Combination therapy with PPARgamma and PPARalpha agonists increases glucose-stimulated insulin secretion in db/db mice

Ken Yajima1,4, Hiroshi Hirose1, Haruhisa Fujita2, Yoshiko Seto2, Hiroshi Fujita2, Kaname Ukeda2, Kiichi Miyashita2, Toshihide Kawai1, Yukihiro Yamamoto1, Takeo Ogawa1, Taketo Yamada3, and Takao Saruta1

1 Department of Internal Medicine, 2 Institute for Advanced Medical Research, and 3 Department of Pathology, Keio University School of Medicine, Tokyo 160-8582; and 4 Department of Internal Medicine, Hamamatsu Red Cross Hospital, Hamamatsu 430-0907, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although peroxisome proliferator-activated receptor (PPAR)gamma agonists ameliorate insulin resistance, they sometimes cause body weight gain, and the effect of PPAR agonists on insulin secretion is unclear. We evaluated the effects of combination therapy with a PPARgamma agonist, pioglitazone, and a PPARalpha agonist, bezafibrate, and a dual agonist, KRP-297, for 4 wk in male C57BL/6J mice and db/db mice, and we investigated glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion. Body weight gain in db/db mice was less with KRP-297 treatment than with pioglitazone or pioglitazone + bezafibrate treatment. Plasma glucose, insulin, triglyceride, and nonesterified fatty acid levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups. Also, PPARgamma agonists ameliorated the diminished GSIS and insulin content, and they preserved insulin and GLUT2 staining in db/db mice. GSIS was further increased by PPARgamma and -alpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful with respect to body weight and pancreatic GSIS in type 2 diabetes with obesity.

peroxisome proliferator-activated receptor; glucose-stimulated insulin secretion; glucolipotoxicity; insulin resistance; type 2 diabetes with obesity


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)gamma agonists have been used for the treatment of type 2 diabetic patients, especially those with obesity, all over the world. They ameliorate insulin resistance of peripheral tissues, such as skeletal muscle, liver, and adipocytes, by various suggested mechanisms. Although some literature has suggested effects of PPARgamma agonists on the pancreas (13, 32), only a few studies have investigated this issue (23, 29, 30). Because the expression of PPARgamma is confirmed not only in rodent (1) but also in human islets (5), an effect of PPARgamma agonists on the pancreas has been suggested.

On the other hand, fibrates are PPARalpha agonists and have been used for patients with dyslipidemia. Some literature has reported that PPARalpha agonists also ameliorated insulin resistance (6, 7, 14, 19, 24, 28, 36), but the effect on insulin secretion is unclear. Dual agonists of PPARgamma and -alpha , KRP-297 (12, 25, 26) and JTT-501 (34), have been developed, and their utility for diabetes and/or metabolic syndrome has been suggested. However, studies of combination therapy with a PPARgamma agonist and a PPARalpha agonist are rare (2, 21), and their effect on insulin secretion has not been reported.

We hypothesized that combination therapy with PPARgamma and -alpha agonists would be useful for reducing lipotoxicity (10, 20, 37) in type 2 diabetes with obesity by augmenting the lipid-lowering effects. We used the PPARgamma agonist pioglitazone (PIO), combination therapy with the PPARgamma agonist PIO and the PPARalpha agonist bezabibrate (P + B), and a dual agonist of PPARgamma and -alpha , KRP-297, in male C57BL/6J mice and male db/db mice as a model of type 2 diabetes with obesity. We measured body weight, food and water intake, and metabolic parameters, and we investigated the effects on the pancreas by determining GSIS with in situ pancreatic perfusion and also by histopathological examination.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Pioglitazone, bezafibrate, and KRP-297 were donated by Takeda (Osaka, Japan), Kissei (Matsumoto, Japan), and Kyorin Pharmaceuticals (Tokyo, Japan), respectively. Normal chow, CE-2, was purchased from Japan Clea (Tokyo, Japan). Pioglitazone, P + B, and KRP-297 were each blended with CE-2 by use of 0.1% carboxymethylcellulose.

Animals. Five-week-old male C57BL/6J mice and male db/db mice were purchased from Japan Clea. Our institution's guidelines for the care and use of laboratory animals were followed. Both mouse strains were fed CE-2 control chow and water ad libitum for 1 wk and each of the following drug-blended chow and water ad libitum for the following 4 wk: PIO [C57BL/6J: 0.02% (wt/wt), 31.3 ± 0.3 mg · kg-1 · day-1; db/db: 0.02% (wt/wt), 34.2 ± 0.4 mg · kg-1 · day-1], P + B [C57BL/6J: 0.02% (wt/wt), 29.2 ± 0.5 mg · kg-1 · day-1; db/db: 0.02% (wt/wt), 33.3 ± 0.4 mg · kg-1 · day-1] + [C57BL/6J: 0.06% (wt/wt), 97.3 ± 1.5 mg · kg-1 · day-1; db/db: 0.07% (wt/wt) 111.0 ± 1.5 mg · kg-1 · day-1], and KRP-297 [C57BL/6J: 0.02% (wt/wt), 30.2 ± 0.3 mg · kg-1 · day-1; db/db: 0.02% (wt/wt), 32.2 ± 0.4 mg · kg-1 · day-1]. Food intake, water intake, and body weight of all mice were measured in the morning 3 to 4 times/wk. Mice were fasted overnight and underwent operation for in situ pancreatic perfusion.

Measurement of metabolic parameters in plasma. Blood from the retroorbital sinus of 10-wk-old mice in a nonfasting state was collected into hematocrit tubes coated with EDTA. Plasma glucose and triglycerides were determined with a Fuji Dry-Chem 5500 (Fuji Film, Tokyo, Japan). Plasma nonesterified fatty acid (NEFA) concentration was determined by the NEFA-C test (Wako Pure Chemical Industries, Osaka, Japan). Plasma insulin concentration was determined by enzyme immunoassay (EIA; Morinaga Institute of Biological Science, Yokohama, Japan).

Pancreatic insulin content. Each pancreatic specimen was homogenized in 1.8 ml of acid ethanol. The homogenates were stored at 4°C for 48 h and then centrifuged at 10,000 rpm for 10 min at 4°C. The supernatants were pooled, and the pellets were then resuspended in 0.2 ml of acid ethanol. After centrifugation, the supernatants were diluted 1:10,000 in 0.1 M PBS containing 0.25% BSA. Insulin concentration was determined by EIA.

In situ pancreatic perfusion. The mice were anesthetized with 50 mg/kg pentobarbital sodium after an overnight fast. The celiac artery and portal vein were cannulated after other vessels had been ligated. Nonrecirculating perfusion was begun at a constant flow rate of 0.5 ml/min. The perfusion medium consisted of Krebs-Ringer bicarbonate buffer containing 3.0% (wt/vol) dextran T-40 (Pharmacia, Uppsala, Sweden), 1.0% (wt/vol) BSA (fraction V, RIA grade; Sigma Chemical, St. Louis, MO), and 20 mM HEPES. A 95% O2-5% CO2 gas mixture was bubbled through the perfusate, and pH was maintained between 7.35 and 7.45. The pancreatic venous effluent was collected into tubes via a catheter in the portal vein at 2-min intervals, frozen immediately, and stored at -20°C for subsequent EIA. To achieve postoperative equilibration, the pancreas was perfused for 15 min with a buffer containing the glucose concentration of the initial 10-min experimental period (5.6 mM). Thereafter, insulin response to a high-glucose concentration (16.7 mM) was examined for 20 min, and glucose concentration was then returned to 5.6 mM for another 20 min.

Histopathological examination. Pancreata from mice in all groups that were not operated on for in situ pancreatic perfusion were fixed in 35-38% Formalin for histopathological studies. The pancreata were embedded in paraffin, sectioned, and then stained with hematoxylin and eosin (H + E). Immunohistochemical studies were also performed using anti-human insulin guinea pig antibodies (Oriental Yeast, Osaka, Japan) and rabbit anti-GLUT2 polyclonal antibody (Chemicon International, Temecula, CA).

Statistical analyses. All results were expressed as means ± SE. Statistical analyses were performed with the Statview program (version 5.0-J, SAS Institute, Cary, NC). Analysis of variance (ANOVA), followed by a post hoc Bonferroni-Dunn multiple comparison test, was used to evaluate differences among groups. A P value <0.05 was considered statistically significant.


    RESULTS
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INTRODUCTION
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RESULTS
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Body weight change, total food intake, and total water intake. The body weight of animals in PPARgamma agonist-treated groups increased more than that of animals in the untreated group in both mouse strains (Table 1). Body weight gain in the KRP-297-treated group was less than that in other treated groups in db/db mice, but not in C57BL/6J mice. Total food intake in the PPARgamma agonist-treated groups was also significantly greater than that in the untreated group in C57BL/6J mice. On the contrary, total food intake of the KRP-297-treated group was the smallest in db/db mice. The PPARgamma agonist-treated groups in C57BL/6J mice showed a mild increase in total water intake, whereas the untreated group in db/db mice showed a marked increase.

                              
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Table 1.   Body weight change, total food intake, and total water intake during 4 wk in C57BL/6J and db/db mice

Metabolic parameters in plasma. Plasma glucose, insulin, triglyceride, and NEFA levels were elevated in untreated db/db mice compared with untreated C57BL/6J mice, and these parameters were significantly ameliorated in the PPARgamma agonist-treated groups (Fig. 1).


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Fig. 1.   Plasma glucose (A), insulin (B), triglyceride (C), and nonesterified fatty acid (NEFA, D) levels at 10 wk for untreated groups [solid bars, n = 5, C57BL/6J mice shown as reference (C57), and open bars, n = 3, (db/db)]; pioglitazone (PIO)-treated group [vertically striped bars, n = 5 (db/db)]; combined pioglitazone- and bezafibrate (P + B)-treated group [checked bars, n = 5 (db/db)]; and KRP-297-treated group [hatched bars, n = 5, (db/db)]. Each value represents a mean ± SE. P < 0.0083 vs. untreated db/db mice.

Pancreatic insulin content. Pancreatic insulin content of untreated db/db mice was almost completely diminished compared with untreated C57BL/6J mice (Fig. 2). Pancreatic insulin content of the PIO- and P + B-treated db/db mice was ameliorated compared with untreated db/db mice, although these differences were not statistically significant (P = 0.051 by ANOVA). The pancreatic insulin content of the KRP-297-treated db/db mice was almost the same as that of untreated C57BL/6J mice.


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Fig. 2.   Pancreatic insulin content at 10 wk for untreated group [solid bar, n = 3 (C57), and open bar, n = 5 (db/db)]; PIO-treated group [vertically striped bars, n = 8 (db/db)]; P + B-treated group [checked bars, n = 8 (db/db)]; and KRP-treated group [hatched bars, n = 5 (db/db)]. Data of untreated C57BL/6J are shown for reference. Each value represents a mean ± SE. P = 0.051 by ANOVA.

In situ pancreatic perfusion. There was no difference in GSIS among the four groups of C57BL/6J mice (Fig. 3A). In db/db mice (Fig. 3B), whereas GSIS in the untreated group was diminished compared with that in C57BL/6J mice, that in the PPARgamma agonist-treated groups was preserved compared with that in the untreated group. GSIS in the PIO-treated group showed no significant difference from that in the untreated group in db/db mice, whereas GSIS in both the P + B- and the KRP-297-treated groups was significantly increased (Table 2).


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Fig. 3.   Glucose-stimulated insulin secretion (GSIS) by in situ pancreatic perfusion in C57BL/6J mice (A) and db/db mice (B): untreated groups (, n = 6 for both C57 and db/db); PIO-treated groups (open circle , n = 6 for both C57 and db/db); P+B-treated groups [, n = 6 (C57) and 5 (db/db)]; KRP-treated groups (triangle , n = 6 for both C57 and db/db). Each value represents mean ± SE (A). Each value of untreated, PIO-treated, and KRP-treated groups represents a mean - SE (B). Each value of P + B-treated group represents a mean + SE (B).


                              
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Table 2.   Glucose-stimulated insulin secretion by in situ pancreatic perfusion in C57BL/6J and db/db mice

Histopathological findings. The islets of untreated db/db mice showed hyperplastic changes (Fig. 4). Insulin in these islets of untreated db/db mice was extremely reduced, and the staining pattern was scattered. Islets in the PIO- and P + B-treated db/db mice showed hyperplasia and almost normal staining of insulin. In islets of the KRP-297-treated db/db mice, the hyperplasia was improved, and the staining of insulin was almost normal. The results of the insulin staining were consistent with those of pancreatic insulin content. GLUT2 was expressed on the beta -cell membrane of normal C57BL/6J mice. Although GLUT2 expression was not detected in untreated db/db mice, it was preserved in PPARgamma agonist-treated db/db mice as well as in C57BL/6J mice. However, there was no significant difference in GLUT2 expression among the PIO-, P + B-, and KRP-297-treated groups of the db/db mice.


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Fig. 4.   Histopathological examination of pancreas from untreated C57BL/6J mice and db/db mice untreated and treated with PIO, P + B, and KRP. H + E, hematoxylin and eosin staining; Insulin, anti-insulin antibody staining; GLUT2, anti-GLUT2 antibody staining. Original magnification: all ×80.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PPARgamma agonists are well suited for the treatment of type 2 diabetic patients with obesity who have severe insulin resistance. However, because PPARgamma is a master regulator of adipocytes (22), its activation may induce increased fat mass and obesity. The degree of body weight gain is dependent on different PPARgamma agonists (18). Also, body weight gain by PPARgamma agonists is reported not only in rodents (11, 27) but also in humans (16, 33), and it is speculated to be due to adipogenesis by the drug effect (8, 31, 35) as well as increased food intake (4, 40, 41). However, the effect of PPAR agonists on insulin secretion is unclear.

In the present study, body weight in the PPARgamma agonist-treated groups increased significantly with increased food intake in C57BL/6J mice. However, weight gain in db/db mice was less with KRP-297 treatment compared with PIO or P + B treatment. We speculate that this is due to the effects of a dual agonist that enhances lipid catabolism and reduces fat mass by PPARalpha activation, as previously suggested (2, 5a). We consider that KRP-297 ameliorated obesity most, so that pancreatic insulin content of the KRP-297-treated group was almost the same as that in normal mice (26), and the islets of the KRP-297-treated group were not hyperplastic in contrast to those of the PIO- or P + B-treated groups.

Also, we have shown in this study that PPARgamma agonists normalized the elevated plasma glucose, insulin, triglyceride, and NEFA levels in db/db mice. However, we did not find an additional effect of a PPARalpha agonist on these parameters. Plasma glucose level in the KRP-297-treated group was the lowest in db/db mice, probably because of the lowest food consumption in addition to the drug effect.

GSIS in C57BL/6J mice showed a good response and was not changed by 4-wk treatment with PPARalpha and/or PPARgamma agonists. In untreated db/db mice, on the other hand, there was no GSIS response, due to insulin shortage resulting from severe insulin resistance, glucotoxicity, and lipotoxicity. Plasma insulin level in untreated db/db mice at this age, however, was higher than that in any other group; the islets of untreated db/db mice secreted insulin against an extremely high plasma glucose level. The stimulatory glucose level of the perfusate was 16.7 mM in our experiments, which was lower than the plasma level of untreated db/db mice. Therefore, we speculate that the islets of untreated db/db mice might be desensitized to glucose and thus did not show a GSIS response at 16.7 mM. However, we have shown reduced insulin staining with hyperplasia of islets, and pancreatic insulin content was barely detectable in untreated db/db mice, both suggesting insulin shortage.

In the present study, GSIS in db/db mice was preserved by PPARgamma agonist treatment. There was amelioration of plasma glucose, insulin, triglycerides, and NEFA levels in these groups. We also observed preserved insulin staining in the islets of the PPARgamma agonist-treated groups, and we concluded that the insulin-saving effects of PPARgamma agonists were mediated by ameliorating insulin resistance. De Souza et al. (3) also suggested amelioration of insulin resistance in Zucker diabetic fatty (ZDF) rats. These authors showed that there was no direct effect of pioglitazone on the pancreas in perifusion experiments. Although P + B and KRP-297 treatments in our study significantly increased GSIS in db/db mice, it is unclear why GSIS was increased by the addition of a PPARalpha agonist. Because there was no difference in plasma glucose, insulin, triglyceride, and NEFA levels among the PIO-, P + B-, and KRP-297-treated groups, increased insulin secretion in the P + B- and KRP-297-treated groups might not be through amelioration of glucolipotoxicity but through increased glucose sensitivity in pancreatic beta -cells.

Lipotoxicity may cause beta -cell abnormalities, loss of GSIS and GLUT2, and triglyceride accumulation (37, 38). Higa et al. (9), using ZDF rats, reported that troglitazone prevented these features of lipotoxicity. Because the peroxisome proliferator response element (PPRE) was identified in the rat GLUT2 gene (promoter) (17), PPARalpha and/or PPARgamma agonists may affect PPRE in the GLUT2 gene in pancreatic beta -cells. Wang et al. (39) suggested that PPARalpha might be one of the transcription factors involved in the direct upregulation of GLUT2 in normal rat islets. Furthermore, Zhou et al. (42) reported that expression of PPARalpha was suppressed in ZDF rat islets. In the present study, GSIS was increased by addition of a PPARalpha agonist, and we evaluated the staining of GLUT2, which is associated with glucose sensing and GSIS. We showed that treatment with PPARgamma agonist preserved GLUT2 in db/db mice. However, it was difficult to identify differences in GLUT2 staining among the PIO-, P + B-, and KRP-297-treated groups. It is possible that a PPARalpha agonist reduced the islet triglyceride content in db/db mice, although we were not able to examine this. Further studies will be needed to clarify the direct effect of PPARalpha on islet function.

To summarize, body weight in PPARgamma agonist-treated groups increased significantly with increased food intake in C57BL/6J mice. In contrast, weight gain in db/db mice was less with KRP-297 treatment compared with PIO- or P + B treatment. PPARgamma agonists ameliorated the diminished GSIS by improving insulin sensitivity in db/db mice, and GSIS was further increased by the combined PPARgamma and PPARalpha agonists. We conclude that combination therapy with PPARgamma and PPARalpha agonists may be more useful than PPARgamma alone with respect to body weight gain and pancreatic GSIS in type 2 diabetes with obesity.


    FOOTNOTES

Address for reprint requests and other correspondence: K. Yajima, Dept. of Internal Medicine, Keio Univ. School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan (E-mail: n62383{at}sc.itc.keio.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajpendo.00149.2002

Received 8 April 2002; accepted in final form 10 December 2002.


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ABSTRACT
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MATERIALS AND METHODS
RESULTS
DISCUSSION
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