Obesity/Type II Diabetes Promotes Function-limiting Changes in Murine Tendons that are not reversed by Restoring Normal Metabolic Function

Abstract

Type II Diabetes (T2DM) negatively alters baseline tendon function, including decreased range of motion and mechanical properties; however, the biological mechanisms that promote diabetic tendinopathy are unknown. To facilitate identification of therapeutic targets we developed a novel murine model of diabetic tendinopathy. Mice fed a High Fat Diet (HFD) developed diet induced obesity and T2DM and demonstrated progressive impairments in tendon gliding function and mechanical properties, relative to mice fed a Low Fat Diet (LFD). We then determined if restoration of normal metabolic function, by switching mice from HFD to LFD, was sufficient to halt the pathological changes in tendon due to obesity/T2DM. However, switching from a HFD to LFD resulted in greater impairments in tendon gliding function than mice maintained on a HFD. Mechanistically, IRβ signaling is decreased in obese/T2DM murine tendons, suggesting altered IRβ signaling as a driver of diabetic tendinopathy. However, knock-down of IRβ expression in S100a4-lineage cells (IRcKO^S100a4) was not sufficient to induce diabetic tendinopathy as no impairments in tendon gliding function or mechanical properties were observed in IRcKO^S100a4, relative to WT. Collectively, these data define a murine model of diabetic tendinopathy, and demonstrate that restoring normal metabolism does not slow the progression of diabetic tendinopathy.

Figure 1

A High Fat Diet induces obesity and Type II Diabetes Mellitus. (A) Male C57Bl/6 J mice were initiated on either a High Fat Diet (HFD) or Low Fat Diet (LFD) at 4 weeks of age. 12 weeks after diet initiation half of the HFD cohort was randomly selected and switched to a LFD. Animals were harvested at 12, 24, 40, and 48 weeks after diet initiation (12–36 weeks after switching to LFD for the HFD-LFD cohort. (B) Body weight was measured in LFD, HFD mice between 12 and 48 weeks after diet initiation. HFD-LFD mice were initiated on a HFD, and switched to a LFD 12 weeks after initiation, as such body weights from these mice were measured only between 24–48 weeks (12–36 weeks after switching to the LFD). (C) At 20 weeks after diet initiation a significant impairment in glucose tolerance was observed in HFD, compared to LFD and HFD-LFD. A sustained increase in glucose levels post-glucose bolus is indicative of impaired glucose tolerance. (D) Quantification of Area under the Curve (AUC) from Glucose Tolerance Test (GTT). (E) Changes in fasting blood glucose were measured after a 5 hr fast between 12–48 weeks after diet initiation. (F) Body fat percentages of LFD, HFD, and HFD-LFD mice at 12 and 48 weeks post diet initiation. *Indicates p < 0.05.

Figure 2

T2DM/obesity alters tendon gliding function and mechanical properties. (A) Measurement of metatarsophalangeal (MTP) joint flexion angle, (B) Gliding Resistance, (C) Maximum load at failure, and (D) Stiffness of the FDL tendon in LFD, HFD and HFD-LFD tendons between12–48 weeks after diet initiation. *Indicates p < 0.05.

Figure 3

T2DM/obesity alters collagen fibril diameter. (A) TEM axial images of the FDL tendon from LFD, HFD and HFD-LFD mice at 40 weeks post diet initiation. Lipid deposits in HFD tendons are noted by black arrows. Scale bars represent 5 microns in 3500x magnification images, and 0.5microns in the 40,000x magnification images. (B) Collagen fibril diameter histograms demonstrating a decrease in median fibril diameter in HFD and HFD-LFD tendons compared to LFD. (C) Collagen fibril diameter distributions with boxplot whiskers spanning data between the 5^th and 95^th percentiles. Data outside this range are plotted as individual points. (D) Collagen fibril density. *Indicates p < 0.05.

Figure 4

Insulin receptor expression and signaling are altered in diabetic tendons. (A) Primary tenocytes isolated from non-diabetic murine tendons demonstrate strong expression of p-Akt relative to vehicle treated tenocytes, indicating activation of IR signaling in tenocytes. (B) Tendons from HFD and LFD mice were stimulated with insulin or vehicle (0.5% BSA in PBS). Activation of IR signaling was observed in LFD tendons based on the increased expression of p-Akt. In contrast, no increase in p-Akt expression was observed in insulin stimulated HFD tendons compared to vehicle treatment indicating blunted sensitivity to insulin in HFD tendons. Blots were probed for p-Akt and imaged, following stripping of the membrane the same blots were then probed for total Akt. Full length blots are presented in Supplementary Figure 1.

Figure 5

IRβ deletion in S100a4-lineage cells does not induce obesity or T2DM. (A) The recombination efficiency of S100a4-Cre in the tendon was visualized using the Ai9 reporter and demonstrates efficient recombination in the tendon. (B) IRcKO^S100a4 decreases IRβ protein expression in tendon, relative to WT. Full length blots are presented in Supplementary Figure 2. (C–E) No changes in (C) body weight, (D) percent body fat, or (E) fasting blood glucose were observed between WT and IRcKO^S100a4 mice at 48 weeks of age. (F–G) IRcKOS100a4 had more efficient restoration of blood levels during the glucose tolerance test (F), and as calculated by quantification of Area Under the Curve from the glucose tolerance test (G).

Figure 6

Deletion of IRβ in S100a4-lineage cells does not impair tendon gliding function, mechanical properties or collagen organization. (A) MTP ROM, (B) Gliding Resistance, (C) Max load at failure and (D) Stiffness were assessed at 48 weeks of age in WT and IRcKO^S100a4 tendons. *Indicates p < 0.05. (E) TEM axial images of the FDL tendon from WT and IRcKO^S100a4 at 48 weeks of age. (F) No changes in collagen fibril diameter were observed between WT and IRcKO^S100a4 tendons. (G) Collagen fibril diameter distributions with boxplot whiskers spanning data between the 5^th and 95^th percentiles. Data outside this range are plotted as individual points. (H) Collagen fibril density of TEM images of tendons from WT and IRcKO^S100a4 mice at 48 weeks of age.