Thrombospondin 1 mediates high-fat diet-induced muscle fibrosis and insulin resistance in male mice

Abstract

Thrombospondin 1 (THBS1 or TSP-1) is a circulating glycoprotein highly expressed in hypertrophic visceral adipose tissues of humans and mice. High-fat diet (HFD) feeding induces the robust increase of circulating THBS1 in the early stages of HFD challenge. The loss of Thbs1 protects male mice from diet-induced weight gain and adipocyte hypertrophy. Hyperinsulinemic euglycemic clamp study has demonstrated that Thbs1-null mice are protected from HFD-induced insulin resistance. Tissue-specific glucose uptake study has revealed that the insulin-sensitive phenotype of Thbs1-null mice is mostly mediated by skeletal muscles. Further assessments of the muscle phenotype using RNA sequencing, quantitative PCR, and histological studies have demonstrated that Thbs1-null skeletal muscles are protected from the HFD-dependent induction of Col3a1 and Col6a1, coupled with a new collagen deposition. At the same time, the Thbs1-null mice display a better circadian rhythm and higher amplitude of energy expenditure with a browning phenotype in sc adipose tissues. These results suggest that THBS1, which circulates in response to a HFD, may induce insulin resistance and fibrotic tissue damage in skeletal muscles as well as the de-browning of sc adipose tissues in the early stages of a HFD challenge. Our study may shed new light on the pathogenic role played by a circulating extracellular matrix protein in the cross talk between adipose tissues and skeletal muscles during obesity progression.

Figure 1.

Thbs1 expression during adipogenesis and HFD-induced obesity. A, Thbs1 expression during the differentiation of 3T3-L1 cells into adipocytes (n = 3, mean ± SEM). The induction of aP2 (Fabp4) and Pparγ is shown for comparison. B, Thbs1 expression in the iWAT, eWAT, and brown adipose tissues isolated from 11-week-old C57BL/6J male mice, respectively (n = 4, mean ± SEM). C, Thbs1 expression in iWAT and eWAT isolated from 11-week-old male mice fed a NFD (open bar)or HFD (closed bar) for 3 weeks (n = 4, mean ± SEM). D, THBS1 protein levels in the sera collected from 11-week-old male mice fed a NFD or HFD for 3 weeks and 15 weeks. The staining with Ponceau S is shown as loading control (Po-S). Representative figures (n = 4 each) are shown. E, Cd36 and Cd47 expression in iWAT and eWAT isolated from male mice fed a NFD (open bar) or a HFD (closed bar) for 3 weeks (n = 8, mean ± SEM). F, The expression of Thbs1, Cd36, and Cd47 in the quadriceps femoris isolated from wild-type male mice fed a NFD or HFD for 3 weeks (n = 8, mean ± SEM). NFD (open bar), HFD (closed bar), *, P <.05.

Figure 2.

Thbs1-null mice are protected from HFD-induced obesity. A, Body weight change during a 45% HFD feeding. WT and Thbs1-null (KO) male mice, fed a 45% HFD beginning at age 8 weeks (n = 8 for each group). B, Fat mass assessed with a nuclear magnetic resonance body composition analyzer (n = 8). C, H&E staining of inguinal white adipose tissues. Upper panel, Eleven-week-old male mice fed normal fat diet (NFD). Lower panel, Eleven-week-old male mice fed a 45% fat diet (HFD) for 3 weeks. Scale, 100 μm. D, The morphometric quantification of adipocyte size in adipose tissue (square micrometers, n = 50 adipocytes per field). E, The number of adipocytes per tissue. Open bar, WT; closed bar, Thbs1 KO mice. F, UCP1-positive adipocytes within iWAT (brown); nuclei are counterstained (blue). G, Western blot of UCP1 in tissue lysates of inguinal adipose tissues (n = 4 for each group). H, The levels of serum glucose (left panel) and insulin (right panel) in mice fed a NFD or HFD for 3 weeks (n = 8, mean ± SEM). *, P < .05 compared with WT.

Figure 3.

Food consumption, physical activity, and oxygen consumption. A, Weekly food consumption of WT (open circle) and Thbs1-null male mice (KO; closed circle) during a 45% HFD challenge. B, Total physical activities of WT and KO male mice under a 45% HFD challenge. C, Oxygen consumption of WT and KO mice adjusted for lean mass (LBM). D, The respiratory quotient (RQ; VCO₂ to VO₂ ratio) of WT and KO mice during a 45% HFD feeding (n = 6, mean ± SEM).

Figure 4.

The loss of Thbs1 protects mice from HFD-induced adipose tissue inflammation. A, H&E staining of eWAT. Upper panel, Eleven-week-old male mice fed a NFD. Lower panel, Eleven-week-old male mice fed a 45% HFD for 3 weeks. Scale, 100 μm. B, HFD-induced crown-like structure is shown with Mac2 staining in eWAT after 28 weeks of HFD feeding. Scale, 200 μm. The higher magnification of WT crown-like structure is shown at the bottom. Scale, 50 μm. C, Gene expression of Emr1 encoding F4/80 in WT or KO eWAT after 28 weeks of a HFD (n = 4). D, Flow cytometry-based cell population analysis. The percentage of the parent cell population is shown for ATMs, M1-like ATMs (M1s), M2-like ATMs (M2s), and double-negative cells. E, The ratio of M1-like to M2-like ATMs in WT and KO mice at 3 and 21 weeks of a HFD (n = 4, mean ± SEM). *, P < .05; **, P < .005.

Figure 5.

Hyperinsulinemic euglycemic clamp study. A, Blood glucose levels of WT and Thbs1-null (KO) mice during hyperinsulinemic euglycemic clamp studies. B, Glucose infusion rate (120 min) (mean ± SEM; WT, n = 10; KO, n = 6; P = .03 at 120 min). C, Glucose flux rate of WT and Thbs1-null (KO) mice under a hyperinsulinemic, euglycemic condition (P = .01 at 120 min). D, Suppression of endogenous glucose production during the study. *, P < .05.

Figure 6.

Tissue glucose uptake. Tissue glucose uptake (Rg) in soleus, gastrocnemius, and vastus lateralis (A) and the iWAT and eWAT (B) [WT, n = 10 (open bars), and Thbs1-null (KO) mice, n = 6 (closed bars); mean ± SEM]. *, P < .05.

Figure 7.

Thbs1-dependent skeletal muscle gene expression. A, The list of Gene Ontology Biological Pathway (GO-BP) pathways significantly different between the two groups. B, A representative heat map shows the expression profile of the genes listed in GO: Collagen (GO: 0005581). C, The expression of collagen family members (Col3, Col6, Col1) in the quadriceps muscles isolated from WT and KO mice after 3 weeks of a NFD or a 45% HFD (n= 8 each, mean ± SEM). D, Picosirius Red staining of quadriceps femoris observed under polarized microscopy. WT mice fed a normal diet (WT NFD), fed a 45% HFD (WT HFD), and Thbs1-KO mice fed HFD (KO HFD). Thinner (green) and thick collagen fibers (yellow-orange) are shown. E, The quantification of the percentage area positive for Picosirius Red staining. Two sections from each mice were analyzed, four mice for each group (mean ± SEM). *, P < .05; **, P < .005; ***, P < .0005.

Figure 8.

THBS1 mediates the link between visceral adiposity and muscle fibrosis/insulin resistance. A HFD rapidly induces expansion of visceral adipose tissue ECM and increases THBS1 levels in circulation. THBS1 may induce muscle fibrosis and insulin resistance as well as debrowning of sc adipose tissues in the early stage of the HFD and adipose tissue inflammation in late stages.