High-cholesterol diet in combination with hydroxypropyl-beta-cyclodextrin induces NASH-like disorders in the liver of rats.

Non-alcoholic fatty liver disease (NAFLD) is a general term for fatty liver disease not caused by viruses or alcohol. Fibrotic hepatitis, cirrhosis, and hepatocellular carcinoma can develop. The recent increase in NAFLD incidence worldwide has stimulated drug development efforts. However, there is still no approved treatment. This may be due in part to the fact that non-alcoholic steatohepatitis (NASH) pathogenesis is very complex, and its mechanisms are not well understood. Studies with animals are very important for understanding the pathogenesis. Due to the close association between the establishment of human NASH pathology and metabolic syndrome, several animal models have been reported, especially in the context of overnutrition. In this study, we investigated the induction of NASH-like pathology by enhancing cholesterol absorption through treatment with hydroxypropyl-beta-cyclodextrin (CDX). Female Sprague-Dawley rats were fed a normal diet with normal water (control group); a high-fat (60 kcal%), cholesterol (1.25 %), and cholic acid (0.5 %) diet with normal water (HFCC group); or HFCC diet with 2 % CDX water (HFCC+CDX group) for 16 weeks. Compared to the control group, the HFCC and HFCC+CDX groups showed increased blood levels of total cholesterol, aspartate aminotransferase, and alanine aminotransferase. At autopsy, parameters related to hepatic lipid synthesis, oxidative stress, inflammation, and fibrosis were elevated, suggesting the development of NAFLD/NASH. Elevated levels of endoplasmic reticulum stress-related genes were evident in the HFCC+CDX group. In the novel rat model, excessive cholesterol intake and accelerated absorption contributed to NAFLD/NASH pathogenesis.

Pathophysiological analysis of uninephrectomized db/db mice as a model of severe diabetic kidney disease.

Diabetic nephropathy, included in diabetic kidney disease (DKD), is the primary disease leading to end-stage renal disease (ESRD) or dialysis treatment, accounting for more than 40% of all patients with ESRD or receiving dialysis. Developing new therapeutics to prevent the transition to ESRD or dialysis treatment requires an understanding of the pathophysiology of DKD and an appropriate animal model for drug efficacy studies. In this study, we investigated the pathophysiology of diabetic kidney disease with type 2 diabetes in uninephrectomized db/db mice. In addition, the nephrectomized db /db mice from 10 weeks to 42 weeks were used to assess the efficacy of long-term administration of the angiotensin-II-receptor antagonist losartan. The blood and urinary biochemical parameters, main pharmacological endpoint of the losartan therapy, were periodically measured. And at the end, histopathological analysis was performed. Uninephrectomized db/db mice clearly developed obesity and hyperglycemia from young age. Furthermore, they showed renal pathophysiological changes, such as increased urinary albumin-creatinine ratio (UACR) (the peak value 3104 ± 986 in 40-week-old mice), glomerular hypertrophy and increased fibrotic areas in the tubulointerstitial tubules. The blood pressure in the losartan group was significantly low compared to the normotensive Vehicle group. However, as expected, Losartan suppressed the increase in UACR (829±500) indicating the medication was sufficient, but the histopathological abnormalities including tubular interstitial fibrosis did not improve. These results suggest that the uninephrectomized db/db mice are useful as an animal model of the severe DKD indicated by the comparison of the efficacy of losartan in this model with the efficacy of losartan in clinical practice.

Immune disorders and sex differences in spontaneously diabetic Torii rats, type 2 diabetic model.

Type 2 diabetes (T2D) is believed to be a non-autoimmune metabolic disorder. However, there are increasing reports that some T2D patients have immune abnormalities. In addition, it is known that there are sex differences in the onset of diabetes and immune responses in humans. Spontaneously Diabetic Torii (SDT) rats, a non-obese T2D model, also have sex differences in the onset of diabetes, but the involvement of immune abnormalities in diabetes is unknown. In this study, we investigated immune abnormalities in SDT rats. Immune cell subset analysis was performed in male and female SDT rats and control Sprague-Dawley (SD) rats at 5, 11, and 17 weeks of age. Male and female SDT rats had swelling of the spleen and lymph nodes and a higher number of T cells and B cells in the blood, spleen, and lymph nodes than SD rats. Only male SDT rats developed diabetes at 17 weeks of age, and the number of classical and non-classical monocytes in the blood and spleen of male SDT rats was higher than that in male SD rats and female SDT rats that did not develop diabetes. Most of these findings were observed before the onset of diabetes (~11 weeks of age), suggesting that classical and non-classical monocytes may contribute to the development of diabetes in male SDT rats. In conclusion, SDT rats may be a useful T2D model involved in immune abnormalities, and further research will help elucidate the pathophysiology of T2D with immune abnormalities and develop new therapeutic agents.

Glomerular hyperfiltration with hyperglycemia in the spontaneously diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model.

Glomerular hyperfiltration is observed in an early stage of kidney diseases including diabetic nephropathy. A better understanding of pathophysiological changes in glomerular hyperfiltration is essential for development of new therapies to prevent kidney disease progression. In this study, we investigated glomerular changes including glomerular filtration rate (GFR) and glomerular size in the Spontaneously Diabetic Torii (SDT) fatty rat, an obese type 2 diabetic model, and we also evaluated pharmacological effects of the sodium glucose cotransporter 2 inhibitor dapagliflozin on the renal lesions. Dapagliflozin was administered to SDT fatty rats from 5 to 17 weeks of age. Blood and urinary biochemical parameters were periodically measured. GFR was determined by transdermal GFR monitor at 16 weeks of age and histopathological analysis was performed at 17 weeks of age. SDT fatty rat developed severe hyperglycemia and exhibited pathophysiological abnormalities in the kidney, such as an increased GFR, glomerular hypertrophy and tissue lesions. Dapagliflozin achieved good glycemic control during the experimental period, inhibited the increase in GFR, and improved histopathological abnormalities in tubules. These results suggest that the SDT fatty rat is a useful model for analyzing the pathogenesis of diabetic nephropathy during its early stage and dapagliflozin improves not only hyperglycemia but also glomerular hyperfiltration and tubule lesions in SDT fatty rat.

Diabetic macular edema-like ocular lesions in male spontaneously diabetic torii fatty rats.

Diabetic macular edema (DME) is a major factor contributing to visual disabilities in diabetic patients, and the number of patients is increasing. Animal models play a key role in the development of novel therapies. In this study, pathophysiological analyses of ocular lesions in Spontaneously Diabetic Torii (SDT) fatty rats were performed. First, vascular endothelial growth factor (VEGF) concentrations in vitreous humor, retinal vascular permeability and retinal thickness were measured in SDT fatty rats (Experiment 1). Furthermore, the pharmacological effects of two anti-diabetic drugs, phlorizin and pioglitazone, on retinal lesions were evaluated (Experiment 2). As results, the SDT fatty rats exhibited VEGF increase in vitreous humor at 8 and 16 weeks of age, and both retinal vascular hyperpermeability and retinal thickening at 16 weeks of age. In particular, the layers between the retinal internal limiting membrane and the outer nuclear layer were thickened. Phlorizin treatment from 4 to 16 weeks of age improved hyperglycemia and normalized retinal thickness; however, the effect of pioglitazone on retinal thickness was not strong despite the normalization of hyperglycemia. These data demonstrate that the male SDT fatty rat is a useful model for developing new therapeutic approaches in DME.

Protein kinase C beta inhibitor prevents diabetic peripheral neuropathy, but not histopathological abnormalities of retina in Spontaneously Diabetic Torii rat.

Spontaneously Diabetic Torii (SDT) rat shows severe ocular complications such as tractional retinal detachment. In the present study, effect of protein kinase C beta (PKCbeta) inhibitor JTT-010 was evaluated to clarify the involvement of PKCbeta in complications of SDT rat. SDT rats were administered JTT-010 (10 or 50 mg/kg/day) for 48 weeks. SDT rats showed delayed oscillatory potentials in electroretinogram. Delayed motor nerve conduction velocity, decreased coefficients of variation of R-R intervals in electrocardiogram and thermal hypoalgesia were also observed. These functional disorders were prevented by administration of JTT-010. Abnormal retinal vascular was formed and the optic disc was protruded in SDT rat; however, JTT-010 did not prevent these hyperglycaemia-induced retinal abnormalities. These findings indicate that PKCbeta is intimately involved in diabetic complications; however, it seems that other factor(s) are primary contributors to histopathological abnormalities in retina. Therefore, PKCbeta inhibitors require concurrent administration of antihyperglycaemic drugs to achieve maximum effect on diabetic complications.

Preventive effects of glycaemic control on ocular complications of Spontaneously Diabetic Torii rat.

AIM: Spontaneously Diabetic Torii (SDT) rat is a new model of non-obese type 2 diabetes. SDT rats show severe ocular complications such as cataracts, tractional retinal detachment with fibrous proliferation and massive haemorrhaging in the anterior chamber. In the present study, blood glucose levels of SDT rats were controlled in order to examine whether these ocular complications are caused by hyperglycaemia. METHODS: SDT rats were treated with an insulin implant to control blood glucose. To evaluate retinal function, we used electroretinograms (ERG) and measured vascular endothelial growth factor (VEGF) concentrations within the aqueous humour. Finally, we studied retinal flat-mounts and trypsin digestion to evaluate vascular abnormalities in SDT rats. RESULTS: Forty-four-week-old SDT rats displayed an increase in VEGF concentrations within the aqueous humour and significant prolongation of the peak latencies in ERG (Sigma(OP(1)-OP(4)); Sprague-Dawley (SD): 146.2 +/- 1.06 ms; SDT: 166.3 +/- 2.38 ms; SDT + insulin: 149.2 +/- 1.83 ms). Retinal flat-mounts of SDT rats showed venous dilation and meandering vascular networks. Furthermore, acellular capillaries were observed in the retinal trypsin digestion. Insulin treatment prevented these ocular abnormalities in SDT rats. CONCLUSIONS: These findings indicate that ocular complications of SDT rats are caused by hyperglycaemia. The features of SDT rats indicate their usefulness for the future study of diabetic retinopathy.

Daily inhibition of postprandial hyperglycaemia with JTT-608, a novel oral antidiabetic agent, ameliorates pancreatic function in neonatally streptozotocin-treated rats.

AIM: Chronic glycaemic control, in particular, the control of postprandial hyperglycaemia, is essential for preventing the development of diabetic complications. We therefore evaluated the chronic treatment effect of a new antidiabetic agent, JTT-608 [trans-4-(methylcyclohexyl)-4-oxobutyric acid], in neonatally streptozotocin-treated rats. METHODS: The rats were maintained with liquid meal three times a day and treated orally with JTT-608 10 min before each meal for 12 weeks. Haemoglobin A1C (HbA1C) and fasting blood glucose levels were measured at 4-week intervals, and effects of JTT-608 on pancreatic function and diabetic complications were examined after dosing period. RESULTS: The postprandial hyperglycaemia was suppressed by JTT-608 administration, and both HbA1C levels and fasting blood glucose levels were reduced during the experimental period. After the treatment period of 12 weeks, JTT-608 further improved the early insulin secretion and the impaired glucose tolerance after meal loading in the diabetic rats. Also, pathological examination revealed that JTT-608 reduced the incidence of the decrease in immunoreactivity of insulin. In examination of other diabetic complications, JTT-608 ameliorated the reduced motor nerve conduction velocities observed in diabetic rats and inhibited the incidence of cataracts with diabetes. CONCLUSIONS: We conclude that a newly developed antidiabetic agent, JTT-608, improves the pancreatic function and prevents the development of diabetic complications by inhibition of daily postprandial hyperglycaemia and could be useful for the treatment of diabetic subjects with impaired insulin secretion.

Novel protein kinase C-beta isoform selective inhibitor JTT-010 ameliorates both hyper- and hypoalgesia in streptozotocin- induced diabetic rats.

AIM: Activation of protein kinase C (PKC) is thought to play an important role in the pathogenesis of diabetic microvascular complications. PKC-beta is elevated in hyperglycaemic conditions, both in vivo and in vitro. In this study, pharmacological effects of a novel PKC-beta isoform selective inhibitor, JTT-010 ((2R)-3-(2-aminomethyl-2,3-dihydro-1H-3a-azacyclopenta(a)inden-8-yl)-4-phenylaminopyrrole-2,5-dione monomethanesulphonate), on diabetic neuropathy were examined. METHODS: PKC inhibitory activity of JTT-010 was evaluated with an enzyme assay. For the in vivo study, streptozotocin (STZ)-induced diabetic rats were treated with JTT-010 for 12 weeks and tail/sciatic nerve conduction velocity (NCV) evaluated. Hyper/hypoalgesia was evaluated using tail-flick and formalin tests. RESULTS: JTT-010 inhibited PKC-betaI and -betaII with IC50 values of 4.0 and 2.3 nm respectively. For other PKC isoforms, IC50 values were 54 nm or greater. In STZ-induced diabetic rats showing a reduction in tail/sciatic nerve conduction velocities, JTT-010 (0.3-3 mg/kg) ameliorated the reduction of these velocities. In a formalin test, STZ-induced diabetic rats had hyperalgesia in the first phase. JTT-010 reduced nociceptive response at doses of 0.1 mg/kg or higher. Furthermore, STZ-induced diabetic rats showed hypoalgesia in the second phase of the formalin test and tail-flick test. JTT-010 also ameliorates these symptoms at doses of 0.1 mg/kg or higher. CONCLUSIONS: These observations suggest that PKC-beta contributes not only to diabetic hyperalgesia, but also to hypoalgesia and also contributes to defects in NCV. PKC-beta inhibitor, JTT-010, may be beneficial in suppressing the development of diabetic nerve dysfunction, including hyperalgesia and hypoalgesia.

Specific inhibition of stretch-induced increase in L-type calcium channel currents by herbimycin A in canine basilar arterial myocytes.

The effects of protein-tyrosine kinase (PTK) and protein-tyrosine phosphatase (PTP) inhibitors on voltage-activated barium currents (I(Ba)) through L-type calcium channels increased by hypotonic solution were investigated in canine basilar arterial myocytes by the whole-cell patch-clamp technique. I(Ba) was elicited by depolarizing step from a holding potential of -80 to +10 mV and identified by using an L-type calcium channel agonist, Bay K 8644 (100 nM), and an L-type calcium channel blocker, nicardipine (1 microM). Hypotonic superfusate induced cell swelling and acted as a stretch stimulus, which reversibly increased peak I(Ba) amplitude at +10 mV. I(Ba) was also decreased by nicardipine (1 microM) under the hypotonic condition. PTK inhibitors such as herbimycin A (30 nM), genistein (10 microM), and lavendustin A (10 microM) decreased I(Ba) enhanced by hypotonic solution. Genistein also decreased I(Ba) in a concentration-dependent manner under the isotonic condition. The inactive genistein analogue daidzein (10 microM) had no effect on I(Ba) under either the isotonic or hypotonic condition. By contrast, herbimycin A did not decrease I(Ba) under the isotonic condition. Sodium orthovanadate (10 microM), a PTP inhibitor, increased I(Ba) under both conditions. The present results suggest that cell swelling by hypotonic solution increases the L-type calcium channel currents in canine basilar artery and that herbimycin-sensitive PTK activity is primarily involved in the enhancement of calcium channel currents.