Role of Plasma Insulin Concentration in Regulating Glucose and Lipid Metabolism in Lean and Obese Zucker Rats

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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 littermates. 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 precipitation 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 concentration (Figure 2). The estimated dose of YM268 for exhibiting a 30% decrease in blood glucose concentration from the pre-treatment level (ED30) was 3.6 mg/kg.

Effects of YM268 on concentrations of blood glucose and plasma insulin in lean and obese Zucker rats Concentrations of blood glucose and plasma insulin in the fed state were measured in nine-week-old lean and obese Zucker rats treated with or without the different doses of YM268 for 4 or 14 d (Figure 3 and 4). YM268 failed to lower blood glucose concentrations signi®cantly in any groups, although blood glucose concentration was not signi®cantly elevated in obese rats. On the other hand, YM268 showed a signi®cant dose-dependent decrease in plasma insulin concentration in both lean and obese Zucker rats. During the course of a 14 d treatment, YM268 had no effect on Figure 3 Blood glucose concentration in lean and obese Zucker body weight in lean and obese Zucker rats (data not rats treated with YM268 for 4 and 14 d. YM268 at doses of 1, 3, 10, and 30 mg/kg were orally administered to lean Zucker rats for shown). 4 (j) and 14 d (u) or to obese Zucker rats for 4 (d) and 14 d (s). Data represent the means Æ SEM of 6 rats. Difference between groups is not signi®cantly different at P > 0.05 (One-way ANOVA).

Figure 2 Hypoglycemic effect of YM268 in spontaneously diabetic KK mice. Hypoglycemic activity of YM268 was evaluated in KK mice based on a decrease in blood glucose level 16 h after the last dosing when administered orally once a day for 4 d. Other Figure 4 Plasma insulin concentration in lean and obese Zucker experimental conditions are shown in the Methods. The pre- rats treated with YM268 for 4 and 14 d. The experimental condi- treatment glucose concentration in this study was tions are shown in Figure 3. Lean Zucker rats: day 4 (j), day 14 17.4 Æ 0.4 mmol/l. Each point with a vertical line represents the (u), obese rats; day 4 (d), day 14 (s). Data represent the mean- mean Æ SEM of 6 mice. **P < 0.01, Signi®cantly different from s Æ SEM of 6 rats. *P < 0.05, **P < 0.01, Signi®cantly different the pre-treatment glucose value (One-way ANOVA, Dunnett's from the corresponding untreated control (One-way ANOVA, multiple range test). Sheffe-S method or Newman±Keuls multiple range test). Insulin sensitizer and lipid metabolism O Noshiro et al 118 Table 1 Effects of YM268 on glucose and insulin concentrations after oral glucose tolerance in fasted lean and obese Zucker rats

Blood glucose Plasma insulin

Lean Obese Lean Obese

mmol=l, 3 h ng=ml, 3 h YM268 0 mg/kg 19.7 Æ 0.2 31.5 Æ 0.8 14.4 Æ 1.3 163.5 Æ 24.2 1 n.d. 29.8 Æ 1.2 n.d. 109.1 Æ 10.5 3 18.4 Æ 0.2 31.3 Æ 2.3 11.7 Æ 1.3 106.4 Æ 17.5 10 18.4 Æ 0.3 20.1 Æ 1.1** 10.2 Æ 0.4* 47.5 Æ 4.3** 30 17.2 Æ 0.5** 18.9 Æ 0.4** 9.9 Æ 0.7* 19.9 Æ 2.6**

Glucose tolerance test was performed on day 11 after overnight fasting. The areas under curve of glucose (mmol=l, 3 h) and insulin (ng=ml, 3 h) after oral glucose load are shown. Results are the means Æ SEM of 5 or 6 observations. *P < 0.05, **P < 0.01, the effect of YM268 is signi®cantly different from the corresponding vehicle control (One-way ANOVA, Dunnett's multiple range test). n.d.: not determined.

concentration was much greater than that in glucose between plasma insulin concentration and either TG concentration in lean and obese Zucker rats treated or NEFA concentration in lean and obese animals with YM268. (Figure 5).

Plasma cholesterol concentrations in YM268-treated Signi®cant correlations of plasma insulin concentration lean and obese Zucker rats with TG and NEFA concentrations in lean and obese Neither total cholesterol nor HDL-cholesterol concen- Zucker rats tration was signi®cantly changed by the administra- In order to clarify the relationship between plasma tion of YM268 to lean or obese Zucker rats for 14 d insulin concentration and lipid metabolism, plasma (Figure 6). In addition, the effects of YM268 on LDL- TG and NEFA concentrations were determined in lean cholesterol concentrations were inconsistent in lean and obese Zucker rats treated with or without YM268. and obese rats. As shown in Figure 7, however, a As shown in Table 2, plasma insulin, TG, and NEFA signi®cant correlation between HDL-cholesterol and levels were 2.3±5.6 times higher in obese rats. YM268 LDL-cholesterol concentrations was observed in administered for 14 d showed a signi®cant dose- obese Zucker rats, but not in lean Zucker rats, indicat- dependent decrease in plasma insulin levels with ing that LDL- and HDL-cholesterol metabolism was ED30 values of 3.0 mg/kg for lean rats and 2.9 mg/ not solely dependent on plasma insulin concentration. kg for obese rats, respectively. Similarly, YM268 lowered TG and NEFA concentrations in lean and obese Zucker rats in a dose-dependent manner. The ED30 of YM268 for lowering TG or NEFA concen- Discussion tration was not altered between lean and obese groups and was closely related to the respective ED30 for lowering plasma insulin concentration. These ®ndings Obesity is frequently associated with compensatory may indicate that the plasma TG and NEFA concen- hyperinsulinemia due to the decreased glucose uptake trations are solely regulated by plasma insulin con- in the peripheral tissue17,18 and decreased ability of centration in lean and obese Zucker rats. This was insulin to reduce hepatic glucose output.3 Hyperinsu- con®rmed by the existence of a signi®cant correlation linemia is often accompanied by abnormalities in

Table 2 Effects of YM268 on triglyceride and non-esteri®ed fatty acid levels in lean and obese Zucker rats

Insulin Triglyceride Non-esterified fatty acid

YM268 Lean Obese Lean Obese Lean Obese

mg=kg ng=ml mmol=dl uEq=I 0 12.3 Æ 2.6 69.9 Æ 7.0 149.7 Æ 18.4 698.8 Æ 108.5 0.354 Æ 0.052 0.830 Æ 0.079 1 n.d. 57.0 Æ 6.2 n.d. 602.1 Æ 68.5 n.d. 0.807 Æ 0.092 3 7.4 Æ 0.9 57.2 Æ 4.3 120.2 Æ 14.8 372.6 Æ 49.9* 0.268 Æ 0.019 0.510 Æ 0.040** 10 5.1 Æ 0.4 23.7 Æ 2.1** 67.9 Æ 2.9* 122.4 Æ 7.3** 0.171 Æ 0.018** 0.279 Æ 0.030** 30 3.6 Æ 0.7** 10.0 Æ 3.1** 41.9 Æ 4.8** 67.8 Æ 6.7** 0.180 Æ 0.020** 0.231 Æ 0.014**

ED30 (mg=kg) 3.0 2.9 4.1 2.1 5.0 3.0 YM268 or vehicle was orally administered to lean and obese Zucker rats for 14 d. Each number shows mean Æ SEM. *P< 0.05, **P < 0.01, compared to untreated control. ED30 equals the dose of YM268 required to induce a 30% reduction in insulin, triglyceride, or non-esteri®ed fatty acid from the corresponding control without YM268. n.d.: not determined. Insulin sensitizer and lipid metabolism O Noshiro et al 119

Figure 6 Plasma cholesterol concentration in YM268-treated lean (A) and obese (B) Zucker rats. Dose relationships were obtained on day 14. Blood samples for cholesterol were col- lected 16 h after the last dosing. Plasma concentrations of total cholesterol (j), HDL-cholesterol (O), and LDL-cholesterol (m) are shown. There is no signi®cant difference from the corresponding untreated control (One-way ANOVA, Sheffe-S method or Newman±Keuls multiple range test).

Figure 5 Correlation of plasma insulin concentration to TG (A) or NEFA level (B) in lean and obese Zucker rats. Points desig- nated by u (lean rats) and d (obese rats) represent the mean - Æ SEM obtained in Table 2. Linear lines were drawn by least- squares ®tting method, A: f(x) 8.95x 4.60, r 0.951, P < 0.01, B: f(x) 9.09x 0.15, r 0.935, P < 0.01. Figure 7 Relationship between plasma concentrations of HDL- cholesterol and LDL-cholesterol in lean and obee Zucker rats. Points represent the means Æ SEM of HDL- and LDL-cholesterol glucose, lipid and lipoprotein metabolism in obese concentrations in lean (u) and obese (d) rats shown in Figure 6. 19,20 Using data obtained from obese rats, linear line was drawn by animal models and human subjects. This metabo- least-squares ®tting method, f(x) 71.49x 104, r 0.897, lically abnormal state may be one of the most impor- P < 0.05. tant risk factors for coronary heart disease.6,7,21 In this study we have examined the role of plasma insulin in regulating glucose and lipid metabolism in obese zone, and BRL 49653 were also reported to be Zucker rats with a marked hyperinsulinemia and effective in this or other insulin-resistant mouse their lean littermates showing normal insulin level. models.10±12,22 YM268 failed to decrease blood glu- The sensitivity of glucose and lipid metabolism in cose concentration signi®cantly in lean and obese response to plasma insulin concentration in lean and Zucker rats, although blood glucose concentration obese Zucker rats was evaluated by the doses of was not signi®cantly elevated in these obese rats YM268, a thiazolidinedione derivative, for exhibiting (Figure 3). This agent, however, dose-dependently a 30% reduction in each parameter. reduced plasma insulin concentration not only in The administration of YM268 to diabetic KK mice obese Zucker rats, but also in lean controls (Figure for 4 d resulted in a marked fall in blood glucose 4). The similar effect of YM268 on plasma insulin concentration in a dose dependent manner (Figure 2). levels was observed in Table 1, where YM268 The ED30 value of YM268 for lowering blood glucose improved glucose tolerance and normalized insulin was 3.6 mg/kg. Troglitazone, pioglitazone, englita- secretion after a glucose load in lean and obese Zucker Insulin sensitizer and lipid metabolism O Noshiro et al 120 rats. These ®ndings are compatible with several reduction in insulin, TG, and NEFA by YM268 is not reports from other laboratories,11,14,19 but are incom- secondary to the reduction in blood glucose. Second, patible with the ®ndings made by de Souza et al23 who the main cause for the abnormalities concerning TG did not observe a decline in insulin concentration in and NEFA metabolism in obese Zucker rats are not pioglitazone-treated normal rats. It is not known the the attenuated insulin sensitivity to lipid metabolism, reason why this difference occured but there is a but the increased pool size of these metabolites. possibility that the ability of insulin sensitizers to Therefore, it is highly possible that these lipid decrease insulin concentration in lean rats is depen- abnormalities are reversed by appropriate dietary or dent on the initial insulin levels which may be pharmacological therapy which reduces the intake or proportional to the degree of insulin resistance present the production of TG and NEFA. in lean animals. Although the mechanism by which YM268 It is unlikely that the effect of YM268 on plasma improves lipid metabolism remains to be fully eluci- insulin levels in lean and obese rats may be secondary dated in this study, plasma NEFA concentration is to the reduction in food intake because YM268 had no known to be one of the factors causing insulin effect on body weight in these animals during the resistance.25 A glucose-fatty acid cycle in muscle course of the experiments (data not shown). Bowen et has been known for many years.26 The oxidation of al19 studied lean and obese Zucker rats and found that NEFA decreases glucose uptake and utilization the treatment with englitazone signi®cantly reduced through substrate competition. Clo®brate or acipimox, insulin concentration in fasted lean and obese rats. which decreases plasma NEFA concentration, Pioglitazone also reduced insulin levels in Wistar fatty improves glucose tolerance or insulin sensitivity in rats and the corresponding lean controls.11 Recently, NIDDM patients.27,28 Therefore, there is a possibility Masuda et al.24 reported the interesting observations that the reduction in NEFA by YM268 is the driving that troglitazone enhanced glucose- or glibenclamide- force for the metabolic changes observed in this study. induced insulin secretion from pancreatic beta cells in Further work will be necessary to examine this pos- vitro at a low concentration but inhibited it at a high sibility. concentration. This agent showed a high af®nity to the In summary, plasma TG and NEFA, but not cho- sulfonylurea receptor but had no effect on KATP lesterol concentration, were dependent on plasma channels. On the other hand, pioglitazone did not insulin concentration in lean and obese Zucker rats. show any effect on glucose-stimulated insulin secre- Insulin sensitivity with respect to TG and NEFA tory responses of islets isolated from normal rats.23 metabolism in obese rats was not different from that Although there has been no data about the effect of in lean rats, indicating that lipid abnormalities in YM268 on insulin secretion in vitro, our preliminary obese Zucker rats are due to the increased pool size ®ndings indicated that it had no acute effect on plasma of the lipids. insulin levels after a single administration in rats (data not shown). Therefore, it is unlikely that the YM268 References as well as other insulin sensitizers reduced plasma 1 Manson JE, Colditz GA, Stampfer MJ, Willett WC, Rosner B, Monson RR, Speizer FE, Hennekens CH. 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