Glucose-fatty acid interaction in skeletal muscle and adipose tissue in insulin resistance.

Insulin resistance (IR) is the result of long-lasting positive energy balance and the imbalance between the uptake of energy rich substrates (glucose, lipids) and energy output. The defects in the metabolism of glucose in IR and type 2 diabetes are closely associated with the disturbances in the metabolism of lipids. In this review, we have summarized the evidence indicating that one of the important mechanisms underlying the development of IR is the impaired ability of skeletal muscle to oxidize fatty acids as a consequence of elevated glucose oxidation in the situation of hyperglycemia and hyperinsulinemia and the impaired ability to switch easily between glucose and fat oxidation in response to homeostatic signals. The decreased fat oxidation results into the accumulation of intermediates of fatty acid metabolism that are supposed to interfere with the insulin signaling cascade and in consequence negatively influence the glucose utilization. Pathologically elevated fatty acid concentration in serum is now accepted as an important risk factor leading to IR. Adipose tissue plays a crucial role in the regulation of fatty acid homeostasis. The adipose tissue may be the primary site where the early metabolic disturbances leading to the development of IR take place and the development of IR in other tissues follows. In this review we present recent evidence of mutual interaction between skeletal muscle and adipose tissue in the establishment of IR and type 2 diabetes.

The impaired response of non-obese hereditary hypertriglyceridemic rats to glucose load is associated with low glucose storage in energy reserves.

The aim of the study was to determine the contribution of skeletal muscle, adipose tissue and liver to the impaired glucose clearance manifesting itself during the initial phase of OGTT in a non-obese animal model of insulin resistance, hereditary hypertriglyceridemic (HHTg) rats. Glucose utilisation and storage in insulin target tissues in vivo and in vitro after a glucose load (3 g/kg b. wt.) administered intragastrically following overnight fasting was compared in adult male HHTg rats and Wistar normotriglyceridemic controls after short-term (2 wk) high-sucrose (70 % calories as sucrose) feeding period. In comparison with normotriglyceridemic controls, in HHTg rats the glucose administration did not stimulate GLUT4 translocation to the plasma membrane in skeletal muscle and adipose tissue that was associated with decreased glucose utilisation by these tissues in vitro. The acute glucose supply did not result in increased glycogen synthesis in the liver and fatty acid synthesis de novo in adipose tissue. On the contrary, the serum glucose, triglyceride and free fatty acid levels remained elevated. In conclusion, in the tissues of HHTg rats, despite the increased insulinemia, the processes leading toward increased glucose utilisation and processes transforming glucose into storage forms, such as triglycerides in adipose tissue and glycogen in skeletal muscle and liver, did not start within this time interval. The combination of the impaired glucose utilisation and the impaired glucose storage in energy reserves leads to higher glycaemia following glucose load in HHTg rats.

The effect of captopril on nitric oxide formation and on generation of radical forms of mitochondrial respiratory chain compounds in ischemic rat heart.

The increase of radical forms of mitochondrial respiratory chain compounds (MRCC) is an indicator of an increased risk of the formation of oxygen radicals. Using electron paramagnetic resonance (EPR), we found an increase of signals corresponding to ubisemichinone radical (.QH) and ironsulfur proteins radical forms (-FeS) of these respiratory chain compounds during ischemia in the isolated perfused rat heart (.QH increased from 1.51 to 3.08, .FeS1 from 1.14 to 2.65 arbitrary units). During the 5-min reperfusion, the signals returned to normoxic levels. In isolated mitochondria exposed to anoxia and reoxygenation the radical forms of .QH and FeS2 changed in a similar manner as in the intact heart. A combination of in vivo captopril treatment and in vitro L-arginine administration significantly decreased the levels of MRCC radicals in the isolated myocardium (.QH from 2.61 to 1.72 and .FeS, from 1.82 to 0.46 under normoxia; .QH from 4.35 to 2.66 and .FeS1 from 1.93 to 1.35 during ischemia). This decrease in MRCC radical forms was associated with increased NO levels in the perfusate, determined as NO2- / NO3-, as well as tissue NO levels determined using EPR as the dinitrosyl iron complex (DNIC). These results provide new information about the cardioprotective effects of ACE inhibitors and L-arginine.

Effect of ACE inhibitor captopril and L-arginine on the metabolism and on ischemia-reperfusion injury of the isolated rat heart.

We investigated the effects of in vivo treatment with the angiotensin-converting enzyme inhibitor (ACE-I) captopril and/or of in vitro administration of L-arginine on the metabolism and ischemia-reperfusion injury of the isolated perfused rat myocardium. Captopril (50 mg/l in drinking water, 4 weeks) raised the myocardial content of glycogen. After 25-min global ischemia, captopril treatment, compared with the controls, resulted in lower rates of lactate dehydrogenase release during reperfusion (8.58 +/- 1.12 vs. 13.39 +/- 1.88 U/heart/30 min, p<0.05), lower myocardial lactate contents (11.34 +/- 0.93 vs. 21.22 +/- 4.28 micromol/g d.w., p<0.05) and higher coronary flow recovery (by 25%), and prevented the decrease of NO release into the perfusate during reperfusion. In control hearts L-arginine added to the perfusate (1 mmol/l) 10 min before ischemia had no effect on the parameters evaluated under our experimental conditions, presumably because of sufficient saturation of the myocardium with L-arginine. In the hearts of captopril-treated rats, L-arginine further increased NO production during reperfusion and the cGMP content before ischemia. Our results have shown that long-term captopril treatment increases the energy potential and has a beneficial effect on tolerance of the isolated heart to ischemia. L-arginine added into the perfusate potentiates the effect of captopril on the NO signaling pathway.

Effect of chronic renal insufficiency on the function and metabolic parameters of the isolated rat heart.

Chronic renal insufficiency (CRI) is often associated with cardiovascular disease; however, its underlying mechanisms are not completely understood. Therefore, in the present study, myocardial functions and metabolic changes were investigated using an animal model of CRI in subtotally nephrectomized rats. In addition, some other parameters, considered risk factors of cardiovascular diseases, were determined. Subtotal nephrectomy led to an elevation in blood pressure (144 +/- 2.8 vs 114 +/- 2.5 mm Hg), left ventricular hypertrophy (290 +/- 12 vs 200 +/- 40 mg/100 g b.w.), hypertriglyceridaemia (2.96 +/- 0.31 vs 0.77 +/- 0.07 mmol/l), and impaired glucose tolerance (AUC 836 +/- 12.4 vs 804 +/- 10.4 mmol x l(-1) x 120 min). Isolated perfused hearts of uraemic rats exhibited diminished basal functions (coronary and aortic flow, stroke volume) by 20-30% compared with the controls. Interestingly, the tolerance of isolated heart to global 20-min no-flow ischaemia was improved in uraemic rats. The most marked differences in heart function recovery during reperfusion concerned aortic flow (90 +/- 2.3 vs 66 +/- 10%) and stroke volume (97 +/- 2.7 vs 68 +/- 5.6% of pre-ischaemic values). Pre-ischaemic myocardial glycogen content was distinctly increased (by 50%) in uraemic rats compared with the controls.

A comparison of the protective effect of a modified StTH solution and HTK-B on the energy and functional status of the isolated rat heart.

Using a model of the isolated beating rat heart, the authors compared the protective effect of St. Thomas Hospital cardioplegic solution enriched with glucose and mannitol (StTH-M) and Bretschneider solution (HTK-B). Results showed that, during 120-minute global ischaemia in cardioplegia, StTH-M was able to maintain levels of high-energy phosphates comparable with those found in a group of hearts perfused with HTK-B at 20 degrees C only when the temperature had been decreased to 12-15 degrees C. Under these conditions, repair of metabolic and functional parameters during post-ischaemic perfusion was also similar in both groups.

A method for slowing down depletion of myocardial energy reserves of the donor heart before transplantation.

One of the methods of donor heart protection against ischemia is a substantial lowering of temperature of the heart perfused with cardioplegic solution (CS). The achieved conservation of energetically rich compounds, however, does not guarantee the full restoration of heart function during reperfusion. Another possibility for heart preservation is repeated application of CS at 20 degrees C. This variant was tested in our experiments on isolated rat hearts perfused under constant pressure with the Krebs-Henseleit solution according to Langendorff. During global ischemia (180 min at 20 degrees C) we applied the St. Thomas Hospital CS lx or 4 x at 60 min intervals. During the ischemia, glycogen, ATP, lactate, Na+, K+ were assessed in the heart. The heart injury was monitored as the release of lactate dehydrogenase (LD) during the 60 min reperfusion. Repeated CS perfusion of the heart during ischemia lowers the contents of lactate and maintains ATP and glycogen content at elevated levels throughout ischemia. The improved condition of the heart after repeated CS application demonstrated as prevention of the gain of Na+ in the cells at the end of ischemia as well as after reperfusion. This was associated with reduced intracellular potassium depletion and LD release into the perfusate.

The phosphate pool of isolated dog heart during global ischaemia: comparison of two cardioplegic solutions with 31P NMR spectroscopy.

31P NMR spectroscopy was used to study the time course of changes in the concentration of high-energy metabolites and intracellular pH in the dog myocardium during hypothermic ischaemia at 9 degrees C in Bretschneider (HTK-B) and St. Thomas' Hospital (StTH) cardioplegic solutions. It was found that ATP and phosphocreatine degrade slowlier in HTK-B than in StTH, with phosphocreatine depletion occurring within 7.9 +/- 1.4 h in HTK-B and within 6.2 +/- 1.4 h in StTH. The values are virtually identical with the time intervals at which ATP concentration falls below the critical level (60% of initial ATP concentration). In agreement with biochemical analysis, a higher concentration of phosphomonoesters was noted until the 180th minute of ischaemia in HTK-B, a finding suggesting more rapid glycogen degradation in HTK-B. Even though HTK-B contains a high concentration of histidine buffer, higher values of intracellular pH were found during ischaemia in StTH. The effect of extracellular concentration of sodium ions on intracellular pH is discussed.

Heart injury in the calcium paradox: the effect of manganese.

Prevention by manganese ions of heart injury induced by the calcium paradox was studied in isolated perfused rat heart. Lactate dehydrogenase (LDH) release, ATP and glycogen content, and 45Ca2+ accumulation were used as markers of the injury. If Mn2+ substituted Ca2+ in the perfusion buffer after Ca2+-free perfusion, LDH release from the heart was inhibited but the inhibition was eliminated by Ca2+ readmission. However, Mn2+ (0.2-2.5 mM), added from the beginning of Ca2+-free perfusion, prevented heart injury at the time of Ca2+ repletion. LDH release and 45Ca2+ accumulation in the myocardium were reduced by 90-99%; ATP, glycogen and water content in the heart as well as perfusion pressure and heart rate remained within control values. The observed protective effect of Mn2+ was proportional to its concentration, and to the duration of Ca2+-free perfusion. A possible explanation for the protective effect of Mn2+ ions can be competition with Ca2+ binding sites related to sarcolemma integrity.

The effect of sodium salicylate and epinephrine on the release of lactate dehydrogenase from isolated rat heart.

The isolated perfused rat heart was used to study the effect of therapeutic concentrations of sodium salicylate and acetylsalicylate with respect to their potential cardioprotective property described in some clinical studies and experiments in vivo. Salicylates were added to the perfusion medium (Krebs-Henseleit buffer plus 5.5 mM glucose) in final concentrations ranging from 0.1 to 3.2 mM. In lower concentrations sodium salicylate reduced release of lactate dehydrogenase from the heart associated with delayed cleavage of endogenous triglycerides and a reduction of heart rate. A significant increase in lactate production, undoubtedly an expression of the uncoupling effect of sodium salicylate noted at 1.6 mM or higher concentration was accompanied by an increased uptake of glucose from the medium and increased coronary flow. In the presence of epinephrine (5.5 microM) sodium salicylate (0.1 and 0.5 mM) reduced only the total number of heart beats. Equimolar doses of acetylsalicylic acid failed to mimick salicylate effects. The results suggest that potentially cardioprotective effects of salicylate followed in these experiments by myocardial membrane leakage may be in part explained by the direct action of salicylate on the myocardium due to its antilipolytic and negative chronotropic effect. We failed to demonstrate this protective effect of salicylate against cardiotoxic doses of exogenous epinephrine.

Myocardial lesions induced by natural catecholamines in vitro.

Isolated rat hearts were perfused using a retrograde technique under constant pressure head or constant coronary flow. The addition of 1-epinephrine or 1-norepinephrine (1 microgram/ml) to the perfusion medium for 1 h caused visible and irreversible morphological changes which usually became apparent after 4 h of perfusion in the form of small, pale, opaque spots or streaks gradually enlarging on the surface or on the cross-section area of the myocardium. Light- and electron-microscopic examination showed a disintegration process analogous to that of myocardial infarction but without the infiltration with blood elements. The structural changes were preceded by an increased release of lactate dehydrogenase into the effluent, the most characteristic metabolic change accompanying myocardial injury. Nevertheless, the underlying mechanism of the cardiotoxic action of catecholamines remains to be clarified; several factors under consideration could be eliminated: hyperlipidemia, trombogenic process, acidity due to enhanced production of lactate, reduced total coronary inflow rate and toxicity of oxidation products of catecholamines.