MG53 Does Not Manifest the Development of Diabetes in db/db Mice

Abstract

MG53 is a member of the TRIM protein family that is predominantly expressed in striated muscles and participates in cell membrane repair. Controversy exists regarding MG53's role in insulin signaling and manifestation of diabetes. We generated db/db mice with either whole-body ablation or sustained elevation of MG53 in the bloodstream in order to evaluate the physiological function of MG53 in diabetes. To quantify the amount of MG53 protein in circulation, we developed a monoclonal antibody against MG53 with high specificity. Western blot using this antibody revealed lower or no change of serum MG53 levels in db/db mice or patients with diabetes compared with control subjects. Neither whole-body ablation of MG53 nor sustained elevation of MG53 in circulation altered insulin signaling and glucose handling in db/db mice. Instead, mice with ablation of MG53 were more susceptible to streptozotocin-induced dysfunctional handling of glucose compared with the wild-type littermates. Alkaline-induced corneal injury demonstrated delayed healing in db/db mice, which was restored by topical administration of recombinant human (rh)MG53. Daily intravenous administration of rhMG53 in rats at concentrations up to 10 mg/kg did not produce adverse effects on glucose handling. These findings challenge the hypothetical function of MG53 as a causative factor for the development of diabetes. Our data suggest that rhMG53 is a potentially safe and effective biologic to treat diabetic oculopathy in rodents.

Figure 1

Commercially available antibody against MG53 detects nonspecific band in serum derived from mg53^−/− mice. A: IHC of MG53 with mAb-MG53 in muscle and heart derived from WT and mg53^−/− mice. B: WB of muscle samples derived from mg53^−/−, WT, and db/db mice. Smaller amounts of muscle sample (0.05–1.00 µg [bottom]) were used to show high sensitivity of mAb-MG53 compared with greater amounts of muscle samples (0.5–10.0 µg) used for WB with Abcam antibody (cat. no. 83302). Various amounts of rhMG53 protein dissolved in the running buffer were used as reference standards. C: Sera derived from mg53^−/− and WT littermates (1 µL per lane; each lane represents a different mouse) were first probed with mAb-MG53 (top). The membrane was stripped and then probed with the Abcam antibody (bottom).

Figure 2

WBs detect lower or no change of serum MG53 levels in db/db mice or patients with diabetes compared with control subjects. A: 0.05–0.20 ng rhMG53 dissolved in mg53^−/− serum was used to establish the linear range for WB with the mAb-MG53, and 1 or 2 µL serum derived from the C57BL/6 mice was loaded onto the same gel. The WB was representative of 15 other samples. B: 1 µL sera derived from WT (lanes 1–5) and db/db littermates (lanes 6–10) (purchased from The Jackson Laboratory [age 14 weeks]) was probed with mAb-MG53. Loading control represents staining of albumin. C: Normalized WB intensities of MG53 in sera were plotted for individual animals. MG53 levels in db/db serum were significantly lower than those in WT serum (**P < 0.001). D: 1 µL sera derived from human individuals without diabetes and with diabetes was probed with mAb-MG53. E: Scatter plot of MG53 levels in sera of humans without diabetes and with diabetes. No significant differences were observed between the two groups (P = 0.113). F: Plot of serum MG53 level vs. fasting blood glucose in both samples of humans without diabetes and samples of humans with diabetes revealed no correlation (R² = 0.048).

Figure 3

Muscle atrophy in db/db mice leads to reduced secretory activity of MG53. A: Skeletal muscles from WT and db/db littermate mice were probed with Abcam antibody (top) and mAb-MG53 (bottom). On average, there was no statistical difference in MG53 protein between WT and db/db muscle. B: Cross section of skeletal muscle derived from WT (left) and db/db (right) mice was stained with mAb-MG53. C: Comparison of soleus muscle mass derived from littermates of WT and db/db mice at 4 months of age (**P < 0.01). D: 20 µL out of a total of 2 mL solution bathing the soleus was loaded onto the gel at different time points after insulin treatment (top). Smaller amounts of MG53 were detected from db/db soleus at all time points. The bottom panel shows loading of the bathing solution normalized to soleus muscle mass. E: Time-dependent secretion of MG53 from WT (red) or db/db (blue) soleus muscle following insulin treatment (**P < 0.01).

Figure 4

Neither ablation of MG53 nor elevation of MG53 impacts glucose hemostasis and insulin sensitivity in db/db mice. A: Body weight measurement of the four mice strains up to 32 weeks of age (WT, n = 13; mg53^−/−, n = 9; db/db, n = 12; and db/db-mg53^−/−, n = 6). These mice were littermates produced through crossbreeding of the db^+/−mg53^+/− pairs. B: GTT measurements at 18 weeks (left) and 30 weeks (right) (WT, n = 11; mg53^−/−, n = 9; db/db, n = 12; and db/db-mg53^−/−, n = 6). GTT of the db/db-mg53^−/− mice showed a trend of reduced glucose tolerance compared with db/db littermates, but the difference did not reach statistical significance. C: ITT measurements at 20 weeks (left) and 32 weeks (right) (WT, n = 11; mg53^−/−, n = 9; db/db, n = 12; and db/db-mg53^−/−, n = 6). D: Quantification of AUC for the data shown in B. E: Body weight of the WT, tPA-MG53, db/db, and db/db-tPA-MG53 littermates was measured up to 32 weeks of age (WT, n = 7; tPA-MG53, n = 6; db/db, n = 7; and db/db-tPA-MG53, n = 5). F: GTT measurements at 18 weeks (left) and 30 weeks (right). G: ITT measurements at 20 weeks (left) and 32 weeks (right). Both sexes of mice were included in the experiments. H: Quantification of AUC for data shown in F.

Figure 5

mg53^−/− mice show compromised glucose handling following STZ administration. A: Fasting blood glucose levels were measured at 1 and 2 weeks following the last dose of STZ injection. B: GTT was conducted at 2 weeks post-STZ. Significant differences were measured at 30 and 45 min (P = 0.002 and 0.008, respectively). C: AUC measurement of B revealed significant differences between WT and mg53^−/− mice (P = 0.025). D: Three weeks post-STZ, ITT measurement revealed no significant difference between WT and mg53^−/− mice.

Figure 6

Compromised corneal wound healing in db/db mice is ameliorated with systemic administration of rhMG53 protein. A: Intensity of fluorescein staining at days 0–3 following alkaline injury to the cornea was presented for WT (top), db/db (middle), and db/db with rhMG53 treatment (bottom). B: Fluorescein intensity at different days after corneal alkaline injury was normalized to day 0. db/db cornea showed delayed healing of the cornea, which was improved with topical administration of rhMG53. C: Flat mounts of the cornea were stained with CD31 to reveal different degrees of vascularization at 14 days post–alkaline-induced injury to the cornea. D: Intensity of CD31 signal was used as index of vascularization of the cornea at 14 days post–alkaline injury (*P < 0.05; **P < 0.01).