Changes in microsomal membrane phospholipids and fatty acids and in activities of membrane-bound enzyme in diabetic rat heart

Diabetes mellitus is associated with alterations in lipid metabolism and cardiac dysfunction despite an absence of coronary arteriosclerotic changes. To investigate mechanisms of cardiac dysfunction in diabetic cardiomyopathy, we studied the relation between activities of membrane-bound enzymes and surrounding phospholipids in rats with diabetes induced with a single intravenous injection of streptozotocin (65 mg/kg). We found that total phospholipid content of sarcoplasmic reticulum membrane increased significantly 8 weeks after treatment with streptozotocin owing to increases in phosphatidylcholine and phosphatidylethanolamine, a decrease in arachidonic acid, and an increase in docosahexaenoic acid in the early stage of diabetes. Sarcolemmal Na⁺/K⁺-ATPase activity and the number of receptors decreased in isolated cardiomyoctes of diabetic rats 8 weeks after streptozotocin administration. The Ca²⁺ uptake of both sarcoplasmic reticulum and mitochondria decreased simultancously in permeabilized, isolated cardiomyocytes from diabetic rats. The depression of membrane-bound enzyme activities was correlated with alterations in phospholipids, which are closely related to the microenvironment of membrane-bound enzymes and influence intracellular Ca²⁺ metabolism. Because these changes in phospholipids and fatty acids were reversible with insulin therapy, they are diabetes-specific and might be a cause of cardiac dysfunction in diabetes.     

The content of glycogen phosphorylase and glycogen in preparations of sarcoplasmic reticulum–glycogenolytic complex is enhanced in diabetic rat skeletal muscle

Aims/hypothesis: We have examined the effect of diabetes and pharmacological insulin treatment on the content of glycogen phosphorylase and glycogen associated with the sarcoplasmic reticulum–glycogenolytic complex from rat skeletal muscle. Methods: Diabetes was induced in rats by streptozotocin injection. Enzymatic activities were measured using spectrophotometric methods. Glycogen phosphorylase was determined measuring the pyridoxal-5' -phosphate content and using polyacrylamide gel electrophoresis. Glycogen content was measured by enzymatic and the phenol sulfuric methods. Results: The content of glycogen phosphorylase associated with the sarcoplasmic reticulum glycogenolytic complex gradually arises after diabetes induction. The content of glycogen phosphorylase was restored to a control value by pharmacological insulin treatment. In addition, the content of glycogen in preparations of sarcoplasmic reticulum–glycogenolytic complex of diabetic animals was also increased, whereas the content of glycogen in total muscle of diabetic rats was similar to that of the control rats. The absolute and relative amount of glycogen associated with sarcoplasmic reticulum seemed to increase in diabetic animals. These effects on the compartmentalisation of glycogen were suppressed by insulin treatment. Additionally, the rate of conversion of glycogen phosphorylase b to a, an index of the phosphorylase kinase activity, was 50 % lower in diabetic rats, increasing the dephosphorylated form of glycogen phosphorylase and, as a consequence, its association with sarcoplasmic reticulum membranes. Conclusion/interpretation: These results suggest that under diabetic conditions, both glycogen phosphorylase and a small percentage of muscle glycogen are relocalized in the sarcoplasmic reticulum–glycogenolytic complex. [Diabetologia (2001) 44: 1238–1246] Received: 5 April 2001 and in revised form: 11 June 2001     

Cardiac ultrastructural changes in streptozotocin-induced diabetic rats: effects of insulin treatment

The effect of insulin treatment on myocardial ultrastructure was studied in experimentally induced diabetic male rats. Rats received an intravenous injection of 60 mg/kg streptozotocin and were tested for glycosuria 3 days later. Half of these animals were then given 3.0 U of protamine zinc insulin daily subcutaneously and again tested for glycosuria. The significant loss in weight observed in the untreated diabetics was prevented in the insulin-treated diabetics. Electron microscopic observation of the left ventricular myocardium from 3 month untreated diabetics revealed mild edema adjacent to the sarcoplasmic reticulum. A more severe form of edema, focal in nature, was also observed. Some distended mitochondria and disrupted banding were associated with these focal areas of edema. Increased lipid levels and intramitochondrial dense staining particles were found in the untreated diabetics. On the other hand, hearts from insulin-treated diabetics did not exhibit these alterations. They did, however, exhibit mitochondrial clumping which was also seen in the untreated diabetics and the same capillary changes. The latter involved a thickening of the lamina densa, loss of the lamina lucida and an increased number of micropinocytotic vesicles in the capillary walls. These results suggest that insulin treatment is capable of preventing only some of the ultrastructural changes induced by streptozotocin-diabetes in rat hearts.     

Lipid peroxidation in early experimental diabetes in rats: effects of diabetes and insulin.

The degree of lipid peroxidation was measured in organs from diabetic rats receiving no treatment, and in those from insulin-treated diabetic rats and controls. Lipid peroxidation was measured as organ content of malondialdehyde, a degradation product of polyunsaturated fatty acids. In the kidney, lipid peroxidation was increased after one week of diabetes; insulin treatment reduced the level of lipid peroxidation to levels lower than seen in controls. In the liver, diabetes caused an increased lipid peroxidation, which could be reversed by insulin; no additional effect of insulin was found. In heart and pancreas no effects of diabetes or insulin were demonstrated. The present paper provides evidence that lipid peroxidation is increased in the early stages of experimental diabetes and is reversible by insulin treatment. Hyperinsulinaemia may, in itself, counteract lipid peroxidation in kidney.     

Effect of streptozotocin-induced diabetes on oxidative energy metabolism in rat kidney mitochondria. A comparative study of early and late effects

AIM: The effects of streptozotocin (STZ)-induced diabetes on oxidative energy metabolism of rat kidney mitochondria were examined at the end of 1 week and 1 month of STZ treatment. METHODS: At the end of 1 week of induction of diabetes, respiration rates with pyruvate + malate and succinate as the substrates increased while those with beta-hydroxybutyrate and ascorbate + TMPD decreased. Respiration with glutamate was not affected. Insulin treatment had no alleviating effect. The changes persisted through 1 month of induction of diabetes and were not corrected by insulin treatment even at this stage. beta-hydroxybutyrate dehydrogenase activity registered significant decrease while the succinate dehydrogenase activity increased in diabetic and insulin-treated diabetic animals whereas only marginal changes were evident in the composition of the cytochromes. RESULTS: The ATPase activity tended to be high in the diabetic groups and was restored by insulin treatment. At both the stages, i.e. early and late stages of diabetes the mitochondria were tightly coupled and the ADP/O ratios were in normal expected ranges. CONCLUSION: Taken together, the results suggest that kidney is the major target tissue to suffer impairment of mitochondrial function with the onset of the disease which persists throughout and that insulin treatment is ineffective in restoring the normal state.     

Subcellular distribution of myocardial 5'-nucleotidase

The controversial subject of the subcellular location of myocardial adenosine production was studied employing density gradient fractionation of heart muscle combined with a novel method for analyzing distribution profiles based on multiple regression (correlation) analysis. Bungarotoxin binding, N-acetyl-beta-D-glucosaminidase, cytochrome c oxidase, NADPH-dependent cytochrome c reductase and lactate dehydrogenase were used as markers for the plasma membrane, lysosomes, mitochondria, sarcoplasmic reticulum and cytosol, respectively. The normalized distribution frequencies (fraction of total) of 5'-nucleotidase in mitochondria, lysosomes, plasma membranes, sarcoplasmic reticulum and cytosol in the 50 x g supernatant of total homogenate of heart muscle were found to be 0, 0.25, 0.44, 0.08 and 0.23, respectively. To increase the resolution power of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied, in which the normalized distribution of 5'-nucleotidase in the homogenate was 0, 0.16 and 0.84 in mitochondria, plasma membranes and lysosomes, respectively. This mainly lysosomal 5'-nucleotidase activity was 61% inhibited by the alpha,beta-methylene analog of ADP, indicating that although the latter has been considered specific to the plasma membrane enzyme, it also inhibits the lysosomal enzyme. The intercellular distribution of 5'-nucleotidase was not studied, but the lack of this enzyme in the mitochondria indicate that the adenosine production observed during mitochondrial AMP production, e.g. during acetate oxidation in intact heart muscle, must involve AMP transport out from the mitochondria.     

Ultrastructure of the pigeon papillary muscle with special reference to the sarcoplasmic reticulum.

The contractile material of the pigeon papillary muscle cell, constitutes one single mass of myofilaments which contains sarcoplasmic tubules and parallel rows of mitochondria and lipid droplets. These rows do not demarcate separate myofibrillar subunits as in the skeletal muscle. The sarcoplasmic reticulum represents a well developed network of tubules of different sizes. Transversely orientated and dilated tubules are present both at the Z line-I band and H-line levels. The tubules at the Z line-I band levels, form belt-like cisternae penetrating the myocardial cell in close proximity to myofilaments and mitochondria. It is suggested that these cisternae, and possibly also the longitudinal parts of the sarcoplasmic reticulum, may function as intracellular storage sites for calcium. The structure and the functional significance of the peripheral couplings is discussed. Transverse tubules and, accordingly, internal couplings are not present in the tissue studied.Zebra bodies are commonly seen, interpolated between the sarcolemma and the contractile material of the cell. The observations indicate that their main function is contractile movements of actin-like myofilaments. It is suggested that these contractions may be likely to exert an adjusting and regulatory effect on the movements of the cellular surface during cardiac activity.     

Sarcoplasmic reticulum membrane and heart development

Intracellular Ca2+ concentrations in cardiac cells are dependent on trans-sarcolemmal Ca2+ fluxes and the ability of sarcoplasmic reticulum to release and take up Ca2+. Ca2+ accumulation by sarcoplasmic reticulum membranes causes muscle to relax, whereas Ca2+ release from sarcoplasmic reticulum initiates contraction. Ca2+ transport by the sarcoplasmic is mediated by a Ca2+-dependent ATPase enzyme. Ca2+ release from sarcoplasmic reticulum may be mediated by a ligant-gated Ca2+ channel. The physiological role of sarcoplasmic reticulum in developing muscle is not well established. In this report we investigated the composition and function of sarcoplasmic reticulum membranes during cardiac myogenesis. Phospholamban, a major phosphoprotein in mature sarcoplasmic reticulum membranes was present during early stages of cardiac myogenesis. The embryonic form of phospholamban was phosphorylated by cAMP-dependent protein kinase but not in the presence of Ca2+ and calmodulin. Ca2+ uptake and Ca2+-dependent ATPase activity were low in fetal sarcoplasmic reticulum compared to adult control membranes, although the apparent affinities of the enzyme for Ca2+ were similar. Sarcoplasmic reticulum vesicles used in these studies had very low levels of plasma membrane and mitochondrial contamination. The amounts of both 110-kDa Ca2+-ATPase and 55-kDa calsequestrin in the sarcoplasmic reticulum membrane were lower in fetal sarcoplasmic reticulum vesicles compared to mature membranes. Ca2+-ATPase and calsequestrin were identified in the isolated sarcoplasmic reticulum vesicles using specific antibodies produced against these membrane proteins. Age-related differences in Ca2+ transport properties of cardiac sarcoplasmic reticulum and in the amount of Ca2+-ATPase and calsequestrin may explain alterations in the regulation of intracellular Ca2+ concentrations in fetal heart muscle. This may relate to the developmental changes observed in myocardial function.     

Phosphorylated intermediate of ATPase of isolated cardiac sarcoplasmic reticulum

Isolated sarcoplasmic reticulum of calf heart was found to incorporate the terminal phosphate of ATP, indicating the presence of a phosphorylated intermediate of ATPase of heart sarcoplasmic reticulum. The incorporation was less for sarcoplasmic reticulum from heart than for that from skeletal muscle. Protein components of heart sarcoplasmic reticulum differed from those of skeletal muscle on acrylamide gel electrophoresis. However, the location of the component of sarcoplasmic reticulum labeled with ³²P of ATP was the same for heart and skeletal muscle. The molecular weight of the labeled component was estimated to be in the range of 90 000 to 100 000.     

Heart mitochondrial function in chronic experimental diabetes in rats

Diabetes was introduced in rats by an intravenous injection of streptozotocin (65 mg/kg). Animals were sacrificed 8 weeks later and mitochondria were isolated from the ventricular tissue by differential centrifugation. The state 3 respiration, oxidative phosphorylation rate and Mg2+-dependent ATPase activities were depressed in mitochondria from diabetic hearts. These changes were partially reversible upon 2 weeks of insulin and fully reversible after 4 weeks of insulin therapy. Mitochondrial calcium uptake but not calcium binding, was decreased in diabetes and this change was fully reversible by 2 weeks of insulin administration. The observed alterations in mitochondrial function could not be explained on the basis of any changes in mitochondrial lipid and protein composition or subcellular contamination. These results indicate the presence of a generalized depression in mitochondrial function in chronic diabetes and such a defect is suggested to contribute in the development of cardiomyopathy at late stages of diabetes.     

Effect of insulin and angiotensin II receptor subtype-1 antagonist on myocardial remodelling in rats with insulin-dependent diabetes mellitus

OBJECTIVES: To assess the role of insulin or an angiotensin II receptor antagonist (losartan), or both, in preventing cardiomyocyte damage in rats suffering from insulin-dependent diabetes mellitus (IDDM), and to correlate it with insulin receptor modulation at the cardiomyocyte, coronary endothelium and skeletal muscle cell level. DESIGN: Animals were divided into groups of normal rats, diabetic rats, and diabetic rats given insulin, each subdivided into a control group and an experimental group treated with losartan. METHODS: The animals were killed 1 month after enrollment to the study. Perfusion of the heart with iodine-125-labelled insulin was carried out for all the groups and the binding kinetics of insulin to its receptors on the coronary endothelial cells and the cardiomyocytes were determined using a physical/mathematical model. In addition, tissue samples from the heart and intercostal skeletal muscle were snap frozen and used for histological, indirect immunofluorescence and western blot analysis. RESULTS: Cardiac muscle from diabetic animals exhibited diffuse cardiomyopathic changes consisting of widespread vacuolation, loss of striation and cellular hypertrophy, which were reduced and even prevented by treatment with insulin and losartan. In addition, losartan seemed to mediate the upregulation of insulin receptor density on cardiomyocytes and skeletal muscle, and increase insulin receptor affinity at the coronary endothelial site. Finally, treatment with losartan induced a significant decrease in glucose concentrations in the diabetic group compared with the appropriate controls. CONCLUSIONS: Addition of losartan to the standard insulin treatment in non-hypertensive animals with IDDM offers new benefits concerning cardiac protection and prevention of damage. This may be attributed, in part, to insulin receptor density and sensitization.     

Mitochondrial dysfunction observed in situ in cardiomyocytes of rats in experimental diabetes.

OBJECTIVE: The aim was to investigate effects of experimental diabetes and insulin treatment on heart myocytes, particularly on the mitochondrial function studied in situ in isolated cardiomyocytes. METHODS: 20 male Sprague-Dawley rats (140-160 g) were made diabetic by intraperitoneal streptozotocin, 70 mg.kg-1. Ten then received daily subcutaneous injections of ultra lente insulin (starting dose of 3 units.d-1) for 7-15 d from the 20th day after streptozotocin. There was a control group of 11 rats. The rats were killed 21-35 d after the induction of diabetes, and heart myocytes were isolated by collagenase digestion. The 45[Ca]2+ uptake of mitochondria in situ in permeabilised myocytes, the transmembrane potential gradient of mitochondria, and the respiration of myocytes, as well as the cell yield and cell [45Ca]2+ uptake, were examined. RESULTS: Mitochondrial uptake of [45Ca]2+ was significantly decreased in the diabetic group compared to control at cytosolic calcium concentrations between 760 nM and 44.6 microM. The mitochondrial potential of diabetic myocytes, estimated from the distribution of [3H]triphenylmethylphosphonium+, was slightly but significantly decreased from the control value. Cell respiration, measured polarographically in the presence of pyruvate and malate or succinate as oxidisable substrates, and with or without 2,4-dinitrophenol, was decreased by diabetes. The rapidly exchangeable [45Ca]2+ content in the myocyte with intact sarcolemmal membrane ("cell Ca2+ uptake") and the yield of cells from heart tissue were also diminished in diabetic rats. These changes were returned to normal by insulin treatment of 7 d or longer. CONCLUSIONS: Insulin deficiency at early stages causes defects of mitochondrial function detectable in situ in cardiomyocytes. This suggests the possibility that such alterations are causative factors in the development of diabetic cardiomyopathy.     

Effect of diabetes and fasting on the uridine triphosphate content and uridine kinase activity of rat cardiac and skeletal muscle.

The influence of diabetes and starvation on uracil nucleotide metabolism in muscle was studied. It was found that the uridine triphosphate (UTP) content of heart and diaphragm muscle was decreased in fasted and streptozotocin-diabetic rats and that insulin treatment of diabetic animals restored the UTP concentration to normal levels. The ATP content of heart tissue was not altered under these conditions. It was also demonstrated that hemidiaphragms from streptozotocin-diabetic rats synthesized less UTP from uridine in vitro than hemidiaphragms from normal animals. Uridine kinase activity of extracts of cardiac and skeletal muscle from fasted and diabetic rats was lower than the activity found in extracts from control animals. It was concluded that uracil nucleotide synthesis by the salvage pathway is decreased in experimental diabetes and fasting.     

Decrease in mitochondrial oxidative protein damage parameters in the streptozotocin-diabetic rat

BACKGROUND: Many authors have shown that hyperglycemia leads to an increase in oxidative protein damage in diabetes. The aim of this study was to reveal the susceptibility of mitochondria from liver, pancreas, kidney, and skeletal muscle of diabetic Sprague-Dawley rats, a model of type 1 diabetes, to oxidative protein damage. METHODS: Mitochondrial fractions were obtained by differential centrifugation. To show the effect of hyperglycemia in promoting oxidative protein damage, we determined mitochondrial protein carbonyl, total thiol, nitrotyrosine, advanced oxidation protein products, and lipid hydroperoxide levels. The levels of the studied markers, except nitrotyrosine, were determined by colorimetric methods. Nitrotyrosine levels were measured by ELISA. All values were compared with those of the controls by using the Mann-Whitney U-test. RESULTS: Nitrotyrosine and lipid hydroperoxide levels were decreased and other parameters were not changed in liver mitochondria of diabetic rats. Protein carbonyl, nitrotyrosine, advanced oxidation protein products, and lipid hydroperoxide levels were decreased and total thiol levels were not changed in pancreas mitochondria of diabetic rats. Only advanced oxidation protein products and lipid hydroperoxide levels were decreased in kidney mitochondria of diabetic rats. The levels of the same parameters were not significantly different in muscle mitochondria of diabetic rats. CONCLUSIONS: The decrease in mitochondrial oxidative protein damage in diabetes may correspond to either an increase in antioxidant defense mechanisms or a different adaptive response of the cells to the increased extramitochondrial oxidative stress in diabetes. The mitochondrial oxidative protein damage-lowering mechanisms in diabetes remain to be clarified.     

Changes in aortic endothelial gene expressions and relaxation responses following chronic short-term insulin treatment in diabetic rats

The purpose of the present study was to examine the relationship between the changes in the expressions of several mRNAs and changes in endothelial function in streptozotocin-induced diabetic and chronic short-term insulin-treated rats. Aortas from later-stage (10 week) diabetics, but not those from their insulin-treated counterparts, showed an impaired endothelial function. We found that the mRNA expressions for 30 genes were significantly upregulated, while those for 13 other genes were downregulated in aortic endothelial cells from diabetes. In later-stage diabetes, chronic insulin treatment ameliorated the endothelial dysfunction and normalized the expressions for 20 out of the 43 genes altered in diabetes. Further, 12 of the remaining 23 genes were altered by high-dose insulin treatment in the controls. In early-stage (1 week) diabetic aortas, which did not show impaired endothelial function, expression changes were shown by only 12/30 and 5/13 of the genes up- or downregulated, respectively, in later-stage diabetes. Thus, in the diabetic aortas endothelial gene expressions and function exhibited time-related changes, and several gene expressions and endothelial function were normalized by insulin treatment. The hyperinsulinemia caused by this treatment may oppose the alterations in some gene expressions and the endothelial proliferation (cell growth-related gene expressions) that occur in established diabetes.     

胰岛素与动脉粥样硬化关系的实验研究

对正常大鼠、糖尿病大鼠、INS治疗的糖尿病大鼠及单纯注射INS的正常大鼠的主动脉进行了形态学的对比观察,并测定了各组大鼠的血糖、血脂、过氧化脂质和血清INS水平。结果显示:糖尿病大鼠的主动脉出现早期动脉粥样硬化(AS)病变,即内膜增厚、内皮下间隙加宽、内弹力板破坏、内膜和中膜浅层平滑肌细胞增生及细胞内脂滴等。INS治疗组大鼠,虽然INS缺乏得到补充,DM状态下的糖、脂质代谢紊乱得到明显纠正,但是主动脉出现类似DM组的病变,且病变程度重于DM组。注射INS的正常大鼠,血清INS水平明显增高,主动脉病变与前两组相比,相对更重。结果表明,INS缺乏的实验性糖尿病大鼠,由于高血糖,高血脂及血小板功能异常等多种因素,可以导致AS的发生;而胰岛素水平过高(绝对或相对的),在DM-AS的发病中,直接或间接的起着重要的促进作用。     

Nerve ischaemia in diabetic rats: time-course of development,effect of insulin treatment plus comparison of streptozotocin and BB models

Summary This study sought to determine the timecourse of development of reduced nerve laser Doppler flux in experimental diabetes and the effect on this anomaly of insulin treatment. In addition, we aimed to compare nerve laser Doppler flux in streptozotocin-and genetically-diabetic BB rat models. Sciatic nerve laser Doppler flux in diabetic rats was variable during the 2 days following streptozotocin injection; from day 4, when the measurement was 80% of control, fluxes fell steadily and formed a plateau at 40% of control values after 4 weeks of diabetes. In a second study, using rats with 4-week streptozotocin-diabetes, sciatic nerve laser Doppler flux was reduced to 44% of the value measured in control rats. Treatment of a parallel group of diabetic rats with insulin, by sustained release implants, prevented this ischaemia, so that nerve laser Doppler flux was 91% of controls. Nerve Doppler flux in BB rats with 6-week genetic diabetes was 57% of a control (non-diabetic) BB group. There were no differences in mean arterial pressures between control and diabetic rats in any of the studies. Heart rates of control and insulin-treated diabetic animals were higher than those of the untreated diabetic group; in the other studies heart rates of diabetic animals were numerically lower than controls, but not significantly so. These observations suggest that sciatic nerves of rats with short-term diabetes, whether induced with streptozotocin or of genetic origin, are markedly ischaemic and that this ischaemia in streptozotocindiabetes is evident within a week of diabetes onset, forms a plateau after 4 weeks and is maintained for at least 2 months. The findings also indicate that treatment of short-term diabetes with insulin can prevent nerve ischaemia.     

Insulin stimulates Akt translocation to mitochondria: Implications on dysregulation of mitochondrial oxidative phosphorylation in diabetic myocardium

Mitochondrial oxidative phosphorylation is the major source of energy in cardiac muscle. In the streptozotocin-induced diabetic (STZ-DM) mice, myocardial oxidative phosphorylation was perturbated and oxidative phosphorylation complex V (ATP synthase) activity was significantly reduced. To determine the independent effects of hyperglycemia and insulin deficiency on the changes of myocardial complex V, we used phlorizin (Ph) to normalize blood glucose in the diabetic mice. Ph treatment did not improve myocardial complex V activity in the STZ-DM mice, whereas insulin treatment normalized myocardial complex V activity in the diabetic mice. Therefore, the reduction of complex V activity was caused by insulin deficiency and not by hyperglycemia in STZ-DM myocardium. Acute insulin stimulation induced phosphorylation of Akt and translocation of Akt to mitochondria in myocardium. Translocation of phospho-Akt to mitochondria was enhanced in the STZ-DM mice and was blunted in the diet-induced diabetic mice. In parallel, insulin activation of complex V was enhanced in the STZ-DM myocardium and suppressed in the diet-induced diabetic myocardium. In vivo inhibition of Akt blocked insulin stimulation of phospho-Akt translocation and blunted activation of complex V. Insulin-activated Akt translocation to mitochondria in cardiac muscle is a novel paradigm that may have important implications on myocardial bioenergetics.