMG-CoA synthase mRNA was re-induced in the intestine and the kidney. The role of hormones and nutrients in the regulation of HMG-CoA synthase gene expression is discussed.