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.     

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.     

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.     

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.     

Endothelins urinary excretion is increased in spontaneously diabetic rats: BB/BB

Previously we reported (1) an increase of endothelin-1,2 (ET) content, in urine of rats made diabetic with streptozotocin (STZ), starting three days and up to 20 weeks from diabetes induction. The increased ET excretion was considered as an early marker of endothelial damage. To ascertain if this phenomenon was present also in a strain of spontaneously diabetic rats, endothelin-1,2 urinary excretion was determined in BB/BB diabetic rats, and their control (BB/WB), at different times after the onset of diabetes, (two, four, six and twelve weeks). BB/BB diabetic rats showed elevated urinary excretion of endothelins as compared to BB/WB control rats, starting two weeks after diabetes onset, and up to twelve weeks. In the same animals, Nerve Conduction Velocity (NCV), was monitored at the same time as an index of the occurrence of a diabetes complication (peripheral neuropathy). NCV resulted to be impaired in the BB/BB diabetic rats as compared to control rats; however the increase of ET in urine, is earlier in comparison to peripheral neuropathy. These data suggest the hypothesis that endothelial damages preceed the overt manifestations of peripheral neuropathy associated to diabetes.     

Fatty acid composition of brain hemispheres of spontaneously hypertensive rats suckled by female Wistar rats

Total lipid fatty acid composition was investigated in brain hemispheres of male Spontaneously Hypertensive Rats (SHR), compared with normotensive Wistar Kyoto rats (WKY) used as controls. Both strains were suckled by adoptive Wistar mothers, and then fed a standard diet after weaning. No difference was observed between the two hemispheres of WKY killed either at 10 or 30 days. In SHR killed at 10 days, the two hemispheres showed differences, SHR left hemispheres exhibiting greater fatty acid composition changes than those of WKY, phenomenon that toned down at 30 days. Hence, SHR pups showed a different total lipid fatty acid composition of their brain hemispheres when compared with their WKY controls, though the two strains received the same diet. Genetically programmed hypertension might be, directly or not, involved in these changes.     

Cardiac dysfunction in isolated perfused hearts from spontaneously diabetic BB rats

The purpose of this investigation was to examine cardiac function and biochemistry in spontaneously diabetic BB rats, a strain in which diabetes occurs spontaneously and closely resembles insulin-dependent diabetes in humans. The study involved two groups: nondiabetic littermates of BB rats and BB diabetic rats treated daily with a very low insulin dose such that the rats were severely hyperglycemic and hyperlipidemic. The hearts from these two groups were isolated and heart function (using isolated perfused working hearts) and biochemistry were examined 6 weeks after the onset of diabetes. BB diabetic rats exhibited a lower calcium-stimulated myosin ATPase activity and depressed left ventricular developed pressure, cardiac contractility, and ventricular relaxation rates compared with BB nondiabetic littermates. These results suggest that the chronically diabetic state in the BB rat produces cardiac changes similar to those demonstrable after chemical diabetes induced by alloxan or STZ, or that seen during human diabetes mellitus.     

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.     

Altered cardiac excitation-contraction coupling in ventricular myocytes from spontaneously diabetic BB rats

Cardiac excitation-contraction (E-C) coupling abnormalities in chemically induced diabetes have been well defined. Heart dysfunction has also been reported in diabetes of genetic origin. The purpose of this study was to determine whether heart dysfunction in genetically predisposed diabetes is attributable to impaired E-C coupling at the cellular level. Myocytes were isolated from 1-yr-old BioBreed (BB) spontaneously diabetic-prone (BB/DP) rats and their diabetic-resistant littermates (BB/DR). Mechanical properties were evaluated by use of a video edge-detection system. Myocytes were electrically stimulated at 0.5 Hz. The contractile properties analyzed included peak shortening (PS), time-to-peak shortening (TPS), time-to-90% relengthening (TR(90)), and maximal velocities of shortening and relengthening (+/-dL/dt). Intracellular Ca(2+) handling was evaluated with fura 2 fluorescent dye. Myocytes from spontaneously diabetic hearts exhibited a depressed PS, prolonged TPS and TR(90), and reduced +/-dL/dt. Consistent with the mechanical response, myocytes from the BB/DP group displayed reduced resting and peak intracellular Ca(2+) concentration associated with a slowed Ca(2+)-transient decay. Furthermore, myocytes from BB/DP hearts were less responsive to increases in extracellular Ca(2+) and norepinephrine and equally responsive to increases in stimulation frequency and KCl compared with those from the BB/DR group. These results suggest that the genetic diabetic state produces altered cardiac E-C coupling, in part, because of abnormalities of the myocyte, similar to that demonstrable after chemically induced diabetes or during human diabetes.     

Secretion of triglyceride and ketogenesis by livers from spontaneous diabetic BB Wistar rats

Livers from male and female BB Wistar spontaneously diabetic rats were perfused in vitro to determine the effects of spontaneously occurring insulin-dependent diabetes on the metabolism of fatty acid. The secretion of triglyceride and the incorporation of [1-14C] oleic acid into perfusate and hepatic triglyceride was reduced by the diabetic state, whereas beta-hydroxybutyrate production and output of total ketone bodies were increased. The spontaneous diabetic Wistar rat clearly is a suitable model to study the derangements induced in lipid/plasma lipoprotein metabolism by the insulin-dependent diabetic state; the data obtained with this model confirm our earlier observations on experimental insulin deficiency induced with alloxan, streptozotocin, and anti-insulin serum.     

Effect of metyrapone on bile flow and bile acid excretion in Wistar rats

The influence of metyrapone on bile flow and excretion of mono-(MBA), di-(DBA) and trihydroxy-(TBA)-bile acids was investigated in adult male Wistar rats after single and repeated pretreatment. MBA were not found in the rat bile. Metyrapone administration (200 mg/kg b.w. i.p.) 1 h before onset of a 3-hour bile collection period diminished bile flow and excretion of DBA and TBA. The relation TBA/DBA was changed towards DBA. Similar results were found after repeated administration 12 h after the last metyrapone injection (4 x 50 mg/kg b.w. i.p. per day for 4 consecutive days). But 60 h after the last metyrapone administration bile flow and the excretion of TBA were enhanced and the TBA/DBA ratio was changed towards TBA. The possible influence of metyrapone on bile acid hydroxylation is discussed and compared with metyrapone action on hydroxylation of foreign compounds.     

Glutamine and glucose metabolism in thymocytes from normal and spontaneously diabetic BB rats.

Metabolism of glutamine and glucose was studied in thymocytes from normal rats and BB rats with the spontaneous autoimmune diabetic syndrome to assess their potential roles as fuels. The major measured products from glucose were lactate and, to a lesser extent, CO2, and pyruvate. Glutamine had no effect on the rates of their production from glucose. Glutamine was metabolized to ammonia, aspartate, glutamate, and CO2, with aspartate being the major product of carbons from glutamine in the absence of glucose. Glucose markedly decreased the formation of ammonia, aspartate, and CO2 from glutamine, but increased that of glutamate, with an overall decrease in glutamine utilization by 55%. More glutamate than aspartate was produced from glutamine in the presence of glucose. The potential production of ATP from glucose was similar to that when glutamine was present alone. However, glucose markedly decreased production of ATP from glutamine, but not vice versa. This resulted in ATP production from glucose being 2.5 times that from glutamine when both substrates were present. The oxidation of glucose to CO2 via the Krebs cycle accounts for 75-80% of glucose-derived ATP production. Cellular ATP levels markedly decreased in the absence of exogenous substrates, but were constant throughout a 2-h incubation in the presence of glutamine, glucose, or both. There were no differences in thymocyte glucose or glutamine metabolism between normal and diabetic BB rats, in contrast to previous findings in peripheral lymphoid organs. Our results suggest that glucose is a more important fuel than glutamine for "resting" thymocytes, again in contrast to the cells of peripheral lymphoid organs in which glutamine is as important as glucose as a fuel.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

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.     

Hepatic microsomal delta 9 desaturation of stearic acid in the spontaneously diabetic female BB rat

delta 9 desaturation of stearic (1-14C) acid has been estimated from incubation of liver microsomes of adult female spontaneously diabetic BB rat, an animal model resembling the spontaneous juvenile diabetes in humans, comparatively to adult female control Wistar rat. The animals were sacrificed, when hyperglycemic, 24 hours after the last insulin injection to the BB rats. Stearic acid delta 9 desaturase activity is drastically depressed in the BB rats when fatty acid composition of liver phospholipids and microsomal total liver lipids are changed in spite of the daily injection of insulin necessary for the BB rats survival.     

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.     

Potential role of antioxidants during ethanol-induced changes in the fatty acid composition and arachidonic acid metabolites in male Wistar rats

Biochemical assessment of liver damage during ethanol-induced stress was done by measuring the activities of serum enzymes, viz., aspartate transaminase (AST) and alkaline phosphatase (ALP), which were significantly elevated in rats fed ethanol. Ethanol administration for a period of 60 days modifies the fatty acid composition, and the analysis of fatty acids showed that there was a significant increase in the concentrations of palmitic acid (16:0), stearic acid (18:0), and oleic acid (18:1) in liver, kidney, and brain, whereas the concentrations of palmitoleic (16:1) and arachidonic acid (20:4) were significantly decreased. The breakdown products of arachidonic acids (20:4), prostaglandins, were elevated. The antioxidants curcumin and N-acetylcysteine (NAC) decreased the activities of serum AST and ALP. Curcumin and NAC decreased the concentrations of fatty acids, viz., palmitic, stearic, and oleic acid, whereas arachidonic acid and palmitoleic acid were elevated. The prostaglandin concentrations were also decreased after curcumin and N-acetylcysteine treatment. Thus the present investigation shows that curcumin and N-acetylcysteine prevent the fatty acid changes produced by ethanol and also reduce the inflammatory response of ethanol by reducing the level of prostaglandins. This revised version was published online in July 2006 with corrections to the Cover Date.