Telmisartan Improves Insulin Resistance Through PPARδ Activation In Mice

Background and Purpose: With the change of lifestyle and diet, the incidence ofobesity, type2diabetes and other metabolic diseases increase year by year. Type2diabetesis mainly caused by insulin secretion insufficient or insulin dysfunction.It is due to thedecreased biological activity of certain amount insulin in the body, including decreasedinsulin sensitivity and reactivity. Insulin resistance are closely related glucose metabolismabnormalities, so it is important to prevention type2diabetes and related metabolicdisorders through improving insulin resistance and glucose metabolism disorders.A type of anti-hypertension drugs, angiotensin II receptor blockers (ARBs), commonlyused to treat hypertension patients combined with diabetes mellitus or diabetic nephropathy.The clinical trials found the ARBs drugs may improve insulin resistance, reduce theincidence of hypertension patients with diabetes.In recent years, some ARBs drugs, such astelmisartan, irbesartan and losartan, are also study about the role of increasing insulinsensitivity.Clinical trials have shown that telmisartan not only improve insulin resistance ofhypertension patients with type2diabetes, stable their blood glucose, but also increaseinsulin sensitivity in patients not with diabetis. Animal experiments also confirmed thattelmisartan improve insulin resistance in obese rats by reducing inflammation.Some angiotensin II type1receptor blockers (ARBs) have been proven to activateperoxisome proliferator-activated receptors (PPARs) effectively. High doses telmisartanincrease the high molecular weight adiponectin levels in diabetic patients with hypertensionand improve insulin resistance by activating PPARγ. The study found that the decreasedsensitivity of increased adiponectin levels and the glucose transported to lipid droplets ininsulin-stimulated adipose tissue caused by telmisartan, when the PPARγ knockout. Incontrast, the muscle specific PPARγ knockout, telmisartan don’t affect adiponectin levels orglucose metabolism, nether in fat nor muscle. Therefore, telmisartan increasing adiponectinlevels and stimulate the glucose utilization in adipose tissue is dependent on PPARγ. Thediabetic hypertensive rats has been improved in addition to the expected preventive antihypertensive effects after telmisartan treatment. Telmisartan also prevent the increase ofserum triglycerides and promote the lipid droplets reduction in the liver tissues. Themechanism may be the restorations of mRNA expressions of PPARγ, PPARδ, PGC1α,adiponectin, adiponectin receptor and glucose transporter4in liver, fat and muscle tissuesby telmisartan.Therefore, telmisartan increased insulin sensitivity have been confirmed bothin clinical and basic researches, but the exact mechanisms of telmisartan playing rolesthrough which molecules or pathways are unclear and in need of further studies.PPARδ (also known as PPARβ) widely express in various tissues, particularly inskeletal muscle with a high level expression. The expression amount is the50and10timeson PPARγ and PPARα respectively. Recent studies indicate PPARδ is important role inglucose metabolism and insulin roles in skeletal muscle. Kramer et al. activates PPARδ inprimary cultured human skeletal muscle cells directly increase fatty acid transportation andglucose uptake, and then promote blood lipid/glucose metabolism and related genesexpressions. PPARδ regulates glucose homeostasis of the whole body confirmed inmuscle-specific PPARδ transgenic mice. Schuler et al. showed PPARδ affects peripheralinsulin sensitivity through the phenotypes of muscle fiber type conversion, obesity and type2diabetes in skeletal muscle-specific PPARδ deletion mice. The PPARδ agonist increasesmitochondria density and the expressions of fatty acid oxidase and peroxisomal β oxidasein mice skeletal muscle. PPARδ agonist GW501516decrease fasting plasma triglyceridesand improve insulin resistance by increasing fatty acid oxidation in skeletal muscle ofmetabolic syndrome patients after two weeks’ treatment. The effect is more obviouscomparing with PPARα agonist GW590735.However, PPARδ-specific agonist GW501516cannot be used for clinic, due to thepharmacological effect is unclear. Therefore, the study about whether ARBs, such astelmisartan, affect PPARδ expressions and functions is very necessary. Since type2diabetes is characterized by insulin resistance in skeletal muscle, we assume thattelmisartan directly affects glucose metabolism in skeletal muscle by PPARδ activation.This study explored the above hypothesis both in vivo and in vitro to provide a theoreticalbasis about prevention of insulin resistance by ARBs in high-risk patients.Nonalcoholic fatty liver is an independent risk factor for cardiometabolic diseases.Currently, lifestyle intervention remains a major treatment approach for fatty liver. Transient receptor potential vanilloid1(TRPV1) channel is a non-selective cation channelwith a preference for calcium ions. TRPV1is able to sense a vast range of endogenousphysical and chemical stimuli and transduce the sensations of noxious heat and painsignaling. Functional TRPV1has also been found in hepatic cells, and up-regulation ofTRPV1predicts a better prognosis for patients with hepatocellular carcinomas. Uncouplingprotein2(UCP2) was reported to play a role in hepatic lipid metabolism, mitochondrialbioenergetics, oxidative stress, apoptosis, and even carcinogenesis. TRPV1activation bydietary capsaicin can increase UCP2expression in adipose tissue. Accordingly, we testwhether TRPV1activation by dietary capsaicin might attenuate events that lead to fattyliver and insulin resistance and identify which mechanism is involved in this process.The study is divided into four parts:1. C2C12cells induced mature. Telmisartantreated C2C12myotubes with the different concentrations or different time. We detected theeffects of telmisartan on the PPARδ expression and activity.2. Long-term telmisartantreated muscle-specific PPARδ knockout (MCK-PPARδ-/-) mice and littermate wild-typemice. We observed the effects of telmisartan on body weights, food intakes, the glucosetolerance and insulin tolerance in mice.3. We detected the glucose uptakes of C2C12cellsand primary cultured skeletal muscle cells of WT mice by telmisartan and insulin acutetreatments. We observed the effects of telmisartan on the expressions of PPARδ, PPARγ,PPARα and insulin signaling pathway molecules of skeletal muscle tissues and cells. Westudy the molecular mechanisms of telmisartan affect glucose metabolism of skeletalmuscle.4. We examined whether the chronic dietary capsaicin has an impact on lipiddeposition in liver tissues by oil-red O staining of the liver in WT and TRPV1-/-mice fedwith normal diet(ND), high-fat diet (HD) and high-fat diet plus capsaicin (HC). Wedetected the TRPV1and UCP2expressions in liver tissues from WT and TRPV1-/-mice byImmunoblots.Materials and Methods:This study includes in vivo and in vitro experiments. MCK-PPARδ-/-and theirlittermate wild-type mice were as the animal model for1to3parts. C57BL/6J andTRPV1-/-mice were as the animal model for the4th part. The C2C12cells and primarycultured skeletal muscle cells of wild-type mice for the study in vitro for1to3parts. TheHepG2cells in vitro for the4thpart. 1. Mouse skeletal muscle lines, C2C12cells were induced to mature cells andtransfected with PPARδ plasmid pAd Track-CMV-PPARδ. After treated with telmisartan,PPARδ, PPARγ and PPARα antagonist, the PPARδ activities of C2C12cells were detectedby Luciferase. The expressions of PPARδ, PPARγ and PPARα were analyzed by westernblot.2.24WT mice and MCK-PPARδ-/-mice respectively were randomly divided intonormal diet and high-fat diet group when they were4-6weeks old. At the20thweek,12high-fat diet mice were randomly selected and treated with telmisartan for10weeks.Weobserved the food intake at the2ndweek and body weight every2weeks. Theintraperitoneal glucose tolerance and insulin tolerance were tested at the20thand30thweek.The acute insulin stimulation test was detected after anesthesia. The bilateral gastrocnemiusmuscles were cut and prepared for the total and membrane proteins. The blood test insulin,triglycerides and total cholesterol were frozen at-70°C.3. We cultured primary skeletal muscle cells of WT mice. We changed serum-freemedium in the cultured C2C12cells and primary skeletal muscle cells and treated withtelmisartan for24hours. The2-deoxyglucose (2-DG) uptake was assayed after acute insulinstimulation.4. After C2C12cells mature, we treated C2C12cells with telmisartan, antagonist ofPPARδ, PI3K, PPARγ and PPARα. The total and membrane proteins were extracted afteracute insulin stimulation. The expressions of Akt, AS160and plasma membrane Glut4aswell as the phosphorylation of Akt and AS160were detected by western blot.5. WT and MCK-PPARδ-/-mice were fed with normal diet, high-fat diet and high-fatdiet plus telmisartan. The expressions of PPARδ, PPARγ and PPARα in gastrocnemius weredetected by western blot. The expressions of Akt, AS160and plasma membrane Glut4aswell as the phosphorylation of Akt and AS160were detected after acute insulin stimulation.6. C57BL/6J (WT) and TRPV1-/-mice were fed with normal diet, high-fat diet andhigh-fat diet plus capsaicin for24weeks. The lipids deposition of liver tissue from WT andTRPV1-/-mice was observed by Oil-red O staining. The expressions of TRPV1and UCP2were detected by western blot.Major Research Results:1. PPARδ was indeed expressed in mice skeletal muscle cells. Telmisartan can activate PPARδ and increase the levels of activity and expressions. The activation of PPARδ inC2C12myotubes by telmisartan was time-and dose-dependent. Telmisartan also increasedthe expressions of PPARγ and PPARα.2. PPARδ, PPARγ and PPARα antagonists can all significantly inhibit the increase ofPPARδ activity in C2C12myotubes induced by telmisartan. PPARδ and PPARα antagonists(GSK0660and GW6471) induced the additive effects and caused enhanced inhibitions ofPPARδ activity in C2C12myotubes. The combination of PPARδ and PPARγ antagonists(GSK0660and GW9662) showed no additive effect.3. We observed the MCK-PPARδ-/-mice and their littermate as wild-type mice. Thebody weight of WT and MCK-PPARδ-/-mice fed with high-fat diet were significantlyhigher than the normal diet group after20weeks. The glucose tolerance and insulintolerance were impaired in WT and MCK-PPARδ-/-mice fed with high-fat diet. Telmisartantreatment reversed high-fat diet-induced weight gain in WT mice. And telmisartan alsodecreased plasma insulin, triglyceride and total cholesterol levels, improve glucosetolerance and increase insulin sensitivity comparing with the WT mice fed with high-fatdiet.4. In the insulin resistant C2C12myotubes induced palmitate, telmisartan significantlyincreased glucose uptakes of C2C12and primary cultured WT mice skeletal musclemyotubes. Also in the insulin resistant C2C12myotubes, the phosphorylation of Akt andAS160and membrane Glut4expression were very weak. Telmisartan enhanced thephosphorylation of Akt and AS160and membrane Glut4expression which can besignificantly reduced by phosphoinositide3-kinase (PI3K) inhibitor LY294002, or PPARδinhibitors GSK0660but not PPARγ inhibitor GW9662and PPARα inhibitor GW6471.5. The in vivo studies showed that the PPARδ expression significantly decreased in theskeletal muscle of WT mice fed with high-fat diet. PPARδ expression restored after10weeks of telmisartan intervention. The expressions of PPARγ and PPARα in the skeletalmuscle were no significant difference beween the high-fat diet group and high-fat diet plustelmisartan group of both the WT and MCK-PPARδ-/-mice. The phosphorylation of Akt andAS160and membrane Glut4expression decreased in the WT high-fat diet mice. Chroniclong-term telmisartan treatment significantly increased the phosphorylation of Akt andAS160and membrane Glut4expression but had no effect on the MCK-PPARδ-/-mice. 6. Functional TRPV1has been detected in hepatocytes and liver tissues. TRPV1activation by capsaicin reduced lipid accumulation and triglyceride level in the liver fromwild type (WT) mice. However, these effects were absent in the liver from TRPV1-/-mice.Chronic dietary capsaicin increased the hepatic uncoupling protein2(UCP2) expression inWT but not in TRPV1-/-mice.Conclusion1. PPARδ existed in mice skeletal muscle cells. Telmisartan can activate PPARδ inC2C12myotubes was time and dose-dependent. Telmisartan also increased the expressionsof PPARγ and PPARα.2. Telmisartan treatment reversed high-fat diet-induced weight gain in WT mice. Andtelmisartan also decreased plasma insulin, triglyceride and total cholesterol levels, improveglucose and insulin tolerancecomparing with the WT mice fed with high-fat diet.3. Telmisartan significantly increased insulin resistant myotubes glucose uptakes.Telmisartan enhanced the phosphorylation of Akt and AS160and membrane Glut4expression in insulin resistant C2C12myotubes. Chronic long-term telmisartan treatmentsignificantly increased the phosphorylation of Akt and AS160and membrane Glut4expression but had no effect on the MCK-PPARδ-/-mice. So telmisartan increases glucoseuptake and inprove insulin sensitivity through activation of PPARδ-mediated PI3Ksignaling in skeletal muscle.4. TRPV1activation by dietary capsaicin prevents high-fat diet-induced fatty liver andinsulin resistance in mice through up-regulation of hepatic UCP2.