SUMMARY

1. The effects of combined treatment with pioglitazone·HCland metformin on diabetes and obesity were investigated inWistar fatty rats, which are hyperglycaemic and hypertriglycer-idaemic and have higher plasma levels of total ketone bodiesthan lean rats.

2. Plasma glucose was significantly decreased when pioglita-zone·HCl or metformin was administered alone and combinedtreatment accentuated this decrease. The administration ofpioglitazone·HCl, but not metformin, also decreased plasma levels of triglyceride and total ketone bodies.

3. The glycogen content of skeletal muscle was not increasedby pioglitazone·HCl or metformin alone, but was increased bycombined treatment (P = 0.003, ANOVA).

4. Pioglitazone·HCl produced increased food intake andbodyweight in hyperphagic Wistar fatty rats; however, con-current administration of metformin significantly amelioratedthese pioglitazone·HCl-induced increases.

5. These results indicate that combined treatment withpioglitazone·HCl and metformin induces a marked hypo-glycaemic effect accompanied by a reduction in plasma levelsof total ketone bodies and prevention of excessive bodyweightgain in Wistar fatty rats. These favourable effects suggest thatthe combination would be beneficial in treating patients withtype 2 diabetes.

Key words: adipose tissue, diabetes mellitus, glycogen, metformin, obesity, pioglitazone·HCl, total ketone bodies, Wistar fatty rat.

INTRODUCTION

Biguanides, such as metformin and buformin, are widely used forthe treatment of obese patients with type 2 diabetes in Europeancountries because they have been shown to induce less lactic acidosis than the most potent biguanide, fenformin, and to improveinsulin resistance. Metformin has also been licensed recently for thetreatment of type 2 diabetes in the US. Clinical studies on metforminin type 2 diabetes have shown that it has the same blood glucose-lowering activity as sulphonylureas,1 decreases fasting blood glucose

and glycosylated haemoglobin (HbA1c) levels in moderately obesepatients,2 is much more effective in lowering blood glucose whenadministered in combination with a sulphonylurea than either agentalone3 and has the same effect on HbA1c as �-glucosidase inhibitors.4

Furthermore, intensive glycaemic control with metformin delays thedevelopment of complications in overweight patients with type 2diabetes.5

Recently, clinical studies on combinations of metformin withinsulin sensitizers have started to emerge.6–8 Pioglitazone·HCl, apotent insulin sensitizer, has been shown to ameliorate hyper-glycaemia, hypertriglyceridaemia and hyperinsulinaemia by reducing peripheral insulin resistance in obese and diabetic animals9–14 and human patients.7,15 We have previously reported that, at doses of 0.3–3 mg/kg per day, pioglitazone·HCl dose-dependently decreases plasma glucose in genetically obese, diabeticWistar fatty rats; a dose of 1 mg/kg per day decreased plasma glucose to 56% of the level observed in controls.11 In a preliminarystudy, we also confirmed that the plasma glucose-lowering effect ofmetformin is dose-dependent over the range 30–300 mg/kg per dayin Wistar fatty rats, with the 300 mg/kg per day dose decreasing thelevel to 78% of that seen in controls (M Suzuki et al., unpubl. obs.,1998). Therefore, in the present study, we investigated the effectsof combined treatment with metformin (300 mg/kg per day) andpioglitazone·HCl (1 mg/kg per day) on diabetes and obesity in Wistarfatty rats.

METHODS

Animals

Male 31-week-old Wistar fatty rats and their lean littermates were obtainedfrom the Laboratory Animal Unit of Takeda Chemical Industries Ltd (Osaka,Japan). All rats were housed in individual metal cages in a room with controlled temperature (23 � 1°C), humidity (55 � 5%) and lighting (lightson from 0800 to 2000 h) and were maintained on a laboratory chow diet (CE-2; Clea, Tokyo, Japan). For the estimation of the combined effects ofpioglitazone·HCl and metformin, Wistar fatty rats were divided into fourgroups (six rats in each group) based on bodyweight and plasma componentsand were given an oral solution containing 0.5% methylcellulose solution(2 mL/kg), pioglitazone·HCl (1 mg/kg per day), metformin (300 mg/kg perday) or both agents combined for 15 days. Wistar lean rats received 0.5%methylcellulose solution orally for 15 days. Bodyweight was measured at 2 or 3 day intervals. On days 0 and 14, blood was withdrawn from a tailvein for the measurement of diabetes-related plasma components (see below).On day 15, rats were killed by exsanguination under ether anaesthesia andthe liver and hindlimb skeletal muscle, together with the inguinal portion of the subcutaneous adipose tissue, the mesenteric adipose tissue and theinterscapular brown adipose tissue, were immediately excised for weighing.The livers and skeletal muscles were then stored at –80°C until required formeasurement of glycogen content.

EFFECTS OF COMBINED PIOGLITAZONE AND METFORMIN ONDIABETES AND OBESITY IN WISTAR FATTY RATS

Masami Suzuki, Hiroyuki Odaka, Noriko Suzuki, Yasuo Sugiyama and Hitoshi Ikeda

Pharmacology Research Laboratories II, Takeda Chemical Industries Ltd, Osaka, Japan.

Correspondence: Masami Suzuki, Pharmacology Research LaboratoriesII, Takeda Chemical Industries Ltd, 2-17-85 Juso-Honmachi, Yodogawa-ku,Osaka 532-8686, Japan. Email: Suzuki_Masami@takeda.co.jp

Received 29 May 2001; revision 24 August 2001; accepted 11 October2001.

Clinical and Experimental Pharmacology and Physiology (2002) 29, 269–274

270 M Suzuki et al.

Measurement of diabetes-associated plasma components

Concentrations of plasma glucose (Glc), triglyceride (TG) and total ketonebodies (T-KB) were measured enzymatically using a Hitachi Autoanalyser7070 (Hitachi, Ibaraki, Japan). Immunoreactive insulin (IRI) was measuredusing a commercially available radioimmunoassay kit (ShionogiPharmaceutical, Osaka, Japan). The HbA1 levels were measured using a commercially available kit (Ropet A1C-S; Nippon Chemipha, Tokyo,Japan).

Determination of hepatic and skeletal muscle glycogenconcentrations

The glycogen concentration in the liver and skeletal muscle was measuredusing a phenol–sulphuric acid extraction technique at room temperature. The liver or skeletal muscle sample (500 mg) was added to 0.5 mL of 30%potassium hydroxide, boiled for 30 min, mixed with 1.2 mL ethanol and centrifuged at 1900 g for 10 min. Distilled water was added to the resultingpellets (5 mL for liver samples and 1 mL for muscle samples), after which20 µL liver suspension or 200 µL muscle suspension was mixed vigorouslywith 200 µL phenol and then with 1 mL sulphuric acid. The mixture was incubated for 30 min. The absorbance of the solution was measured at 490 nm and the Glc concentration was determined using a standard Glc solution (Boehringer Mannheim GmbH, Mannheim, Germany).

Materials

Pioglitazone·HCl (lot no. M-683–044) was synthesized at Takeda ChemicalIndustries Ltd. Metformin (lot no. 84H0451) was purchased from SigmaChemical (St Louis, MO, USA). All reagents were purchased from WakoPure Chemical Industries (Osaka, Japan).

Statistical analysis

All data are presented as the mean�SEM. A two-way analysis of variance(ANOVA) was performed to examine the individual effects of pioglitazone·HCl

and metformin and any interaction between the effects of the two agents.Differences in mean values between the fatty rat control group and all other groups were analysed by Dunnett’s test. Differences in mean valuesbetween two specific groups (the fatty rat control and normal lean groups,or the pioglitazone·HCl alone and pioglitazone·HCl plus metformin groups)were analysed by Student’s t-test. Regression analysis was also performedusing data on food intake and bodyweight gain or inguinal adipose tissueweight in the fatty rats.

RESULTS

Effects of combined treatment on diabetes-associatedplasma components

At the beginning of the study, the Wistar fatty rats were hypergly-caemic (Glc 21.7 mmol/L), hypertriglyceridaemic (TG 329 mg/dL)and hyperinsulinaemic (insulin 8.3 nmol/L) and their plasma T-KBlevel (144 µmol/L) was 1.7-fold higher than that in age-matched normal lean rats.

Data on plasma Glc, HbA1 and IRI levels on day 14 are presentedin Fig. 1. When administered alone, pioglitazone·HCl and metformincaused plasma Glc to decrease to 57 and 78% of the control level,respectively. Pioglitazone·HCl alone also caused HbA1 to decreaseto 87% of the control level, whereas metformin alone had no effecton HbA1. During combined treatment, plasma Glc and HbA1 werereduced to 38 and 82% of their respective control levels. Two-wayANOVA confirmed that both pioglitazone·HCl and metformin had asignificant individual effect on plasma Glc (F(1,20) = 94.01, P < 0.001;and F(1,20) = 23.13, P < 0.001, respectively), but detected no inter-action between pioglitazone·HCl and metformin (F(1,20) = 0.126, P = 0.726). The IRI tended to decrease during combined treatment,falling to 70% of the control level; however, neither of the agentsaffected IRI levels when administered alone.

Fig. 1 Effects of 14 days oral treatment with pioglitazone·HCl (1 mg/kg per day), with ( ) or without ( ) metformin (300 mg/kg per day), on (a) plasmaglucose, (b) glycosylated haemoglobin (HbA1) and (c) insulin levels in male Wistar fatty rats. Data are the mean�SEM (n = 6). *P < 0.01 compared withthe Wistar fatty rat control group ( ) by Dunnett’s test; †P < 0.01 compared with the Wistar fatty rat control group by Student’s t-test; §P < 0.05 comparedwith the pioglitazone·HCl group by Student’s t-test. (�), metformin alone; (�), Wistar lean rats.

Combined pioglitazone and metformin 271

Changes in plasma TG and T-KB over the 14 day treatment periodare presented in Fig. 2. Metformin alone did not affect TG, but produced an increase in T-KB on day 7. Pioglitazone·HCl causedTG to decrease to 51% of the control value on day 7 and to 59%on day 14, but combination with metformin resulted in no furtherreduction in TG. In contrast, while pioglitazone·HCl alone causedT-KB to decrease to 82% of the control value on day 7 and to 74%on day 14, combined treatment with metformin caused T-KB todecrease to 77% of the level seen with metformin alone on day 7and to 85% on day 14. Two-way ANOVA using the values obtainedon day 14 revealed that pioglitazone·HCl caused a significantdecrease in T-KB (F(1,20) = 10.38, P = 0.004), whereas metformin didnot (F(1,20) = 3.487, P = 0.077) and that there was no interactionbetween the two agents(F(1,20) = 0.645, P = 0.431).

Effects of combined treatment on bodyweight, foodintake and liver and adipose tissue weights

Metformin produced only a slight increase in bodyweight, whereasmarked weight gain was apparent with pioglitazone·HCl (Fig. 3).However, concurrent administration with metformin significantlysuppressed the pioglitazone·HCl-induced increase in bodyweight.

Wistar fatty rats are hyperphagic and the administration of pioglita-zone·HCl further increased their food intake (to 130% of the control level). Although metformin alone did not affect food intake,when it was administered concurrently with pioglitazone·HCl, thepioglitazone·HCl-induced increase in food intake was suppressedto 110% of the control level. Liver weight was not affected by treat-ment with either of the agents (Fig. 4). Pioglitazone·HCl produceda slight, non-significant rise in inguinal and mesenteric adipose tissue weight, increasing values to 117 and 114% of those observedin controls, respectively. The gain in interscapular brown adiposetissue weight was much more marked, reaching 250% of the control value. However, concurrent administration with metforminopposed the pioglitazone·HCl-induced increase in inguinal adiposetissue weight. During combined treatment, the weight of this tissuewas 91% of that recorded for the pioglitazone·HCl alone group.Accordingly, while the two-way ANOVA showed that neither pioglita-zone·HCl nor metformin alone had any significant effect on inguinaladipose tissue weight (F(1,20) = 0.905, P = 0.353; and F(1,20) = 0.006,P = 0.941, respectively), a slight interaction between the two agentswas detected with regard to their effect on inguinal adipose tissueweight (F(1,20) = 3.081, P = 0.095). Possible correlations between foodintake and bodyweight gain or inguinal adipose tissue weight were

Fig. 3 Effects of 15 days oral treatment with pioglitazone·HCl (1 mg/kg per day), with ( ) or without ( ) metformin (300 mg/kg per day), on (a) foodintake and (b) bodyweight gain in male Wistar fatty rats. Data are the mean�SEM (n = 6). On day 0, the bodyweight of the Wistar fatty and lean rats was669 � 37 and 481 � 17 g, respectively and no significant differences between the Wistar fatty rat control group ( ) and any of the treated fatty rat groupswas detected by Dunnett’s test. *P < 0.05, **P < 0.01 compared with the Wistar fatty rat control group by Dunnett’s test; †P < 0.01 compared with the Wistarfatty rat control group by Student’s t-test; §P < 0.05, §§P < 0.01 compared with the pioglitazone·HCl group by Student’s t-test. (�), metformin alone; (�),Wistar lean rats.

Fig. 2 Effects of 14 days oral treatment with pioglitazone·HCl (1 mg/kg per day), with (�) or without (�) metformin (300 mg/kg per day) on (a) plasmatriglyceride (TG) and (b) total ketone body (T-KB) levels in male Wistar fatty rats. Data are the mean�SEM (n = 6). *P < 0.05, **P < 0.01 compared withthe Wistar fatty rat control group (�) by Dunnett’s test; †P < 0.01 compared with the Wistar fatty rat control group by Student’s t-test. (�), metformin alone;(�), Wistar lean rats.

272 M Suzuki et al.

also examined in the treated and untreated fatty rat groups. A signifi-cant positive correlation was found between food intake and body-weight gain (r = 0.846, P < 0.001), but not between food intake andinguinal adipose tissue weight (r = 0.274, P = 0.195).

Effects of combined treatment on liver and skeletalmuscle glycogen content

The mean glycogen content of the livers of Wistar fatty rats was1.7-fold higher than that in normal lean rats at baseline and treat-ment with metformin and/or pioglitazone·HCl had no effect on thisvalue (Fig. 5). In contrast, the baseline glycogen content of the skeletal muscle of Wistar fatty rats was 4.3-fold higher than that innormal lean rats and metformin or pioglitazone·HCl produced a

slight decrease when administered alone. However, combined treat-ment with metformin and pioglitazone·HCl tended to increase muscular glycogen content (to 142% of the control value). The two-way ANOVA revealed a significant interaction between the twoagents with regard to their effect on the muscular glycogen content(F(1,20) = 11.577, P = 0.003), although neither pioglitazone·HCl normetformin alone had any significant effect (F(1,20) = 0.670, P = 0.423;and F(1,20) = 3.964, P = 0.060, respectively).

DISCUSSION

In the present study, pioglitazone·HCl and metformin showed a combined effect on both plasma Glc and HbA1 in Wistar fatty rats.

Fig. 4 Effects of 15 days oral treatment with pioglitazone·HCl (1 mg/kg per day), with ( ) or without ( ) metformin (300 mg/kg per day), on (a) liverand (b–d) adipose tissue weights in male Wistar fatty rats. MAT, mesenteric adipose tissue; IAT, inguinal adipose tissue; BAT, interscapular brown adiposetissue. Data are the mean�SEM (n = 6). *P < 0.01 compared with the Wistar fatty rat control group ( ) by Dunnett’s test; †P < 0.01 compared with theWistar fatty rat control group by Student’s t-test. (�), metformin alone; (�), Wistar lean rats.

Fig. 5 Effects of 15 days oral treatment with pioglitazone·HCl (1 mg/kg per day), with ( ) or without ( ) metformin (300 mg/kg per day), on the glycogen content of the (a) liver and (b) skeletal muscle in male Wistar fatty rats. Data are the mean�SEM (n = 6). No significant differences between the Wistar fatty rat control group ( ) and any of the other groups was detected by Dunnett’s test. *P < 0.01 compared with the Wistar fatty rat control groupby Student’s t-test; †P < 0.05 compared with the pioglitazone·HCl group by Student’s t-test. (�), metformin alone; (�), Wistar lean rats.

Combined pioglitazone and metformin 273

The two-way ANOVA confirmed that pioglitazone·HCl and metformineach had a significant effect on plasma Glc and that there was nointeraction between the two agents, suggesting that the effect of thecombination of pioglitazone·HCl and metformin on plasma Glc isadditive. These results are in good agreement with those of a recentclinical study, which reported that combined therapy with troglita-zone, another insulin sensitizer, and metformin additively decreasedfasting and post-prandial plasma Glc levels in patients with type 2diabetes.6

Pioglitazone·HCl and other thiazolidinedione derivatives areknown to exert their hypoglycaemic effect by reducing insulin resistance in insulin-targeted tissues, without increasing insulinsecretion. However, the molecular mechanisms by which theseagents reduce insulin resistance remain unclear. Recent studies haveshown that thiazolidinedione derivatives, including pioglita-zone·HCl, troglitazone and rosiglitazone, are high-affinity ligandsfor peroxisome proliferator-activated receptor-� (PPAR�).16–19 Inmice, PPAR�2 is predominantly expressed in adipose tissue, but isalso expressed in skeletal muscle.20 In adipose tissue, PPAR� playsa role in the induction of adipocyte differentiation.21,22 Okuno et al.reported that troglitazone causes an increase in the number of smalladipocytes in Zucker fatty rats23 and considered the conversion oflarge adipocytes into small ones to be the mechanism by which thiazolidinediones reduce insulin resistance, because large adipo-cytes produce such insulin resistance-related substances as tumournecrosis factor (TNF)-� and non-esterified fatty acids (NEFA),whereas small ones do not. Recently, we found that treatment ofWistar fatty rats with pioglitazone·HCl for 4 days decreased theTNF-� content of skeletal muscle, which uses large amounts of glucose. This reduction in the TNF-� content was paralleled by afall in the plasma Glc level. Thus, we concluded decreased TNF-�production in skeletal muscle to be another potential mechanismunderlying the hypoglycaemic activity of thiazolidinediones.24

Many papers concerning the mechanisms underlying the hypogly-caemic effect of metformin have been published. Among otherthings, metformin suppresses Glc and amino acid uptake in the intes-tine, inhibits alanine uptake and Glc output by increasing lactic acidlevels in the liver and increases the number of insulin receptors andpromotes Glc uptake in skeletal muscle and adipose tissues.25–33

Because strict control of blood Glc and HbA1c levels suppresses anddelays the development of diabetic complications in patients withtype 2 diabetes,5,34 combined treatment using two hypoglycaemicagents with different mechanisms of action, such as pioglitazone·HCland metformin, may be useful.

Although metformin showed no hypotriglyceridaemic activity inthe present study, pioglitazone·HCl caused a marked decrease in TGlevels, consistent with those reported previously in several strainsof mice and rats, including Wistar fatty rats.10,11 Pioglitazone·HClappears to increase expression of the lipoprotein lipase gene viaPPAR� or to enhance lipoprotein lipase activity, thereby causing theTG level to decrease, as has been suggested previously.35,36 Adecrease in the high baseline concentration of plasma T-KB was also induced in Wistar fatty rats by the administration of pioglita-zone·HCl, but not metformin. According to the two-way ANOVA,there was no interaction between the two agents in this respect. Theadipose tissues of Wistar fatty rats show enhanced lipolysis10 andplasma NEFA levels are therefore increased.12 This results in anincrease in acetyl-CoA, because NEFA is converted to acetyl-CoAin mitochondria. Pioglitazone·HCl administration has been shown

to cause both a decrease in NEFA12 and an increase in glycolysisvia acetyl-CoA.10 Thus, the decrease in T-KB induced by pioglita-zone·HCl may be due to a reduction in acetyl-CoA. This action of pioglitazone·HCl may lead to the amelioration of ketoacidosis in diabetic patients.

Treatment with pioglitazone·HCl caused an increase in body-weight, as well as in inguinal and mesenteric adipose tissue weights,in Wistar fatty rats. However, concurrent administration of met-formin significantly reduced the pioglitazone·HCl-induced gain inbodyweight and tended to suppress the pioglitazone·HCl-inducedincrease in inguinal adipose tissue weight. Because there was signifi-cant positive correlation between food intake and bodyweight gain,the prevention of bodyweight gain can be explained by the suppres-sion of hyperphagia. However, there was no correlation betweenfood intake and inguinal adipose tissue weight. Thus, the reductionin inguinal adipose tissue weight gain may not be due simply to sup-pressed hyperphagia. Clinical studies have shown that the admin-istration of metformin results in decreased food consumption andbodyweight in patients with type 2 diabetes.37,38 Because obesity isa well-known risk factor for the development of insulin resistance,the weight loss or suppression of excessive bodyweight gain inducedby combined treatment with pioglitazone·HCl and metformin islikely to be beneficial. In the present study, pioglitazone·HCl alsocaused an increase in interscapular brown adipose tissue weight.Pioglitazone·HCl has been shown previously to increase the expres-sion of uncoupling protein-1 and -3 mRNA.39 Therefore, the changein thermogenesis produced by brown adipose tissue may form partof the mechanism by which this agent reduces insulin resistancethrough the reduction of adipose tissue weight.

Neither metformin nor pioglitazone·HCl affected the glycogencontent of the liver in Wistar fatty rats. In contrast, a slight decreasein the skeletal muscle glycogen content was observed followingtreatment with metformin or pioglitazone·HCl alone. However, com-bined treatment caused a marked increase in the glycogen contentof the skeletal muscle and the two-way ANOVA revealed a signifi-cant interaction between the two agents in this respect. Metforminadministration has been reported to induce an increase in the glyco-gen content of skeletal muscle, but not of the liver, in KK mice.40

This occurred because only glycogen synthetase activity wasincreased in the skeletal muscle, whereas the activities of both glyco-gen synthetase and phosphorylase were increased in the liver.40

Therefore, the increase in the glycogen content of the skeletal muscle of Wistar fatty rats treated concurrently with both agents may have been due to increased glycogen synthetase activity inducedby metformin. Although pioglitazone·HCl caused an increase in thesynthesis of lipids from Glc, the combined treatment induced anincrease in the synthesis of glycogen from Glc, suggesting that theeffect of metformin overcame that of pioglitazone·HCl. It would belogical to assume that this change in Glc metabolism could play arole in the suppression of bodyweight gain.

The results presented here indicate that concurrent administrationof pioglitazone·HCl and metformin induces a marked hypoglycaemiceffect, accompanied by a reduction in the plasma T-KB level andsuppression of excessive bodyweight gain, in Wistar fatty rats. Inhumans, the dose of pioglitazone·HCl used in clinical practice is 15 or 30 mg, while that of metformin is 0.5–3 g. The doses used inthe present study reflected this ratio; thus, we considered a metformindose of 300 mg/kg per day to be suitable for use in combination with pioglitazone·HCl 1 mg/kg per day. Our findings suggest that

274 M Suzuki et al.

combined therapy with these two agents would be beneficial in thetreatment of type 2 diabetes.

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