Delivery of adiponectin gene to skeletal muscle using ultrasound targeted microbubbles improves insulin sensitivity and whole body glucose homeostasis

Abstract

Numerous studies have shown that adiponectin confers antidiabetic effects via both insulin-like and insulin-sensitizing actions. The majority of adiponectin in circulation is derived from adipocytes; however, other tissues such as skeletal muscle can produce adiponectin. This study was designed to investigate the functional significance of adiponectin produced by skeletal muscle. We encapsulated the adiponectin gene in lipid-coated microspheres filled with octafluoropropane gas that were injected into the systemic circulation and destroyed within the microvasculature of skeletal muscle using ultrasound. We first demonstrated safe and successful targeting of luciferase and green fluorescent protein reporter genes to skeletal muscle using this approach and then confirmed efficient overexpression of adiponectin mRNA and oligomeric protein forms. Glucose tolerance test indicated that overexpression of adiponectin in skeletal muscle was able to improve glucose intolerance induced by feeding mice a high-fat diet (HFD), and this correlated with improved skeletal muscle insulin signaling. We then performed hyperinsulinemic-euglycemic clamp studies and demonstrated that adiponectin overexpression attenuated the decreases in glucose infusion rate, glucose disposal, and increase in glucose appearance induced by HFD. Ultrasound-targeted microbubble destruction (UTMD) delivery of adiponectin to skeletal muscle also enhanced serum adiponectin levels and improved hepatic insulin sensitivity. In conclusion, our data show that UTMD efficiently delivers adiponectin to skeletal muscle and that this improves insulin sensitivity and glucose homeostasis.

Fig. 1.

Reporter gene and adiponectin gene delivery to skeletal muscle by ultrasound-targeted microbubble destruction (UTMD). We encapsulated the plasmid encoding luciferase reporter in microbubbles that were infused systemically to wild-type mice and targeted expression to skeletal muscle via applying ultrasound to one hind leg while the other hind leg served as a control. Skeletal muscle tissues were then collected, and homogenates were assayed for luciferase activity [relative luciferase units (RLU)/μg protein] (A). A similar experiment was performed using luciferase reporter gene under control of a myogenin-driven promoter (B). We then infused a plasmid containing green fluorescent protein reporter, and muscle cryostat sections were prepared to analyze green fluorescence (C). Representative collapsed “xy projections” image of skeletal muscle from enhanced green fluorescent protein (eGFP) plasmid transfection is shown. After these validation steps, we delivered the adiponectin gene to skeletal muscle using this approach, and muscle homogenates were analyzed by immunoblotting for adiponectin expression. Shown is a representative Western blot obtained from SDS-PAGE to examine adiponectin oligomers (D, top) and total adiponectin in which all samples were reduced to generate only monomer (D, bottom). fAd, full-length adiponectin. Quantitation of high molecular weight (HMW), hexameric and total adiponectin detected upon Western blotting is shown in E, F, and G, respectively. Quantitative data represent means ± SE. *P < 0.05 vs. control; n = 3–6 mice in each group.

Fig. 2.

Glucose tolerance test (GTT). Wild-type mice were fed either commercial chow (Chow) or 60% high-fat diet (HFD) at the age of 6 wk. After 6 wk HFD, mice were subjected to either pGL3 [empty vector (EV)] or adiponectin (fAd) plasmid transfection at hind leg skeletal muscle mediated by UTMD. Seven days post-UTMD, ip glucose tolerance test (IPGTT) was performed after 5–6 h fasting on three animal groups: chow-fed UTMD-EV, 60% HF-fed UTMD-EV, and 60% HF-fed UTMD-fAd. A: IPGTT curves (mmol/l) B: area under the curve (AUC). Data represent means ± SE. *P < 0.05 vs. chow-fed UTMD-EV and #P < 0.05 vs. 60% HF-fed UTMD-EV; n = 4–5.

Fig. 3.

Hyperinsulinemic-euglycemic clamp studies following UTMD transfection in chow and diet-induced obese mice. Wild-type mice were fed either commercial chow or 60% high-fat (HF) diet at the age of 6 wk. After 12 wk HF diet, we used UTMD to deliver either pGL3 (EV) or fAd plasmid to hind leg skeletal muscles. Seven days post-UTMD, hyperinsulinemic-euglycemic clamp was performed to assess whole body glucose homeostasis on three animal groups: chow-fed UTMD-EV, 60% HFD UTMD-EV, and 60% HFD UTMD-fAd. Jugular vein and carotid artery catheters were embedded in animals 4 days before the hyperinsulinemic-euglycemic clamp procedure, and clamps were performed on animals after 5–6 h starvation. Blood samples were collected during the clamp procedure, and calculations were made based on the radioactivity readings from serum to represent whole body glucose metabolism. A: glucose infusion rate (GIR) (mg·kg⁻¹·min⁻¹). B: glucose turnover rate (post-to-basal ratio). R_a, glucose appearance rate; R_d, glucose disappearance rate; basal, before insulin clamp; post, after insulin clamp. C: glycolytic rate (mg·kg⁻¹·min⁻¹). Data represent means ± SE. *P < 0.05 vs. Chow (UTMD-EV) group and #P < 0.05 vs. 60% HF-fed UTMD-EV. βP < 0.05 vs. basal (before insulin clamp) within the same diet and treatment group; n = 4–5.

Fig. 4.

Insulin stimulated signaling and expression of adiponectin receptor isoforms and adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif (APPL) isoforms in skeletal muscle. Mice were fed either commercial chow or 60% HF diet at the age of 6 wk. After 12 wk HF diet, we used UTMD to deliver either pGL3 (EV) or fAd plasmid to hind leg skeletal muscles. Seven days post-UTMD, animals were starved 5–6 h, and insulin was administered as a bolus injection (4 U/kg body wt) via tail vein. Fifteen minutes after injection skeletal muscle tissues were collected, and homogenates were prepared and then analyzed by Western blotting. Representative Western blot of phospho (p)-protein kinase B (Akt) [threonine-308 (T308, A), serine-473 (S473, C), total Akt2 (A and C)] and quantitative analysis [T308 (B), S473 (D), Akt2 (E)]. F: representative Western blot for adiponectin receptor (AdipoR) 1, AdipoR2, APPL1, and APPL2. Data represent means ± SE. *P < 0.05 vs. chow-fed UTMD-EV and #P < 0.05 vs. 60% HF-fed UTMD-EV; n = 5–6.

Fig. 5.

Serum adiponectin, insulin, and adiponectin signaling, lipid analysis in liver. Mice were fed either commercial chow or 60% HF diet at the age of 6 wk. After 12 wk HF diet, we used UTMD to deliver either pGL3 (EV) or fAd plasmid to hind leg skeletal muscles. Serum samples were collected pre- and post-UTMD procedure. Seven days post-UTMD, animals were starved 5–6 h, and insulin was administered as a bolus injection (4 U/kg body wt) via tail vein. Fifteen minutes after injection, liver tissue was collected, and homogenates were prepared and then analyzed by Western blotting. Representative Western blot of p-Akt [T308 (A), S473 (C), total Akt2 (A and C)] and quantitative analysis [T308 (B), S473 (D)]. E: representative Western blot for AdipoR1, AdipoR2, APPL1, and APPL2. F: liver triglyceride content (nmol/μg protein). G-K: representative Western blot and quantitative analysis of serum adiponectin level. Data represent means ± SE. *P < 0.05, chow-fed UTMD-EV vs. 60% HFD UTMD-EV, #P < 0.05, 60% HFD UTMD-EV vs. 60% HFD UTMD-fAd, and αP < 0.05 vs. pre-UTMD; n = 4–5.