Hyaluronan accumulates with high-fat feeding and contributes to insulin resistance

Abstract

Increased deposition of specific extracellular matrix (ECM) components is a characteristic of insulin-resistant skeletal muscle. Hyaluronan (HA) is a major constituent of the ECM. The hypotheses that 1) HA content is increased in the ECM of insulin-resistant skeletal muscle and 2) reduction of HA in the muscle ECM by long-acting pegylated human recombinant PH20 hyaluronidase (PEGPH20) reverses high-fat (HF) diet-induced muscle insulin resistance were tested. We show that muscle HA was increased in HF diet-induced obese (DIO) mice and that treatment of PEGPH20, which dose-dependently reduced HA in muscle ECM, decreased fat mass, adipocyte size, and hepatic and muscle insulin resistance in DIO mice at 10 mg/kg. Reduced muscle insulin resistance was associated with increased insulin signaling, muscle vascularization, and percent cardiac output to muscle rather than insulin sensitization of muscle per se. Dose-response studies revealed that PEGPH20 dose-dependently increased insulin sensitivity in DIO mice with a minimally effective dose of 0.01 mg/kg. PEGPH20 at doses of 0.1 and 1 mg/kg reduced muscle HA to levels seen in chow-fed mice, decreased fat mass, and increased muscle glucose uptake. These findings suggest that ECM HA is a target for treatment of insulin resistance.

FIG. 1.

HF feeding increases muscle HA content, and treatment with PEGPH20 in mice reduces HA in muscle. A: Immunohistochemical detection of HA in gastrocnemius of chow- and HF-fed mice. Data are quantified by measuring the integrated intensity of the staining and normalized to chow-fed mice. n = 5–6. *P < 0.05 vs. chow. B: Chow-fed mice were intravenously injected with PEGPH20, and HA content in gastrocnemius muscle was assessed by immunohistochemistry over time after the bolus. Data are normalized to time 0. n = 3. *P < 0.05 vs. 0 h. C: Plasma PEGPH20 activity was measured in mice from B to determine the half-life. n = 3. D: Immunohistochemical detection of HA in gastrocnemius muscle of vehicle- or chronic PEGPH20–treated DIO mice. Data are normalized to vehicle-treated HF-fed mice. n = 6. *P < 0.05 vs. vehicle HF. All data are represented as mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)

FIG. 2.

Treatment of PEGPH20 in DIO mice causes a transient body weight loss. DIO mice were treated with either vehicle or PEGPH20 for 24 days. Body weight (A), food consumption (B), fat mass (C), and lean mass (D) were assessed over time. n = 10. *P < 0.05 vs. vehicle and ^#P < 0.05 vs. day 0. EE (E) and physical activity (F) were assessed in mice for 3 days after the first injection of vehicle or PEGPH20. n = 6. *P < 0.05 vs. vehicle. All data are represented as mean ± SEM.

FIG. 3.

Chronic, but not acute, treatment of PEGPH20 increases insulin sensitivity in DIO mice. DIO mice received one (acute) or multiple (chronic) intravenous injections of vehicle or PEGPH20. IC_v was performed 3 days after the injection to determine blood glucose (A and E); GIR (B and F); Rg (C and G), an index of muscle glucose uptake; and endogenous glucose production (EndoRa) and glucose disappearance rate (Rd) (D and H). n = 4–8. *P < 0.05 vs. vehicle. All data are represented as mean ± SEM.

FIG. 4.

Effects of chronic PEGPH20 on muscle in DIO mice. A: Gastrocnemius muscle was collected at the end of the IC_v. Tyrosine phosphorylation of IRS1 and IRS1-associated p85 were assessed by immunoprecipitation and Western blotting. B: Protein expression of Akt and phosphorylated Akt was assessed by Western blotting in muscle homogenates. C: Protein expression of collagen IV (ColIV) and CD31 were measured by immunohistochemistry in gastrocnemius muscle collected at the end of the IC_v. ColIV expression was measured by the integrated intensity of staining. Muscle vascularity was determined by counting CD31-positive structures. D: Cardiac output to muscle was assessed by microspheres that were injected to the arterial catheter after the IC_v. Data were normalized to vehicle. n = 4–8. *P < 0.05 vs. vehicle. E–G: Soleus and EDL muscles were isolated from mice chronically treated with vehicle or PEGPH20 for 24 days. In vitro glucose uptake was measured using 2-[³H]deoxyglucose on day 27. n = 7–9. *P < 0.05 vs. vehicle and #P < 0.05 vs. basal. All data are represented as mean ± SEM. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (A high-quality color representation of this figure is available in the online issue.)

FIG. 5.

Chronic PEGPH20 treatment decreases adipocyte size and gene expression of the inflammatory markers. A: Immunochemical detection of HA and Toluidine Blue O staining in the perigonadal adipose tissue of vehicle and chronic PEGPH20-treated DIO mice. B: Adipocyte size was determined by manual measurements of adipocyte diameters of at least 100 adipocytes per mouse. C: Gene expression in perigonadal adipose tissue. Data were normalized to 18S expression. Mθ, macrophage; M1, proinflammatory markers; M2, anti-inflammatory markers. D: Liver triglyceride content in vehicle- and chronic PEGPH20-treated DIO mice. n = 6–8. *P < 0.05 vs. vehicle. All data are represented as mean ± SEM. (A high-quality color representation of this figure is available in the online issue.)

FIG. 6.

Dose-dependent effect of chronic PEGPH20. DIO mice were treated with vehicle or PEGPH20 at 0.001, 0.01, 0.1, and 1 mg/kg for 24 days. Body weight (A), body composition changes (B), food consumption (C), EE (D), and physical activity (E) were measured over the treatment. n = 4–6. *P < 0.05 vs. vehicle. IC_v was performed 3 days after the last injection. Blood glucose (F), GIR (G), and muscle Rg (H) during the IC_v were determined. n = 4–6. ξP < 0.05 vs. vehicle; *P < 0.05 vs. vehicle. I: Dose-dependent effect of chronic PEGPH20 on HA content in the gastrocnemius of DIO mice. Dashed lines indicate HA content in chow-fed vehicle mice or HF-fed vehicle mice. Filled areas indicate the SEM of individual line values. n = 4–5. y = −0.44log(x) + 0.622. r² = 0.9581. All data are represented as mean ± SEM. SVL, superficial vastus lateralis.