Captopril normalizes insulin signaling and insulin-regulated substrate metabolism in obese (ob/ob) mouse hearts

The goal of this study was to determine whether inhibiting the renin-angiotensin system would restore insulin signaling and normalize substrate use in hearts from obese ob/ob mice. Mice were treated for 4 wk with Captopril (4 mg/kg x d). Circulating levels of free fatty acids, triglycerides, and insulin were measured and glucose tolerance tests performed. Rates of palmitate oxidation and glycolysis, oxygen consumption, and cardiac power were determined in isolated working hearts in the presence and absence of insulin, along with levels of phosphorylation of Akt and AMP-activated protein kinase (AMPK). Captopril treatment did not correct the hyperinsulinemia or impaired glucose tolerance in ob/ob mice. Rates of fatty acid oxidation were increased and glycolysis decreased in ob/ob hearts, and insulin did not modulate substrate use in hearts of ob/ob mice and did not increase Akt phosphorylation. Captopril restored the ability of insulin to regulate fatty acid oxidation and glycolysis in hearts of ob/ob mice, possibly by increasing Akt phosphorylation. Moreover, AMPK phosphorylation, which was increased in hearts of ob/ob mice, was normalized by Captopril treatment, suggesting that in addition to restoring insulin sensitivity, Captopril treatment improved myocardial energetics. Thus, angiotensin-converting enzyme inhibitors restore the responsiveness of ob/ob mouse hearts to insulin and normalizes AMPK activity independently of effects on systemic metabolic homeostasis.     

The antianginal drug trimetazidine shifts cardiac energy metabolism from fatty acid oxidation to glucose oxidation by inhibiting mitochondrial long-chain 3-ketoacyl coenzyme A thiolase

Trimetazidine is a clinically effective antianginal agent that has no negative inotropic or vasodilator properties. Although it is thought to have direct cytoprotective actions on the myocardium, the mechanism(s) by which this occurs is as yet undefined. In this study, we determined what effects trimetazidine has on both fatty acid and glucose metabolism in isolated working rat hearts and on the activities of various enzymes involved in fatty acid oxidation. Hearts were perfused with Krebs-Henseleit solution containing 100 microU/mL insulin, 3% albumin, 5 mmol/L glucose, and fatty acids of different chain lengths. Both glucose and fatty acids were appropriately radiolabeled with either (3)H or (14)C for measurement of glycolysis, glucose oxidation, and fatty acid oxidation. Trimetazidine had no effect on myocardial oxygen consumption or cardiac work under any aerobic perfusion condition used. In hearts perfused with 5 mmol/L glucose and 0.4 mmol/L palmitate, trimetazidine decreased the rate of palmitate oxidation from 488+/-24 to 408+/-15 nmol x g dry weight(-1) x minute(-1) (P<0.05), whereas it increased rates of glucose oxidation from 1889+/-119 to 2378+/-166 nmol x g dry weight(-1) x minute(-1) (P<0.05). In hearts subjected to low-flow ischemia, trimetazidine resulted in a 210% increase in glucose oxidation rates. In both aerobic and ischemic hearts, glycolytic rates were unaltered by trimetazidine. The effects of trimetazidine on glucose oxidation were accompanied by a 37% increase in the active form of pyruvate dehydrogenase, the rate-limiting enzyme for glucose oxidation. No effect of trimetazidine was observed on glycolysis, glucose oxidation, fatty acid oxidation, or active pyruvate dehydrogenase when palmitate was substituted with 0.8 mmol/L octanoate or 1.6 mmol/L butyrate, suggesting that trimetazidine directly inhibits long-chain fatty acid oxidation. This reduction in fatty acid oxidation was accompanied by a significant decrease in the activity of the long-chain isoform of the last enzyme involved in fatty acid beta-oxidation, 3-ketoacyl coenzyme A (CoA) thiolase activity (IC(50) of 75 nmol/L). In contrast, concentrations of trimetazidine in excess of 10 and 100 micromol/L were needed to inhibit the medium- and short-chain forms of 3-ketoacyl CoA thiolase, respectively. Previous studies have shown that inhibition of fatty acid oxidation and stimulation of glucose oxidation can protect the ischemic heart. Therefore, our data suggest that the antianginal effects of trimetazidine may occur because of an inhibition of long-chain 3-ketoacyl CoA thiolase activity, which results in a reduction in fatty acid oxidation and a stimulation of glucose oxidation.     

Inhibition of insulin gene expression by long-term exposure of pancreatic beta cells to palmitate is dependent on the presence of a stimulatory glucose concentration

Long-term exposure of pancreatic beta cells to elevated levels of fatty acids (FAs) impairs glucose-induced insulin secretion. However, the effects of FAs on insulin gene expression are controversial. We hypothesized that FAs adversely affect insulin gene expression only in the presence of elevated glucose concentrations. To test this hypothesis, isolated rat islets were cultured for up to 1 week in the presence of 2.8 or 16.7 mmol/L glucose with or without 0.5 mmol/L palmitate. Insulin release, insulin content, and insulin mRNA levels were determined at the end of each culture period. Palmitate increased insulin release at each time point independently of the glucose concentration. In contrast, insulin content was unchanged in the presence of palmitate at 2.8 mmol/L glucose, but was markedly decreased in the presence of 0.5 mmol/L palmitate and 16.7 mmol/L glucose after 2, 3, and 7 days of culture. In the presence of a basal concentration of glucose, insulin mRNA levels were transiently increased by palmitate at 24 hours but were unchanged thereafter. In contrast, palmitate significantly inhibited the stimulatory effects of 16.7 mmol/L glucose on insulin mRNA levels after 2, 3, and 7 days. To determine whether the inhibitory effect of palmitate on glucose-stimulated insulin mRNA levels was associated with decreased insulin promoter activity, HIT-T15 cells were cultured for 24 hours in 11.1 mmol/L glucose in the presence or absence of palmitate, and insulin gene promoter activity was measured in transient transfection experiments using the insulin promoter-reporter construct INSLUC. INSLUC activity was decreased more than 2-fold after 24 hours of exposure to 0.5 mmol/L palmitate. We conclude that long-term exposure of pancreatic beta cells to palmitate decreases insulin gene expression only in the presence of elevated glucose concentrations, in part through inhibition of insulin gene promoter activity.     

High levels of fatty acids delay the recovery of intracellular pH and cardiac efficiency in post-ischemic hearts by inhibiting glucose oxidation

OBJECTIVES: This study was designed to determine if the fatty acid-induced increase in H(+) production from glycolysis uncoupled from glucose oxidation delays the recovery of intracellular pH (pH(i)) during reperfusion of ischemic hearts. BACKGROUND: High rates of fatty acid oxidation inhibit glucose oxidation and impair the recovery of mechanical function and cardiac efficiency during reperfusion of ischemic hearts. METHODS: pH(i) was measured by 31P nuclear magnetic resonance spectroscopy in isolated working rat hearts perfused in the absence (5.5 mmol/l glucose) or presence of 1.2 mmol/l palmitate (glucose+palmitate). Glycolysis and glucose oxidation were measured using [5-3H/U-14C]glucose. RESULTS: When glucose+palmitate hearts were subjected to 20 min of no-flow ischemia, recoveries of mechanical function and cardiac efficiency were significantly impaired compared with glucose hearts. Glucose oxidation rates were significantly lower in glucose+palmitate hearts during reperfusion compared with glucose hearts, whereas glycolysis rates were unchanged. This resulted in an increase in H(+) production from uncoupled glucose metabolism, and a decreased rate of recovery of pH(i) in glucose+palmitate hearts during reperfusion compared with glucose-perfused hearts. Dichloroacetate (3 mmol/l) given at reperfusion to glucose+palmitate hearts resulted in a 3.2-fold increase in glucose oxidation, a 35% +/- 3% decrease in H(+) production from glucose metabolism, a 1.7-fold increase in cardiac efficiency and a 2.2-fold increase in the rate of pH(i) recovery during reperfusion. CONCLUSIONS: A high level of fatty acid delays the recovery of pH(i) during reperfusion of ischemic hearts because of an increased H(+) production from glycolysis uncoupled from glucose oxidation. Improving the coupling of glucose metabolism by stimulating glucose oxidation accelerates the recovery of pH(i) and improves both mechanical function and cardiac efficiency.     

Palmitate potentiation of glucose-induced insulin release: a study using 2-bromopalmitate

The mechanisms whereby fatty acids (FA) potentiate glucose-induced insulin secretion from the pancreatic beta cell are incompletely understood. In this study, the effects of palmitate on insulin secretion were investigated in isolated rat islets. Palmitate did not initiate insulin secretion at nonstimulatory glucose concentrations, but markedly stimulated insulin release at concentrations of glucose > or = 5.6 mmol/L. At concentrations of palmitate > or =0.5 mmol/L, the important determinant of the potency of the FA was its unbound concentration. At total concentrations < or = 0.5 mmol/L, both the total and unbound concentrations appeared important. Surprisingly, 2-bromopalmitate did not affect palmitate oxidation, but significantly diminished palmitate esterification into cellular lipids. Neither methyl palmitate, which is not activated into a long-chain acyl-CoA ester, nor 2-bromopalmitate affected glucose-stimulated insulin release. Further, 2-bromopalmitate partly inhibited the potentiating effect of palmitate. These results support the concept that FA potentiation of insulin release is mediated by FA-derived signals generated in the esterification pathway.     

Accelerated glycolysis and greater postischemic dysfunction in hypertrophied rat hearts are independent of coronary flow

BACKGROUND: After ischemia, glycolysis and dysfunction are greater, while coupling of glucose oxidation to glycolysis is lower in hypertrophied hearts than in nonhypertrophied hearts. OBJECTIVE: To test the hypothesis that accelerated glycolysis, reduced coupling of glucose oxidation to glycolysis and increased postischemic dysfunction in hypertrophied hearts compared with nonhypertrophied hearts occur in the absence of differences in coronary flow. MATERIALS AND METHODS: Function, glycolysis and glucose oxidation were measured in isolated working control and hypertrophied rat hearts studied for 30 min before, and for 40 min after no flow global ischemia for 20 min under conditions in which coronary flow was comparable between the two groups. The hearts were perfused with 1.2 mmol/L palmitate, 5.5 mmol/L [5-3H/U-14C]-glucose, 0.5 mmol/L lactate, 100 mU/L insulin at a preload of 11.5 mmHg, and an afterload of 60 mmHg in control hearts or 80 mmHg in hypertrophied hearts. RESULTS: Despite comparable rates of coronary flow, functional recovery was lower in hypertrophied hearts than in control hearts. The rates of glycolysis were accelerated in hypertrophied hearts, while glucose oxidation did not significantly differ between the two groups. As a result, the coupling of glucose oxidation to glycolysis was lower in hypertrophied hearts than in control hearts. CONCLUSIONS: Increased postischemic dysfunction, accelerated glycolysis and reduced coupling of glucose oxidation to glycolysis in hypertrophied hearts compared with control hearts cannot be accounted for by differences in coronary flow. These data provide support for the concept that alterations in glucose metabolism contribute to the exaggerated postischemic dysfunction of hypertrophied hearts.     

Glucose stimulation of insulin secretion in islets of fed and starved rats and its dependence on lipid metabolism

The influence of a physiologic range of palmitate concentrations (0, 0.25, 0.5, and 1.0 mmol/L) on glucose ability to modify insulin secretion, (U-14C) palmitate oxidation, and (U-14C) glucose incorporation into lipids has been studied in islets isolated from either fed or 48-hour starved rats. Palmitate potentiated the insulin response of fed islets to glucose in a particular dose-related manner. Glucose stimulated secretion was accompanied by a decreased palmitate oxidation and an increased (U-14C) glucose incorporation into di-, tri-acylglycerols, and predominantly into phospholipids. These metabolic parameters showed also a positive dependence on palmitate concentration. Starvation increased islet capacity to oxidize palmitate, rendered it insensitive to glucose inhibition, and inhibited both (U-14C) glucose incorporation into all lipid fractions and sugar induced insulin release. The stimulation of islet lipid synthesis by glucose seems to be limited by the exogenous supply of fatty acids and their rate of oxidation. As judged from (U-14C) glucose incorporation data, the rate of phospholipid biosynthesis showed a significant and positive correlation with insulin secretion. This metabolic pathway might provide islet cells with some lipid intermediates (diacylglycerol and/or specific phospholipids) that have been considered as possible mediators of the calcium messenger system.     

Fatty acid induced insulin resistance in rat-1 fibroblasts overexpressing human insulin receptors: impaired insulin-stimulated mitogen-activated protein kinase activity

Summary Saturated fatty acids cause insulin resistance but the underlying molecular mechanism is still unknown. We examined the effect of saturated non-esterified fatty acids on insulin binding and action in transfected Rat-1 fibroblasts, which over-expressed human insulin receptors. Incubation with 1.0 mmol/l palmitate for 1–4 h did not affect insulin binding, insulin receptor autophosphorylation, insulin-stimulated tyrosine kinase activity toward poly(Glu⁴:Tyr¹), pp185 and Shc phosphorylation and PI3-kinase activity in these cells. However, the dose response curve of insulin-stimulated glucose transport was right-shifted. Palmitate inhibited the maximally insulin-stimulated mitogen activated protein (MAP) kinase activity toward synthetic peptide to 7 % that of control. The palmitate treatment influenced neither cytosolic protein kinase A activity nor cAMP levels. These results suggested that 1) palmitate did not inhibit the early steps of insulin action from insulin binding to pp185 or Shc phosphorylation but inhibited insulin-stimulated MAP kinase, and that 2) palmitate decreased insulin sensitivity as manifested by inhibited insulin-stimulated glucose uptake. In conclusion, the mechanism of saturated non-esterified fatty acid induced insulin resistance in glucose uptake may reside at post PI3-kinase or Shc steps, including the level of MAP kinase activation. [Diabetologia (1997) 40: 894–901] Received: 15 January 1997 and in revised form: 9 April 1997     

Differential mechanisms of glucose and palmitate in augmentation of insulin secretion in mouse pancreatic islets

Aims/hypothesis. To assess the possible importance of saturated fatty acids in glucose amplification of K⁺ _ATP channel-independent insulin secretion. Methods. Insulin release from perifused pancreatic islets of NMRI mice was determined by radioimmunoassay. Results. In the presence of K⁺ (20 mmol/l) and diazoxide (250 μmol/l), which stimulates Ca²⁺ influx and opens K⁺ _ATP channels, palmitate (165 μmol/l total; 1.2 μmol/l free) increased insulin secretion at 3.3, 10 and 16.7 mmol/l glucose while glucose (10; 16.7 mmol/l) did not increase insulin secretion. In the presence of K⁺ (60 mmol/l) and diazoxide (250 μmol/l), glucose (10; 16.7 mmol/l) stimulation of K⁺ _ATP channel-independent insulin secretion increased, whereas the effectiveness of palmitate (165 μmol/l total; 1.2 μmol/l free) on insulin secretion at both 3.3, 10 or 16.7 mmol/l glucose was reduced. Palmitate thereby mimicked the stimulatory pattern of the protein kinase C activator, 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l), which also failed to increase insulin secretion at maximum depolarising concentrations of K⁺ (60 mmol/l). Furthermore, the protein kinase C inhibitor calphostin C (1 μmol/l), led to a complete suppression of the effects of both palmitate (165 μmol/l total; 1.2 μmol/l free) and myristate (165 μmol/l total; 2.4 μmol/l free) stimulation of glucose (16.7 mmol/l)-induced insulin secretion. Calphostin C (1 μmol/l), however, failed to affect insulin secretion induced by glucose (16.7 mmol/l). Conclusion/interpretation. These data suggest that glucose could increase insulin secretion independently of saturated fatty acids like palmitate and myristate, which amplify glucose-induced insulin secretion by activation of protein kinase C. [Diabetologia (2001) 44: 738–746] Received: 30 October 2001 and in revised form: 31 January 2001     

In vitro study of human skeletal muscle strips: effect of nonesterified fatty acid supply on glucose storage

To examine the effect of increased nonesterified fatty acid concentration on glucose storage in human muscle, an in vitro method for study of glycogen synthesis in this tissue has been established. Muscle strips obtained from rectus abdominus during elective abdominal surgery were clamped at resting length, and adenosine triphosphate/total adenosine nucleotide ratios remained constant for 3 hours ex vivo. Leakage of enzyme markers of muscle damage was minimal, and electron microscopy showed preserved myofibril ultrastructure. Insulin stimulation brought about a dose-dependent increase in rates of glycogen synthesis with a half-maximal effect at 9 x 10(-10) mol/L insulin. In 15 consecutive studies, basal rates of glycogen synthesis were 4.1 +/- 0.5, 3.2 +/- 0.7, and 3.0 +/- 0.3 nmol glucose/25 mg/h in the absence of palmitate, with 1.4 mmol/L and 2.8 mmol/L palmitate, respectively. Insulin-stimulated rates of glycogen synthesis were 8.6 +/- 1.2, 6.0 +/- 1.8, and 5.8 +/- 0.8 nmol glucose/25 mg/h. Thus, increasing fatty acid concentrations decreased rates of glycogen synthesis both basally and with insulin stimulation. The insulin signal itself was not affected as the percentage stimulation over basal rates remained approximately constant in the presence or absence of fatty acid (2.1-, 1.9- and 1.9-fold, respectively). Insulin sensitivity in vivo is usually expressed as absolute rates of glucose uptake during euglycemic hyperinsulinemia, and if plasma fatty acid elevation were to be studied in vivo an erroneous conclusion may be reached of resistance to hormone action per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

High rates of residual fatty acid oxidation during mild ischemia decrease cardiac work and efficiency

It is unknown what effects high levels of fatty acids have on energy metabolism and cardiac efficiency during milder forms of ischemia. To address this issue, isolated working rat hearts perfused with Krebs-Henseleit solution (5 mM glucose, 100 μU/mL insulin, and 0.4 (Normal Fat) or 1.2 mM palmitate (High Fat)) were subjected to 30 min of aerobic perfusion followed by 30 min of mild ischemia (39% reduction in coronary flow). Both groups had similar aerobic function and rates of glycolysis, however the High Fat group had elevated rates of palmitate oxidation (150%), and decreased rates of glucose oxidation (51%). Mild ischemia decreased cardiac work (56% versus 40%) and efficiency (29% versus 11%) further in High Fat hearts. Palmitate oxidation contributed a greater percent of acetyl-CoA production during mild ischemia in the High Fat group (81% versus 54%). During mild ischemia glycolysis remained at aerobic levels in the Normal Fat group, but was accelerated in the High Fat group. Triglyceride, glycogen and adenine nucleotide content did not differ at the end of mild ischemia, however glycogen turnover was double in the High Fat group (248%). Addition of the pyruvate dehydrogenase inhibitor dichloroacetate to the High Fat group resulted in a doubling of the rate of glucose oxidation and improved cardiac efficiency during mild ischemia. We demonstrate that fatty acid oxidation dominates as the main source of residual oxidative metabolism during mild ischemia, which is accompanied by suppressed cardiac function and efficiency in the presence of high fat.     

GPR119 regulates genetic markers of fatty acid oxidation in cultured skeletal muscle myotubes

Gene knockout and agonist studies indicate that activation of the G protein-coupled receptor, GPR119, protects against diet-induced obesity and insulin resistance. It is not known if GPR119 activation in skeletal muscle mediates these effects. To address this uncertainty, we measured GPR119 expression in skeletal muscle and determined the effects of PSN632408, a GPR119 agonist, on the expression of genes and proteins required for fatty acid and glucose oxidation in cultured myotubes. GPR119 expression was readily detected in rat skeletal muscle and mRNAs were induced by 12 weeks of high-fat feeding. Treatment of cultured mouse C₂C₁₂ myotubes with 5 μM PSN632408 or 0.5 mM palmitate reduced expression of mRNAs encoding fatty acid oxidation genes to similar extents. More so, treatment with PSN632408 decreased AMPKα (Thr172 phosphorylation) activity in the absence of palmitate and ACC (Ser79 phosphorylation) activity in the presence of palmitate. In human primary myotubes PSN632408 decreased expression of PDK4 and AMPKα2 mRNA in myotubes derived from obese donors. These data suggest GPR119 activation in skeletal muscle may impair fatty acid and glucose oxidation.     

Stimulatory short-term effects of free fatty acids on glucagon secretion at low to normal glucose concentrations

While free fatty acids (FFA) are well known as insulin secretagogues, their effects on pancreatic alpha cells have been mostly neglected. In the present study we therefore systematically analyzed the glucagon metabolism of rat pancreatic islets under the influence of FFA. Primary islets were incubated in the presence or absence of 200 micromol/L albumin-complexed palmitate or oleate at 2.8 mmol/L versus 16.7 mmol/L glucose and glucagon secretion was monitored over 8 hours. In addition to these time-course experiments, dose dependency of palmitate-induced effects was tested by a 2-hour incubation with 50 to 300 micromol/L albumin-complexed palmitate at 2.8 mmol/L and 5.6 mmol/L glucose. Apart from glucagon secretion, intracellular immunoreactive glucagon and cellular preproglucagon-mRNA (PPG-mRNA) content were determined from the remaining cell lysates. FFA, especially palmitate, induced a significant and dose-dependent increase of glucagon secretion (in average 2-fold above control) during the first 120 minutes of incubation at low to normal glucose (2.8 and 5.6 mmol/L). There was no significant glucagonotropic effect of FFA at concomitant 16.7 mmol/L glucose. Intracellular glucagon as well as cellular PPG-mRNA content were found to be dose-dependently diminished by palmitate when compared with untreated controls at 5.6 mmol/L glucose. The present analysis therefore points to a new role for FFA as a nutritient secretagogue and a modulator of alpha-cellular glucagon metabolism.     

Curcumin improves insulin resistance in skeletal muscle of rats

Background and aims

Curcumin has been reported to lower plasma lipids and glucose in diabetic rats, and to decrease body weight in obese rats, which may partly be due to increased fatty acid oxidation and utilization in skeletal muscle.

Methods and results

Diabetic rats induced by high-fat diet plus streptozotocin (STZ, 30 mg/kg BW) were fed a diet containing 50, 150, or 250 mg/kg BW curcumin for 7 wk. Curcumin dose-dependently decreased plasma lipids and glucose and the dose 150 mg/kg BW appeared to be adequate to produce a significant effect. Curcumin supplementation reduced glucose and insulin tolerance measured as areas under the curve. L6 myotubes were treated with palmitate (0.25 mmol/L) in the presence of different levels of curcumin for 24 h in our in vitro experiment. Curcumin at 10 μmol/L was adequate to cause a significant increase in 2-deoxy-[³H]d-glucose uptake by L6 myotubes. Curcumin up-regulated expression of phosphorylated AMP-activated protein kinase (AMPK), CD36, and carnitine palmitoyl transferase 1, but down-regulated expression of pyruvate dehydrogenase 4 and phosphorylated glycogen synthase (GS) in both in vivo and in vitro studies. Moreover, curcumin increased phosphorylated acetyl COA carboxylase in L6 myotubes. The effects of curcumin on these enzymes except for GS were suppressed by AMPK inhibitor, Compound C. LKB1, an upstream kinase of AMPK, was activated by curcumin and inhibited by radicicol, an LKB1 destabilizer.

Conclusion

Curcumin improves muscular insulin resistance by increasing oxidation of fatty acid and glucose, which is, at least in part, mediated through LKB1-AMPK pathway.     

Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids

Summary Prolonged exposure of islets to fatty acids results in a lowered glucose set-point for insulin secretion. We examined the mechanism in islets cultured for 24 h with 0.25 mmol/l palmitate. As expected, insulin secretion at 2.8 and 8.3 mmol/l glucose was increased in the palmitate-treated islets as opposed to no change at 27.7 mmol/l glucose. Co-culturing with 0.05 μg/ml Triacsin C, an inhibitor of long chain acyl-CoA synthetase, blocked this effect. Glucose utilization and oxidation showed the same pattern as insulin secretion, with the step-up for both measurements being fully manifest at 2.8 mmol/l glucose. Glucokinase K_m and V_max measured in islet extracts were unaffected by the palmitate. In contrast, hexokinase V_max was increased by 25–35 % in both the cytoplasmic and mitochondrial-bound pools. Our data suggest prolonged exposure to fatty acids increased beta-cell hexokinase activity, thereby modifying the kinetics of glucose entry into the metabolic pathway and glucose-induced insulin secretion. The cellular mediator is likely an increased level of long chain fatty acyl-CoA esters. [Diabetologia (1997) 40: 392–397] Received: 5 November 1996 and in revised form: 7 January 1997     

Unbound rather than total concentration and saturation rather than unsaturation determine the potency of fatty acids on insulin secretion.

Isolated mouse islets were used to compare the effects of three saturated (myristate, palmitate and stearate) and three unsaturated (oleate, linoleate and linolenate) long-chain fatty acids on insulin secretion. By varying the concentrations of fatty acid (250-1250 micromol/l) and albumin simultaneously or independently, we also investigated whether the insulinotropic effect is determined by the unbound or total concentration of the fatty acids. Only palmitate and stearate slightly increased basal insulin secretion (3 mmol/l glucose). All tested fatty acids potentiated glucose-induced insulin secretion (10-15 mmol/l), and the following rank order of potency was obtained when they were compared at the same total concentrations: palmitate approximately = stearate > myristate > or = oleate > or = linoleate approximately = linolenate. The effect of a given fatty acid varied with the fatty acid to albumin molar ratio, in a way which indicated that the unbound fraction is the important one for the stimulation of beta cells. When the potentiation of insulin secretion was expressed as a function of the unbound concentrations, the following rank order emerged: palmitate > myristate > stearate approximately = oleate > linoleate approximately = linolenate. In conclusion, the acute and direct effects of long-chain fatty acids on insulin secretion are due to their unbound fraction. They are observed only at fatty acid/albumin ratios higher than those normally occurring in plasma. Saturated fatty acids are stronger insulin secretagogues than unsaturated fatty acids. Unbound palmitate is by far the most potent of the six common long-chain fatty acids.     

AMP-activated protein kinase control of energy metabolism in the ischemic heart

Myocardial ischemia produces an energy-deficient state in heart muscle, which if not corrected can lead to cardiomyocyte death. AMP-activated protein kinase (AMPK) is a key kinase that can increase energy production in the ischemic heart. During ischemia a rapid activation of AMPK occurs, resulting in an activation of both myocardial glucose uptake and glycolysis, as well as an increase in fatty acid oxidation. This activation of AMPK has the potential to increase energy production, thereby protecting the heart during ischemic stress. However, at clinically relevant high levels of fatty acids, ischemia-induced activation of AMPK also stimulates fatty acid oxidation during and following ischemia. This can contribute to ischemic injury secondary to an inhibition of glucose oxidation, which results in a decrease in cardiac efficiency. As a result, AMPK activation has the potential to be either beneficial or harmful in the ischemic heart.     

高浓度葡萄糖、脂肪酸和糖皮质激素对PDX-1表达和胰岛素分泌功能的影响

SD大鼠胰岛细胞分别在含5.6mmol／L葡萄糖、33mmol／L葡萄糖、0．6mmol／L软脂酸或20nmol／L地塞米松培养液中培养3d，RIA测定基础和葡萄糖刺激后胰岛素分泌量，免疫组化法测定细胞内胰十二指肠同源盒蛋白1（PDX-1）蛋白表达和原位杂交法测定细胞内PDX-1 mRNA表达。结果显示高浓度葡萄糖、脂肪酸和糖皮质激素对PDX-1蛋白表达和胰岛素分泌均有抑制作用。     

Palmitate-mediated alterations in the fatty acid metabolism of rat neonatal cardiac myocytes

During ischemia and reperfusion, increased palmitate oxidation is associated with diminished function of the myocardium. Palmitate, but not oleate, has been implicated in the induction of apoptosis in isolated neonatal rat ventricular myocytes. We report that extended incubation (20 h) of cultured neonatal rat cardiomyocytes, in the presence of palmitate, causes a decrease in the ability of these cells to oxidize fatty acids, an increase in cellular malonyl-CoA and a decrease in the activity of 5' AMP-activated protein kinase (AMPK) compared to myocytes incubated in the presence of oleate. While palmitate decreases the oxidative metabolism of fatty acids, it increases the formation of intracellular triglyceride and ceramide. Increased ceramide formation is associated with an increase in apoptosis in many cell systems and we also observe an increase in caspase-3 like activity and DNA-laddering in these cells. At the onset of cardiac failure, a switch in myocardial substrate utilization from fatty acids to glucose occurs. Our data suggest that decreased palmitate oxidation in cardiac myocytes in culture may signal the initiation of programmed cell death and ceramide elevation previously documented in ischemic, reperfused hearts.     

Antiketogenic effect of glucose per se in vivo in man and in vitro in isolated rat liver cells

The in vivo effect of glucose per se on blood ketone bodies, glycerol, and nonesterified fatty acids (NEFA) has been investigated in five normal (60 hours fasted) men receiving a somatostatin (SRIF) infusion (500 micrograms/h-1). When glycemia was raised over 10 mmol/L for 180 minutes by exogenous IV glucose infusion, neither insulin nor C peptide increase. NEFA and glycerol returned to fasting value in 40 minutes and remained stable. Ketone bodies decreased continuously and were significantly below the fasting values at the end of the study (1.3 +/- 0.3 mmol/L v 2.2 +/- 0.4 mmol/L, P less than 0.05). In order to ascertain whether glucose has been acting only on lipolysis or also on the liver ketogenic capacity, its effect was studied in vitro on isolated liver cells from 24-hour starved rats incubated with various amounts of palmitate. Glucose (30 mmol/L) did not affect the maximal ketogenic capacity (80 mumol/g (w/w)/h) measured with 1.6 mmol/L palmitate but increased the apparent palmitate K 0.5 for ketogenesis from 0.16 to 0.3 mmol/L. At physiologic free fatty acids concentration (0.22 mmol/L), glucose decreased ketogenesis by 90%. The effect was time-dependent, maximum after 30 minutes of incubation. Half-maximum inhibition by glucose was obtained at 6 mmol/L, a concentration at which lactate production was unaffected. These results suggest that glucose per se inhibits ketogenesis in vivo by acting probably both on lipolysis and on liver ketogenic capacity.