ABSTRACT

Angiotensin II receptor blocker (ARB) reduces the incidence of type-2 diabetes mellitus. In this study, I examined the effects of candesartan on metabolic syndrome by using diet-induced obesity (DIO)-C57BL/6J mice and diabetic db/db mice. One of the ARBs, candesartan ameliorated insulin resistance, increased both the plasma adiponectin and the plasma levels of high molecular weight (HMW) adiponectin and decreased the expression levels of inflammatory genes in white adipose tissue (WAT). In addition, candesartan decreased the adipocyte sizes of WAT in these mice. Moreover, candesartan increased the mRNA expression levels of adiponectin, adiponectin receptors, AdipoR1 and AdipoR2, and peroxisome proliferator-activated receptor (PPAR)γ in WAT of db/db mice. To assess the contribution of adiponectin signaling to the effects of candesartan on amelioration of insulin resistance, I next examined by using Adipor1/r2 double-knockout mice. Candeartan ameliorated insulin resistance in wild-type mice, but not in Adipor1/r2 double-knockout mice. Candesartan increased the plasma adiponectin levels and the plasma levels of HMW adiponectin in both wild-type mice and Adipor1/r2 double-knockout mice. Candesartan increased the mRNA expression levels of PPARγ, decreased those of inflammatory genes, and decreased oxidative stress in WAT of wild-type mice, but not in those of Adipor1/r2 double-knockout mice. Furthermore, candesartan decreased the adipocyte sizes of WAT in wild-type mice, but not in those of Adipor1/r2 double-knockout mice. In conclusion, the effects of candesartan on amelioration of insulin resistance might be partly through increase of HMW adiponectin and activation of adiponectin receptors (AdipoRs).

INTRODUCTION

Obesity is a major risk factor for the development of hypertension and is also the principal risk factor for insulin resistance and the development of type 2 diabetes. Hypertension in obese individuals is further complicated by the concomitant presence of insulin resistance.

Angiotensin converting enzyme (ACE) inhibitors or ARBs, which are blockers of the rennin-angiotensin system, have been shown to increase insulin sensitivity and are now widely used as therapy for hypertension in diabetic patients. Angiotensin II (Ang II) is considered the major final mediator of the renin-angiotensin system. The actions of Ang II have been implicated in many cardiovascular conditions, such as hypertension, atherosclerosis, coronary heart disease, restenosis and heart failure. Ang II can act through two different receptors: Ang II type 1 (AT1) receptor and Ang II type 2 (AT2) receptor which are members of the G-protein-coupled receptor family. Ang II regulates the production of adipokines. Ang II increases the expression and the release of pro-inflammatory cytokines, and reduces plasma levels and gene expression of adiponectin, an insulin-sensitizing and anti-inflammatory adipokine.

Adiponectin is a hormone secreted by adipocytes that acts as a major anti-diabetic and anti-atherogenic adipokine. Adiponectin acts through two main receptors, AdipoR1 and AdipoR2. AdipoR1 and AdipoR2 are key players in the physiological and pathophysiological significance of adiponectin, and are involved in the regulation of glucose and lipid metabolism, inflammation and oxidative stress.

PPARγ is one of the key regulators of glucose homeostasis. Activation of PPARγ by agonists such as thiazolidinediones stimulates lipid storage in adipocytes, thereby reducing lipotoxicity in liver and skeletal muscle. In addition, PPARγ activation increases small adipocytes, thereby increasing the insulin-sensitizing hormone adiponectin and reducing tumor necrosis factor (TNF)-α, which induces insulin sensitivity. It has previously been reported that a PPAR γ agonist, pioglitazone, increased secretion of total and HMW adiponectin. It has been previously reported that decreased HMW adiponectin plays a crucial and causal role in obesity-linked insulin resistance and metabolic syndrome. Importantly, under pathophysiological conditions, such as obesity and diabetes, only HMW adiponectin was decreased; therefore, strategies to increase only HMW adiponectin may be a logical approach to provide a novel treatment modality for obesity-linked diseases, such as insulin resistance and diabetes.

Candesartan, one of the ARBs, has been reported to improve insulin sensitivity. Moreover, Koh et al reported that candesartan therapy increased serum adiponectin levels and insulin sensitivity in hypertensive patients. In this study, I examined the effects of candesartan in DIO-C57BL/6J mice, diabetic db/db mice and differentiated 3T3-L1 adipocytes, and I investigated the mechanisms by which they improve insulin resistance, especially in WAT. Moreover, I used Adipor1/r2 double-knockout mice to investigate whether candesartan might be capable of ameliorating insulin resistance in the absence of adiponectin signaling.

RESULTS

To examine the anti-diabetic effects of candesartan, I treated DIO-C57BL/6J mice with candesartan. I examined the effects of candesartan on the improvement of glucose intolerance and insulin resistance, using oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Plasma glucose levels during both test and plasma insulin levels during OGTT in candesartan-treated mice were significantly lower than those in vehicle-treated mice, suggesting that candesartan treatment ameliorated insulin resistance in DIO-C57BL/6J mice. Moreover, candesartan significantly increased the plasma adiponectin levels, in particular of HMW adiponectin.

Candesartan significantly increased the mRNA expression levels of Cu,Zn-superoxide dismutase (SOD)1 in WAT, whereas it decreased the mRNA expression levels of TNF-α and monocyte chemoattractant protein (MCP)-1 in WAT and plasma MCP-1 levels. In addition, candesartan decreased the adipocyte sizes of WAT in DIO-C57BL/6J mice.

Next, to examine the anti-diabetic effects of candesartan on severe diabetic model, I treated db/db mice with candesartan. Plasma glucose levels during OGTT and ITT in candesartan-treated mice were significantly lower than those in vehicle-treated mice, suggesting that candesartan treatment ameliorated insulin resistance in db/db mice. Moreover, candesartan increased both the plasma adiponectin and the plasma levels of HMW adiponectin in db/db mice.

Candesartan increased the mRNA expression levels of adiponectin, AdipoR1 and AdipoR2 in WAT. Moreover, candesartan significantly increased the mRNA expression levels of PPARγ and tended to decrease those of MCP-1 in WAT. In addition, candesartan decreased the adipocyte sizes of WAT in db/db mice.

As described above, candesartan increased the plasma adiponectin levels, in particular of HMW adiponectin, in DIO-C57BL/6J mice and db/db mice. To further investigate the contribution of adiponectin signaling to the effects of candesartan on amelioration of insulin resistance, I examined whether or not candesartan would ameliorate insulin resistance in Adipor1/r2 double-knockout mice. Candeartan ameliorated insulin resistance in wild-type mice, but not in Adipor1/r2 double-knockout mice. Candesartan increased the plasma adiponectin levels and the plasma levels of HMW adiponectin in both wild-type mice and Adipor1/r2 double-knockout mice. On the other hand, candesartan increased the mRNA expression levels of PPARγ and SOD1, and decreased those of TNF-α and MCP-1 in WAT of wild-type mice, but not in those of Adipor1/r2 double-knockout mice. Moreover, candesartan decreased the adipocyte sizes of WAT in wild-type mice, but not in those of Adipor1/r2 double-knockout mice.

Finally, to investigate the direct effect of candesartan on adiponectin expression, I treated differentiated 3T3-L1 adipocytes with candesartan. Candesartan significantly increased the mRNA expression levels of adiponectin, AdipoR2 and glucose transporter 4. In addition, candesartan tended to increase the mRNA expression levels of PPARγ.

DISCUSSION

I examined the effects of candesartan on insulin resistance and metabolic syndrome in vivo and in vitro. Candesartan ameliorated insulin resistance in DIO-C57BL/6J mice and db/db mice. In the current study, I showed for the first time that candesartan significantly increased both the plasma adiponectin levels and the plasma levels of HMW adiponectin in DIO-C57BL/6J mice and db/db mice. Candesartan also significantly increased the mRNA expression levels of adiponectin, AdipoR1 and AdipoR2 in WAT of db/db mice.

To assess the contribution of adiponectin signaling to the effects of candesartan on amelioration of insulin resistance, I next examined by using Adipor1/r2 double-knockout mice. Candeartan ameliorated insulin resistance in wild-type mice, but not in Adipor1/r2 double-knockout mice. These results indicate that adiponectin signaling is casually involved in the candesartan-mediated amelioration of insulin resistance. Interestingly, candesartan increased the plasma adiponectin levels and HMW adiponectin in both wild-type and Adipor1/r2 double-knockout mice. Moreover, candesartan reduces expression levels of proinflammatory adipokines, increased expression levels of SOD1 involved in reduction of oxidative stress and indeed reduced oxidative stress in WAT dependently on AdipoRs actions. These data suggest that candesartan increases adiponectin independently of AdipoRs, and also that candesartan ameliorate insulin resistance dependently on AdipoRs actions.

To investigate the direct effect of candesartan on adiponectin expression, I next examined the effects of candesartan in differentiated 3T3-L1 adipocytes. Candesartan significantly increased the mRNA expression levels of adiponectin and AdipoR2 in differentiated 3T3-L1 adipocytes. These results imply that increasing of HMW adiponectin and AdipoR2 by treatment of candesartan might alter insulin sensitivity and metabolism of adipocytes in a paracrine manner, in part.

In conclusion, the effects of candesartan on amelioration of insulin resistance might be partly through increase of HMW adiponectin and activation of adiponectin receptors (AdipoRs).

本研究は、アンジオテンシンII受容体拮抗薬カンデサルタンのインスリン抵抗性改善効果のメカニズムを明らかにするため、種々の動物モデル、3T3-L1脂肪細胞を用いて、特に、脂肪細胞への作用、アディポカインの是正に焦点を当てて検討したものであり、下記の結果を得ている。

1. 高脂肪食を負荷して食餌依存性に肥満を誘導したC57BL/6Jマウスにカンデサルタンを経口投与し、インスリン抵抗性改善効果を生化学的、分子生物学的手法を用いて検討した。インスリン抵抗性、耐糖能障害の改善作用については、経口糖負荷試験、インスリン負荷試験により評価した。血中アディポネクチン濃度はELISA法により測定し、高分子量アディポネクチンの測定にはWestern blotting法を、各臓器における遺伝子発現解析にはReal-time PCR法を用いた。さらに、白色脂肪組織の脂肪細胞サイズも測定した。結果、カンデサルタンは摂食量、体重に影響を与えずに、血中アディポネクチン濃度、高分子量アディポネクチンの上昇、炎症性サイトカインの発現量を減少させて、耐糖能異常、インスリン抵抗性を改善させた。 また、カンデサルタンにより白色脂肪組織の脂肪細胞サイズは減少した。

2. 次に、過食による肥満・糖尿病のモデルであるdb/dbマウスにカンデサルタンを経口投与し、同様の検討を行った。結果、db/dbマウスにおいても、カンデサルタンは摂食量、体重に影響を与えずに、血中アディポネクチン濃度、高分子量アディポネクチンの上昇、炎症性サイトカインの発現量を減少させて、耐糖能異常、インスリン抵抗性を改善させ、白色脂肪細胞サイズを小型化させた。さらに、db/dbマウスにおいては、カンデサルタンは脂肪組織におけるperoxisome proliferator-activated receptor (PPAR)γ、アディポネクチン受容体AdipoR1、AdipoR2 の発現量を増加させた。

3. カンデサルタンのインスリン抵抗性改善効果の解析をさらに詳細に行うために、分化した3T3-L1成熟脂肪細胞へカンデサルタンを処置し、遺伝子発現解析を行った。結果、カンデサルタンは、アディポネクチン、AdipoR2、PPARγの遺伝子発現を上昇させた。

4. 以上の結果より、カンデサルタンの耐糖能障害、インスリン抵抗性改善作用にアディポネクチン/ AdipoRシグナルが関与している可能性が考えられた。そこで、カンデサルタンの抗糖尿病作用メカニズムにアディポネクチン/AdipoRシグナルが関与しているかどうかをより詳細に検討するために、AdipoR1・R2ダブル欠損マウスを用いて検討を行った。結果、野生型マウスおよびAdipoR1・R2ダブル欠損マウスにおいて、カンデサルタンは血中アディポネクチン濃度を上昇させた。 野生型マウスにおいて、カンデサルタンは、脂肪組織における炎症性サイトカインの発現量を低下させ、また、酸化ストレスを軽減させて、耐糖能障害、インスリン抵抗性を改善させたがAdipoR1・R2ダブル欠損マウスにおいては、カンデサルタンの上記の効果は認められなかった。 また、野生型マウスにおいて、カンデサルタンは、PPARγ mRNA発現量を増加させたが、AdipoR1・R2ダブル欠損マウスでは変化しなかった。 さらに野生型マウスにおいて、カンデサルタンは、脂肪細胞サイズを減少させたが、AdipoR1・R2ダブル欠損マウスでは変化しなかった。

今回の結果より、カンデサルタンが血中のアディポネクチン濃度を増加させ、脂肪組織におけるアディポネクチン受容体を介し、炎症性サイトカインの発現低下、またPPARγ を増加させ、酸化ストレス消去に関わるSOD1の発現増加を介し、実際に脂肪組織の酸化ストレスを軽減させた。以上より、カンデサルタンのインスリン抵抗性改善メカニズムの一部にアディポネクチン/AdipoRシグナルが関与している可能性が示唆された。

以上、本論文は、カンデサルタンの抗糖尿病作用メカニズムに対するアディポネクチン/AdipoRシグナルの関与を、AdipoR1・R2ダブル欠損マウスを用いて初めて検討したものである。本研究は、未だ明らかとなっていないカンデサルタンのインスリン抵抗性改善作用のメカニズムの解明に重要な貢献をなすと考えられ、学位の授与に値するものと考えられる。