Obese Otsuka Long-Evans Tokushima Fatty Rats Research Articles

Synthetic protease inhibitor camostat prevents and reverses dyslipidemia, insulin secretory defects, and histological abnormalities of the pancreas in genetically obese and diabetic rats

Background Otsuka Long-Evans Tokushima Fatty (OLETF) rat, a model of type 2 diabetes, lacks the expression of cholecystokinin-1 receptor mRNA and exhibits inflammation and degeneration of the pancreas and eventually develops insulinopenic diabetes. Protease inhibitors are known to modulate inflammatory response and fibrosis as well as inhibit proteases activity. Aim To examine the effects of long-term treatment with camostat, a synthetic protease inhibitor, on metabolic and histopathological changes in the islets of OLETF rats. Method OLETF rats were fed either camostat-containing food (200 mg/100 g) from 12 or 28 weeks of age to 72 weeks of age, or fed standard rat diet. Results Camostat-fed rats gained less weight or lost weight, although they consumed more food than the control rat when food intake was adjusted for body weight. Camostat reduced visceral adipose depots and fasting serum concentrations of triglyceride, free fatty acids, cholesterol, glucose, and insulin. Pancreatic insulin content in camostat-treated rats was significantly higher than in control rats. Immunohistochemistry revealed marked suppression of expressions of tumor necrosis factor α, interleukin 1 β, interleukin 6, and α-smooth muscle actin in the islets of camostat-treated rats, compared with control rats. Histologically, disruption of the islets and pancreatic fibrosis were noted in control rats but not in camostat-fed rats. Conclusion Our findings suggest that camostat prevents and reverses obesity, hyperinsulinemia, hyperglycemia, and hyperlipidemia and markedly inhibits inflammation, fibrosis, and disruption of the islets in the genetically obese diabetic OLETF rats.

α-Lipoic acid prevents diabetes mellitus in diabetes-prone obese rats

Several lines of evidence have suggested that triglyceride accumulation in skeletal muscle and pancreatic islets is causally related to type 2 diabetes mellitus. We recently showed that α-lipoic acid (ALA), a potent antioxidant and cofactor of mitochondrial respiratory enzymes, reduces body weight of rodents by suppressing food intake and increasing energy expenditure. We sought to determine if ALA can prevent the development of diabetes mellitus in obese Otsuka Long–Evans Tokushima Fatty (OLETF) rats. Most (78%) untreated OLETF rats showed glycosuria at 40 weeks of age, but this was completely prevented by ALA. Compared with untreated OLETF rats, ALA reduced body weight and protected pancreatic β-cells from destruction. ALA also reduced triglyceride accumulation in skeletal muscle and pancreatic islets. These results indicate that ALA prevents diabetes mellitus in obese diabetes-prone rats by reducing lipid accumulation in non-adipose tissue as well as in adipose tissue.

Bezafibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, prevents pancreatic degeneration in obese and diabetic rats.

Damage to the exocrine pancreas has been observed in patients and animals with hyperlipidemia and hyperglycemia. Bezafibrate, a peroxisome proliferator-activated receptor (PPAR)-alpha activator, has been shown to improve lipid and glucose metabolism, and to interfere with the inflammatory response. To examine the effects of bezafibrate on exocrine pancreas in hyperlipidemic obese and diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats that have no cholecystokinin-1 receptor gene expression. One group of rats (n = 8) received a bezafibrate-rich diet (150 mg/100 g normal chow) from 12 weeks of age until 30 weeks of age, whereas a control group (n = 8) received standard rat chow. Bezafibrate treatment significantly reduced serum triglyceride, total cholesterol, and free fatty acids levels and significantly increased the pancreatic wet weight (1,145 +/- 54 vs 874 +/- 33 mg/rat, p < 0.01), and protein (169 +/- 7 vs 128 +/- 11 mg/pancreas p < 0.01) and enzyme contents in the pancreas compared with those in untreated control rats. Immunohistochemical studies of the pancreas showed that expression of proinflammatory cytokines such as tumor necrosis factor-alpha, interleukin-1beta and interleukin-6, and alpha-smooth muscle actin in bezafibrate-treated rats was greatly suppressed compared with that in the untreated control rats. The histopathologic changes such as vacuolar degeneration and tubular complexes observed in the control rat pancreas were markedly improved in bezafibrate-treated rats. Our results suggest that bezafibrate reduces hyperlipidemia, inhibits pancreatic inflammation, and prevents pancreatic degeneration in obese and diabetic OLETF rats.

Substitution of Dmo1 with normal alleles results in decreased manifestation of diabetes in OLETF rats.

Dmo1 (Diabetes Mellitus OLETF type I) is a major quantitative trait locus for dyslipidaemia, obesity and diabetes phenotypes in the Otsuka Long Evans Tokushima Fatty (OLETF) rat strain. To evaluate possible metabolic and pathological improvements generated by correction of the Dmo1 genetic pathway, we produced congenic lines, in which both OLETF Dmo1 alleles are replaced by the F344-derived genome. Congenic animals were produced by introgressing F344-derived Dmo1 alleles into the OLETF rat. Congenic animals of the fourth generation (BC4) were intercrossed to obtain F1 animals (BC4:F1). Animals of the next generation, BC4:F2, were used for this study. We used 23 BC4:F2 males harbouring homozygous replacement of the OLETF Dmo1 region with the F344-derived genome. Seven animals with OLETF-derived Dmo1 alleles were used as controls. Dmo1-F344/F344 congenic rats showed significant decreases in body weight, abdominal fat weight, serum triacylglycerols, total cholesterol, food consumption and blood glucose after glucose loading (13%, 39%, 45%, 27%, 18% and 27% respectively; p < 0.05) compared with Dmo1-OLETF/OLETF animals. Furthermore, histopathological analysis of the kidney showed that mesangial sclerosis, hyalin deposits and deposition of PAS-positive substance were significantly lower in Dmo1-F344/F344 animals (p < 0.05). Improvements in metabolic parameters and histopathological scores show that correction of the Dmo1 genetic pathway in the diabetic and mildly obese OLETF rat strain produces wide-ranging therapeutic effects. Thus, this pathway might represent a new drug target also applicable to humans.

Responsivity to NPY and melanocortins in obese OLETF rats lacking CCK-A receptors

Otsuka Long–Evans Tokushima Fatty (OLETF) rat lacking CCK-A receptors are hyperphagic and obese. Previous work has demonstrated alterations in neuropeptide Y (NPY) and proopiomelanocortin (POMC) mRNA expression in ad libitum fed OLETF rats compared to lean Long–Evans Tokushima Otsuka (LETO) controls. In order to determine whether alterations in sensitivity to central peptides involved in overall feeding control may contribute to the hyperphagia and obesity in OLETF rats, we assessed OLETF and LETO rats feeding responses to lateral ventricular infusions of NPY (1 and 3.2 nmol), the melanocortin 3/4 agonist MTII (0.1 and 0.32 nmol) and the melanocortin receptor antagonist SHU-9119 (0.25 and 0.5 nmol). At a 3-h time point, NPY increased food intake in both OLETF and LETO rats. OLETF rats were more sensitive, having significant increases at both NPY doses and a greater increase at the higher dose. The melanocortin agonist MTII decreased intake in both LETO and OLETF rats. At the 20-h time point, the magnitude of suppression was greater in OLETF rats. SHU-9119 increased food intake in both groups. OLETF rats were more sensitive with larger relative increase and longer-lasting effects at the lower dose. These results are consistent with demonstrated alterations in neuropeptide gene expression in OLETF rats and indicate that alterations in responsivity to NPY and melanocortin signaling are unlikely to contribute to their hyperphagia and obesity.

Normalization of circulating leptin levels by fasting improves the reproductive function in obese OLETF female rats

In order to examine a possible detrimental effect of hyperleptinemia on the reproductive system, we examined whether a decrease in circulating leptin levels by fasting affects the estradiol/progesterone-induced luteinizing hormone (LH) and prolactin (PRL) surges in genetically obese OLETF (Otsuka-Long-Evans-Tokushima-Fatty) rats. Experiments were performed on both normally fed and 3-day starved groups from ovariectomized OLETF rats and their controls LETO (Long-Evans-Tokushima-Otsuka). Starved LETO rats, whose leptin levels were less than 0.5ng/ml, did not show a significant surge of either LH or PRL. Normally fed OLETF rats, whose leptin levels were 9.7 ± 1.8ng/ml, showed a significant but small surge for both LH and PRL. Interestingly, starved OLETF rats, whose leptin levels (4.1 ± 0.7ng/ml) were similar to those in normally fed LETO rats (3.3 ± 0.4ng/ml), had significantly greater surges of both hormones than normally fed OLETF group. This study demonstrates for the first time that the normalization of circulating leptin levels in female OLETF rats augments the steroid-induced LH and PRL surges, and also suggests adeleterious effect of hyperleptinemia on the reproductive axis.

Effect of obesity and troglitazone on expression of two glutathione peroxidases: Cellular and extracellular types in serum, kidney and adipose tissue

To determine the effect of obesity on expression of cellular- (C-) and extracellular (EC-) glutathione peroxidase (GPX) in serum, kidney and adipose tissue, we measured GPX in serum, kidneys and adipose tissue of the obese Otsuka-Long-Evans-Tokushima Fatty (OLETF) rat and its lean counterpart (LETO). We also investigated the effect of troglitazone. Five each of OLETF and LETO rats were fed diet with or without 0.2% troglitazone for 10 days. Final body weight, kidney weight, blood glucose and serum tumor necrosis factor-α (TNF-α) level were higher in OLETF rats than in LETO rats. Serum and kidney GPX activities were higher, but adipose tissue GPX activity was lower, in OLETF rats than in LETO rats. Troglitazone treatment decreased adipose tissue GPX activity and abolished overproduction of TNF-α in OLETF rats. Immunoblot analysis, for the first time, revealed that both obesity and troglitazone suppressed the protein signals for C-GPX and EC-GPX in adipose tissue. Serum protein carbonyl groups were increased in OLETF rats and troglitazone completely blocked this increase. Increased serum GPX activity in obese rat was due to the increased secretion of EC-GPX from the kidney. Troglitazone protected against the enhanced oxidative stress induced by obesity independently of the serum GPX concentration.

Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats

The Otsuka Long-Evans Tokushima fatty (OLETF) rat is an animal model of type 2 diabetes, characterized by abdominal obesity, insulin resistance, hypertension, and dyslipidemia. To elucidate the underlying molecular mechanism of obesity and its related complications, we used representational difference analysis and identified the genes more abundantly and specifically expressed in the visceral adipose tissue (VAT) of obese OLETF rats compared with the diabetes-resistant counterpart, that is, Long-Evans Tokushima Otsuka (LETO) rats. By Northern blot analysis, we confirmed the differential expression of 13 genes, including 3 novel genes. The upregulated expression of well-characterized lipid metabolic enzymes, such as lipoprotein lipase, phosphoenolpyruvate carboxykinase, and cholesterol esterase, were observed in VAT of OLETF rats. We demonstrated the differential expression of secreted proteins in VAT of OLETF rats, such as thrombospondin 1 and contrapsin-like protease inhibitor. In contrast to lipid enzymes, the secreted proteins revealed exclusive mRNA expression and they were not detected in VAT of LETO rats. Furthermore, the novel genes OL-16 and OL-64 were also expressed specifically in VAT of OLETF rats and were absent in that of LETO rats and other tissues, including subdermal and brown adipose tissues. The C-terminal partial amino acid sequence of OL-64 revealed that it showed ~40% homology with α1-antitrypsin and it seemed to be a new member of the serine proteinase inhibitor (SERPIN) gene family. VAT of OLETF rats had a unique gene expression profile, and the accumulated VAT-specific known and novel secreted proteins may play a role(s) in the pathogenesis of obesity and its related complications.—K. Hida, J. Wada, H. Zhang, K. Hiragushi, Y. Tsuchiyama, K. Shikata, and H. Makino. Identification of genes specifically expressed in the accumulated visceral adipose tissue of OLETF rats. J. Lipid Res. 2000. 41: 1615–1622.

Effects of central and peripheral injection of leptin on food intake and on brain Fos expression in the Otsuka Long-Evans Tokushima Fatty rat with hyperleptinaemia.

Hyperleptinaemia is observed in obese animals and humans, suggesting that leptin resistance rather than leptin deficiency is a characteristic feature of obesity. This study was designed to determine whether peripherally or centrally administered leptin is effective on the short-term food intake and expression of Fos protein in the hypothalamus in the Otsuka Long-Evans Tokushima Fatty (OLETF) or Long-Evans Tokushima Otsuka (LETO) rat, as a control. The OLETF rat exhibits a polygenic syndrome of hyperphagia, obesity, hyperinsulinaemia, and hyperglycaemia. Male OLETF rats of 5, 8, and 14 weeks of age became heavier than LETO rats. Serum leptin concentrations were not significantly different between LETO and OLETF rats at the age of 5 weeks, but in 8- and 14-week-old OLETF rats were increased to 3.4 and 2.9 times those of LETO rats, respectively. The 8-week-old OLETF and LETO rats were given intraperitoneal (i.p.) injections with recombinant mouse leptin to measure the kinetics. There was a dramatic increase in plasma leptin concentration at 1 h, a decline by 3 h, and the concentrations 6 h after injection were similar to the basal levels. There were no significant difference between OLETF and LETO rats. In LETO rats at 5, 8 and 14 weeks of age, i.p. injection of leptin significantly decreased food intake. Whereas 5-week-old OLETF rats responded to leptin with a decrease in food intake, 8- and 14-week-old OLETF rats became resistant to peripherally administered leptin. In contrast, intracerebroventricular (i.c.v.) injections of leptin were very effective in inhibiting food intake in both OLETF and LETO rats at 14 weeks of age. Intraperitoneal injection of leptin in the LETO rats at each age increased the number of Fos-positive nuclei detected in the ventromedial hypothalamic (VMH), the dorsomedial hypothalamic (DMH) and arcuate nuclei, whereas there was no significant increase in the number of cells expressing c-fos protein in the hypothalamus of the 8- and 14 week-old OLETF rats with hyperleptinaemia. On the other hand, increased expression of c-fos protein in the VMH, DMH and arcuate nuclei following i.c.v. injection of leptin was observed in both OLETF and LETO rats at 5, 8 and 14 weeks of age. These data demonstrated that obese OLETF rats are peripherally leptin resistant, while they retain sensitivity to centrally administered leptin.

Increased hepatic glucose production and decreased hepatic glucose uptake at the prediabetic phase in the Otsuka Long-Evans Tokushima Fatty rar model

To investigate the time course of the hepatic glucose metabolism in non—insulin-dependent diabetes (NIDDM), we measured hepatic glucose production (HGP) and first-pass uptake of portal glucose infusion by the liver (HGU) using dual-tracer methods in a NIDDM model, Otsuka Long-Evans Tokushima Fatty (OLETF) rats, and in normal controls, Long-Evans Tokushima Otsuka (LETO) rats, at 8, 14, and 28 weeks of age (n = 5, respectively). The fasting plasma glucose level in OLETF rats was significantly higher than in LETO rats at 28 weeks of age (8.9 ± 1.7 v 6.3 ± 0.4 mmol/L, P < .01), while there was no significant difference at 8 and 14 weeks. Hyperinsulinemia in OLETF rats appeared at ≥8 weeks of age. Basal HGP was significantly higher in OLETF than in LETO rats at 8 and 28 weeks (8 weeks, 12.7 ± 1.7 v 9.4 ± 1.8 mg · kg −1 · min −1, P < .05; 28 weeks, 10.9 ± 1.6 v 7.1 ± 1.3 mg · kg −1 · min −1, P < .01). At 14 weeks, basal HGP was not sinificantly different between OLETF and LETO rats. However, at all study points, HGU during a portal glucose infusion was significantly lower in OLETF than in LETO rats (8 weeks, 0.9 ± 0.2 v 2.3 ± 0.5, P < .01; 14 weeks, 0.8 ± 0.3 v 1.4 ± 0.3, P < .05; 28 weeks, 0.7 ± 0.2 v 1.4 ± 0.3 mg · kg −1 · min −1, P < .01). Fasting plamsa free fatty acid (FFA) levels were not significantly different between OLETF and LETO, except at 8 weeks. Suppression of plasma FFA levels by endogenous insulin during a portal glucose infusion was impaired in OLETF rats compared with LETO rats. In summary, this study demonstrates that derangement of hepatic glucose handling, such as increased basal HGP and decreased HGU, is observed in obese NIDDM model OLETF rats at the prediabetic phase when hyperglycemia is still not apparent. Furthermore, these derangements may be accompanied by impaired lipid metabolism.

Obesity is necessary but not sufficient for the development of diabetes mellitus

To investigate whether inheritance or obesity plays a more important role in the development of non—insulin-dependent diabetes mellitus (NIDDM), female Otsuka-Long-Evans-Tokushima Fatty (OLETF) rats, which possess the diabetogenic gene, ODB-1, and Long-Evans-Tokushima-Otsuka (LETO) rats, which have no ODB-1, were compared. Neither strain becomes obese and diabetic when bred ordinarily. Female OLETF rats and male and female LETO rats were assigned to two groups of 20 rats each. Obesity was induced in one group by feeding a high-energy “cafeteria” diet (group D), and the other group was given standard chow (group C). Twenty male OLETF rats were used as NIDDM positive controls. At 25 weeks of age, the mean body weight of group D male LETO and female OLETF rats increased at a rate similar to that of male OLETF rats; female LETO rats did not show increased body weight. The incidence of diabetes mellitus in obese female OLETF rats in group D and positive control male OLETF rats was the same (80%). Only 30% of obese male LETO rats in group D developed diabetes mellitus. The insulin response to intravenous glucose in group D female OLETF rats was the highest for all groups but not sufficient to decrease blood glucose levels. In female OLETF rats, glucose infusion rate (GIR) during a euglycemic-hyperinsulinernic clamp test in group D was decreased to 50% of the group C value and tissue glucose uptake as determined by 3H-glucose infusion was significantly decreased in muscle. In male LETO rats, group D GIR was mildly decreased (80% of group C value) compared with the GIR of female OLETF rats. For obese group D female OLETF rats, abdominal fat increased more with obesity than in their male LETO counterparts. GIR was inversely correlated with the weight of abdominal fat when the data of all groups of animals were combined. The expression of GLUT4 mRNA and its protein level in adipose and muscle tissues and tumor necrosis factor alpha (TNF-α) protein in adipose tissue were not significantly different between group D and group C of both strains. In conclusion, the incidence of diabetes in female OLETF rats that possess the diabetogenic gene was significantly greater than in the LETO strains that do not possess the gene, in the presence of excess adiposity.