Direct Angiotensin II Type 2 Receptor Stimulation Ameliorates Insulin Resistance in Type 2 Diabetes Mice with PPARγ Activation

Objectives The role of angiotensin II type 2 (AT2) receptor stimulation in the pathogenesis of insulin resistance is still unclear. Therefore we examined the possibility that direct AT2 receptor stimulation by compound 21 (C21) might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes (T2DM) with PPARγ activation, mainly focusing on adipose tissue. Methods T2DM mice, KK-Ay, were subjected to intraperitoneal injection of C21 and/or a PPARγ antagonist, GW9662 in drinking water for 2 weeks. Insulin resistance was evaluated by oral glucose tolerance test, insulin tolerance test, and uptake of 2-[3H] deoxy-D-glucose in white adipose tissue. Morphological changes of adipose tissues as well as adipocyte differentiation and inflammatory response were examined. Results Treatment with C21 ameliorated insulin resistance in KK-Ay mice without influencing blood pressure, at least partially through effects on the PPARγ pathway. C21 treatment increased serum adiponectin concentration and decreased TNF-α concentration; however, these effects were attenuated by PPARγ blockade by co-treatment with GW9662. Moreover, we observed that administration of C21 enhanced adipocyte differentiation and PPARγ DNA-binding activity, with a decrease in inflammation in white adipose tissue, whereas these effects of C21 were attenuated by co-treatment with GW9662. We also observed that administration of C21 restored β cell damage in diabetic pancreatic tissue. Conclusion The present study demonstrated that direct AT2 receptor stimulation by C21 accompanied with PPARγ activation ameliorated insulin resistance in T2DM mice, at least partially due to improvement of adipocyte dysfunction and protection of pancreatic β cells.


Introduction
The angiotensin (Ang) II type 1 (AT 1 ) receptor mediates the major effects of Ang II in the pathogenesis of insulin resistance and subsequent type 2 diabetes mellitus (T2DM) [1]. AT 1 receptor blockers (ARBs) are known to improve insulin resistance and reduce the new onset of diabetes [2][3][4][5][6]. When the AT 1 receptor is blocked by ARBs and unbound Ang II can act on the Ang II type 2 (AT 2 ) receptor, stimulation of the AT 2 receptor might be involved in the effects of ARBs. AT 2 receptor stimulation appears to antagonize the signaling activated by AT 1 receptor stimulation in various tissues [7]; however, the role of AT 2 receptor stimulation in metabolic disorders is still unclear. We demonstrated that there was no apparent difference in insulin-mediated glucose uptake into skeletal muscle between wild-type and AT 2 receptor null mice, whereas insulin-induced glucose uptake in white adipose tissue in AT 2 receptor null mice was significantly lower than that of control mice. 3 It was reported that AT 2 receptor-dependent Ang II signaling increases adipose cell mass and glucose intolerance, thereby participating in the deleterious effects of a high-fat diet [8]. Mitsuishi et al demonstrated that the Ang II-induced reduction in muscle mitochondria in mice was partially, but significantly, reversed by blockade of either the AT 1 receptor or AT 2 receptor, associated with increased fat oxidation, decreased muscle triglyceride, and improved glucose tolerance [9]. In terms of mitochondria, Abadir et al recently presented evidence of age-related changes in mitochondrial Ang II receptor expression, i.e., increased mitochondrial AT 1 receptor and decreased AT 2 receptor density, which was reversed by chronic treatment with an ARB, and demonstrated that activation of the mitochondrial AT 2 receptor increased NO production and probably attenuated aging [10]. The availability of selective agonists of the AT 2 receptor could be useful in helping to resolve some of the apparently conflicting results regarding the role of the AT 2 receptor in various pathological conditions. The recent development of selective non-peptidic AT 2 receptor agonists has provided new important tools for further evaluation of the roles of AT 2 receptor stimulation in pathophysiological conditions and should relaunch interest in studying the intracellular effects and regulation of this receptor in various tissues [11,12].
Adipose tissue as an endocrine organ plays a crucial role in the pathogenesis of insulin resistance and the onset of type 2 diabetes. Adipose tissue contains renin, angiotensinogen and angiotensinconverting enzyme (ACE), which results in increased production of Ang II as a local regulator of adipose tissue functions. Previous reports indicated that blockade of AT 1 receptor stimulation attenuated adipocyte dysfunction; however, the effects of AT 2 receptor stimulation on adipose tissue functions are not yet clear. We reported that the weight of both epididymal and retroperitoneal adipose tissue in ApoE knockout (KO) mice with AT 2 receptor deletion (AT 2 /ApoEKO) was greater than that in ApoEKO mice after a high-cholesterol diet. In adipose tissue of AT 2 /ApoEKO mice, the adipocyte number was decreased and the expression of peroxisome proliferator-activated receptor gamma (PPARc), CCAAT-enhancer-binding protein a (C/EBPa, and adipocyte Protein 2 (aP2) was lower than that in ApoEKO mice, in association with an increase in nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, suggesting that AT 2 receptor stimulation in adipose tissue is involved in the improvement of adipocyte differentiation and adipose tissue dysfunction in an atherosclerotic model [13]. It was previously reported that the AT 2 receptor also mediates the effect of Ang II to induce the production and release of prostacyclin from adipocytes, which in turn stimulates differentiation of adipose precursor cells [14,15]. Yvan-Charvet L et al also reported that adipocyte hypertrophy and increased lipogenic gene expression induced by adipose angiotensinogen overproduction was prevented by deletion of the AT 2 receptor [16]. These results led us to examine the roles of direct AT 2 receptor stimulation by compound 21 (C21) in adipose tissue in terms of glucose intolerance using type 2 diabetic mice KK-Ay.
PPARc, a nuclear transcription factor, plays an important role in regulation of adipocyte differentiation, insulin resistance, and inflammation [17]. In addition, large clinical studies have demonstrated that a PPARc agonist had beneficial effects on not only glycemic control but also preventing atherosclerotic disease [18][19][20][21]. Possible crosstalk between PPARc and the AT 1 receptor such as a decrease in AT 1 receptor promoter activity and AT 1 receptor expression by PPARc activation has been suggested [22,23]. It has also been reported that AT 1 receptor blockade decreases NF-kB activation, with PPARc activation in the vasculature [24]. AT 1 receptor stimulation activates ERK, and PPARc stimulation inhibits this ERK activation in VSMC [25]. On the other hand, it is reported that Ang II induces PPARc activation in PC12W cells via AT 2 receptor activation [26]. Accordingly, we hypothesized that AT 2 receptor stimulation by C21 might contribute to possible insulin-sensitizing/anti-diabetic effects in type 2 diabetes, with PPARc activation. It is also clinically important to investigate the possibility that crosstalk between AT 2 receptor stimulation and PPARc activation may regulate adiopocyte function and insulin resistance, and therefore we also examined this possibility in this study.

Animal and treatment
Eight-week-old adult male KK-Ay mice (CLEA, Tokyo, Japan) were used in this study. They were housed in an air conditioned room at 25uC with a 12-h light/12-h dark cycle. They were given a standard diet (MF, Oriental Yeast, Tokyo, Japan) and water ad libitum. Compound 21 was provided by Vicore Pharma (Gothenburg, Sweden). KK-Ay mice were subjected to intraperitoneal injection of C21 (10 mg/kg per day) dissolved in 100 ml of phosphate buffered saline (PBS) for 2 weeks. Some KK-Ay mice treated with C21 were concurrently administered GW9662, a PPARc antagonist, at a dose of 0.35 mg/kg per day in drinking water. The control mice were given intraperitoneal injection of an equivalent volume of vehicle as the experimental group. Systolic blood pressure (SBP) was measured by the tail-cuff method (MK-2000ST, Muromachi Kikai, Co. Ltd., Tokyo, Japan) as described previously [27]. All procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and reviewed and approved by the Animal Studies Committee of Ehime University.

Oral glucose tolerance test and insulin tolerance test
Oral glucose tolerance test (OGTT) was performed after 16hour overnight fasting [28]. Glucose solution (2 g/kg) was administered orally, and a small amount of blood was obtained from the orbital sinus or tail vein without anesthesia at 0, 30, 60 and 120 min after glucose loading. For insulin tolerance test (ITT), mice were given an intraperitoneal injection of 0.5 U/kg insulin solution (NovoRapid, Novo Nordisk, Bagsvaerd, Denmark) after 4-hour fasting. Blood glucose concentration was determined by glucose dehydrogenase pyrroloquinolinequinone method (Freestyle, Nipro, Osaka, Japan). Serum insulin concentration was measured by enzyme-linked immunosolvent assay (ELISA) (Ultra Sensitive Rat Insulin kit, Morinaga Institute of Biological Science, Kanagawa, Japan).

Morphological analysis of adipose tissue and pancreatic tissue
Epididymal and retroperitoneal white adipose tissue was taken, fixed with formalin, and paraffin-embedded sections were prepared [29]. After staining the sections with fuchsin, adipocyte number in three microscopic fields was counted and expressed as the cell number per mm 2 . Pancreatic tissue was taken similarly, fixed with formalin, and paraffin-embedded sections were stained with Gomori's aldehyde-fuchsin to visualize b cell granules in islets [30]. The ratio of stained area (b cell area) was calculated using computer-imaging software (Densitograph; ATTO Corporation). Values were obtained from five different mice in each group.

Measurement of rate constant of net tissue uptake of 2-[ 3 H]deoxy-D-glucose
Uptake of 2-[ 3 H]deoxy-D-glucose (2-[ 3 H]DG) in peripheral tissues was measured as previously reported [31]. Epididymal and retroperitoneal white adipose tissue was rapidly dissected and weighed. The rate constant of net tissue uptake of 2-[ 3 H]DG was calculated as described previously [32].

DNA-binding activity of PPARc in adipose tissue
Nuclear extract from adipose tissue was prepared using a nuclear extract kit (Sigma-Aldrich, Japan) after homogenization of adipose tissue with a glass homogenizer according to the manufacturer's protocol. DNA-binding of PPARc was determined with a DNA-binding ELISA kit (TransAM TM PPARc, Active Motiv, Carlsbad, CA). The kit is designed to detect PPARc protein in nuclear extract, which binds to PPARc response element immobilized at the bottom of the 96-well plate, using specific antibodies conjugated to horseradish peroxidase. Five micrograms of protein from each nuclear extract was applied to the assay, and DNA-binding of PPARc was determined.

Statistical analysis
All values are expressed as mean 6 standard error in the text and figures. Data were evaluated by analysis of variance (ANOVA) followed by post-hoc analysis for multiple comparisons. A difference with p,0.05 was considered significant.

Effect of compound 21 on blood glucose, serum insulin, adiponectin, and TNF-a concentrations
Treatment of KK-Ay with an AT 2 receptor agonist, C21 (10 mg/kg per day), for 14 days did not change systolic blood pressure compared with that in control KK-Ay mice. (data not shown) The increase in body weight of KK-Ay was attenuated with C21 treatment, whereas this effect of C21 was attenuated by GW9662 administration ( Table 1 and 2). Food intake was not different in each group. Treatment with C21 significantly reduced blood glucose concentration and serum insulin concentration in the fed condition compared with those in control KK-Ay mice ( Figure 1A and 1B). The decreases of blood glucose and serum insulin concentrations by C21 treatment were attenuated by coadministration of GW9662, whereas treatment with GW9662 alone did not affect blood glucose and serum insulin concentrations. We also observed that C21 treatment increased serum adiponectin concentration, but decreased serum TNF-a concentration compared with those in control KK-Ay mice ( Figure 1C and 1D). GW9662 treatment attenuated the C21-mediated increase of serum adiponectin concentration and the decrease of serum TNF-a concentration, whereas treatment with GW9662 alone did not affect them.

Effect of compound 21 on glucose intolerance and insulin sensitivity
In OGTT, the basal blood glucose concentration after 16-hour fasting did not differ between each group, whereas the peak of the glucose rise in response to a glucose load was lower and the decrease of blood glucose concentration was faster in mice treated with C21 compared with control and GW9662-treated KK-Ay mice (Figure 2A). Serum insulin concentration was lower in mice treated with C21 compared with control and GW9662-treated mice. Moreover, we observed that C21 treatment increased serum insulin concentration at 30 minutes after a glucose load ( Figure 2B). The decrease of blood glucose concentration and  the increase of serum insulin concentration after a glucose load by C21 treatment were attenuated by GW9662 administration (Figure 2A and 2B). In ITT, the decrease of blood glucose concentration by insulin injection was further enhanced in the C21-treated group, while this increase of insulin response by C21 treatment was inhibited by GW9662 treatment ( Figure 2C).

Effect of compound 21 on glucose uptake in adipose tissue of KK-Ay mice
Administration of C21 significantly increased 2-[ 3 H]DG uptake in white adipose tissue with or without insulin stimulation ( Figure 3). The enhancement of 2-[ 3 H]DG uptake after insulin injection by C21 was attenuated by co-administration of GW9662 in white adipose tissue, whereas treatment with GW9662 alone did not affect 2-[ 3 H]DG uptake.

Adipose tissue weight and adipocyte size after treatment with compound 21
To examine the possible mechanism of the C21-mediated improvement of glucose intolerance in KK-Ay mice, we focused on adipose tissue. Treatment with C21 for 14 days significantly decreased the ratio of epididymal and retroperitoneal adipose tissue weight to body weight, whereas this ratio became higher with GW9662 co-treatment (Table 3, Figure 4A and 4B). On histological analysis, mean adipocyte size in white adipose tissue was smaller in the C21-treated group, and consequently the number of adipocytes was increased by C21 treatment ( Figure 4C, 4D and 4E). The C21-mediated decrease in adipocyte size and increase in adipocyte number were attenuated by GW9662 administration, while GW9662 treatment alone did not influence adipocyte size or number.
Effect of compound 21 on expression of adipocyte differentiation markers and DNA-binding activity of PPARc in white adipose tissue Treatment with C21 increased the mRNA expression of PPARc in epididymal and retroperitoneal adipose tissue of KK-Ay mice ( Figure 5A). We next examined DNA-binding activity of PPARc in these adipose tissues of KK-Ay mice and observed that DNAbinding activity of PPARc was enhanced in C21-treated mice ( Figure 5B). These increases in PPARc expression and DNAbinding activity of PPARc were inhibited by co-administration of GW9662, whereas treatment with GW9662 did not influence these parameters. The expression of adipocyte differentiation markers, such as C/EBPa and aP2, was increased by C21 treatment ( Figure 5C and 5D). GW9662 administration attenuated the C21-mediated increase in expression of C/EBPa and aP2, whereas treatment with GW9662 alone did not influence this.
Effect of compound 21 on expression of adiponectin, inflammatory cytokines, and angiotensin II receptors in white adipose tissue C21 treatment enhanced mRNA expression of adiponectin in epididymal and retroperitoneal adipose tissue compared with that in control KK-Ay mice, and this increase was attenuated by coadministration of GW9662 ( Figure 6A). Treatment with C21 decreased mRNA expression of inflammatory cytokines, such as TNF-a, IL-6, and MCP-1, which was inhibited by GW9662 treatment (Figure 6B, 6C, and 6D). In contrast, treatment with GW9662 alone did not change mRNA expression of these inflammatory markers. There was no significant difference in

Recovery of b cell number in pancreas after compound 21 treatment
We observed that C21 treatment increased insulin concentration after a glucose load, and we assumed that C21 could prevent pancreatic b cell damage in KK-Ay. The number of b cells in pancreas islets detected by aldehyde-fuchsin staining was increased in C21-treated mice compared with control and GW9662-treated mice ( Figure 7A and 7B). This increase in b cell number in the pancreas by C21 treatment was attenuated by GW9662 administration, while treatment with GW9662 alone did not influence these parameters.

Discussion
The roles of AT 2 receptor stimulation in the pathogenesis of insulin resistance and functions of adipose tissue in T2DM are still an enigma. Most studies addressing the involvement of the AT 2 receptor in a variety of experiments have been performed in genetically altered, either AT 2 receptor-deficient or AT 2 receptoroverexpressing mice with or without ARB treatment, and  unfortunately these experimental approaches have yielded conflicting results. Moreover, PD123319, a widely used AT 2 receptor antagonist, acts nonselectively at a higher dose [33] and likewise, CGP42112, a widely used AT 2 receptor agonist, has both agonistic and antagonistic actions depending on its dose used. These findings made it difficult to properly assess AT 2 receptor actions, especially in vivo. For example, the effect of the AT 2 receptor on cardiac function after myocardial infarction is controversial, due to lack of an experimental tool to directly stimulate the AT 2 receptor under in vivo conditions [34]. A novel non-peptide AT 2 receptor agonist, C21, is a nonpeptidergic agent that has high affinity and selectivity for the AT 2 receptor [35]. Therefore, it can be expected that C21 will allow the effects of the AT 2 receptor in cardiac hypertrophy to be studied by means of direct, selective AT 2 receptor stimulation, which will hopefully help to overcome current controversies [36]. Consistent with this, Kaschina et al reported that treatment with C21 significantly improved systolic and diastolic ventricular function in the rat after myocardial infarction, suggesting that direct AT 2 receptor stimulation may be a novel therapeutic approach to improve cardiac function after myocardial infarction through anti-apoptotic and anti-inflammatory mechanisms [37]. In keeping with these results, we used C21 to elucidate the roles of direct AT 2 receptor stimulation in the pathogenesis of insulin resistance in T2DM, mainly focusing on adipose tissue.
We observed that treatment with a non-hypotensive dose of C21 decreased adipose tissue weight/body weight, and that C21 treatment decreased blood glucose and serum insulin concentrations in non-fasting condition in KK-Ay mice. It is well known that insulin resistance is a major metabolic feature of obesity and plays  a key role in the etiology of T2DM. We also showed that C21 treatment attenuated the rise of blood glucose concentration after a glucose load, with an increase of serum insulin concentration. Consistent with these results, we demonstrated that C21 treatment increased serum adiponectin and decreased TNF-a concentrations, and enhanced adipocyte differentiation, with increases in mRNA expression of PPARc, C/EBPa and aP2, and PPARc activation. These results, including a previous report, [13] suggest that AT 2 receptor stimulation could enhance adipocyte differentiation, thereby resulting in the improvement of glucose intolerance. However, it should be noted that the lack of hypotensive effects as judged by the tail cuff method does not completely exclude a possible effect of C21 on blood pressure because the tail cuff method does not give accurate readings of diastolic pressure nor does it not provide for 24 hour measurements of blood pressure. Many lines of evidence have indicated that obesity is closely linked to a chronic inflammatory state, which contributes to metabolic disorders [38]. Adipose tissue is thought to directly exaggerate insulin resistance and to trigger inflammation by secreting adipokines such as IL-6 and TNF-a [1]. Thus, adipose tissue dysfunction can tend toward to a chronic inflammatory state through imbalance of pro-inflammatory and anti-inflammatory activities [39]. Moreover, inflammatory cytokines regulate the differentiation of adipocytes and their function, resulting in worsening of insulin resistance. For instance, TNF-a inhibits adipogenesis by preventing the induction of PPARc and C/EBPa expression [40]. We observed that C21 treatment increased serum adiponectin concentration and decreased serum TNF-a concen-tration, and that administration of C21 enhanced mRNA expression of adiponectin and decreased mRNA expression of TNF-a, IL-6 and MCP-1 in white adipose tissue. Moreover, TNFa is known to directly inhibit insulin signaling, resulting in insulin resistance [41]. Consistent with these findings, we observed that C21 treatment significantly increased 2-[ 3 H]DG uptake in white adipose tissue. These results are consistent with the possibility that AT 2 receptor stimulation by C21 and or some other actions of C21 may attenuate inflammation in white adipose tissue and thereby help enhance adipocyte differentiation and improve insulin resistance. Addressing more detailed mechanisms is important for future clinical application of direct AT 2 receptor stimulation.
AT 2 receptor activation is reported to induce PPARc activation in PC12W cells [26], leading us to hypothesize that AT 2 receptormediated improvement of glucose intolerance is at least in part due to PPARc activation. We observed that AT 2 receptor stimulation by C21 enhanced not only PPARc expression but also DNAbinding activity of PPARc in white adipose tissue of KK-Ay, suggesting that the possible crosstalk between AT 2 receptor stimulation and PPARc activation could be involved in the attenuation of insulin resistance in concert with the regulation of adipocyte differentiation and functions. Accordingly, we demonstrated that C21-mediated improvement of insulin resistance, adipocyte differentiation, and inflammation in adipose tissue were blunted by PPARc blockade with GW9662, supporting the idea that direct AT 2 receptor stimulation by C21 ameliorated insulin resistance in T2DM model mice at least partially due to PPARc activation. In contrast, in adipose tissue, the possibility exists that   decreased PPARc expression improved insulin sensitivity, as demonstrated in PPARc-deficient mice [42]. Moreover, decreasing PPARc activity by a specific antagonist was shown to improve insulin sensitivity, accompanied by adipocyte hypotrophy and hyperplasia as observed in PPARc heterozygous mice [43]. These apparent discrepancies might be dependent on differences in experimental models. It would be intriguing to investigate the roles of possible interaction of the AT 2 receptor and PPARc in the pathogenesis of diabetes.
We observed that C21 treatment increased serum insulin concentration in response to a glucose load. Both AT 1 and AT 2 receptors are present in various pancreatic tissues, and Ang II has been proposed to have important roles in the pancreas, such as regulating islet b cell insulin biosynthesis and glucose-stimulated insulin release [44]. AT 1 blockade by ARBs has been reported to have beneficial effects on the pancreas in various diabetes model mice [45][46][47]. However, the role of the AT 2 receptor in the pancreas is still unclear. Therefore, we examined the possibility that AT 2 receptor stimulation could protect against b cell damage in the diabetic pancreas and increase insulin production and secretion. We demonstrated that C21 treatment increased the number of b cells in pancreas islets compared with that in control KK-Ay mice. We speculate the reason why the C21 reduced insulin levels in the fed state but increased insulin levels in the OGTT might be due to the preservation of pancreatic b cell number and enhancement of glucose uptake in white adipose tissues, resulting in increases in insulin secretory response and decreases in insulin resistance. The detailed mechanisms of AT 2 receptor-mediated pancreatic b cell protection in diabetic mice remain to be elucidated. One possible mechanism is a C21mediated decrease in TNF-a, since TNF-a is known to induce apoptotic cell death in mouse primary b cells and insulinoma cell lines [48], and inhibit glucose-stimulated insulin transcription and secretion observed in the HIT-T15 pancreatic b cell line [49]. We also showed that treatment with GW9662 attenuated the C21mediated increase of serum insulin concentration after a glucose load and the preservation of b cell number in the pancreas in KK-Ay. Activation of PPARc has been shown to regulate transcription of several key b cell genes involved in glucose sensing, b cell development, glucose-stimulated insulin secretion, and insulin gene transcription. Activation of these targets leads to enhanced insulin secretion and gene expression in the diabetic state [50]. These data, including our results, suggest that the possible crosstalk between AT 2 receptor stimulation and PPARc activation may also be involved in pancreatic b cell function.
It should be acknowledged that the current studies do not prove that any of the metabolic effects of C21 are necessarily mediated by AT 2 receptor stimulation because we have not excluded the possibility that C21 might be functioning as a direct agonist of PPAR isoforms including PPARc and PPARa and PPARd. Future studies will be required to determine the extent to which the metabolic effects of C21 are mediated by its interaction with AT 2 as opposed to its possible interaction with other receptors such as PPARc, PPARa, and PPARd, etc.
Taken together, the current results demonstrate that the AT 2 agonist C21 improves insulin resistance in T2DM mice in association with enhanced adipocyte differentiation and possibly function, and with protection of b cells in pancreas islets. These effects of C21 were blunted by PPARc blockade, suggesting a role for PPAR activation in mediating the metabolic actions of C21. The extent to which the metabolic effects of C21 are mediated by its direct interaction with AT 2 and or by direct interaction with other receptors such as PPARc, PPARa, and PPARd remains to be determined. In any case, molecules like C21 may hold promise as potential therapeutic options for the management of insulin resistance and T2DM in the future.