Long-term Aerobic Exercise Improves Vascular Insulin Sensitivity And Alleviates Hypertension

Background:There are at least 270 million hypertensive patients in China, accounting for 20% of all hypertensive patients globally and contributing to more than 1.5 million deaths per year. In the worldwide, approximately 1 billion people have hypertension. Hypertension is called A Silent Killer to those patients. Prehypertension is defined as BP in between 120~139/80~89 mm Hg. Prehypertension is truly highly prevalent recent years, with 38.9%~41.3% of the population suffered from it in China. Prehypertension independently increases the risk of hypertension and subsequent cardiovascular events. Lifestyle modifications or drug intervention that can delay progression from prehypertension to hypertension would be of great value.Impaired insulin signaling has been found to be associated with hypertension. Previous studies demonstrated the relationship between hypertension and systemic insulin resistance including hyperglycemia and hyperinsulinemia. More importantly, our laboratory found that vascular insulin resistance, which is characterized by blunted insulin-induced vasorelaxation or imbalance of insulin signaling, occurs before hypertensive in y SHRs, suggesting that vascular insulin resistance is an important pathophysiological change occurs before hypertension and may cause impaired cardiovascular structure and function. However, how reduced vascular insulin sensitivity occurs in prehypertension was not clear.Adiponectin is an adipocytokine secreted by adipose tissue and is normally present in plasma. Adiponectin is reported as a potent insulin enhancer linking adipose tissue and glucose metabolism. It also exerts roles of anti-inflammation and cardiovascular protection. Clinical observation has revealed that hypoadiponectinemia is significantly associated with hypertension, suggesting that reduced adiponectin is one of the independent risk factors for hypertension and also a predictor for the development of hypertension. However, the relationship between adiponectin levels and vascular insulin resistance before hypertension has never been identified. On the other hand, meta-analysis of randomized controlled trials shows chronic dynamic aerobic endurance training reduces BP. Therefore, regular physical exercise is broadly recommended by current European and American hypertension guidelines as first-line therapy. Endurance training decreases BP, in part, through reducing systemic peripheral vascular resistance. However, underlying or alternate mechanisms involved in the BP lowering effects of exercise remain largely elusive. The effect of exercise on vascular insulin resistance is unclear.So, the aims of the study are: 1) to evaluate whether and how hypoadiponectinemia influences insulin-induced vasodilation of resistance vessels using normotensive y SHRs; 2) to investigate whether exercise training beginning at the prehypertensive stage mitigate hypertension by enhancing vascular insulin sensitivity and the mechanism.Methods:1. Animal blood pressure was measured Using Softron BP-98 A system. Fasting blood glucose and insulin levels were measured with the use of a blood glucose meter((Lifescan, USA) and an RIA test kit, respectively. Insulin sensitivity using the quantitative insulin sensitivity check index(QUICKI) was calculated using the following formula: QUICKI = 1/[log(I0) + log(G0)], where I0 is fasting insulin(μU/ml), and G0 is fasting glucose(mg/dl). Serum adiponectin levels were measured using an adiponectin Elisa kit.2. Functional assessment of murine mesenteric arterioles. Mesenteric arteriole segments were isolated and mounted in a temperature-controlled myograph. An optimal passive tension(~2 m N) was applied for 1 hour before the experiments were started. Mesenteric arteriolar segments were precontracted with 1 μmol/L PE. A dose-response curve was obtained by cumulative addition of ACh and insulin. Relaxation at each concentration was measured.3. The swim training were performed on SHRs in the exercised groups. Animals were trained free of loading, 5 days/wk for 10 wk in a 60 cm × 60 cm tank filled with 50 cm depth of water at 33~35℃. All training sessions took place during the morning hours(9:00 AM–11:00 AM).4. Total RNA was extracted and synthesized to c DNA using the reverse transcription reagent kit. Expression analysis of the reported genes was performed by real-time PCR. Protein samples were extracted and the concentrations were measured using a BCA method. Protein expression and phosphorylation were detected by Western blot analysis.5. Total NO production in culture medium was determined by a NO detective kit with a modified Griess reaction method. Levels of ET-1 in the tissue and media were measured with a sensitive ELISA kit.6. Human umbilical vein endothelial cells(HUVECs) were grown in endothelial cell basal medium. HUVECs were transfected with si RNA or scramble control by Lipofe ctamine? 2000 following the manufacturer’s instructions. At 24 h, 48 h or 72 h after transfection, cells were collected and the efficiency of gene knockdown was detected by Western blot analysis.Results:1. Young 4-week SHRs(y SHRs) had no significant difference in systolic blood pressure, or systemic insulin sensitivity as assessed by QUICKI when compared with their age-matched Wistar-Kyoto(WKY) counterparts(y WKY). Both sodium nitroprusside(SNP, an endothelium-independent vasodilator) and acetylcholine(ACh, an endothelium-dependent vasodilator) stimulated vasodilation of mesenteric arterioles were comparable between y SHRs and y WKY rats. The ability of insulin to cause dose-dependent vasorelaxation was significantly impaired in vascular segments from y SHRs than those from y WKY rats(26.59 ± 2.53 % vs. 45.59 ± 4.22 %, P < 0.05). Insulin-induced phosphorylations of e NOS and Akt were both significantly impaired but insulin-stimulated phosphorylation of ERK1/2 was significantly enhanced in y SHRs arteries than that of y WKY rats, suggesting impaired inulin sensitivity in resistance vessels from y SHRs whose blood pressure had not been increased.2. Serum adiponectin concentration and adiponectin m RNA level in adipose tissue from y SHRs were both significantly lower than that in y WKY rats. The transcript and protein levels of Adipo R1 and Adipo R2 in mesenteric arteries were decreased in y SHRs. Furthermore, APPL1 transcription and protein levels in mesenteric arteries were downregulated in y SHRs.3. After 1 week of treatment with recombinant human globular domain of adiponectin(g Ad), insulin-induced vasorelaxation was partially restored in vascular segments from y SHRs. Insulin-induced phosphorylations of Akt and e NOS were markedly improved while phosphorylated ERK1/2 and ET-1 m RNA level were reduced in g Ad treated y SHRs. Western blotting analysis revealed that g Ad treatment markedly increased APPL1 expression, consistent with APPL1 m RNA level detected in mesenteric arteries.4. Homozygous adiponectin KO(APN-/-) mice whose serum adiponectin concentration and adiponectin m RNA level in adipose tissue were significantly reduced. APN-/-mice also showed a reduced endothelial function and impaired sensitivity of insulin in inducing vasorelaxation. APPL1 m RNA and protein levels in the mesenteric arteries were all decreased. In addition, insulin-induced phosphorylations of e NOS and Akt were both significantly reduced and insulin-stimulated ERK1/2 phosphorylation was significantly increased in arteries of APN-/- mice compared with WT. Interestingly, g Ad treatment for 1 week upregulated APPL1 expression and subsequently reverse the insulin-stimulated signaling and vasodilation in mesenteric arteries of APN-/- mice.5. Treatment with g Ad increased APPL1 expression in a dose- and time-dependent fashion in HUVECs with scrambled si RNA, but not si RNA of Adipo R1 and Adipo R2. Incubation with g Ad significantly upregulated APPL1, cytoplasm LKB1 levels, and AMPK activation; reduced PTEN expression; thus increased insulin-induced Akt/e NOS phosphorylations, and decreased phosphorylation of ERK1/2. This was accompanied by increased NO release and decreased ET-1 level in the culture medium. Suppression of APPL1 expression mediated by si RNA in HUVECs significantly blocked its downstream LKB1 translocation from nuclear to cytoplasm, changed AMPK activation, PTEN expression, and consequently inhibited the insulin-sensitizing effects induced by g Ad.6. SHRs were subjected to 10 wk of exercise training(SHR-EX). SBP in SHR-EX was markedly reduced compared with that in SHR-SED, suggesting the effects of exercise on reducing high BP. It was also observed that SHRs showed greater systemic insulin sensitivity after 10 wk of swim training compared with their sedentary counterparts. The ability of insulin to cause dose-dependent vasorelaxation was significantly impaired in vascular segments from SHR-SED when compared with samples from WKY rats. This indicates that resistance vessels of SHRs have impaired vasodilator actions in response to insulin. Importantly, 10 wk of exercise markedly improved vascular insulin sensitivity as evidenced by increased vasodilator response to insulin.7. GRK2 expression was significantly increased in SHR-SED when compared with samples from WKY rats. Importantly, GRK2 expression was significantly decreased in SHRs after exercise for 10 wk when compared with samples from SHR-SED. Furthermore, 10 wk of exercise opposed enhancement of GRK2 activity in SHRs as assessed by phosphorylation of GRK2 at Ser670. Insulin-evoked phosphorylation of vascular Akt at Thr308/Ser473 and phosphorylation of e NOS at Ser1177 was markedly decreased in SHR-SED when compared with samples from WKY rats. Conversely, insulin-induced phosphorylation of both vascular Akt and e NOS were enhanced in SHR-EX.8. GRK2 knockdown by si RNA significantly increased insulin-stimulated Akt and e NOS phosphorylation. Similarly, preincubation with a relatively specific GRK2 inhibitor also enhanced the vasodilator actions of insulin as well as insulin-stimulated Akt and e NOS activation in arterioles from the SHR-SED group. Thus GRK2 inhibition by several methods mimicked the effect of exercise to improve vascular insulin sensitivity in SHRs.9. The augmentation of insulin-induced vasodilation in SHR after 10-wk exercise training was opposed by delivery of GRK2 using Chariot reagent to the arteriole segments that presumably resulted in overexpression of GRK2. Importantly, after Chariot-mediated delivery of GRK2, insulin-induced activation of Akt and e NOS were also significantly reduced. Thus GRK2 overexpression opposed the effect of exercise on improving vascular insulin sensitivity in SHRs and provides further evidence that GRK2 mediates the effects of exercise to improve vasodilator actions of insulin that help to reduce BP.Conclusions:1. Hypoadiponectinemia contributes to vascular insulin resistance by inducing low-APPL1 expression and differentially modulating the activation of Akt/e NOS/NO and ERK1/2/ET-1 in vascular endothelium in normotensive y SHRs.2. Exercise training beginning at the prehypertensive stage in SHRs ameliorates hypertension via opposing development of vascular insulin resistance, which is at least partly attributable to exercise-induced downregulation of GRK2 in resistance vessels in SHRs. Therefore, restoration of endogenous adiponectin production, lifestyle interventions including physical activity and treatment targeting APPL1 or GRK2 may have potential therapeutic value in the prevention and alleviation of vascular insulin resistance and hypertension.