| 1. Physical Exercise Ameliorates Insulin Resistance:The Role of GRK4Regulating Dopamine D1Receptor in Skeletal MuscleType2diabetes mellitus (T2DM) should be an acknowledged risk to the health of individuals and the economies of society. Insulin resistance (IR), one of the main pathophysiological of T2DM, is one of the most important precursors and contributors to cardiovascular disease. High calorie diets, minimal physical activity and associated obesity, has been thought the environmental factors leading to IRAs the main targeted organ of insulin, skeletal muscle use the80%blood glucose under the effect of insulin, and exercise are one of the major physiological stimuli of glucose uptake into this tissue and one of the major non-medication therapies to ameliorate IR for the T2DM patients. Meanwhile, with exercise, the plasma dopamine concentration also increases, and perhaps increases the expression and function of dopamine receptor. Besides, a dopamine D1-like receptor agonist, fenoldopam, has been identified to improve peripheral insulin sensitivity in streptozotocin-induced type2diabetic rats, and the other D1agonist, SKF38393, shows similar effect. Dopamine D1-like receptor might be an associated molecule in glucose and insulin homeostasis. Dopamine D1-like receptor might be a novel pathway to regulate the insulin sensitivity after physical train. To test the hypothesis, we studied the effect of dopamine D1receptor, a dominance isoform of D1-like receptor, on insulin sensitivity in skeletal muscle after exercise.Moreover, the G protein-coupled receptor kinase (GRK) is the main regulator to G protein-coupled receptor (GPCR). The dopamine receptor familiy, as one kind of GPCR, is regulated by GRKs. Various GRKs including GRK2, GRK3and GRK5, are associated with insulin resistance. However, whether the GRK4expresses in skeleton muscle and plays a role in insulin resistance is unknown. Thus, the interaction of GRK4and dopamine D1receptor is studied in this research. 1.1MethodsThe experiments were conducted in four parts. In the first part, we observed the protein and mRNA expression and location of dopamine D1receptor in skeleton muscle and cell by immunostaining and immunoblotting.In second part, we observed the alleviation of1to4weeks treadmill exercise to insulin resistence and the blocking effect of D1-like receptor antagonist, SCH23390. The protein expression of dopamine D1receptor in skeleton muscle was also measured by immunoblotting. Moreover, an insulin resistance model of skeleton muscle cell was performed by palmitic acid (PA). We observed the increasing effect of electrical pulse stimulation (EPS)-induced cell constriction to glucose uptake and the blocking effect of SCH23390. The membrane expression of dopamine D1receptor in skeleton muscle cell was also measured.Furthermore, the mechanism of dopamine D1receptor regulating insulin signal pathway was studied in third part. We observed the effect of different concentration fenoldopam to glucose uptake and the effect of fenoldopam to Akt phosphorylation and GLUT4translocation.In fouth part, we observed the protein and mRNA expression and location of GRK4in skeleton muscle and skeleton cell by immunohistochemisty staining, immunoblotting and RT-PCR. Further studies focused on the interaction between GRK4and dopamine D1receptor in skeleton muscle. The interaction between GRK4and dopamine D1receptor was measured by confocal laser scanning with or without insulin resistence and EPS-induced constriction. Moreover, the protein expression was measured in skeleton muscle tissue from the GRK4transgenic mice, in which the activity of GRK4is increased.1.2Result1.2.1Localization of dopamine D1receptor in the skeleton muscle cellsTo investigate the localization of dopamine D1receptor, skeleton muscle cells were stained by anti-dopamine D1receptor antibody. Dopamine D1receptors were expressed in skeleton muscle cells by immunohistochemistry. Dopamine D1receptors (≈74kDa) bands were found in lysate of skeleton muscle cells and skeletal muscle tissue.1.2.2. Exercise ameliorates insulin resistance and the blocking effect of dopamine D1-like antagonist SCH23390 Exercise reduced plasma glucose level and serum insulin level in a time-dependent manner. The glucose level was significantly lower than the sedentary STZ-treat group. The serum insulin level of STZ-treat mice after1to4weeks exercise was lower than the sedentary STZ-treat mice.1to4weeks exercise also decreased HOMA-IR and ameliorated insulin resistance. The dopamine D1receptor antagonist, SCH23390, significantly increased the glucose level, insulin level and HOMA-IR of STZ-treat mice after treadmill training, blocked the effect of exercise to IR. As compared with the STZ-treat mice without treadmill training, the expression of dopamine D1receptor at protein levels began to increase at1week exercise and exhibited a time-dependent increase following exercise, and peaked at4weeks.Besides, the skeleton muscle cells were stimulated by a pulse generator in vitro for15min,30min, lh with or without D1antagonist SCH23390. EPS made the level of glucose uptake increase, which were significantly higher than PA-treated cells without EPS after1h EPS. SCH23390impaired the contraction-induced glucose uptake and significantly decreased the glucose level by incubation with the cells for1h. Meanwhile, the surface expression of dopamine D1receptor at protein levels began to increase at15min EPS and exhibited a time-dependent increase following exercise, and peaked at1hour.1.2.3. Dopamine D1-like receptor agonist fenoldpam increases glucose uptake of skeleton muscle cells via Akt-Glut4pathway.To investigate the role of dopamine D1receptor in glucose uptake, the skeleton muscle cells treated by PA (24h) were incubated with four concentrations of fenoldpam (10-8～10-5mol/L) for30min or24h. The [3H]-2-deoxy-D-glucose uptake was impaired by the effect of PA. Meanwhile,10-6-10-5mol/L fenoldpam incubation for30min and10-7-10-5mol/L fenoldpam incubation for24h significantly increased the [3H]-2-deoxy-D-glucose uptake in the PA-treat cells. PA reduced the phosphorylation level of Akt at basic state and blocked the stimulatory effect of insulin on phosphorylation of Akt. The D1-like agonist, fenoldpam, increased the phosphorylation level of Akt in PA-treated skeleton muscle cells, and improved the insulin reactivity of Akt phosphorylation. Fenoldpam also significantly increased the relative fraction of GLUT4in the membrane with or without insulin.1.2.4. The interaction between GRK4and dopamine D1receptor in skeleton muscleGRK4were expressed in skeleton muscle cells and skeleton muscle tissue by immunohistochemistry. D1receptors (≈74kDa) bands were found in lysate of skeleton muscle cells and skeletal muscle tissue. The expression of GRK4was decreased in insulin resistance C2C12cells. lh-EPS induced cell constriction did not change the GRK4protein expression but increased the membrane distribution of D1receptors. Furthermore, the increased GRK4activity decreased the D1receptors expression.1.3Conclusion1.3.1Dopamine D1receptor are expressed in skeleton muscle.1.3.2Long-term exercise increases the dopamine D1receptor protein expression in skeleton muscle; while acute constriction of skeleton muscle induces the surface expression of the dopamine D1receptor increasing.1.3.3Dopamine D1receptor increases the level of Akt phosphorylation, and the translocation of GLUT4to cell membrane, involved in the regulation of insulin sensitivity.1.3.4GRK4is expressed in skeleton muscle. The interaction between GRK4and dopamine D1receptor is involved in the regulation of insulin sensitivity.2. The GRK4Enhances the AT1R-mediated Vascular Activity in HypertensionEssential hypertension constitutes a major risk factor for stroke, myocardial infarction, and heart and kidney failure. The kidney, vasculature, and nervous system govern the long-term control of blood pressure by regulating sodium homeostasis, peripheral resistance, and central arterial stiffness. Most hormones and humoral factors regulate blood pressure via their receptors, including G protein-coupled receptors (GPCRs). Abnormal G protein-coupled receptor kinase (GRK) function has the potential to affect receptor-regulated biological responses in many physiological and pathological conditions, including hypertension. The GRK family plays an important role in the regulation of blood pressure. GRK4is distinguished from other members of the GRK family by its constitutive activity and limited tissue expression. The GRK4variants65L,142V, and486V are associated with essential hypertension in ethnically distinct populations.Increased activity of the renin-angiotensin system is important in the pathogenesis of hypertension. GRK4interacts with the renin-angiotensin system to regulate blood pressure. Increased renal expressions of both GRK4and angiotensin type1receptor (AT1R) contribute to the increased blood pressure in SHRs. Conduit and resistance arterial vessels are important in the regulation of blood pressure and myocardial function. Whether or not GRK4and the ATIR interact in the aorta and other arteries in regulating vascular smooth muscle function is not known. Thus, the interaction of GRK4and AT1R in VSMC is studied in this research.2.1MethodsThe experiments were conducted in three parts. In the first part, we observed the protein and mRNA expression and location of GRK4in artery by immunostaining, immunoblotting and RT-PCR.In second part, we observed the effect of Ang Ⅱ and ARB (candesartan) to blood pressure and vascular activity of GRK4. A142V transgenic mice and the expression of AT1R in artery from GRK4. A142V transgenic mice.Furthermore, the mechanism of GRK4regulating AT1R in VSMC transduced with hGRK4142V was studied in third part. At first, we observed the stimulation effect of Ang Ⅱ and the blocking effect of ARB to intracellular calcium concentration. Besides, the protein and mRNA expression were measured by immunoblotting and RT-PCR. And we also tested the level of AT1R protein degradation and AT1R phosphorylation. Furthermore, the interaction between GRK4and AT1R was measured by co-immunoprecipitation and confocal scanning. As a regulator of AT1R promoter activity, we measured NF-B binding to the AT1R promoter. And the AT1R expression was measured after blocking NF-B with an NF-B inhibitor, BAY11-7082.2.2Result2.2.1Expression of GRK4in arteryWe first determined GRK4is expressed in the aorta by immunofluorescence, immunoblotting, and RT-PCR. Immunofluorescence microscopy showed GRK4staining in the tunica media and adventitia of aortae from SD rats and C57BL/6J mice. GRK4expression was also found with immunoblotting; specific GRK4(54kDa,60kDa,65kDa) bands were found in A10cells, which were attenuated, especially the60kDa band, after transduction with the specific GRK4siRNA. RT-PCR showed the expected125bp GRK4band, based on the primers.To confirm the GRK4expression in the adventitia, we checked the GRK4expression in fibroblasts and adipocytes by immunoblotting and RT-PCR. We found that both fibroblasts and adipocytes expressed GRK4. Removal of the adventitia did not affect the Ang II-mediated vasoconstriction, indicating that the GRK4in the adventitia did not take participate in the Ang II-mediated vasoconstriction.2.2.2Blood pressure and vascular activity in hGRK4142V transgenic mice and artery AT1R expressionTo further investigate the physiological role of the GRK4-regulated AT1R expression, we studied AT1R expression and function in hGRK4WT and hGRK4142V transgenic mice. Anesthetized hGRK4.142V transgenic mice had higher systolic, diastolic and mean blood pressures than anesthetized hGRK4. WT transgenic mice. The intravenous infusion of Ang II caused a greater increase in SBP in hGRK4.142V than hGRK4WT transgenic mice while the intravenous infusion of candesartan caused a greater decrease in blood pressure in hGRK4142V than hGRK4WT transgenic mice.We also studied the vasoconstrictor effect of Ang II on the aorta from hGRK4142V and hGRK4. WT transgenic mice. The vasoconstriction caused by Ang II was greater in hGRK4.142V than hGRK4. WT transgenic mice in the presence or absence of the endothelium. The AT1R blocker, candesartan (10-6M) blocked the vasoconstrictor effect of Ang II, in both transgenic mice such that there was no longer any difference between the two mouse strains. AT1R expression in aorta was higher in hGRK4.142V than hGRK4WT transgenic mice.2.2.3Regulation by GRK4of AT1R expression and function in A10cellsTo determine the effect of hGRK4on AT1R expression, we used A10cells transduced with hGRK4142V. The intracellular calcium concentration after stimulation with Ang II was higher in hGRK4142V-than hGRK4WT-transduced cells. Then we found AT1R protein and mRNA expressions were higher in hGRK4.142V than GRK4. WT cells; AT1R protein degradation was lower in hGRK4142V-than GRK4. WT-transduced cells. In addition, AT1R phosphorylation was lower in hGRK4142V-than hGRK4WT-transduced cells.An additional study found a co-localization and co-immunoprecipitation between GRK4and AT1R; the co-immunoprecipitation of GRK4and AT1R was less in hGRK4142V-than hGRK4. WT-transduced cells, which could be a factor in the decreased phosphorylation of AT1R in hGRK4142V-transduced cells. As a regulator of AT1R promoter activity, we measured NF-B binding to the AT1P promoter and found it higher in hGRK4142V-than hGRK4. WT-transduced cells. Blockade of NF-B with an NF-B inhibitor, BAY11-7082, inhibited the increase in AT1R expression in hGRK4.142V-transduced cells, indicating that NF-B was involved in the positive regulation of AT1R expression by hGRK4.142V.2.3Conclusion2.3.1GRK4is expressed in VSMCs of the aorta.2.3.2GRK4142V increases the Ang Ⅱ-mediated vasoconstriction, lead to higher blood pressure.2.3.3GRK4.142V increases arterial AT1R receptor expression and function, which may be involved in the abnormalities of conduit vessels in essential hypertension. |
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