| BackgroundObesity represents one of the main health problems in modern societies and has long been related to insulin resistance and increased cardiovascular diseases. Recent evidence from animal studies has shown that insulin resistance plays a common causal role in the development of endothelial dysfunction. Insulin, in addition to its essential metabolic actions, has important vascular actions involving stimulation of the production of nitric oxide(NO) from endothelia, leading to vasodilation and increased blood flow in tissue perfusion. The integrity of endothelial cell insulin signaling plays a critical role in determining vessel endothelial function and NO bioavailability in response to conventional agonists. Therapeutic interventions that improve endothelial function and/or insulin sensitivity attenuate metabolic and cardiovascular abnormalities in animal and clinical investigations. However, the upstream molecules regulating endothelial insulin sensitivity have not been clarified. Mitochondrial reactive oxygen species(mt ROS) has recently been implicated as key regulators of vascular homeostatic functions under basal conditions in a variety of vascular beds, where, in particular, they contribute to endothelium-dependent vasodilation, whereas excessive mt ROS generation is considered to cause deleterious vascular cell signaling and subsequent endothelial dysfunction. Sirtuin 3(SIRT3) is a mitochondrial NAD+-dependent deacetylase that governs mitochondrial metabolism and reactive oxygen species(ROS) homeostasis. SIRT3 has been reported to be closely involved in regulation of mitochondrial function in metabolic syndrome, aging and pulmonary arterial hypertension. Recent studies reported that SIRT3 protects endothelial cells from high glucose-induced cytotoxicity and Ang II-induced endothelial dysfunction. However, its contribution to endothelial homeostasis and vascular function remains unknown. We found impaired insulin-induced mesenteric vasorelaxation in obese patients that is associated with reduced vascular SIRT3 expression. We further identified SIRT3 as a critical protector against obesity-induced vascular insulin resistance and resultant endothelial dysfunction.Aims1. To determine whether or not SIRT3 is involved in HFD-induced endothelial insulin resistance and endothelial dysfunction. 2. To further explore the underlying mechanism of HFD-induced endothelial insulin resistance associated with SIRT3 deficiency.Methods1. Twelve morbid obese subjects underwent bariatric surgery in Xijing Hospital, with a body mass index(BMI) ≥ 30 kg/m2, HOMA-IR ≥ 3.8. Subjects with history or clinical evidence of cardiovascular diseases(congestive heart failure, ischemic heart disease and hypertension) were excluded, but not those with type 2 diabetes or dyslipidemia. Control samples were obtained from the eight subjects(BMI﹤30 kg/m2, HOMA-IR﹤3.8) who underwent abdominal surgery(hiatus hernia repair, achalasia or cholecystectomies). Exclusion criteria for this control group were: diabetes, hypertension, history or clinical evidence of cardiovascular disease and any other condition that might interfere with the progress of the study. Insulin resistance was estimated by calculating the score for Homeostasis model assessment of insulin resistance(HOMA-IR). Small arteries(approximately 2 mm length) were isolated from visceral fat obtained from insulin-resistant morbid obese and non-obese human subjects during surgery. 2. At the age of 8 wk, male SIRT3 knock out(KO) and wild-type(WT) mice were randomized into two groups and maintained on a normal diet(ND) or a high fat diet(HFD) for 24 wk. Mice were sacrificed and the descending aorta was carefully excised. The contractile force was recorded using a Power Lab Chart v 7.2.1 program. Aortic rings were precontracted with phenylephrine(PE, 10-5 M). Endothelium-dependent vasorelaxation evoked by acetylcholine(ACh, 10-10 to 10-5 M) and insulin(INS, 10-9 to 10-6 M) and endothelium-independent vasorelaxation evoked by cumulative sodium nitroprusside(SNP, 10-10 to 10-5 M) were expressed as percent contraction determined by the percentage of inhibition to the precontracted tension. 3. Extracting vascular protein from human subjects and mice. Western blot was used to detect the level of SIRT3, Akt, phospho(p)-Akt(Ser473), p-e NOS(Ser1177), e NOS, SOD2, acetyl-SOD2(Lys68) or β-actin. Real-time PCR was used to measure the level of SIRT3 m RNA and 18 S r RNA served as an endogenous control. 4. The endothelial cells were exposed to palmitate(500 μM) for 24 h. For the insulin stimulation experiments, the cells were treated with 100 n M insulin for 20 min. Intracellular NO was examined with NO sensitive fluorescent dye diaminofluorescein-2 diacetate(DAF2 DA) and cells were imaged by a Nikon Eclipse Ti-E inverted fluorescence microscope. Total nitric oxide production(NOx) in media supernatant of cultured cells was determined by measuring the concentration of nitrite, a stable metabolite of NO with a modified Griess reaction method. 5. Small interfering RNA(si RNA) specifically targeting SIRT3 m RNA and their scramble RNA were transfected into HUVECs to down-regulate SIRT3 expression. The HUVECs were infected with lentivirus(Lv)-SIRT3 or control Lv-p CMV at a multiplicity of infection(MOI) of 10 to restore SIRT3 expression. The mitochondria-targeted O2?--sensitive fluorophore Mito SOX Red was used to detect the mt ROS and cells were imaged using a confocal microscope.Results1. Vascular SIRT3 protein expression was significantly lower in morbid obese subjects compared with non-obese control subjects. More importantly, small mesenteric arteries from morbid obese subjects showed a significant reduction in the vasodilation response to insulin at a physiological concentration of 10-10 M compared with control subjects. 2. SIRT3 m RNA and protein expression of aorta significantly decreased in mice fed with 12-wk-HFD and dropped to about 25% after 24-wk-HFD feeding compared with ND feeding. More importantly, there was a significant reduced vasorelaxation to insulin in aorta from HFD-feeding mice compared with mice fed with ND, while no difference was observed in vasorelaxation to SNP between the two groups. 3. Western blot revealed that palmitate excess had no effects on the basal phosphorylation or protein levels of Akt and e NOS in endothelial cells. However, under insulin stimulation, phosphorylation of Akt(Ser473) and e NOS(Ser1177) were decreased significantly in palmitate-treated cells followed by decreased NO production, indicating that endothelial response to insulin was reduced. Compared with vehicle-treated cells, there was a moderate but not significant reduction of SIRT3 expression at 4 and 8 h of palmitate exposure. However, we observed a nearly 25% and 50% reduction in SIRT3 protein expression at 16 and 24 h of palmitate-exposure, respectively.4. SIRT3 si RNA markedly reduced SIRT3 protein expression by about 80%. SIRT3 knockdown resulted in a significant decrease in insulin-stimulated Akt and e NOS phosphorylation in endothelial cells. Accordingly, a remarkably reduced insulin-stimulated NO production was observed in SIRT3-knockdown cells. There were no significant differences in the total protein levels of Akt and e NOS between SIRT3-knockdown and negative control cells. HUVECs with Lv-SIRT3 showed a significant increase in SIRT3 expression compared with Lv-p CMV- infected cells. Importantly, overexpression of SIRT3 by Lv-SIRT3 transduction abolished the palmitate-induced inhibition on both insulin signaling and insulin-stimulated NO production. 5. SIRT3 deficiency significantly impaired IPGTT and IPITT in mice on HFD compared with HFD-induced WT mice. There were no significant differences in vascular relaxations to insulin or ACh between SIRT3 KO and WT mice on ND. However, SIRT3 KO mice fed with HFD for 24 wks showed a more pronounced impairment of vascular response to both insulin and ACh compared with obese mice. Endothelium-independent relaxations to SNP were similar in all groups. 6. Compared with WT mice on HFD, SIRT3 KO mice on HFD showed a significant mitochondrial O2?- increase in the aorta as detected by Mito SOX fluorescence. Both palmitate expousure and si RNA-mediated knockdown of SIRT3 resulted in a marked elevation in mitochondrial O2?- generation while overexpression of SIRT3 by Lv-SIRT3 infection significantly inhibited palmitate excess-induced mitochondrial O2?-overproduction in endothelial cells. SIRT3 knockdown led to a reduction in insulin-stimulated NO release which was abolished by elimination of mt ROS with Mito TEMPO. Pretreatment with Mito TEMPO significantly alleviated the impairment of endothelium-dependent relaxation to insulin in the aortae from SIRT3 KO mice on HFD. Besides, a significant increased acetylation of SOD2(Lys68) was observed in aorta from WT mice on HFD compared with the WT mice on ND. However, there was no difference in total level of SOD2 protein betweem the two groups.ConclusionsSIRT3 deficiency and resultant mt ROS overproduction leads to endothelial insulin resistance and contributes to endothelial dysfunction in obesity. Overexpression of SIRT3 improves endothelial insulin sensitivity through inhibiting mitochondrial oxidative stress. These findings reveal a novel role for SIRT3 in regulating endothelial insulin sensitivity and provide further compelling support for SIRT3 as a potential therapeutic target for obese-related vascular diseases. |
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