Propofol (Diprivan®) and Intralipid® exacerbate insulin resistance in type-2 diabetic hearts by impairing GLUT4 trafficking.

BACKGROUND: The IV anesthetic, propofol, when administered as fat emulsion-based formulation (Diprivan) promotes insulin resistance, but the direct effects of propofol and its solvent, Intralipid, on cardiac insulin resistance are unknown. METHODS: Hearts of healthy and type-2 diabetic rats (generated by fructose feeding) were aerobically perfused for 60 minutes with 10 µM propofol in the formulation of Diprivan or an equivalent concentration of its solvent Intralipid (25 µM) ± insulin (100 mU•L). Glucose uptake, glycolysis, and glycogen metabolism were measured using [H]glucose. Activation of Akt, GSK3ß, AMPK, ERK1/2, p38MAPK, S6K1, JNK, protein kinase Cθ (PKCθ), and protein kinase CCßII (PKCßII) was determined using immunoblotting. GLUT4 trafficking and phosphorylations of insulin receptor substrate-1 (IRS-1) at Ser307(h312), Ser1100(h1101), and Tyr608(hTyr612) were measured. Mass spectrometry was used to determine acylcarnitines, phospholipids, and sphingolipids. RESULTS: Diprivan and Intralipid reduced insulin-induced glucose uptake and redirected glucose to glycogen stores in diabetic hearts. Reduced glucose uptake was accompanied by lower GLUT4 trafficking to the sarcolemma. Diprivan and Intralipid inactivated GSK3ß but activated AMPK and ERK1/2 in diabetic hearts. Only Diprivan increased phosphorylation of Akt(Ser473/Thr308) and translocated PKCθ and PKCßII to the sarcolemma in healthy hearts, whereas it activated S6K1 and p38MAPK and translocated PKCßII in diabetic hearts. Furthermore, only Diprivan phosphorylated IRS-1 at Ser1100(h1101) in healthy and diabetic hearts. JNK expression, phosphorylation of Ser307(h312) of IRS-1, and PKCθ expression and translocation were increased, whereas GLUT4 expression was reduced in insulin-treated diabetic hearts. Phosphatidylglycerol, phosphatidylethanolamine, and C18-sphingolipids accumulated in Diprivan-perfused and Intralipid-perfused diabetic hearts. CONCLUSIONS: Propofol and Intralipid promote insulin resistance predominantly in type-2 diabetic hearts.

Loss of Intralipid®- but not sevoflurane-mediated cardioprotection in early type-2 diabetic hearts of fructose-fed rats: importance of ROS signaling.

BACKGROUND: Insulin resistance and early type-2 diabetes are highly prevalent. However, it is unknown whether Intralipid® and sevoflurane protect the early diabetic heart against ischemia-reperfusion injury. METHODS: Early type-2 diabetic hearts from Sprague-Dawley rats fed for 6 weeks with fructose were exposed to 15 min of ischemia and 30 min of reperfusion. Intralipid® (1%) was administered at the onset of reperfusion. Peri-ischemic sevoflurane (2 vol.-%) served as alternative protection strategy. Recovery of left ventricular function was recorded and the activation of Akt and ERK 1/2 was monitored. Mitochondrial function was assessed by high-resolution respirometry and mitochondrial ROS production was measured by Amplex Red and aconitase activity assays. Acylcarnitine tissue content was measured and concentration-response curves of complex IV inhibition by palmitoylcarnitine were obtained. RESULTS: Intralipid® did not exert protection in early diabetic hearts, while sevoflurane improved functional recovery. Sevoflurane protection was abolished by concomitant administration of the ROS scavenger N-2-mercaptopropionyl glycine. Sevoflurane, but not Intralipid® produced protective ROS during reperfusion, which activated Akt. Intralipid® failed to inhibit respiratory complex IV, while sevoflurane inhibited complex I. Early diabetic hearts exhibited reduced carnitine-palmitoyl-transferase-1 activity, but palmitoylcarnitine could not rescue protection and enhance postischemic functional recovery. Cardiac mitochondria from early diabetic rats exhibited an increased content of subunit IV-2 of respiratory complex IV and of uncoupling protein-3. CONCLUSIONS: Early type-2 diabetic hearts lose complex IV-mediated protection by Intralipid® potentially due to a switch in complex IV subunit expression and increased mitochondrial uncoupling, but are amenable to complex I-mediated sevoflurane protection.

The mechanism of Intralipid®-mediated cardioprotection complex IV inhibition by the active metabolite, palmitoylcarnitine, generates reactive oxygen species and activates reperfusion injury salvage kinases.

BACKGROUND: Intralipid® administration at reperfusion elicits protection against myocardial ischemia-reperfusion injury. However, the underlying mechanisms are not fully understood. METHODS: Sprague-Dawley rat hearts were exposed to 15 min of ischemia and 30 min of reperfusion in the absence or presence of Intralipid® 1% administered at the onset of reperfusion. In separate experiments, the reactive oxygen species (ROS) scavenger N-(2-mercaptopropionyl)-glycine was added either alone or with Intralipid®. Left ventricular work and activation of Akt, STAT3, and ERK1/2 were used to evaluate cardioprotection. ROS production was assessed by measuring the loss of aconitase activity and the release of hydrogen peroxide using Amplex Red. Electron transport chain complex activities and proton leak were measured by high-resolution respirometry in permeabilized cardiac fibers. Titration experiments using the fatty acid intermediates of Intralipid® palmitoyl-, oleoyl- and linoleoylcarnitine served to determine concentration-dependent inhibition of complex IV activity and mitochondrial ROS release. RESULTS: Intralipid® enhanced postischemic recovery and activated Akt and Erk1/2, effects that were abolished by the ROS scavenger N-(2-mercaptopropionyl)glycine. Palmitoylcarnitine and linoleoylcarnitine, but not oleoylcarnitine concentration-dependently inhibited complex IV. Only palmitoylcarnitine reached high tissue concentrations during early reperfusion and generated significant ROS by complex IV inhibition. Palmitoylcarnitine (1 µM), administered at reperfusion, also fully mimicked Intralipid®-mediated protection in an N-(2-mercaptopropionyl)-glycine -dependent manner. CONCLUSIONS: Our data describe a new mechanism of postconditioning cardioprotection by the clinically available fat emulsion, Intralipid®. Protection is elicited by the fatty acid intermediate palmitoylcarnitine, and involves inhibition of complex IV, an increase in ROS production and activation of the RISK pathway.

Early mitochondrial dysfunction in glycolytic muscle, but not oxidative muscle, of the fructose-fed insulin-resistant rat.

Although evidence that type 2 diabetes mellitus (T2DM) is accompanied by mitochondrial dysfunction in skeletal muscle has been accumulating, a causal link between mitochondrial dysfunction and the pathogenesis of the disease remains unclear. Our study focuses on an early stage of the disease to determine whether mitochondrial dysfunction contributes to the development of T2DM. The fructose-fed (FF) rat was used as an animal model of early T2DM. Mitochondrial respiration and acylcarnitine species were measured in oxidative (soleus) and glycolytic [extensor digitorum longus (EDL)] muscle. Although FF rats displayed characteristic signs of T2DM, including hyperglycemia, hyperinsulinemia, and hypertriglyceridemia, mitochondrial content was preserved in both muscles from FF rats. The EDL muscle had reduced complex I and complex I and II respiration in the presence of pyruvate but not glutamate. The decrease in pyruvate-supported respiration was due to a decrease in pyruvate dehydrogenase activity. Accumulation of C14:1 and C14:2 acylcarnitine species and a decrease in respiration supported by long-chain acylcarnitines but not acetylcarnitine indicated dysfunctional ß-oxidation in the EDL muscle. In contrast, the soleus muscle showed preserved mitochondrial respiration, pyruvate dehydrogenase activity, and increased fatty acid oxidation, as evidenced by overall reduced acylcarnitine levels. Aconitase activity, a sensitive index of reactive oxygen species production in mitochondria, was reduced exclusively in EDL muscle, which showed lower levels of the antioxidant enzymes thioredoxin reductase and glutathione peroxidase. Here, we show that the glycolytic EDL muscle is more prone to an imbalance between energy supply and oxidation caused by insulin resistance than the oxidative soleus muscle.

Infarct-remodelled hearts with limited oxidative capacity boost fatty acid oxidation after conditioning against ischaemia/reperfusion injury.

AIMS: Infarct-remodelled hearts are less amenable to protection against ischaemia/reperfusion. Understanding preservation of energy metabolism in diseased vs. healthy hearts may help to develop anti-ischaemic strategies effective also in jeopardized myocardium. METHODS AND RESULTS: Isolated infarct-remodelled/sham Sprague-Dawley rat hearts were perfused in the working mode and subjected to 15 min of ischaemia and 30 min of reperfusion. Protection of post-ischaemic ventricular work was achieved by pharmacological conditioning with sevoflurane. Oxidative metabolism was measured by substrate flux in fatty acid and glucose oxidation using [(3)H]palmitate and [(14)C]glucose. Mitochondrial oxygen consumption was measured in saponin-permeabilized left ventricular muscle fibres. Activity assays of citric acid synthase, hydroxyacyl-CoA dehydrogenase, and pyruvate dehydrogenase and mass spectrometry for acylcarnitine profiling were also performed. Six weeks after coronary artery ligation, the hearts exhibited macroscopic and molecular signs of hypertrophy consistent with remodelling and limited respiratory chain and citric acid cycle capacity. Unprotected remodelled hearts showed a marked decline in palmitate oxidation and acetyl-CoA energy production after ischaemia/reperfusion, which normalized in sevoflurane-protected remodelled hearts. Protected remodelled hearts also showed higher ß-oxidation flux as determined by increased oxygen consumption with palmitoylcarnitine/malate in isolated fibres and a lower ratio of C16:1+C16OH/C14 carnitine species, indicative of a higher long-chain hydroxyacyl-CoA dehydrogenase activity. Remodelled hearts exhibited higher PPARα-PGC-1α but defective HIF-1α signalling, and conditioning enabled them to mobilize fatty acids from endogenous triglyceride stores, which closely correlated with improved recovery. CONCLUSIONS: Protected infarct-remodelled hearts secure post-ischaemic energy production by activation of ß-oxidation and mobilization of fatty acids from endogenous triglyceride stores.