International Journal of Obesity (1997) 21, 115±121 ß 1997 Stockton Press All rights reserved 0307±0565/97 $12.00 Role of plasma insulin concentration in regulating glucose and lipid metabolism in lean and obese Zucker rats O Noshiro, R Hirayama, A Shimaya, T Yoneta, K Niigata, and H Shikama Metabolic Diseases Research, Pharmacology Laboratories, Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co., Ltd. Tsukuba, Ibaraki 305, Japan OBJECTIVE: To determine the role of plasma insulin concentration in regulating glucose and lipid metabolism in insulin-resistant obese Zucker rats and to compare obese rats with lean controls with respect to changes in insulin sensitivity. DESIGN: Animal study of lean and obese rats with or without insulin sensitizer, YM268. ANIMALS: Nine week old male lean (Fa/±) and obese (fa/fa) Zucker rats. MEASUREMENTS: Plasma glucose, insulin, triglyceride (TG), non-esteri®ed fatty acid (NEFA), cholesterol at baseline and after 14 d, the dose of YM268 for exhibiting a 30% decrease in each parameter (ED30). RESULTS: Insulin, TG, and NEFA concentrations were approximately 2±6 times higher in obese rats. YM268 had no effect on glucose but decreased insulin in lean and obese rats with ED30 of 3.0 and 2.9 mg/kg. YM268 also reduced TG and NEFA in lean and obese rats [ED30 (mg/kg): lean; 4.1 (TG), 5.0 (NEFA), obese; 2.1, 3.0]. A signi®cant correlation of either TG or NEFA level to insulin was established in lean and obese rats. CONCLUSION: Plasma TG and NEFA, but not cholesterol concentration, are dependent on plasma insulin in lean and obese Zucker rats, and insulin sensitivity with respect to TG and NEFA metabolism in obese rats may not be different from that in lean rats. Keywords: obese Zucker rat; plasma insulin concentration; lipid metabolism; insulin sensitizer thiazolidinedione; YM268 Introduction aggravated by the multiple effects of abnormalities in glucose and lipid metabolism. Several insulin sensitizers are reported to amelio- Obesity is frequently associated with insulin resis- rate insulin resistance in the genetically obese ani- tance, decreased insulin action on stimulating glucose mals, such as obese Zucker rats, Wistar fatty rats, ob/ utilization in the peripheral tissue1,2 and decreased ob mice and KK mice.10±12 Unlike other known ability of insulin to reduce hepatic glucose output.3 hypoglycemic agent such as sulfonylureas, insulin Insulin resistance is often accompanied by elevated sensitizers do not stimulate insulin secretion from concentrations of plasma insulin and abnormalities in the pancreas.10 One of the thiazolidinedione deriva- lipid and lipoprotein metabolism, such as hypertrigly- tives, pioglitazone, decreased hyperglycemia, hyper- ceridemia and low concentrations of high-density insulinemia, and lipids abnormalities in obese Zucker lipoprotein (HDL) cholesterol.4,5 These metabolic rats, KKAy mice and Wistar fatty rats.11,13,14 This abnormalities may be risk factors for non-insulin- agent, however, had no effect on blood glucose con- dependent diabetes mellitus (NIDDM) and coronary centrations in the corresponding lean littermates heart disease.6,7 Although there have been many whereas plasma insulin levels were signi®cantly low- reports showing a linkage between insulin resistance ered.11,14 Although the mechanisms by which thiazo- and glucose intolerance in diabetic animals,8 Kodama lidinedione derivatives improve insulin resistance are et al clearly demonstrated that the linkage between not entirely understood, a wide variety of mechanisms them was not always observed because insulin resis- has so far been proposed. Pioglitazone was reported to tance occurred before the manifestation of hypergly- ameliorate insulin resistance by increasing insulin- cemia in young db/db mice.9 Furthermore, lipoprotein stimulated autophosphorylation of insulin receptor abnormalities often precede the onset of NIDDM by and its kinase activity,15 whereas this agent caused many years and persist despite achievement of eugly- no increases in autophosphorylation and kinase activ- cemia. NIDDM and coronary heart disease are often ity of the receptor in the corresponding lean litter- mates. Under the same conditions, plasma insulin and Correspondence: Dr H Shikama triglyceride (TG) concentrations were signi®cantly Received 22 May 1996; revised 2 September 1996; accepted 7 October 1996 decreased by this agent although plasma glucose Insulin sensitizer and lipid metabolism O Noshiro et al 116 concentration remained unchanged. These ®ndings 3, and 10 mg/kg. The hypoglycemic activity was with insulin sensitizers may not indicate that such evaluated by blood glucose concentration in the fed changes in insulin and lipid metabolism in normogly- state 16 h after the last dosing. Obese Zucker rats at cemic rats are through insulin receptor kinase activity nine weeks of age were orally given YM268, sus- and/or improved glycemic control. pended in 0.5% methylcellulose, at doses of 1, 3, 10, To further elucidate the mechanism of insulin and 30 mg/kg for 14 d. The corresponding lean rats resistance, we have determined the role of plasma also received the same drug regimen, except the insulin in regulating glucose and lipid metabolism in lowest dose (1 mg/kg) was omitted. On day 11 after lean and obese Zucker rats and have compared obese overnight fasting, glucose tolerance and insulin secre- rats with lean controls with respect to insulin sensi- tion were evaluated with the use of a 3-h oral glucose tivity. Lean and obese Zucker rats were treated with or tolerance test. The 40% glucose solution (5 ml/kg) without YM268, a thiazolidinedione derivative, for was given orally, and blood samples for the determi- 14 d. The dose of YM268 required to cause a 30% nation of blood levels of glucose and insulin were decrease in insulin, TG, or non-esteri®ed fatty acid drawn from the tip of the tail vein with heparinized (NEFA) concentrations (ED30) in these animals was haematocrit tubes at 0, 30, 60, 120, and 180 min. The obtained using dose-response curves based on least effects of YM268 on glucose or insulin concentration squares ®tting method. The ED30 values were used to after glucose load were evaluated by the product of compare insulin sensitivity with respect to glucose glucose or insulin concentration by time interval (the and lipid metabolism in lean and obese rats. area under courve of glucose or insulin). On day 15 from the start of drug treatment, all Zucker rats in the fed state were bled for the determination of blood glucose, insulin, TG, NEFA, cholesterol, and HDL- Methods cholesterol concentrations. In order to obtain ED30 value of YM268 for insulin, TG, or NEFA in lean or obese Zucker rats, the logarithm of the dose of Materials YM268 vs plasma concentration of each parameter YM268, bis {4-[(2,4-dioxo-5-thiazolidinyl) methyl]- was linearized by least-squares ®tting (SAS Software, phenyl} methane (Figure 1) was synthesized at Yama- Tokyo, SAS Institute Japan). The regression equation nouchi Pharmaceutical Co., Ltd. (Tsukuba, Japan). All was used to determine the ED30. The experimental other reagents were of analytical grade commercially protocol was approved by the local animal ethics available. committee for animal studies. Animals Analytical procedures Male KK mice were purchased from CLEA Japan Blood glucose was analyzed by glucose oxidase (Tokyo, Japan) and housed in individual cages and fed method (Glucose mono test, Boehringer Mannheim, on moderately high-calorie laboratory chow (CMF, Tokyo, Japan) and insulin was determined with radio- 373 kcal/100 g, Oriental Yeast Industry, Tokyo, immunoassay (Incstar, Stillwater, MN, USA) using rat Japan). KK mice, more than 40 g of body weight, insulin as a standard. The intra- or inter-assay varia- were used in this study. Male obese (fa/fa) Zucker rats tion for immunoassay for insulin was 6.4 or 8.5% and their lean littermates (Fa/±) at seven weeks of age when 12.5 ng/ml of insulin was used. NEFA and TG were obtained from Charles River Laboratories (Wil- concentrations were determined by colorimetric mington, MA, USA) and allowed free access to assays with kits purchased from Wako Pure Chemi- laboratory chow (CE-2, 341 kcal/100 g, CLEA Japan, cals (Osaka, Japan), and cholesterol concentration was Tokyo, Japan). The animals were kept in a 12 : 12 h determined with a kit purchased from Boehringer light-dark cycle. Mannheim (Cholesterol mono test, Tokyo, Japan) and HDL-cholesterol was determined after precipita- tion of low density lipoprotein (LDL) and very low Experimental design density lipoprotein (VLDL) by heparin and Mn.16 Male KK mice showing blood glucose levels of more In this study LDL-cholesterol level was de®ned as than 11 mmol/l were dosed with YM268, or vehicle follows: LDL-cholesterol (cholesterol) ± (HDL-cho- orally once a day for 4 d. Doses of this agent were 1, lesterol). Statistical analysis Data are given as the mean Æ SEM. Comparisons between experimental groups were made using one- way ANOVA followed by any one of Dunnett's multiple range test, Sheffe-S-method and Newman± Keuls multiple range test. Differences were accepted Figure 1 Chemical structure of YM268. as signi®cant at the P < 0.05 level. Insulin sensitizer and lipid metabolism O Noshiro et al 117 Results Effects of YM268 on glucose and insulin concentrations after oral glucose tolerance in lean and obese Zucker rats Hypoglycemic effects of YM268 in spontaneously A signi®cant reduction in both glucose and insulin diabetic KK mice concentrations after a glucose load was observed in Oral administration of multiple doses of YM268 to fasted lean and obese rats treated with YM268 (Table spontaneously diabetic, fed KK mice for 4 d resulted 1). However, the magnitude of reduction in insulin in a dose-dependent fall in blood glucose concentra- tion (Figure 2). The estimated dose of YM268 for exhibiting a 30% decrease in blood glucose concen- tration from the pre-treatment level (ED30) was 3.6 mg/kg.