Glucose and Lipid Metabolism in Spontaneously Diabetic Torii Rat

The impairment of glucose metabolism may lead to type 2 diabetes and the malfunction of lipid metabolism contributing to metabolic syndrome, which increases risk of heart disease, vascular disease, and type 2 diabetes. Our previous study provided evidence for involvement of hepatic glucose metabolic disorder in onset and progress of diabetes in Spontaneously Diabetic Torii (SDT) rats. As it is increasingly important to elucidate the mechanism of onset of diabetes and to develop drugs to prevent diabetic pathological progress, SDT rat should offer a highly useful model of spontaneous diabetic onset for such studies. In addition, abnormality in triglyceride (TG) absorption and impaired lipid catabolism antecedent to hypoinsulinemia/hyperglycemia seem to cause postprandial hypertriglyceridemia in SDT rat; hence, the characteristics of lipid metabolism in SDT rat can be useful in studies of diabetic hypertriglyceridemia and TG metabolism.


INTRODUCTION
In recent years, great attention has been focused on high risks of development of cardiovascular diseases in non-diabetic patients with glucose metabolic disorder and of diabetes in patients with glucose metabolic disorder.Similarly, hypertriglyceridemia is one of the major abnormalities found in diabetes with insulin deficiency [1], and is thought closely related to coronary heart disease and atherosclerosis, important complications of diabetes [2,3].Failures in any of the metabolic processes can lead to serious health problems: the impairment of glucose metabolism may lead type 2 diabetes, and the malfunction of lipid metabolism contributes to metabolic syndrome, which is a cluster of conditions that puts people at increased risk of heart disease, vascular disease, and type 2 diabetes.These epidemiological situations make it important to study diabetic pathogenesis to prevent progress to diabetes.
Among studies on the developmental stage of diabetes, particularly little has been published on hepatic glucose metabolism and triglyceride (TG) metabolism.In our previous studies, we focused on the liver, the major organ involved in glucose metabolism, to investigate changes in hepatic glucose metabolism from pre-diabetic to diabetic stages, with the aim of identifying the molecules involved in onset of diabetes [4].Furthermore, we examined the contribution of TG absorption and clearance to diabetic hypertriglyceridemia in Spontaneously Diabetic Torii (SDT) rats [5].SDT rat is a model of non-obese type 2 diabetes with impaired secretion of insulin [6,7].In male SDT rats, plasma glucose levels spontaneously increased around 16 weeks of age (Fig. 1A).The plasma insulin levels of SDT rats at 20 weeks or older age were significantly lower than *Address correspondence to this author at the Japan Tobacco Inc., Central Pharmaceutical Research Institute, Biological/Pharmacological Research Laboratories, 1-1 Murasaki-cho, Takatsuki, Osaka 569-1125, Japan; Tel: +81 72 681 9700; Fax: +81 72 681 9722; E-mail: tomohiko.sasase@jt.comthose of age-matched Sprague-Dawley (SD) rats (Fig. 1B).Significant decreases in body weight were observed in diabetic SDT rats.The plasma TG and total cholesterol levels remained unchanged at 16 weeks of age, but plasma TG level increased after 30 weeks of age (Fig. 1C).Interestingly, increased plasma TG level elevation after oil loading was observed in SDT rats before hyperglycemia/ hypertriglyceri-demia [5], as was impaired glucose tolerance in pre-diabetic state [8].Most of all, the characteristic that onset of diabetes is delayed until around 20 weeks of age makes this animal a highly effective model in which to pursue the mechanism of development of diabetes.

GLUCOSE METABOLISM
Glucokinase (GK) plays a critical role in metabolism of glucose in liver because it appears at the first step of glycolysis [9].In SDT rat, decreased mRNA expression and activity of GK were observed from pre-diabetic 16 weeks of age [4], suggesting that a decrease in GK is partly responsible for a mild increase in blood glucose in SDT rat.Suppressed GK is found in NIDDM patients [10,11] and some diabetic animal models (e.g.Zucker Diabetic Fatty (ZDF) rat [12], streptozotocin (STZ)-induced rat [13]); however, elevation of GK is reported in other diabetic animals [14][15][16].Although how the difference is caused has not been clarified, it may be supposed that suppressed insulin is always accompanied by suppressed GK level.
Glycogen synthase (GS) and glycogen phosphorylase (GP) are rate-limited key enzymes of glycogen metabolism [17,18].Glycogen content was decreased in pre-diabetic SDT rats, and the decrease was more pronounced at older age [4].A decreased GS activity and a moderate decrease in glycogen store preceded development of diabetes in SDT rat.In diabetic state, GP activity and mRNA expression in liver were also decreased in SDT rats.GP activity is considered to be decreased as a consequence of elevation of serum glucose and reduction of glycogen stock.GP is known to exert high activity in response to attenuated insulin signal due to insulin deficiency, and low expression or activity in association with an increase in blood glucose, a decrease in glycogen store, an enhancement of gluconeogenesis [17], and an increase in glucagon/insulin ratio [19].The glycogen metabolism pathway in diabetic SDT rats is broken down, thus glycogen can not play a role in glucose supply [4].At least an increase in blood glucose and a decrease in glycogen store are considered to be applicable to the SDT model.Insulin treatment ameliorates hyperglycemia and prevents diabetic complications in SDT rats [20][21][22][23].However, exogenous insulin itself has affected glucose metabolism in liver, and thus a drug that directly decreases blood glucose maybe useful to study effects of hypergycemia in metabolism.Phlorizin, a specific inhibitor of sodium-coupled glucose transporter (SGLTs), is reported to cause excretion of glucose into urine and lower blood glucose levels in several diabetic animal models [24,25].SDT rats treated with phlorizin for 4 weeks from 20 weeks of age showed  decreased blood glucose and TG, and insulin level was maintained (Fig. 2A-C).As result of phlorizin treatment, down-regulated expression of hepatic GK, GS, and GP mRNAs were recovered (Fig. 2D-F).These results support the idea that the hyperglycemia directly controls expression of hepatic enzymes responsible for glycogen metabolism.

LIPID METABOLISM
Decreased signals of insulin and leptin cause diabetic hyperphagia [29], and explains for hypertriglyceridemia in diabetes.In diabetic state, hyperphagia leads to intestinal hypertrophy [30], which contributes to increased cholesterol synthesis and lipid absorption.Therefore, the impaired chylomicron catabolism is closely associated with diabetic hypertriglyceridemia.In addition, overproduction of the TGrich lipoprotein may contribute to diabetic hypertriglyceridemia. Earlier studies indicated that increased TG absorption from the small intestine contributes to hypertriglyceridemia in diabetes [31][32][33].
SDT rat shows marked hypertriglyceridemia concomitant with hyperglycemia/hypoinsulinemia [6,7] (Fig. 1C).The major increase of TG in the circulation of SDT rats occurs in the TG-rich lipoprotein fraction chylomicron [5].Lower plasma leptin level followed by hyperphagia [5] seems to cause intestinal hypertrophy in SDT rats [34] (Fig. 3).The small intestine plays a predominant role in the absorption of dietary lipids.Abnormal expression and/or activity of enzymes involved in intestinal TG synthesis and chylomicron assembling such as acyl-CoA:monoacylglycerol acyltransferase 2 (MGAT2), acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1), and microsomal triglyceride transfer protein (MTP) have been observed in some in vitro and in vivo models of diabetes [35][36][37].SDT rats have increased intestinal mRNA of these enzymes and transfer protein [5] in addition to intestinal hypertrophy.Therefore, it is conceivable that increased intestinal mRNAs of these enzymes and transfer protein related to postprandial TG absorption are caused by extreme hypoinsulinemia/hyperglycemia.
Plasma TG and chylomicron levels are increased in diabetic animal models [38][39][40][41].After olive oil loading, SDT rats showed higher plasma TG level compared to that of normal rats [5].SDT rats also showed increased TG in lymph chylomicron and plasma TG accumulation after oil loading in diabetic state, indicating that postprandial TG absorption from intestine is increased in SDT rats in diabetic state (Fig. 4A, B).Interestingly, sustained plasma TG level elevation after oil loading was also observed in SDT rats before hyperglycemia/hypertriglyceridemia.In contrast, TG in lymph chylomicron and plasma TG accumulation after oil loading was not different from those of SD rats at this age (Fig. 4A, B).Significant delay in TG clearance was observed from 8 weeks of age (Fig. 5A, B).These results clearly indicate impaired TG catabolism of SDT rats from prediabetic stage and excess TG absorption at diabetic stage [5].In a model of impaired insulin secretion (such as STZinduced diabetic rats), the high TG levels are probably due to low TG clearance because of reduced adipose tissue lipoprotein lipase (LPL) activity [42].Lower LPL expression affects impaired TG catabolism in SDT rats (Fig. 5C).Diabetic SDT rats showed reduced hepatic TG secretion (Fig. 4C) as same as STZ rats.Plasma insulin level in STZ rats was negatively correlated with lipids and apoB in plasma and TG-rich lipoprotein [43].Also, Mason et al. reported that decreased TGSR in STZ rats was improved by insulin treatment [44].Therefore, the hypoinsulinemic environment may cause decreased TGSR in SDT rats.This characteristic on TGSR is different from hypertriglyceridemic model with insulin resistance and hyperglycemia that shows increased VLDL-TG.Hepatocytes from obese Zucker rats displayed higher total VLDL-TG secretion [45] and acute glucose infusion elevated plasma TG concentration [46].

CONCLUSION
Our previous study provided evidence for involvement of hepatic glucose metabolic disorder in onset and progress of diabetes in SDT rat [4].As it is increasingly important to elucidate the mechanism of onset of diabetes and to develop drugs to prevent diabetic pathological progress, SDT rat should offer a highly useful model of spontaneous diabetic onset for such studies.In addition, abnormality in TG absorption and impaired lipid catabolism antecedent to hypoinsulinemia/hyperglycemia seem to cause postprandial hypertriglyceridemia in SDT rat [5]; hence, the characteristics of lipid metabolism in SDT rat can be useful in studies of diabetic hypertriglyceridemia and TG metabolism.SDT rats show impaired glucose tolerance and insulin response to oral glucose loading in pre-diabetic state [8].The relation- ship between impaired lipid catabolism and impaired glucose tolerance in the pre-diabetic state also seems to offer some keys towards clarification of the onset mechanism of diabetes.