Candesartan, an angiotensin-II receptor blocker, ameliorates insulin resistance and hepatosteatosis by reducing intracellular calcium overload and lipid accumulation

Insulin resistance is a major contributor to the pathogenesis of several human diseases, including type 2 diabetes, hypertension, and hyperlipidemia. Notably, insulin resistance and hypertension share common abnormalities, including increased oxidative stress, inflammation, and organelle dysfunction. Recently, we showed that excess intracellular Ca2+, a known pathogenic factor in hypertension, acts as a critical negative regulator of insulin signaling by forming Ca2+-phosphoinositides that prevent the membrane localization of AKT, a key serine/threonine kinase signaling molecule. Whether preventing intracellular Ca2+ overload improves insulin sensitivity, however, has not yet been investigated. Here, we show that the antihypertensive agent candesartan, compared with other angiotensin-II receptor blockers, has previously unrecognized beneficial effects on attenuating insulin resistance. We found that candesartan markedly reduced palmitic acid (PA)-induced intracellular Ca2+ overload and lipid accumulation by normalizing dysregulated store-operated channel (SOC)-mediated Ca2+ entry into cells, which alleviated PA-induced insulin resistance by promoting insulin-stimulated AKT membrane localization and increased the phosphorylation of AKT and its downstream substrates. As pharmacological approaches to attenuate intracellular Ca2+ overload in vivo, administering candesartan to obese mice successfully decreased insulin resistance, hepatic steatosis, dyslipidemia, and tissue inflammation by inhibiting dysregulated SOC-mediated Ca2+ entry and ectopic lipid accumulation. The resulting alterations in the phosphorylation of key signaling molecules consequently alleviate impaired insulin signaling by increasing the postprandial membrane localization and phosphorylation of AKT. Thus, our findings provide robust evidence for the pleiotropic contribution of intracellular Ca2+ overload in the pathogenesis of insulin resistance and suggest that there are viable approved drugs that can be repurposed for the treatment of insulin resistance and hypertension.


Glucose tolerance test and insulin tolerance test
IP-GTT was performed by intraperitoneal injection of glucose (1 g /kg body weight) after a 16 h fast. Blood was collected from a small incision at the tip of the tail at 0, 15, 30, 60, and 90 min after glucose injection. Blood glucose levels were measured with a blood glucometer (Accu-Check Aviva, Roche). IP-ITT was performed by intraperitoneal injection of insulin (0.75 U/kg body weight; Actrapid Penfill, Novo Nordisk) after a 4 h fast. Blood glucose levels were measured at 0, 15, 30, 60, and 120 min after insulin injection. HOMA-IR was calculated using the following formula: (fasting insulin × fasting glucose)/405.

Determination of body composition and liver fat content
The body composition of mice was measured using a nuclear magnetic resonance imaging technique (Minispec Live Mice Analyzer (LF50), Bruker) at the end of the experiments. Frozen liver tissues were homogenized in modified RIPA buffer. Triglyceride contents and total proteins in liver lysates were measured with AU480 biochemistry.
Triglyceride contents were normalized by the total protein concentration.

Serum and urine biochemical assay
At the end of the experiments, the mice were fasted for 16 h and refed with an HFD before receiving isoflurane. Abdominal aorta blood was obtained. Liver, kidney, and adipose tissues were dissected and stored at −80°C. Serum biochemical analyses of ALT, AST, TG, total cholesterol, LDL-cholesterol, and HDL-cholesterol were performed as described previously using a Model AU-480 (Beckman Coulter, Fullerton, CA, USA).

Monitoring of subcellular localization of AKT PH domain
CHO cells or HepG2 cells were transfected with cDNA encoding hAKT PHD (residues 1-144) using Lipofectamine (Life Technologies) as described previously 1 . To monitor the subcellular localization of endogenous AKT, we subjected liver sections to antigen retrieval with 10 mM Tris/HCl (pH 9.0) for 20 min, followed by treatment with serum-free protein blocking solution (DAKO) for 30 min and permeabilization with 0.5% Tween 20 for 15 min. Then, we incubated the liver sections with an anti-AKT PHD antibody (SKB1, #05-591, Sigma-Aldrich) followed by a FITC-conjugated secondary antibody. After mounting, the liver sections were imaged with an LSM 980 laser-scanning confocal microscope (Carl Zeiss). Images were analyzed with ZEN Software (Carl Zeiss).
The AKT PH mCherry adenovirus (5 × 10 8 plaque-forming units) was delivered by systemic tail-vein injection into C57BL/6J mice after 8 weeks of HFD feeding and 2 weeks of treatment with 1.0 mg/kg candesartan or vehicle, as described previously 2 . Seven days after adenoviral infection, the livers were collected from the mice following an overnight fast and subsequent refeeding with HFD for 4 h. For immunofluorescence, formalin-fixed liver sections or cells were plated onto slide glass and counterstained with DAPI to visualize the nuclei. After mounting, the sections were imaged with a Zeiss LSM 700 laserscanning confocal microscope (Carl Zeiss) and analyzed with ZEN Software (Carl Zeiss).
Cells were attached to coverslips and incubated with 4 μM fura-2 AM and 0.05% pluronic acid for 15 min at room temperature in the dark. The dye-loaded cells were then placed on an inverted microscope and perfused with physiological salt solution solutions to detect [Ca 2+ ]i. The [Ca 2+ ]i was determined by the fura-2 ratio of dual excitation wavelengths 340 nm and 380 nm (emission wavelength is 530 nm). The emitted fluorescence was monitored by a CCD camera (Q-imaging) attached to an inverted microscope (Nikon, Japan) and analyzed with a Metafluor system (Molecular Devices, USA). For measurement of Ca 2+ influx, cells were stimulated with the sarco/endoplasmic reticulum Ca 2+ ATPase inhibitor cyclopiazonic acid (CPA) in Ca 2+ -free solution to deplete Ca 2+ stores and then administered 5 mM Ca 2+ -containing PBS.