Resistin Induces Hypertension and Insulin Resistance in Mice via a TLR4-Dependent Pathway

Resistin, an adipokine involved in insulin resistance (IR) and diabetes, has recently been reported to play a role in cardiovascular events. However, its effect on blood pressure (BP) and the underlying mechanisms remain unclear. In the present study, we showed that resistin induces hypertension and IR in wild type (WT) mice, but not in tlr4−/− mice. Resistin upregulated angiotensinogen (Agt) expression in WT mice, whereas it had no effect on tlr4−/− mice, or in mice treated with the angiotensin-converting enzyme inhibitor perindopril. Real-time PCR and chromatin immunoprecipitation further confirmed that resistin activates the renin-angiotensin system (RAS) via the TLR4/P65/Agt pathway. This finding suggested an essential role of resistin in linking IR and hypertension, which may offer a novel target in clinic on the study of the association between diabetes and hypertension.

Resistin activates the renin-angiotensin system by upregulating Agt expression. To investigate the mechanistic basis for resistin-induced hypertension, the mRNA levels of certain BP-regulatory genes were measured. Resistin significantly upregulated Agt mRNA expression in the liver of WT mice, whereas it had no effect in tlr4 −/− mice (Fig. 3A). Plasma ANG II level, the active form of Agt, was not significantly different, although a trend toward an increase in resistin-treated mice was observed (Fig. 3B). Similar mRNA levels of angiotensin-converting-enzyme (ACE), endothelial nitric oxide synthase (eNOS), and endothelin receptors A (ETA) and B (ETB) were detected in the lungs of WT and tlr4 −/− mice ( Fig. 3C-F). In addition, renin (Ren), angiotensin-converting-enzyme 2 (ACE2) and angiotensin II receptor type 1a (Agtr1a) levels were not affected by resistin in WT mice (data not shown). Similar results were obtained in in vitro studies. After 24 h of resistin treatment, Agt mRNA was significantly upregulated in HepG2 cells (Fig. 3H), whereas endothelin-1 (ET-1) and eNOS levels remained constant in EA.hy 926 endothelial cells (Fig. 3I). Subsequently, siRNA was used to inhibit tlr4 expression. After 24 h, Agt expression was detected in HepG2 cells. The data showed siRNA dramatically suppressed tlr4 expression and this effect blocked the stimulation effect of resistin on Agt expression ( Fig. 3G-H). These data indicated that resistin specifically stimulates Agt expression in the liver and this effect is TLR4-dependent.
As a precursor of Angiotensin I (ANG I), Agt is crucial to the renin-angiotensin system (RAS), which is known as a classical blood pressure regulation system. Therefore, the signal transduction pathway of resistin was further examined by blocking the RAS using the ACE inhibitor perindopril (peri). Resistin had no effect on SBP and DBP in WT mice pre-treated with perindopril ( Fig. 4A), and plasma glucose levels were also unchanged ( Fig. 4B). Moreover, resistin-induced upregulation of Agt was inhibited by pre-treatment with perindopril (Fig. 4C). These findings indicate that resistin-induced hypertension is dependent on the activation of the RAS.
Resistin induces Agt expression by activating the TLR4/P65 pathway. In our previous study, resistin upregulated p65 mRNA and protein expression 13 . Consistently, in the present study, hepatic p65 expression was upregulated in WT mice treated with resistin, but not in tlr4 −/− or perindopril-treated mice (Fig. 4D). Deb et al. identified a p65 binding site in the mouse Agt promoter, implying that p65 can directly activate Agt transcription 14 . To determine whether p65 is involved in resistin-induced Agt upregulation, we used ChIP to quantify the occupancy of p65 on the Agt promoter. Our data showed that resistin treatment increased binding of p65 to the Agt promoter in WT mice, but this effect was abolished in tlr4 −/− mice (Fig. 4E). These results support that resistin activates the RAS through the TLR4/P65/Agt pathway.

Discussion
Epidemiological studies have demonstrated that patients with diabetes are more likely to develop hypertension, which increases their risk for CVD 2 . Moreover, as a major pathological feature of type 2 diabetes, IR has often been reported occur in the people with essential hypertension, suggesting these two pathological conditions may develop through a shared pathway 3,15 . Resistin has been suggested to induce IR and is associated with the development of CVD 8 . However, the role of resistin in the regulation of BP and the development of diabetes and hypertension remains unclear. In the present study, we investigated the effect of resistin on BP and IR. Our data showed that resistin induces both IR and hypertension in mice and these effects are TLR4-dependent.
Studies in rodents and humans have suggested that resistin promotes IR 6,7 , although contradictory findings have also been reported 16,17 . In the present study, plasma glucose and insulin levels, as well as HOMA-IR, were elevated in WT mice after resistin treatment (Fig. 1C-E), confirming that resistin induces IR. To investigate the role of resistin in the development of hypertension, WT and tlr4 −/− mice were treated with resistin for 6 days. Both SBP and DBP increased in WT mice, whereas no changes in BP were observed in tlr4 −/− mice, indicating (C) Plasma insulin levels; (D) HOMA-IR was calculated using the following formula: fasting glucose (mmol/L) × fasting insulin (mU/ml)/22.5. Retn group was injected with 400 ng/day resistin, while the control group was injected with PBS. Injections were performed once per day for 6 consecutive days. Data are presented as mean ± sd (n = 9). *P < 0.05, **P < 0.01. that resistin likely regulates BP through TLR4-mediated signaling. In addition, resistin had no effect on plasma glucose and insulin levels or HOMA-IR in tlr4 −/− mice (Fig. 2B-D), suggesting that resistin-induced IR is also TLR4-dependent.
Previous studies have implicated resistin in the regulation of certain vasoconstrictors and vasodilators 9,18-20 . Here, we linked resistin with RAS, which is a hormone system involved in BP regulation. As a precursor of ANG II, Agt is released from the liver and converted into ANG I through the action of renin. Then, ANG I is converted to ANG II (a major RAS effector) by ACE 21,22 . An increase in Agt expression could cause an increase in ANG II and activation of RAS, leading to an elevation in BP. In the present study, Agt expression in the mouse liver was significantly upregulated by resistin treatment (Fig. 3A). Pre-treatment of mice with the ACE inhibitor perindopril abolished resistin-induced hypertension and IR, suggesting that the action of resistin is inhibited when the RAS is blocked. There is considerable evidence demonstrating the activation of the NF-κ B pathway by resistin [23][24][25] and resistin was previously found to enhance p65 mRNA and protein expression in the liver 13 . As a major transcription factor in the NF-κ B pathway, p65 (Rel A) can directly bind to the promoter of Agt and thereby upregulate Agt expression 14 . Therefore, we presumed that resistin-induced upregulation of Agt expression could be mediated by p65. Quantification of mRNA levels indicated that resistin upregulated the expression of p65 in WT mice, but not in tlr4 −/− or perindopril-treated mice (Fig. 4D), corresponding to the upregulation of Agt expression. ChIP data showed that resistin promoted the binding of p65 to the Agt promoter in WT but not tlr4 −/− mice (Fig. 4E), indicating that resistin upregulates Agt expression via the TLR4/p65 pathway.
Unlike mice, in human, resistin is produced mainly in macrophages rather than in adipocytes 26 , therefore, whether its effect on rodents is translatable to human is not fully confirmed 27,28 . However, an elevated resistin level has been observed in the patient with IR or type 2 diabetes in many clinical studies and in the patients with essential hypertension according to some studies 29,30 . This also fits to the finding in our mouse model on the importance of resistin linking IR and hypertension. However, dual to the complexity in human epidemiology, it's difficult to address whether resistin is an active player in the development of those pathological conditions especially hypertension in diabetes patients. Therefore, some literatures suggest effect of resistin on diabetic hypertension may be through elevated ET-1 or reduced eNOS 31,32 which we didn't observed in our mouse model (Fig. 3D) as well as in vitro study (Fig 3F). Here, we have found the role of resistin in the activation of RAS in our mouse model which offered another possible explanation for the association of resistin with diabetes and hypertension.
Taken together, our data indicate that resistin induces IR and hypertension in mice via a mechanism dependent on TLR4 and RAS, suggesting that resistin is a crucial factor linking diabetes and hypertension. The results of the present study support the existence of a common pathway underlying the development of diabetes and hypertension, and provide a novel animal model to further investigate this issue.

Methods
Animals. Male C57BL/10 (wild-type, WT) and C57BL/10ScN (TLR4 gene deleted type, tlr4 −/− ) mice (7-8 weeks old) received daily intravenous injections of PBS or 400 ng/day of recombinant mouse resistin (R&D Systems, Minneapolis, MN, USA) for six consecutive days. For the renin-angiotensin system (RAS) blocking experiment, mice were pre-treated one day prior to the start of the injection regimen with the angiotensin-converting enzyme (ACE) inhibitor perindopril (5 mg/kg; mBbio, Nanjing, China) by oral administration, and perindopril was administered 30 min before each injection. BP was measured with a tail-cuff system (BP-98A; Gene & I Co. Ltd, Beijing, China). Retro-orbital blood was collected after 12 h of food withdrawal and mice were directly sacrificed afterwards. Plasma glucose levels were determined by the glucose oxidase method using a glucose determination kit (Applygen Technologies Inc, Beijing, China). Insulin and resistin levels were measured with an insulin ELISA kit (Xinqidi Biological Technology Co. Ltd, Wuhan, China).All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of Hubei wild-type mice pre-treated with perindopril (Peri). BP was measured before resistin treatment (day 0, D0) and after 6 days of resistin treatment (day 6, D6); (B) Plasma glucose levels in mice exposed to different treatments; (C) Agt and (D) p65 expression in mice exposed to different treatments and in different mouse lines; (E) Binding of p65 to the Agt promoter was determined by chromatin immunoprecipitation. Retn group was injected with 400 ng/day resistin, while the control group was injected with PBS. Perindopril (5 mg/kg/day) was administered orally for 7 days (animals were treated as described in Materials and Methods). Data are presented as mean ± sd (n = 8). *P < 0.05, **P < 0.01.