Chronotropic function in spontaneously diabetic BB rats

In the present study, isolated atrial function in spontaneously diabetic BB Wistar rats maintained for 12 weeks on a low (BB-LI) and a high (BB-HI) dosage of insulin was examined. Basal atrial rates were unchanged in the diabetic animals, relative to nondiabetic littermates (ND-BB) or Wistar controls. The BB-HI animals were euglycemic and responded to isoproterenol in a similar manner to the ND-BB and Wistar control animals. In contrast, BB-LI animals remained hyperglycemic and exhibited lower responses to the maximum chronotropic effects of isoproterenol. Plasma thyroid hormone levels were unaltered in the BB-diabetic animals. These results therefore reveal an absence of bradycardia and hypothyroidism in spontaneously diabetic BB rats, in contrast to previous observations in streptozotocin diabetic rats. However, a decrease in chronotropic response to isoproterenol was still noted in the BB-LI animals. These findings suggest that decreased positive chronotropic effect of isoproterenol in diabetes may not be a direct consequence of altered thyroid status.     

Glucose and glutamine metabolism in rat macrophages: enhanced glycolysis and unaltered glutaminolysis in spontaneously diabetic BB rats.

Metabolism of glutamine (Gln, 2 mM) and glucose (5 mM) was studied in vitro in isolated resident peritoneal macrophages from both normal (BBn) and spontaneously diabetic BB (BBd) rats. The major products from Gln were ammonia, glutamate, CO2 and to a lesser extent aspartate. Glucose decreased (P less than 0.01) the production of ammonia, CO2 and aspartate from Gln by 34-60%, but had no effect on the amount of glutamate accumulated. The major products from glucose were lactate and to a much lesser extent pyruvate and CO2. Gln decreased (P less than 0.01) 14CO2 production from [U-14C]glucose by 19-28%, increased (P less than 0.01) pyruvate production by 35-49%, but had no effect on lactate production. The fraction of glucose metabolized via the pentose phosphate pathway (PC) was less than 5%. There were no significant differences in Gln metabolism between BBn and BBd macrophages. The production of lactate and pyruvate and the flux from glucose into the PC were increased (P less than 0.01) by 2.4, 1.8 and 1.5-fold, respectively, in BBd cells. Increased macrophage glucose metabolism was also observed in diabetes-prone BB (BBdp) rats at 75-80 days but not at 50 days of age. In the presence of both Gln and glucose, potential ATP production from glucose was 2- and 4-times that from Gln, respectively, in BBn and BBd cells. Lactate production was the major pathway for glucose-derived ATP generation. These results demonstrate (a) glycolysis and flux from glucose through the pentose phosphate pathway are enhanced with no alteration in glutaminolysis in BBd macrophages; and (b) glucose may be a more important fuel than Gln for macrophages, particularly in BBd rats. The increased glucose metabolism may be associated with functional activation of the macrophages that have been proposed to be involved in beta-cell destruction and the development of diabetes.     

Genes of SHR rats protect spontaneously diabetic BB/OK rats from diabetes: lessons from congenic BB.SHR rat strains

Diabetes in BB rats share many common features with human type 1 diabetes. One of them is the complex and polygenic nature of disease. Analysis of cross hybrids of diabetic BB/OK rats and rats of different diabetes-resistant strains has demonstrated that beside the MHC genes, Iddm1 and the lymphopenia, Iddm2, additional non-MHC genes are involved in diabetes development. To study the importance of the non-MHC genes, Iddm4 and Iddm3, two congenic BB.SHR rat strains were generated by recombining a segment of the SHR chromosome 6 (Iddm4; termed BB.6S; 15cM) or chromosome 18 (Iddm3; termed BB.18S; 24cM) into the BB/OK background by serial backcrossing and marker-aided selection. The characterization of both congenic strains demonstrates a drastic reduction of diabetes frequency in comparison to the BB/OK strain (86% vs 14% and 34%). It is supposed that diabetes protective genes of SHR must be located on both chromosomal segments and that these suppress the action of the essential and most important genes of diabetes development in the BB/OK rat, Iddm1, and Iddm2.     

Serum lipids and lipoprotein composition in spontaneously diabetic BB Wistar rats

Serum lipid and lipoprotein composition in spontaneously diabetic BB Wistar rats, nondiabetic littermates, and control Wistar rats was studied to elucidate diabetes-related abnormalities of lipoprotein composition. Serum total triglycerides and pre-beta-lipoprotein concentrations of insulin-treated spontaneously diabetic BB and nondiabetic littermate rats were significantly higher than those of control Wistar rats. Serum cholesterol and HDL cholesterol concentrations of spontaneously diabetic BB and nondiabetic littermate rats did not differ from controls. Concentrations of very low density lipoproteins (VLDL), low density lipoproteins (LDL), and high density lipoproteins (HDL) of spontaneously diabetic BB and nondiabetic littermate rats were higher than those of normal rats. With sodium dodecylsulfate-polyacrylamide gel electrophoresis it was observed that the spontaneously diabetic BB and nondiabetic littermate rat VLDL contained higher percentages of apoE relative to total apoC when compared with control Wistar rats. With isoelectric focusing, apoC-II relative percentages in VLDL and HDL of both spontaneously diabetic BB and nondiabetic littermate rats were higher than apoC-II proportions in VLDL and HDL of controls. Apolipoprotein A-I of the control rat HDL showed four isoforms that focused at pI 5.8 (17.3%), 5.75 (30.6%), 5.65 (31.8%), and 5.55 (20.5%); however, the spontaneously diabetic BB and nondiabetic littermate rat HDL apoA-I was mainly represented by two isoforms that focused at pI 5.8 and 5.75. VLDL of both diabetic and nondiabetic BB rats contained higher levels of acidic apoE isoforms compared to their counterparts in control Wistar rats. Although HDL cholesterol concentrations of spontaneously diabetic BB rats remained normal, protein concentrations were higher resulting in a low cholesterol/protein ratio in HDL suggesting that the cholesterol-carrying capacity of spontaneously diabetic BB rat HDL could be less than normal and may be due to an abnormal apoA-I composition. Quantitative alterations of lipid and lipoprotein composition appear in the BB Wistar rat when compared to the Wistar rat, but some of the changes are more pronounced in the spontaneously diabetic BB Wistar rat.     

Lactate production is the major metabolic fate of glucose in splenocytes and is altered in spontaneously diabetic BB rats.

Enhanced glucose metabolism is necessary to support the activation and proliferation of lymphocytes. To define further quantitatively the metabolic fates of glucose and assess glucose utilization both in normal cells and in an autoimmune disease with abnormal lymphocytes, [U-14C]glucose conversion into 14CO2 and the production of lactate and pyruvate were measured in splenocytes. Cells from non-diabetes-prone (BBn) and spontaneously diabetic (BBd) rats were studied both freshly isolated 'resting' and cultured for 96 h with and without concanavalin A (Con A) stimulation. (1) Lactate was confirmed to be the major end product in both freshly isolated (53% of utilized glucose) and unstimulated cultured (62% of utilized glucose) cells from BBn animals studied at (2-8) x 10(6) cells/ml concentration. The use of concentrations from 10 x 10(6) to 300 x 10(6) cells/ml resulted in progressively less lactate production per 10(6) splenocytes. (2) Cells from BBd animals after stimulation with Con A incorporated less [3H]thymidine and produced significantly less lactate (155 +/- 14 versus 305 +/- 24 nmol/2 h per 10(6) cells) than did BBn cells (P less than 0.05). (3) However, more lactate (101 +/- 8 versus 78 +/- 6 nmol/5 h per 10(6) cells) was produced by 'resting' cells from BBd animals compared with BBn (P less than 0.03), and this difference was sustained after 4 days in culture. (4) Significantly greater amounts of pyruvate were produced by BBd than by BBn cells, particularly when stimulated with Con A, suggesting an alteration in the availability of reducing equivalents in BBd cells. (5) These results are consistent with prior metabolic as well as immunological 'activation' of cells in vivo in the BB diabetic animals.     

The metabolism of glucose in diaphragm muscle from normal rats, from streptozotocin-treated diabetic rats and from rats treated with anti-insulin serum

1. The metabolism of [U-¹⁴C]glucose by the isolated diaphragm muscle of normal rats, rats rendered diabetic with streptozotocin and rats with transitory insulin deficiency after an injection of anti-insulin serum was studied. 2. The incorporation of [¹⁴C]glucose into glycogen and oligosaccharides was significantly decreased in the diabetic diaphragm muscle and in the muscle from rats treated with anti-insulin serum. 3. Neither diabetes nor transitory insulin deficiency influenced the oxidation of glucose, or the formation of lactate and hexose phosphate esters from glucose. 4. Insulin fully restored the incorporation of glucose into glycogen and maltotetraose in the diabetic muscle, but the incorporation into oligosaccharides, although increased in the presence of insulin, was significantly lower than the values obtained with normal diaphragm in the presence of insulin.     

Effects of parenteral palatinose on glucose metabolism in normal and streptozotocin diabetic rats

The present experiment was carried out to investigate the metabolism of palatinose (6-O-alpha-D-glucopyranosyl-D-fructose) in the rat. The bolus injection of palatinose (0.5 g/kg) in the tail vein of normal and streptozotocin (STZ) diabetic rats caused significant increments in glucose and insulin concentrations. However, in severe STZ diabetic rats (greater than 300 mg/dl of fasting plasma glucose) no significant change in glucose and insulin concentrations was observed. In liver perfusion, the gradual decrease in glucose output from the normal and mild STZ diabetic rat livers perfused with 20 mM Krebs-Ringer-Tris buffer pH 7.4 was prevented by the addition of 5.5 mM palatinose in the perfusate and fructose was detected in the effluent during the palatinose infusion. The results indicate that palatinose is metabolized to glucose and fructose in both normal and diabetic rat tissues, and this causes the increase in blood glucose concentration. On the other hand, the direct stimulatory effect of insulin release from pancreatic B-cell was not observed when the palatinose was infused into the isolated perfused rat pancreas. The study suggest that palatinose administered parenterally is metabolized by tissues and expected to be used as a source of fluid and energy supply.     

Glutamine and glucose metabolism in bovine blood lymphocytes.

1. Glutamine and glucose metabolism was studied in bovine blood lymphocytes incubated at 37 degrees C in the presence of Krebs-Ringer bicarbonate buffer (pH 7.4) containing 1 mM [U-14C]glutamine and 5 mM [U-14C]glucose, respectively. 2. The major metabolic products from glutamine were ammonia, glutamate, and to a lesser extent, aspartate and CO2. Glucose was metabolized mainly to lactate and, to a lesser extent, pyruvate and CO2. These findings indicate incomplete oxidation of glutamine and glucose carbons in bovine blood lymphocytes. 3. Glucose provided three-fold greater amounts of energy to bovine blood lymphocytes than did glutamine on the basis of their measured end-products. Glycolysis accounted for 50% of glucose-derived ATP production. 4. Our findings suggest similar metabolic patterns of glutamine and glucose in lymphocytes between ruminants and non-ruminant species (e.g. rats). However, in contrast to rat peripheral lymphocytes, glucose, rather than glutamine, was a major energy substrate for bovine blood lymphocytes.     

Cardiac function in spontaneously diabetic BB rats treated with low and high dose insulin

Cardiac abnormalities observed in animals with drug-induced diabetes may be due to the direct cardiotoxic effect of the drugs or factors not related to the diabetic state. The purpose of this investigation was to examine cardiac sarcoplasmic reticular (SR) calcium transport and heart function in the BB rat, a strain in which diabetes occurs spontaneously and clearly resembles insulin-dependent diabetes in humans. Complete insulin withdrawal for 2 or 4 days from BB diabetic rats leads to a spectrum of metabolic derangements including a loss of body weight, hyperglycemia, and elevated triglyceride levels confirming the insulin dependence of this model. The present study involved treating BB diabetic rats with a low (hyperglycemic) and high (normoglycemic) insulin dose for 12 weeks after the detection of glycosuria. The hearts from these animals were then isolated, and SR Ca2+ transport and heart function (using isolated perfused working hearts) were examined and compared with BB nondiabetic littermates or Wistar controls. Strain-related differences were found in ATP-dependent SR Ca2+ transport between the Wistar and BB rats. There were, however, no significant diabetes-related differences in SR Ca2+ transport between the low dose insulin treated diabetic group (LD) and the high dose insulin treated diabetic group (HD) or the nondiabetic littermates. Plasma lipid concentrations of the LD and HD BB rats and nondiabetic littermates were also generally higher than those of control Wistar rats indicating strain-related but not diabetes-related differences. In addition, there were no differences in cardiac function between the LD and BB nondiabetic littermates or Wistar controls.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycogen metabolism in white and red muscle or normal and diabetic rats. The glycogen concentration and glycogen synthesis from glucose.

The amount of glycogen and its synthesis from glucose was studied in white muscle (extensor digitorum longus -- EDL) and red muscle (soleus -- SOL) of normal rats and rats with alloxan diabetes by the anthrone method. The amount of glycogen was higher in the white muscle of normal rats, both after a 24 hours' fast (0.37+/-0.02 mg/g as against 0.29+/-0.01 mg/g in the SOL) and with feeding ad libitium (0.72+/-0.05 mg/g as against 0.58+/-0.03 mg/g in the SOL). After a 24 hours' fast, the glycogen content of both muscles was non-significantly higher in alloxan-diabetic rats than in normal animals, whereas in diabetic animals fed ad libitum it was significantly lower than in normal rats fed in the same manner (0.54+/-0.07 mg/g in the EDL and 0.33+/-0.03 mg/g in the SOL). The difference between the glycogen content of the white and red muscle of diabetic rats was also in favour of the white muscle. Muscle glycogenesis from intragastrically administered glucose was higher in the red muscle in all the experimental groups. In normal fed ad libitum the glycogen content of the EDL did not change after glucose administration, but in the SOL it rose from 0.58+/-0.03 to 0.83+/-0.05 mg/g. In fasting (24 hours) normal rats it rose sharply in both muscles, from 0.037+/-0.02 to 0.57+/-0.03 mg/g in the EDL and from 0.29+/-0.01 to 0.87+/-0.06 mg/g in the SOL. In fasting (24 hours) diabetic animals, the glycogen content rose after glucose in the SOL only, from 0.36+/-0.01 to 0.66+/-0.06 mg/g. The differences found in glycogen synthesis in the white and red muscle of normal and diabetic rats are discussed mainly from the aspect of the existence of a relationship between the glycogen concentration and glycogen synthetase activity.     

Acute insulin withdrawal contributes to ischemic heart failure in spontaneously diabetic BB Wistar rats

The contribution of poor metabolic control to myocardial ischemic failure was determined in isolated working hearts from insulin-dependent BB Wistar rats. Removal of insulin treatment 24 h prior to study (uncontrolled diabetic rats) resulted in significant increases in serum glucose, serum fatty acids, and myocardial triglyceride, compared with animals in which insulin treatment was not withheld (insulin-treated diabetic rats). Isolated working hearts obtained from these two groups were subjected to a 40% reduction in coronary flow in the presence of a maintained metabolic demand (hearts were paced at 200 beats/min and perfused at an 80 mmHg (1 mmHg = 133.3 Pa) left aortic afterload, 11.5 mmHg left atrial preload). Within 15 min of ischemia, a significant deterioration of mechanical function occurred in the uncontrolled diabetic rats, whereas function was maintained in the insulin-treated diabetic rats. Oxygen consumption by the two groups of hearts was similar prior to the onset of ischemia and decreased during ischemia in parallel with the work performed by the hearts. This suggests that the accelerated failure rate in uncontrolled diabetic rat hearts is unlikely a result of an increased oxygen requirement. These data are a direct demonstration that acute changes in metabolic control of the diabetic can contribute to the severity of myocardial ischemic injury.     

Evidence for insulin dependent hepatic microsomal gamma-linolenic acid chain elongation in spontaneously diabetic Wistar BB rats.

We studied hepatic microsomal gamma-linolenoyl-CoA elongation and fatty acid composition of liver microsomes in spontaneously diabetic Wistar BB rats. The liver microsomal gamma-linolenoyl-CoA elongation was decreased in diabetic Wistar BB rats during both normo- and hyperglycemic periods and restored during the hypoglycemic period following insulin treatment. These results are in agreement with our previously reported data on linoleic acid delta 6 and delta 5 desaturations and support the non-parallel relationship between the chain elongation system and the glycemia. The fatty acid composition of BB rat liver microsomes was only partially consistent with the gamma-linolenoyl-CoA elongation activity at the different periods of glycemia, probably because factors other than elongation impairments were involved in the evolution of fatty acid composition.     

Glomerular function in spontaneously diabetic rats.

This study was undertaken to determine whether hyperfiltration exists at the single nephron level and whether albumin excretion is increased early in the course of diabetes in Biobreeding rats. Diabetic rats were studied at 8-12 weeks after the onset of diabetes. Control animals were age-matched, diabetes-resistant rats. Urinary and tubular fluid albumin concentrations were measured by polyacrylamide gel electrophoresis. Clearance and micropuncture techniques were used to determine whole kidney and single nephron glomerular filtration rate, renal blood flow, and glomerular capillary pressure. The urinary albumin excretion rate (1.3 +/- 0.1 mg/24 hr) and the tubular fluid albumin concentration (4.7 +/- 0.7 mg/dl) in the diabetic group were significantly elevated when compared with urinary albumin excretion (0.9 +/- 0.1 mg/24 hr) and tubular fluid albumin concentration (2.5 +/- 0.5 mg/dl) in the control group. There were no significant differences in glomerular hemodynamics (whole kidney or single nephron glomerular filtration rate or glomerular capillary pressure) between diabetic and control rats. The kidney weight and kidney weight to body weight ratio were significantly higher in diabetic rats when compared with control rats. Early diabetes in Biobreeding rats is characterized by mild albuminuria and increased kidney size, but not glomerular hyperfiltration.     

Assessment of the antidiabetic activity of epicatechin in streptozotocin-diabetic and spontaneously diabetic BB/E rats

(–)-Epicatechin has previously been suggested to rapidly reverse alloxan diabetes in rats. We have assessed the therapeutic value of the compound in two further animal models of insulin-dependent diabetes mellitus, namely streptozotocin - diabetic rats and the spontaneously diabetic BB/E rat . There was no indication of a reversal of established diabetes in either the streptozotocin-diabetic or the spontaneously diabetic BB/E rats. Moreover, epicatechin also failed to halt the progression of the disease in prediabetic BB/E rats. Earlier claims of the potential use of epicatechin as an antidiabetic agent must therefore be treated with some caution.     

Serum metabolite signature predicts the acute onset of diabetes in spontaneously diabetic congenic BB rats

The clinical presentation of type 1 diabetes is preceded by a prodrome of beta cell autoimmunity. We probed the short period of subtle metabolic abnormalities, which precede the acute onset of diabetes in the spontaneously diabetic BB rat, by analyzing the serum metabolite profile detected with combined gas chromatography/mass spectrometry (GC/MS) and liquid chromatography/mass spectrometry (LC/MS). We found that the metabolite pattern prior to diabetes included 17 metabolites, which differed between individual diabetes prone (DP) BB rats and their age and sex matched diabetes resistant (DR) littermates. As the metabolite signature at the 40?days of age baseline failed to distinguish DP from DR, there was a brief 10-day period after which the diabetes prediction pattern was observed, that includes fatty acids (e.g. oleamide), phospholipids (e.g. phosphocholines) and amino acids (e.g. isoleucine). It is concluded that distinct changes in the serum metabolite pattern predict type 1 diabetes and precede the appearance of insulitis in spontaneously diabetic BB DP rats. This observation should prove useful to dissect mechanisms of type 1 diabetes.     

Ultrastructural changes in the gracile nucleus of the spontaneously diabetic BB rat.

The present study describes the structural changes in the gracile nucleus of the spontaneously diabetic BB rat. At 3-7 days post-diabetes, axons, axon terminals and dendrites showed electron-dense degeneration. Degenerating axons were characterized by swollen mitochondria, vacuolation, accumulation of glycogen granules, tubulovesicular elements, neurofilaments and dense lamellar bodies. Degenerating axon terminals consisted of an electron-dense cytoplasm containing swollen mitochondria, vacuoles and clustering of synaptic vesicles. These axon terminals made synaptic contacts with cell somata, dendrites and other axon terminals. Degenerating dendrites were postsynaptic to normal as well as degenerating axon terminals. At 1-3 months post-diabetes, degenerating electron-dense axons, axon terminals and dendrites were widely scattered in the neuropil. Macrophages containing degenerating electron-dense debris were also present. At 6 months post-diabetes, the freshly degenerating neuronal elements encountered were similar to those observed at 3-7 days. However, there were more degenerating profiles at 6 months post-diabetes compared to the earlier time intervals. Terminally degenerating axons were vacuolated and their axoplasm appeared amorphous. It is concluded that degenerative changes occur in the gracile nucleus of the spontaneously diabetic BB rat.     

Abnormal thymocyte maturation in spontaneously diabetic BB rats involves the deletion of CD4-8+ cells.

The object of this study was to further characterize the pathophysiology of the peripheral T lymphopenia in the BB rat. Towards this end, surface markers on unseparated thymocytes and purified thymocyte subsets from age- and sex-matched diabetes-resistant (BBn) and diabetes-prone (BBd) rats were analyzed by two-color flow cytometry. The proportions of thymocytes falling into each of the four main phenotypic subsets were comparable in BBn (n = 9) and BBd (n = 8) rats: respectively, 4.6 +/- 0.6% and 4.4 +/- 0.8%, CD4-8-; 68.1 +/- 1.9% and 71.1 +/- 3.2%, CD4+8+; 18.3 +/- 1.5% and 15.4 +/- 2.3%, CD4+8-; 9.1 +/- 0.9% and 9.1 +/- 1.0%, CD4-8+. In addition, absolute numbers of thymocytes were not significantly different. The levels of expression of CD4, TCR-alpha beta within each thymocyte subset were comparable in BBn and BBd animals as were the anti-TCR-induced proliferative responses of their CD4+8- and CD4-8+ thymocytes. However, phenotypic abnormalities within the CD4-8+ thymocyte subset of the BBd rat were found. A very significant (p less than 0.005) deletion of mature CD4-8+, TCR-alpha beta + thymocytes and a proportional increase (p less than 0.005) of immature CD4-8+, TCR-alpha beta low thymocytes. Moreover, a twofold decrease of CD8 expression by mature CD4-8+ thymocytes was observed in BBd animals. These results suggest that an impaired thymic maturation contributes to the peripheral T lymphopenia of the BBd rat.     

Natural cytotoxic autoantibody against thymocytes in spontaneously hypertensive rats

A strain of SHR rats, which spontaneously develops hypertension and periarteritis nodosa, had a decreasing number of rosette-forming T cells in their thymuses and a progressive decline in cellular immune functions by aging. They were found to produce natural thymocytotoxic autoantibody (NTA) detected by a complement-dependent cytotoxicity test. This autoantibody occurred from 1 month of age and throughout life; the incidence was more than 60% of SHR rats at any age. Thymocytes from all six rat strains tested showed similarly high sensitivity to NTA but none of the strains tested produced NTA except the SHR strain. Rosette-forming thymocytes of WKA rats, which were separated by Ficoll gradient, showed much higher cytotoxic sensitivity to NTA than did whole thymocytes and nonrosetting thymocytes. The cytotoxicity of NTA was weak or negative for spleen cells, lymph node cells, bone marrow cells, and blood lymphocytes of WKA rats. However, the cytotoxic activity of NTA was completely absorbed with the thymus, spleen, lymph node cells, and brain homogenates and was partially absorbed with bone marrow cells, but not with liver and kidney homogenates. NTA in SHR rats was an IgM-globulin as determined by sensitivity to 2-ME treatment and by Sephadex G-200 column chromatography. These results suggest that NTA is responsible for the selective suppression of T-cell functions in SHR rats.