Untreated Streptozotocin-diabetic Rats Research Articles

Oxidative Stress Triggers Cardiac Fibrosis in the Heart of Diabetic Rats

Diabetic cardiomyopathy is characterized by myocyte loss and myocardial fibrosis, leading to decreased elasticity and impaired contractile function. The study examines the downstream signaling whereby oxidative stress, induced by hyperglycemia, leads to myocardial fibrosis and impaired contractile function in the left ventricle of diabetic rats. It also examines the effects of dehydroepiandrosterone (DHEA), which prevents the oxidative damage induced by hyperglycemia in experimental models. DHEA was administered for 6 wk in the diet [0.02%, wt/wt)] to rats with streptozotocin-induced diabetes. Oxidative balance, advanced glycated end products (AGEs) and AGE receptors, transcription factors nuclear factor-kappaB and activator protein-1, and profibrogenic growth factors (connective tissue growth factor and TGFbeta1) were determined in the left ventricle of treated and untreated streptozotocin-diabetic rats. Structural and ultrastructural changes, and the contractile force developed by electrically driven papillary muscles, under basal conditions and after stimulation with isoproterenol, were also evaluated. Oxidative stress induced by hyperglycemia increased AGEs and AGE receptors and triggered a cascade of signaling, eventually leading to interstitial fibrosis. DHEA treatment, by improving oxidative balance, counteracted the enhanced AGE receptor activation and increase of profibrogenic factors and restored tissue levels of collagen I, collagen IV, and fibronectin to those of control animals. Moreover, DHEA completely restored the contractility of isolated papillary muscle. Oxidative stress led to cardiac fibrosis, the most important pathogenetic factor of the heart's impaired functional integrity in diabetes. Structural and ultrastructural changes and impairment of muscle function induced by experimental diabetes were minimized by DHEA treatment.

ACE-inhibition is superior to endothelin A receptor blockade in preventing abnormal capillary supply and fibrosis of the heart in experimental diabetes.

There is little information whether cardiac capillary supply is deranged in diabetes. Hyperglycaemia is a potent stimulus for endothelin-1 (ET-1) production. We therefore hypothesised that increased ET-1 production in Streptozotocin-induced Type 1 diabetes causes abnormalities of cardiac capillaries and the aorta. To this end we compared the effects of an ET receptor A blocker (ETA-RB) with that of an ACE-inhibitor (ACE-i) or their combination in rats with Streptozotocin (STZ) diabetes. Sprague Dawley rats were injected with 65 mg STZ i.v. and subsequently developed diabetes. Rats were left untreated or received daily either the ACE-i Trandolapril, the ETA-RB Darusentan or a combination of both. After 6 months the experiment was terminated and the heart and the aorta were investigated using quantitative morphological techniques. ACE-i but not ETA-RB lowered blood pressure in STZ Type 1 diabetic rats. Capillary length density was lower in untreated STZ diabetic rats (2932+/-128 mm/mm3) compared to non-diabetic control rats (3410+/-252 mm/mm3). Treatment with ACE-i (3568+/-431 mm/mm3), but not with ETA-RB (2893+/-192 mm/mm3), prevented the decrease in capillary supply. Volume density of the myocardial interstitium was higher in untreated STZ diabetic rats (0.86+/-0.04%) compared to non-diabetic control rats (0.36+/-0.06%). In all three intervention groups the values were lower (ACE-i: 0.53+/-0.05%, ETA-RB: 0.7+/-0.08% and combination: 0.69+/-0.1). Our study identifies a capillary defect of the heart in STZ diabetes, i.e. decreased capillary supply. This abnormality was reversed by ACE-i, but not by ETA-R blockade. A similar trend, although not complete normalisation, was seen in cardiac fibrosis.

Immunoneutralization of endogenous glucagon-like peptide-2 reduces adaptive intestinal growth in diabetic rats.

Supraphysiological doses of glucagon-like peptide-2 (GLP-2) have been shown to induce intestinal growth by increasing villus height and crypt depth and by decreasing apoptosis, but a physiological effect of GLP-2 has not yet been demonstrated. Earlier, we found elevated levels of endogenous GLP-2 in untreated streptozotocin diabetic rats associated with marked intestinal growth. In the present study, we investigated the role of endogenous GLP-2 for this adaptive response. We included four groups of six rats: (1) diabetic rats treated with saline, (2) diabetic rats treated with non-specific antibodies, (3) diabetic rats treated with polyclonal GLP-2 antibodies and (4) non-diabetic control rats treated with saline. All animals were treated with once daily intraperitoneal injections for 13 days and killed on day 14. Diabetic rats treated with saline or non-specific antibodies had a significantly (P<0.01) increased area of mucosa (13.00+/-0.64 and 13.37+/-0.60 mm(2), respectively) in the proximal part of the small intestine compared with non-diabetic controls (7.97+/-0.70 mm(2)). In contrast, diabetic rats treated with GLP-2 antibodies had a significantly (P<0.01) smaller increase in area of mucosa in the proximal part of the small intestine (10.84+/-0.44 mm(2)). Antibody treatment had no effect on body weight, blood glucose concentrations and food intake. Thus, blocking of endogenous GLP-2 in a model of adaptive intestinal growth reduces the growth response, providing strong evidence for a physiological growth factor function of GLP-2.

Chronic intraperitoneal insulin delivery, as compared with subcutaneous delivery, improves hepatic glucose metabolism in streptozotocin diabetic rats

We have previously shown that chronic insulin treatment by the intraperitoneal route normalizes the elevated glucose production (GP) in streptozotocin (STZ) diabetic rats, while insulin delivered by the subcutaneous route only partially normalizes GP. To investigate the biochemical mechanism of the effect of chronic insulin delivery by either route on hepatic glucose metabolism, we measured the hepatic activity of glucose 6-phosphatase (G6Pase) and glucokinase (GK). Four groups of rats were used: (1) nondiabetic rats (N, n = 7), (2) untreated STZ diabetic rats (D, n = 8), (3) diabetic rats treated intraperitoneally (IP, n = 6), or (4) subcutaneously (SC, n = 8) (both 3 U of insulin/d). Glucose levels, higher in D, were normalized by insulin treatment regardless of route. Peripheral insulin levels were lowest in D and highest in SC as expected (N, 162 ± 18 pmol/L; D, 66 ± 12; IP, 360 ± 96; SC, 798 ± 198). STZ diabetes resulted in a 10-fold decrease in GK (P < .001), and a 2-fold increase in G6Pase activity (P < .01). Both intraperitoneal and subcutaneous treatments normalized G6Pase activity. In contrast, with subcutaneous but not intraperitoneal treatment, GK activity was still 35% less than normal (SC v N, P < .05). Glucose 6-phosphate (G6P) levels did not differ among the groups. In summary: (1) the increase in GP in D reflected increased activity of G6Pase and reduced activity of GK, (2) the partial suppression of GP with subcutaneous insulin treatment reflected correction of increased G6Pase activity, but only partial correction of low GK activity, and (3) the normalization of GP with intraperitoneal insulin treatment reflected correction of both increased G6Pase activity and low GK activity. Our current studies indicate that chronic intraperitoneal insulin treatment is superior to subcutaneous treatment with regard to hepatic glucose metabolism. Copyright © 2000 by W.B. Saunders Company

Effect of pyridoxine and insulin administration on brain glutamate dehydrogenase activity and blood glucose control in streptozotocin-induced diabetic rats

Blood glucose level and kinetic parameters of glutamate dehydrogenase (GDH) were measured in the cerebellum, brain stem and cerebral cortex of control, insulin treated, pyridoxine treated, pyridoxine and insulin treated and untreated streptozotocin-diabetic rats. The combined administration of insulin and pyridoxine was found to be better in controlling the hyperglycaemia. Insulin with pyridoxine treatment brought back the increased maximal velocity of GDH during diabetes to control state. Also, there was an increase in Michaelis-Menten constant. These results suggest that pyridoxine and insulin together serve a better control for diabetes.

Effect of insulin on the altered thyroid function and adrenergic responsiveness in the diabetic rat

We have previously reported the tail skin temperature response to isoproterenol as being significantly attenuated in male rats 4 weeks after the induction of diabetes with streptozotocin. The current study evaluated the time course of this altered adrenergic responsiveness and the role of thyroid hormone and insulin treatment in the tail skin temperature response. In the first study, the tail skin temperature response to isoproterenol was monitored weekly, for 6 weeks. The tail skin temperature response was similar in control and diabetic animals after 1 week of streptozotocin treatment. However, after 2, 4, 5, and 6 weeks of diabetes the tail skin temperature response was significantly reduced. Total T3, T4, and free T4 concentrations were also significantly reduced in these diabetic animals. In a second study, the effects of graded doses of insulin treatment on thyroid hormone levels was assessed. The reduced thyroid hormone concentrations observed in untreated streptozotocin-diabetic rats were restored towards control levels in animals receiving the lowest dose of insulin (1 U/day), whereas higher doses of insulin were required to more closely restore euglycemia and lower glycated hemoglobin. A subsequent study, utilizing a 1-U/day dose of insulin, resulted in a normalization of the tail skin temperature response to isoproterenol, during 8 weeks of treatment in the treated diabetic rat. In a final study utilizing the spontaneous BB diabetic rat maintained on daily insulin treatment, no differences in the tail skin temperature response or thyroid hormone levels were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies of renal autoregulation in pancreatectomized and streptozotocin diabetic rats

We studied renal autoregulation in pancreatectomized Munich-Wistar diabetic rats and in their sham-operated controls. In a second experiment we studied renal autoregulation in untreated and insulin treated streptozotocin diabetic Munich-Wistar rats and their nondiabetic controls. In the first experiment the diabetic rats had higher baseline renal blood flows (RBF). There was a fall in renal vascular resistance (RVR) and sustained RBF in both diabetic and control rats as renal perfusion pressures (RPP) was reduced from 130 and 110 mm Hg. As RPP was reduced from 110 and 80 mm Hg, there was no significant Change in RVR in control rats and RBF began to fall. Below RPP of 80 mm Hg RVR rose and RBF fell sharply in these rats. In contrast, there was a progressive fall in RVR as RPP was lowered to 60 mm Hg in the diabetic rats and, thus, RBF was much better sustained in these animals. In the second experiment the plasma glucose level was 502 +/- 52 mg/dl (X +/- SD) in the untreated diabetic rats and only modestly reduced to 411 +/- 49 mg/dl in the insulin treated animals. Untreated streptozotocin diabetic rats had moderately reduced and insulin-treated diabetic rats had mildly reduced baseline RVR and RBF. However, in these animals as in the pancreatectomized rats the increases in RVR noted in control rats at subautoregulatory RPPs were not seen. Thus, regardless of whether baseline RBFs were increased or decreased, diabetic rats sustained RBF at markedly reduced RPPs far more efficiently than did nondiabetic rats. The pathogenesis of these abnormalities in diabetic rats was not learned in these studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Cation permeability of the blood-brain barrier in streptozotocin-diabetic rats

Decreased sodium permeability across the blood-brain barrier occurs in streptozotocin-treated rats after 2 weeks of diabetes. To establish whether this is a phenomenon specific for cations, the blood-brain barrier permeability for sodium, potassium and calcium was studied with an arterial integral uptake technique. Experiments were performed in control rats and, after two weeks after diabetes induction, in untreated streptozotocin-diabetic rats and in insulin-treated streptozotocin rats. In untreated diabetes, the neocortical blood-brain barrier permeability for sodium decreased by 35% (5.2 +/- 1.7 vs 3.4 +/- 1.1 10(-5).cm3.s-1.g-1) and potassium permeability by 39% (19.8 +/- 5.7 vs 12.1 +/- 3.9 10(-5).cm3.s-1.g-1), whereas no differences in calcium permeability occurred. Insulin treatment was associated with an increase in the blood-brain barrier permeability to sodium (4.8 +/- 1.0 10(-5).cm3.s-1.g-1) as compared to untreated diabetes (3.4 +/- 1.1 10(-5).cm3.s-1.g-1). It is concluded that the observed changes in sodium and potassium permeability cannot be caused by electrostatic membrane changes. More specific abnormalities of the transport of sodium and potassium across the blood-brain barrier are likely to occur; disturbances in the sodium-potassium-pump activity could account for such alterations.

Effects of continuous subcutaneous insulin infusion on renal morphology in experimental diabetes. II. Glomerular basement membrane thickness.

Stereological studies were performed on renal glomeruli from control rats, untreated streptozotocin-diabetic rats and diabetics treated by subcutaneous infusion of insulin for 16 weeks. Estimates of glomerular basement membrane thickness were obtained by dividing membrane volume by membrane surface area. In addition, arithmetic and harmonic mean thicknesses were calculated from orthogonal intercept lengths, and used to provide information on the variability of basement membrane thickness. In untreated diabetics, both arithmetic and harmonic mean thicknesses were significantly greater than values found in control animals. The thickening of the basement membrane appeared to be generalized rather than markedly focal. All thickness variables were normalized by insulin infusion therapy. These findings underline the importance of good glycaemic control and early intervention in experimental diabetes and one of its main complications.

Glycogen repletion in different skeletal muscles from diabetic rats.

It was hypothesized that chronically untreated streptozotocin-diabetic rats may compensate for the detrimental effect of insulin deficiency on glycogen restoration following muscular work. Glycogen concentration ([GLY]) was reduced by in situ stimulation of the sciatic nerve of anesthetized normal (N) and diabetic (D) Sprague-Dawley albino rats. Glycogen repletion occurred most rapidly within 30 min after stimulation. By 2 h all N muscles returned to basal [GLY]. D muscles repleted glycogen at a rate 45-75% of normal and not all of the D muscle returned to basal [GLY] by 8 h poststimulation. Neither the partial reduction in diabetic hyperglycemia by using phlorizin nor the acute further lowering of diabetic hypoinsulinemia by using insulin antibody affected glycogen restoration in D muscles. Acute insulin replenishment resulted in restoration of normal [GLY] in D muscles within 2 h poststimulation. These findings are not consistent with the proposed hypothesis and indicate insulin is necessary to have normal rates of glycogen restoration following muscular activity.

Absent or delayed preovulatory luteinizing hormone surge in experimental diabetes mellitus.

The proestrus preovulatory luteinizing hormone (LH) surge was absent or delayed in more than 56% of untreated streptozotocin-diabetic rats. Absence of LH surge was associated with anovulation. Insulin treatment for 10-14 days restored the diminished surge and ovulation frequency. Pituitary LH release in response to exogenous gonadotropin-releasing hormone administration in diabetic rats was not different from controls. Impaired hypothalamic function may comprise the basis for the increased incidence of infertility in diabetes mellitus.

Normalization of hepatic lysosomal autophagy in streptozotocin diabetic rats after pancreatic transplantation

This study was performed to determine whether the enhanced hepatic autophagocytosis occurring in diabetes mellitus could be reversed by pancreatic transplantation despite the persistence of hyperglucagonemia in transplant recipients. Twenty weeks after pancreatic transplantation, hepatic lysosomes and autophagic vacuoles were quantitated morphometrically in untreated streptozotocin-diabetic rats and in streptozotocin-diabetic rats treated by pancreatic transplantation. Hyperglycemia and hypoinsulinemia in untreated diabetic rats were corrected after pancreatic transplantation. However, the peripheral plasma glucagon concentration remained significantly higher in transplant recipients than in non-diabetic controls. Light microscopic morphometry revealed no significant effect of the diabetic state or its reversal on the volume of the single hepatocyte or the volume density and numerical density of hepatocytes. However, electron microscopic examination showed that the number and volume density of lysosomes and autophagic vacuoles, which were significantly increased in diabetic rats, returned to control values after pancreatic transplantation. It is concluded that chronic hyperglucagonemia does not preserve enhanced hepatic autophagocytosis when the diabetic hypoinsulinemia is corrected by the pancreatic transplant.

Abnormalities of pancreatic somatostatin secretion corrected by in vivo insulin treatment of streptozotocin-diabetic rats.

In islets removed from untreated streptozotocin-diabetic rats, the somatostatin-containing cells were unresponsive to changes in glucose concentration, while they did respond to raised cyclic AMP levels. By contrast, in vivo insulin treatment restored the glucose sensitivity of the somatostatin secreting cells. In addition, in vivo insulin treatment resulted in a lowering of the high basal somatostatin secretion rate found in islets from untreated diabetic rats. These changes were made without any alteration of islet insulin content or enhancement of the in vitro insulin secretion. These results indicate that there is not a basic defect in the glucoreceptor of the somatostatin cell in diabetes.

Hepatocyte and Kupffer cell functions during liver regeneration in streptozotocin-diabetic rats.

The insulinoprivic influence of acute severe streptozotocin diabetes on liver regeneration in rats was evaluated by determining liver weights as well as hepatocyte and Kupffer cell functional capacities. Functional capacities were assessed by bromosulfophthalein uptake for hepatocytes and carbon phagocytosis for Kupffer cells. Evaluation immediately after partial hepatectomy revealed a 66% reduction of liver mass, a 63% decrease in hepatocyte bromosulfophthalein removal, and a 65% decline in Kupffer cell carbon phagocytosis. Per cent recovery at 48-hr posthepatectomy was considerably greater for carbon phagocytosis than for bromosulfophthalein removal by regenerating livers. This apparent difference in functional recovery was likely due in part to enhanced non-Kupffer cell carbon phagocytosis. No significant differences of the three regeneration indices were noted for untreated streptozotocin-diabetic rats compared to nondiabetic animals. However, insulin administration to fasted streptozotocin diabetics significantly stimulated liver regeneration above that of untreated fasted rats and almost equivalent to that of pair-fed animals. Fasted rats had in general slower liver regeneration than pair-fed animals as expected. Furthermore, insulin administration to fasted nondiabetic rats after partial hepatectomy caused severe hypoglycemia and resulted in a further depression of liver regeneration.

Long-term effects of untreated diabetes on the arterial wall in rat. An ultrastructural study.

Arterial ultrastructure was examined in twelve untreated streptozotocin-diabetic rats. Five showed severe changes in muscular (coronary, tibial, pulmonary) arteries two months after induction of diabetes. The basement membrane of smooth muscle cells was patchily thickened in these arteries. No similar changes were seen in muscular arteries of the other seven diabetic rats or in 10 control rats. No lesions were found in elastic arteries (aorta, main pulmonary artery) of any diabetic or control animals. Plasma non-esterified fatty acids, total ketone bodies, and glucose levels were higher in the diabetic rats with arterial changes (p less than 0.01) than in the diabetic animals without such changes. The molar ratio of non-esterified fatty acid to albumin in plasma ranged between 2.3-3.3 in the diabetic rats with arterial lesions, 1.4-1.8 in those without such features, and 0.6-1.1 in the controls. The excess of non-esterified fatty acid in plasma during insulin deficiency could be an important factor in the initiation of arterial changes in this model of experimental diabetes.

Glucose and ATP levels in pancreatic islet tissue of normal and diabetic rats.

It has been suggested that the hyperglucagonemia observed in diabetic animals and man may be due to an impairment of glucose uptake and metabolism by the alpha-cells resulting in a decreased production of ATP. To test this hypothesis glucose, ATP, glucagon, and insulin were measured in pancreatic islets of normal and alloxan or streptozotocin diabetic rats. Two experimental approaches were used. In the first, the pancreas was perfused in vitro for assessing insulin and glucagon release due to 10 mM amino acids with and without 5 mM glucose. These perfusions were performed in the presence and absence of insulin. After perfusion, the pancreas was frozen and processed for analysis of islet glucose, ATP, insulin, and glucagon content. The second approach was to investigate the islet sucrose, urea, and glucose spaces together with ATP, insulin, and glucagon content in vivo in normal and in insulin-treated and untreated streptozotocin diabetic rats. Perfusion of the pancreas in vitro with 5 mM glucose resulted in higher glucose content of normal islets than in alloxan and streptozotocin diabetic islets. Similarly in the in vivo studies, the intracellular glucose space of the streptozotocin diabetic islets was 30% the value found in normals. In the in vivo experiments, despite the relatively small intracellular glucose space of alpha-cell islets, the ATP content of these islets was only 15-20% lower than the ATP content of normal islets. In the in vitro experiments, perfusion with glucose resulted in ATP contents of alpha-cell islets and of normal mixed alpha-beta-cell islets which were indistinguishable. However, the ATP content of alpha-cell islets was maintained for prolonged periods in the absence of glucose in contrast to mixed islets, composed primarily of beta-cells, in which the ATP level decreased by 45% when glucose-free medium was perfused for sustained periods. Finally, insulin infused in high concentrations or administered to the diabetic animal had no effect on the glucose spaces or the ATP contents of normal or alpha-cell islets. It can be calculated that in vivo the intracellular glucose level of islets from streptozotocin treated rats is approximately 15 mM. Since in normals an extracellular glucose concentration of this magnitude inhibits stimulated glucagon release completely, it would seem unlikely that a lack of intracellular glucose is the cause of the apparent glucose "blindness" of the alpha-cells in diabetes. In fact, in perfusion studies as little as 2.5 mM free intracellular glucose was sufficient to suppress glucagon secretion from diabetic alpha-cells. The results of the ATP measurements clearly eliminate a possible energy deficit of diabetic alpha-cells as cause of the apparent glucose resistance of alpha-cells.