Effect of sex hormones on the onset of diabetic syndrome in WBN/Kob rats]

The effects of sex hormone on diabetic conditions were investigated in WBN/Kob strain rats, i.e., castrated or spayed, hormone-treated, and non-treated rats. The effects of sex hormone on glycosuria, body-weight change, glucose tolerance and histopathology of the pancreas were compared among these animals. There were no abnormal changes in these parameters in the non-treated females and estrogen-treated males. The glycosuria began to be observed from the age of about 30 weeks in the non-treated group and from the age of 52 weeks in the castrated group. In the female animals, this symptom began to appear from the age of 55 weeks in the testosterone-treated group and from the age of 72 weeks in the spayed group. Before the onset of the diabetic symptoms, glucose tolerance was impaired in these animals. Body weights of the castrated and estrogen-treated males were lower than that of the non-treated males, especially in the estrogen-treated males. Those of the spayed and testosterone-treated females were much heavier than that of the non-treated females. Testosterone treatment accelerated body-weight gain in the spayed female animals. Histopathological examination of the pancreas revealed atrophy of the aciner tissue and atrophy and disappearance of the islet cells similar to those of the non-treated WBN/Kob male rats in the castrated males, spayed females and testosterone-treated females. However, these changes were not observed in the non-treated females or estrogen-treated males. These findings suggest that female hormone suppressed the onset of hyperglycemia along with glycosuria and male hormone accelerates the onset of hyperglycemia in the WBN/Kob rats.     

A new diabetic strain of rat (WBN/Kob)

A new, spontaneously occurring diabetic syndrome has been observed in the aged males of an inbred strain of Wistar rats, WBN/Kob. The main clinical sign, glycosuria, was first detected at about 60 weeks of age, and thereafter some animals developed hyperlipidaemia and gradual emaciation. Prior to the onset of glucosuria, male rats showed impaired glucose tolerance after a glucose load at 21 weeks of age. The histopathologic lesions of the pancreas in the diabetic males consisted of multifocal fibrosis, decreased in number and size of islets and atrophy of exocrine tissue. Multifocal inflammatory foci of varying stages were the main pancreatic lesion in prediabetic male rats. This inflammatory change was detected even in 12-week-old rats and tended to occur around the islets. Therefore focal fibrosis and the decrease in the number and size of islets were considered to result from post-inflammatory scarring. The maturity-onset of this syndrome and the impaired glucose tolerance in younger animals suggested that diabetes mellitus of this rat strain is insulin-independent type II. However, the histological lesions of the pancreas were somewhat different from previous reports of both type I and II diabetes mellitus in man and animals.     

Characterization of a novel congenic strain of diabetic fatty (WBN/Kob-Lepr(fa)) rat

The WBN/Kob-Lepr^fa rat is a new congenic strain for the fa allele of the leptin receptor gene (Lepr). Homozygous (fa/fa) WBN/Kob-Lepr^fa rats provide a model of non-insulin-dependent diabetes with obesity. Here, we describe the characteristics of this new animal model in detail. At 7 weeks of age, both male and female obese WBN/Kob rats showed inflammatory cell infiltration of the pancreas that suggested pan-pancreatitis and an abnormal OGTT. At 3 months of age, both male and female obese WBN/Kob rats developed overt diabetes mellitus associated with severe chronic pancreatitis. In contrast, lean female WBN/Kob rats do not develop pancreatitis or diabetes. In WBN/Kob rats, this mutation might promote the onset of severe pancreatitis, leading to the rapid development of diabetes mellitus.     

Electrocardiographical findings of WBN/Kob rats

Electrocardiographical (ECG) investigations were carried out on 4 and 12 week old WBN/Kob and Wistar male rats. In comparison with Wistar rats which showed ECG findings typical of those of normal rats, WBN/Kob rats showed significantly larger QRS complex amplitude, smaller T wave amplitude, longer QRS duration, and longer QT interval at 4 weeks of age. This tendency became clearer at 12 weeks of age. There were no changes in serum inorganic ion concentrations or cardiac histopathology suggestive of these ECG changes in WBN/Kob rats. These ECG findings in WBN/Kob rats are considered to be a hereditary characteristic of the strain.     

Insulin-Induced Hypoglycemic Peripheral Motor Neuropathy in Spontaneously Diabetic WBN/Kob Rats

Intensive insulin therapy can lead to hypoglycemia, with patients sometimes developing hypoglycemic neuropathy. Spontaneously diabetic Wistar Bonn Kobori (WBN/Kob) rats develop diabetic peripheral motor neuropathy characterized by segmental demyelination and axonal degeneration. We examined the short-term effects of hypoglycemia on neuropathic changes in these rats. Spontaneous diabetic WBN/Kob rats received insulin implants for 40 d and were divided into 3 groups based on blood glucose levels: group N, normoglycemic to slightly hyperglycemic (150 to 250 mg/dL); group H, hypoglycemic to slightly hyperglycemic (50 to 200 mg/dL); and group D, nontreated spontaneously diabetic (350 to 420 mg/dL). Conduction velocity was measured in sciatic–tibial motor nerves; these nerves also underwent qualitative and quantitative histomorphologic analysis. Conduction velocity was not significantly different in N, D, and H groups. Morphologic analysis of the sciatic nerves of H rats showed severe changes, including axonal degeneration, myelin distention, and endoneurial fibrosis, that tended to occur in large, myelinated fibers. N and D rats showed relatively mild changes. The degree and distribution of degenerated nerve fibers in H rats were significantly higher than in N and D rats. These results suggest that hypoglycemia of less than 50 mg/dL induced severe peripheral neuropathy. Hypoglycemic lesions differed from the hyperglycemic lesions in diabetic WBN/Kob rats. This rat strain is an appropriate model for investigating the hypoglycemic peripheral neuropathy that can be associated with a diabetic condition.Peripheral neuropathy is a leading complication of diabetes mellitus. Although its exact pathogenesis is not fully understood, chronic hyperglycemia and resultant microenvironmental changes in peripheral nerve tissue contribute to the development of neuropathy.⁵ Therefore, intensive insulin therapy is needed to prevent such complications in patients with type 1 diabetes. However, intensive insulin therapy can lead to hypoglycemia, with patients sometimes developing hypoglycemic peripheral neuropathy.⁸Although experimental hypoglycemic peripheral neuropathy has been studied by using animal models of type 1 diabetes, few studies have included morphologic analyses.^4,7,14,15 These studies showed that hypoglycemia causes axonopathy involving both degenerative and regenerative events. However, hyperglycemic peripheral neuropathy characterized by axonal atrophy has also been induced in diabetic animal models, such that the hyperglycemic changes in these models were similar to hypoglycemic changes. Diabetic WBN/Kob rats spontaneously develop diabetic peripheral motor neuropathy characterized by segmental demyelination and secondary axonal degeneration.^12,13,19 Morphologic changes in diabetic peripheral motor neuropathy are characterized by various degenerative and regenerative changes in myelin sheath, demyelination, and a shift toward axons of smaller diameter. Therefore, WBN/Kob rats may be useful for distinguishing hyperglycemic from hypoglycemic changes. In addition, the threshold of hypoglycemia that induces the morphologic and clinical changes characteristic of peripheral neuropathy in diabetic animals remains unclear.¹⁴ In the present study, we investigated the effects of short-term hypoglycemia on peripheral neuropathic changes in diabetic WBN/Kob rats.     

Circadian Neuroendocrine Role in Age-Related Changes in Body Fat Stores and Insulin Sensitivity of the Male Sprague-Dawley Rat

A role for circadian neuroendocrine rhythms in the age-related development of obesity and insulin resistance was investigated in the male Sprague-Dawley rat. The phases and amplitudes of the plasma rhythms of several metabolic hormones (i.e. corticosterone, prolactin, insulin, and triiodothyronine) differed in lean, insulin-sensitive (3-week-old rats). insulin-resistant (8-week-old rats) and obese, insulin-resistant (44-week-old rats) animals. Simulation of the daily rhythms of endogenous corticosterone and prolactin by daily injections of the hormones at times corresponding to the peak levels found in 3-week-old rats reversed age-related increases in insulin resistance and body fat in older (5-6-month-old) rats. Ten such daily injections of corticosterone and prolactin in 12-14-week-old rats produced long-term reductions in body fat stores (30%). plasma insulin concentration (40%'). and insulin resistance (60%) (determined by a glucose tolerance test) measured 11-14 weeks after the treatment. Alterations in circadian neuroendocrine rhythms may account for age-related changes in carbohydrate and lipid metabolism in the male Sprague-Dawley rat, and resetting of these rhythms by appropriately timed daily injections of corticosterone and prolactin may help maintain metabolism characteristic of younger animals.     

Circadian Neuroendocrine Role in Age-Related Changes in Body Fat Stores and Insulin Sensitivity of the Male Sprague-Dawley Rat

A role for circadian neuroendocrine rhythms in the age-related development of obesity and insulin resistance was investigated in the male Sprague-Dawley rat. The phases and amplitudes of the plasma rhythms of several metabolic hormones (i.e. corticosterone, prolactin, insulin, and triiodothyronine) differed in lean, insulin-sensitive (3-week-old rats). insulin-resistant (8-week-old rats) and obese, insulin-resistant (44-week-old rats) animals. Simulation of the daily rhythms of endogenous corticosterone and prolactin by daily injections of the hormones at times corresponding to the peak levels found in 3-week-old rats reversed age-related increases in insulin resistance and body fat in older (5-6-month-old) rats. Ten such daily injections of corticosterone and prolactin in 12-14-week-old rats produced long-term reductions in body fat stores (30%). plasma insulin concentration (40%″). and insulin resistance (60%) (determined by a glucose tolerance test) measured 11-14 weeks after the treatment. Alterations in circadian neuroendocrine rhythms may account for age-related changes in carbohydrate and lipid metabolism in the male Sprague-Dawley rat, and resetting of these rhythms by appropriately timed daily injections of corticosterone and prolactin may help maintain metabolism characteristic of younger animals.     

Effects of ovariectomy on indices of insulin resistance, hypertension, and cardiac energy metabolism in middle-aged spontaneously hypertensive rats (SHR).

Insulin resistance is a risk factor for coronary heart disease. The protection of young women from coronary events is sharply reduced with menopause. To assess the impact of menopause on glucose tolerance, insulin resistance, body weight gain, heart size, and cardiac energy metabolism, we studied 28-week-old female SHR and Wistar-Kyoto (WKY) rats, who were either ovariectomized (SHR(OVX) and WKY(OVX)) or sham-operated (SHR(SHAM) and WKY(SHAM)). Animals underwent blood-pressure measurement and an oral glucose tolerance test (OGTT). Hearts were weighed and assayed for metabolic enzyme activities. Female SHR were 33 % lighter and hypertensive (+ 36 mmHg), with 33 % larger hearts (when corrected for body weight differences) compared to WKY. Although ovariectomized animals of both strains were heavier overall than their sham-operated counterparts, when heart weights were corrected for body weight, both OVX strains had lighter hearts than both SHAM strains. Glucose and insulin responses during OGTT were similar between the four groups; however, free fatty acid (FFA) responses were approximately 50 % greater in SHR than WKY, although less in SHR(OVX) than SHR(SHAM). WKY(OVX) demonstrated 8 % lower ventricular hexokinase activity than WKY(SHAM), which may reflect reduced cardiac glucose utilization. We also noted 16 % higher citrate synthase activity in WKY hearts. In conclusion, the insulin resistance characteristic of younger SHR is blunted in middle-aged female rats, although FFA responses remain elevated. Ovariectomy did not alter in vivo glucose tolerance in this group; however, sex hormones may be important in maintaining normal heart size and the potential for cardiac glucose metabolism.     

Postrest contraction in the ventricular papillary muscle of spontaneously diabetic WBN/Kob rat.

In the present study, we investigated the characteristics of the postrest contraction (PRC) in chronic diabetic ventricular muscle. We used WBN/Kob rats of 7-8 weeks as the spontaneously diabetic animal and Wistar rats of 7-8 weeks as the control. We found: (1) No significant differences were seen in the amplitude, the contracting speed, and the relaxing speed of electrically stimulated twitch tension between control and WBN/Kob rats. In addition, the relationship between amplitude of twitch tension and stimulus cycle lengths (0.2-5 sec) was very similar in both animals. (2) The ratios of the first twitch tension (T1) of PRC with various rest intervals (5-600 sec) to the steady-state tension (Tss) were significantly smaller in the diabetic rats than in the controls. (3) When the preparation was stimulated at shorter cycle lengths, the recovery process of PRC was separated into at least two components (fast and slow components). In the diabetic rats, the time constant (tau) of both components was significantly longer than in controls. (4) After caffeine (10(-3) M) treatment, tau of the fast component in the control rats became longer, whereas it remained unchanged in diabetic rats. These findings suggest a dysfunction of the intracellular calcium handling system in spontaneously diabetic heart that is likely to include impaired calcium sequestration and/or extrusion.     

Effect of adrenalectomy on in vivo glucose metabolism in insulin resistant Zucker obese rats

Lean (Fa/?) and obese (fa/fa) Zucker rats were adrenalectomized (ADX) in order to assess the contribution of adrenal hormones to insulin resistance of the obese Zucker rat. Glucose utilization was measured using an insulin suppression test. Sham-operated obese rats gained almost twice as much weight as sham-operated lean littermates. However, body weight gain of ADX animals was comparable in both genotypes. It was significantly less than that of the respective sham-operated controls. Body weight differences can be accounted for almost entirely by a marked loss of adipose tissue. Although insulin resistance may be attributable to obesity in part, steroid hormones are thought to be directly antagonistic to insulin for glucose metabolism. Adrenalectomy resulted in a decrease in serum glucose concentrations for both lean and obese Zucker rats compared with their respective sham-operated groups. Serum insulin concentration of lean ADX rats was 23% of sham-operated controls; in obese ADX rats, it was 9% of controls. Elevated levels of steady state serum glucose (SSSG) levels in sham-operated obese rats demonstrate a marked resistance to insulin induced glucose uptake compared with sham-operated lean animals. Adrenalectomy caused a marked improvement in insulin sensitivity of obese rats. The hyperglycemic SSSG levels of the obese rats were reduced 2.5 times by ADX. These results indicate that insulin resistance of Zucker obese rats can be ameliorated by ADX, suggesting adrenal hormones contribute to insulin resistance in these animals.     

Appearance and metabolic development of diabetes mellitus in the sand rat, Psammomys obesus

The aim of this study was to examine the long-term effects of synthetic chow diet on the metabolic pattern of diabetic syndrome in a large group of sand rats. Few animals had a fulminating reaction with markedly decreased glucose tolerance, low plasma insulin levels and death within 3-4 weeks. But the most of sand rats developed obesity and elevated plasma insulin levels. From the third month, 40% of sand rats presented a diabetic syndrome with hyperinsulinemia, hyperglycemia, markedly decreased glucose tolerance and insulin resistance. Plasma lipids were increased; the lipid and glycogen accumulation in the liver was high. So this diabetic syndrome can be compared to maturity onset diabetes. If this synthetic chow diet lasted more than 6 months, the most of animals lost considerable weight with a strong lipid depletion of fat stores. Serum immunoreactive insulin levels fall and the blood glucose rose over 500 mg/100 ml with glycosuria and ketonuria . The elevated triglyceride content of plasma and the lipid deposits in the liver were exaggerated; glycogen had disappeared. Animals developed an overtly insulin- dependent diabetes, the latter phase of the disease. The sand rat appears to us as a potentially interesting model for investigation both maturity onset and ketotic-type diabetic syndrome.     

Secretory apparatus assessed by analysis of pancreatic secretory stress protein expression in a rat model of chronic pancreatitis

Secretory stress proteins (SSP) are a family of proteins including isoforms of pancreatitis-associated protein (PAP) and pancreatic stone protein (PSP/reg). In vitro exposure to trypsin results in the formation of insoluble fibrillar structures. SSP are constitutively secreted into pancreatic juice at low levels. The WBN/Kob rat is a model for chronic pancreatitis, displaying focal inflammation, destruction of the parenchyma and changes in the architecture of the acinar cell; the synthesis and secretion of SSP are also increased. We have investigated the secretory apparatus by SSP immunohistochemistry at the light- and electron-microscopical (EM) levels. Immunocytochemistry of PSP/reg in Wistar control rats reveals low levels, with individual acinar cells exhibiting high immunoreactivity in zymogen granules. PAP is not detectable. In the WBN/Kob rat, PSP/reg and PAP immunoreactivity is markedly increased. Double immunofluorescence for PSP/reg and PAP I or II demonstrates that these proteins colocalize to the same cell. Acinar cells change their secretory architecture by fusion of zymogen granules and elongation of the fused organelles. The immunogold technique has demonstrated an increase of SSP in zymogen granules in WBN/Kob rats. PSP/reg-positive zymogen granules fuse to form elongated structures with fibrillar contents. An extensive PSP/reg-positive fibrillar network is established in the cytosol. Extracellular fibrils have been observed in several ductules. Thus, SSP-derived fibrils form concomitantly with acinar damage in the WBN/Kob rat. Based on the known tryptic cleavage site of SSP, the in vivo generation of fibrils is presumably the result of premature trypsin activation.     

Diabetes enhances dental caries and apical periodontitis in caries-susceptible WBN/KobSlc rats

Many epidemiologic studies have suggested that diabetes may be an important risk factor for periodontal disease. To determine whether diabetes induces or enhances periodontal disease or dental caries, dental tissue from diabetic male and nondiabetic female WBN/KobSlc rats and male and female age-matched nondiabetic F344 rats was analyzed morphologically and morphometrically for these 2 types of lesions. Soft X-ray examination revealed that the incidence and severity of both molar caries and alveolar bone resorption were much higher in male WBN/KobSlc rats with chronic diabetes than in nondiabetic female rats of the same strain. Histopathologic examination showed that dental caries progressed from acute to subacute inflammation due to bacterial infections and necrosis in the pulp when the caries penetrated the dentin. In the most advanced stage of dental caries, inflammatory changes caused root abscess and subsequent apical periodontitis, with the formation of granulation tissue around the dental root. Inflammatory changes resulted in resorption of alveolar bone and correlated well with the severity of molar caries. Our results suggest that diabetic conditions enhance dental caries in WBN/KobSlc rats and that periodontal lesions may result from the apical periodontitis that is secondary to dental caries.     

Gender-dependent expression of pancreatic proteins in streptozotocin-induced diabetic rats

To elucidate gender-dependent protein regulation and molecular abnormalities in streptozotocin (STZ)-induced diabetes, we compared differentially expressed pancreatic proteins between male and female diabetic rats and their healthy controls using a 2-DE-based proteomic approach. In animal experiments, we found that females exposed to STZ displayed greater susceptibility towards diabetes development due to lower insulin secretion and severe β-cell damage. It was also accompanied with more impaired regulation of sex hormones, lower glucose tolerance, and higher blood glucose levels compared to male diabetic rats. Among 748 detected protein spots ranging in mass from 6 to 240 kDa between pH 3 and 10, a total of 42 proteins showed significant sexually-dimorphic regulation patterns between male and female diabetic rats. Proteomic data revealed that male and female rats displayed prominent gender-dimorphic differential regulation of pancreatic proteins involved in glycolysis, the citric acid cycle, amino acid synthesis, lipid metabolism, insulin biosynthesis, β-cell regeneration, cell signaling, as well as antioxidative and cellular stress defense. In conclusion, the current proteomic study revealed that severely impaired protein regulation in the pancreas, at least in part, is responsible for increased susceptibility of female rats to STZ-induced diabetes.     

Diabetic peculiarity of the ALS-Ay and ALR-Ay strains

ALS and ALR mice were developed as mouse models of alloxan-induced diabetes. These strains do not show spontaneous onset of diabetes. When an obesity gene (Ay) was introduced to these two strains, severe diabetic conditions occurred spontaneously in the produced ALS-Ay and ALR-Ay strains. These strains were examined body weight gain, food consumption, water consumption, urinary sugar content, ketone body level and blood sugar level, and subjected to glucose tolerance test. As a result, in comparison with ALS mice, male ALS-Ay mice showed no obesity and very low tolerance to the glucose tolerance test performed 24 weeks after birth. The level of insulin secretion was 5.0 microU/ml or less, showing hardly any secretory reaction. On the other hand, female ALS-Ay mice were obese and showed no marked decrease in glucose tolerance. The level of insulin secretion was high, and the secretory reaction was strong. In ALR-Ay strain, both male and female mice were obese and showed diabetic conditions similar to those of ALS-Ay mice, though the severity tended to be lower. The characteristic features of diabetic conditions in these mice suggest that these strains, particularly ALS-Ay, may serve as useful new-type models of diabetes.     

Quantitative Trait Locus Analysis for Chronic Pancreatitis and Diabetes Mellitus in the WBN/Kob Rat

A causative gene mutation is still undefined in approximately half of patients with hereditary pancreatitis, and no genetic factor has been identified in most patients with sporadic chronic pancreatitis. To identify a pancreatitis-associated gene, we performed a quantitative trait locus (QTL) analysis for the traits of chronic pancreatitis and diabetes mellitus in WBN/Kob rats. We identified two highly significant QTLs for chronic pancreatitis and/or hyperinsulinemia on chromosomes 7 and X. These QTLs were located on completely different chromosomal regions from those of causative genes that have been reported for human chronic pancreatitis: PRSS1, CFTR, and SPINK1. For these QTLs, prevalences of the WBN/Kob allele significantly increased in the rats with chronic pancreatitis. These findings indicate that chronic pancreatitis in WBN/Kob rats is controlled by multiple genes, and a genetic analysis in WBN/Kob rats might be useful for gene targeting for human chronic pancreatitis.     

Effects of ovariectomy and constant light on responsiveness to estrogen in the rat

The hypothesis that the responsiveness of sexual behavior and LH secretion to exogenous gonadal steroid treatment is dependent on the endogenous steroid environment existing prior to treatment was tested in female rats. The major finding was that estrogen was more effective in stimulating lordosis behavior when treatment was commenced immediately after ovariectomy than when it was delayed for 6 weeks. This indicates that the sensitivity of behavior regulating mechanisms in the female rat declines after removal of the “activating” hormones, as previously reported for testosterone in the male. Similar results were obtained in groups of animals whose pattern of steroid secretion prior to ovariectomy had been changed by 2 months' exposure to constant light. The constant illumination itself showed no significant effect on behavioral responsiveness in spayed estrogen-treated rats. Results are also reported for plasma LH determinations and uterine weights in each of the experiments. Plasma LH levels were found to be lower under conditions of constant as compared to cycling light, both in spayed untreated and spayed estrogen-treated animals.     

Sex-linked changes in immunoreactive glucose-6-phosphate dehydrogenase in rat liver

The level of hepatic immunoreactive glucose-6-phosphate dehydrogenase protein was found to correlate well with the enzyme activity in adult rats fed the stock laboratory diet in a variety of hormonal conditions. The amount of immunoreactive protein and enzyme activity was 2-fold greater in sexually mature female rats compared with aged matched male animals. However, this difference was absent in diabetic animals, and furthermore although triiodothyronine administration to the diabetic male rat could restore the level of enzyme activity to that of the normoglycaemic animal, it was much less effective in the female animal. In contrast, administration of insulin to the normoglycaemic animal increased the level of glucose-6-phosphate dehydrogenase in the female, but was without effect in the male. These results are discussed in relation to the possible role of thyroid status and steroid sex hormones in the regulation of hepatic glucose-6-phosphate dehydrogenase.