Sexually dimorphic diet-induced insulin resistance in obese tissue inhibitor of metalloproteinase-2 (TIMP-2)-deficient mice

Abstract

Circulating levels of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitor of metalloproteinases (TIMPs), are altered in human obesity and may contribute to its pathology. TIMP-2 exerts MMP-dependent (MMP inhibition and pro-MMP-2 activation) and MMP-independent functions. To assess the role of TIMP-2 in a murine model of nutritionally induced obesity, weight gain in wild-type and TIMP-2 deficient [knockout (KO)] mice fed a chow or high-fat diet (HFD) was determined. The effects of diet on glucose tolerance and insulin sensitivity, as well as pancreatic β-cell and adipocyte physiology, were assessed. Chow-fed TIMP-2 KO mice of both sexes became obese but maintained relatively normal glucose tolerance and insulin sensitivity. Obesity was exacerbated on the HFD. However, HFD-fed male, but not female, TIMP-2 KO mice developed insulin resistance with reduced glucose transporter 2 and pancreatic and duodenal homeobox 1 levels, despite increased β-cell mass and hyperplasia. Thus, although β-cell mass was increased, HFD-fed male TIMP-2 KO mice develop diabetes likely due to β-cell exhaustion and failure. TIMP-2 mRNA, whose expression was greatest in sc adipose tissue, was down-regulated in HFD-fed wild-type males, but not females. Furthermore, HFD increased membrane type 1-MMP (MMP-14) expression and activity in male, but not female, sc adipose tissue. Strikingly, MMP-14 expression increased to a greater extent in TIMP-2 KO males and was associated with decreased adipocyte collagen. Taken together, these findings demonstrate a role for TIMP-2 in maintaining extracellular matrix integrity necessary for normal β-cell and adipocyte physiology and that loss of extracellular matrix integrity may underlie diabetic and obesogenic phenotypes.

Fig. 1.

Obesity and hyperphagia in TIMP-2 KO mice. A, Regardless of diet type, TIMP-2 KO mice weighed significantly more than WT mice. Weight loss corresponds to IPGTT (G) and IPITT (I). B, Average daily food consumption over the 3-month study revealed that TIMP-2 KO mice were hyperphagic (M, P < 0.0001; F, P = 0.01). C, Chow-fed male TIMP-2 KO mice were more hyperphagic than female TIMP-2 KO mice. The decline in food consumption after d 80 was not due to obesity-induced autoregulation of feeding but a response to overnight starvation for ITT (see weight loss in A). D, HFD consumption in male TIMP-2 KO mice significantly spiked after complete cage change but not replacing bedding alone. HFD-fed female TIMP-2 KO mice showed dramatically increased food consumption initially but remained hyperphagic throughout the diet and also had increased food intake upon cage change. n = 8; **, P ≤ 0.01; ##, P < 0.0001.

Fig. 2.

Diet-induced metabolic dysfunction in TIMP-2 KO mice. A, At the study start, plasma glucose, insulin, and leptin levels were comparable in WT and TIMP-2 KO mice, suggesting that KO mice were not overtly diabetic and TIMP-2 KO hyperphagia was not due to reduced leptin. B, At the study termination, even though chow-fed TIMP-2 KO mice weighed considerably more than WT mice, plasma glucose, insulin, and leptin levels were comparable. C, In contrast, HFD-fed TIMP-2 KO mice of both sexes were hyperglycemic, hyperinsulinemic, and hyperleptinemic at the study termination. n = 4–8 per sex, genotype, and diet. *, P < 0.05; **, P ≤ 0.01; #, P ≤ 0.001; ##, P < 0.0001.

Fig. 3.

Sex-specific insulin resistance in TIMP-2 KO mice. Although chow-fed TIMP-2 KO mice displayed normal glucose tolerance (A), HFD-fed TIMP-2 KO mice of both sexes were glucose intolerant (B). Male TIMP-2 KO mice were more intolerant than female KO mice, because many readings exceeded the glucometer's 600 mg/dl maximum. C, Fasting and glucose-stimulated insulin levels were increased in chow-fed male, but not female, TIMP-2 KO mice. D, Fasting and glucose-stimulated insulin levels were increased in HFD-fed TIMP-2 KO mice of both sexes. Although chow-fed TIMP-2 KO mice displayed normal insulin responsiveness (E), only male HFD-fed TIMP-2 KO mice were insulin resistant (F). n = 4–8; *, P < 0.05; **, P ≤ 0.01; #, P ≤ 0.001; ##, P < 0.0001.

Fig. 4.

β-Cell compensation to diet-induced obesity in TIMP-2 KO mice. A, β-Cell mass was increased in male TIMP-2 KO, but not WT, HFD-fed mice (genotype, P = 0.003; diet, P = 0.004). B, β-Cell proliferation was unaltered in chow-fed TIMP-2 KO mice but increased in HFD-fed TIMP-2 KO mice to a greater extent than HFD-fed WT mice (diet, P < 0.0001; genotype, P = 0.0001). C, Male TIMP-2 KO islets in vivo displayed decreased Pdx1 expression. D, Both insulin and GLUT2 expression were decreased in TIMP-2 KO islets in vivo. E, Western blot analysis of isolated islets confirmed decreased Pdx1 and GLUT2 expression observed in vivo. F, HFD had no effect on TIMP-2 expression in isolated islets (Western blotting) or islets in vivo (immunofluorescence). Scale bars, 50 μm. n = 4; *, P < 0.05; #, P < 0.001; ##, P < 0.0001.

Fig. 5.

Increased inflammation in male TIMP-2 KO adipose tissue. A, TIMP-2 mRNA expression was significantly decreased in response to HFD within male WT sc and visceral (visc) but not epididymal (epi) or brown adipose tissue. B, Although increased adipose tissue mass was not detected in chow-fed TIMP-2 KO mice (M, P = 0.44; F, P = 0.53) with the technique used (i.e. dissected wet tissue weight), adipose mass was significantly increased in HFD-fed TIMP-2 KO mice. C, In contrast to the pancreas, increased adipose mass was associated with adipocyte hypertrophy in both chow- and HFD-fed male TIMP-2 KO mice. D, Subcutaneous adipose tissue histology revealed by hematoxylin and eosin staining. Scale bar, 100 μm. E, HFD increased vascularization (as revealed by Western blot analysis of PECAM-1 expression) comparably in WT and TIMP-2 KO male sc adipose tissue but did not alter vascularization in female mice. F, Quantitative real-time PCR revealed increased transcriptional adipokine (adiponectin and leptin), macrophage (EMR1 and MCP-1), and proinflammatory cytokine (IL-6 and TNFα) expression, suggesting increased inflammation in male HFD-fed TIMP-2 KO adipose tissue. G, HFD induced macrophage infiltration (as revealed by an increased number of Mac-2 immunoreactive cells) to a greater extent in male TIMP-2 KO than WT sc adipose tissue. Scale bar, 100 μm. n = 8 (B), n = 4 (all others); *, P < 0.05; **, P ≤ 0.01; #, P ≤ 0.001; ##, P < 0.0001.

Fig. 6.

Sex-specific increased collagenolysis in response to diet-induced obesity. A, HFD induced increased pro-MT1-MMP expression and MT1-MMP activity in male, but not female, sc adipose tissue. MT1-MMP activity was increased a greater extent in TIMP-2 KO mice. B, MT1-MMP immunoreactivity in sc adipose tissue. C, Brightfield and polarization microscopy photomicrographs of Sirius Red staining demonstrating decreased connective tissue and pericellular adipocyte collagen integrity in HFD-fed male sc tissue, consistent with increased MT1-MMP activity. Scale bars, 100 μm. n = 4; **, P ≤ 0.01; #, P ≤ 0.001.