Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice



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Adipose tissue macrophages have been proposed as a link between obesity and insulin resistance. However, the mechanisms underlying these processes are not completely defined. Calpains are calcium-dependent neutral cysteine proteases that modulate cellular function and have been implicated in various inflammatory diseases. To define whether activated calpains influence diet-induced obesity and adipose tissue macrophage accumulation, mice that were either wild type (WT) or overexpressing calpastatin (CAST Tg), the endogenous inhibitor of calpains were fed with high (60% kcal) fat diet for 16 weeks. CAST overexpression did not influence high fat diet-induced body weight and fat mass gain throughout the study. Calpain inhibition showed a transient improvement in glucose tolerance at 5 weeks of HFD whereas it lost this effect on glucose and insulin tolerance at 16 weeks HFD in obese mice. However, CAST overexpression significantly reduced adipocyte apoptosis, adipose tissue collagen and macrophage accumulation as detected by TUNEL, Picro Sirius and F4/80 immunostaining, respectively. CAST overexpression significantly attenuated obesity-induced inflammatory responses in adipose tissue. Furthermore, calpain inhibition suppressed macrophage migration to adipose tissue in vitro. The present study demonstrates a pivotal role for calpains in mediating HFD-induced adipose tissue remodeling by influencing multiple functions including apoptosis, fibrosis and inflammation.

Accumulation of adipose tissue macrophages has been proposed as a major link between obesity, insulin resistance and type 2 diabetes1. However, the mechanisms underlying these processes are still remain elusive. Calpains are calcium dependent intracellular cysteine proteases that tightly regulate their substrate proteins through limited proteolysis2. The two major isoforms, calpain-1 and -2, are expressed ubiquitously, whereas the other isoforms (e.g. -3, -9) are tissue-specific2. Activated calpain by calcium damages cells by selectively degrading intracellular proteins, including signaling proteins (e.g., cyclin-dependent kinase, protein kinase C)3,4, cytoskeletal proteins (e.g., talin, spectrin)5,6, and transcription factors (e.g., c-Jun, IkB)7,8,9. Calpains play a critical role in cellular apoptosis through activation of both caspase-dependent and caspase-independent pathways10,11. Calpains are also involved in acute inflammatory processes via the activation of nuclear factor kappa B (NF-kB)12. Since calcium-induced calpain activation is an irreversible reaction, calpains are tightly regulated by calpastatin (CAST), which is an endogenous inhibitor that binds strongly to calpains13. CAST contains four tandem repeats of a calpain-inhibitory domain, and each CAST molecule is capable of inhibiting more than one calpain molecule14. Calpains have been implicated to play a critical deleterious role in endothelial dysfunction15, hypertrophy and fibrosis16, and intestinal macrophage activation in experimental colitis17.

Recently, using a pharmacological inhibitor and calpain-1 deficient mice, we demonstrated that calpain inhibition significantly attenuated inflammatory processes present in aortic vascular diseases such as angiotensin II (AngII)-induced abdominal aortic aneurysms (AAA) and atherosclerosis in mice18,19. The beneficial effect of calpain inhibition was associated with reduction of macrophage accumulation, NF-kB mediated inflammation, and cytoskeletal protein, filamin A, fragmentation in the aorta19. However, the functional contributions of calpain activation in chronic adipose tissue inflammation, and macrophage infiltration under the condition of obesity remains to be elucidated.

Using CAST transgenic mice, we demonstrate that calpain inhibition transiently improved glucose tolerance and resulted in decreased adipose tissue macrophage accumulation in obese mice. Furthermore, calpain inhibition significantly modulated adipose tissue remodeling by influencing multiple functions including adipocyte apoptosis, fibrosis, inflammation and migratory properties of macrophages.

Figure 1
inflammation, Calpain Inhibition Attenuates Adipose Tissue Inflammation and Fibrosis in Diet-induced Obese Mice

Abundance of calpain protein and its activity was increased in obese adipose tissue. (A) Calpain-1, -2, -4, CAST and β-actin protein were detected by Western blotting in epididymal white adipose tissue (EpiWAT) extracts of wild type mice fed either a LFD or HFD for 16 weeks (n = 3). (B) Cross-sections from the EpiWAT of LFD and HFD fed wild type mice were immunostained with anti-mouse calpain-2 (red color). Arrows indicate positive immunostaining. (C) Calpain activity was measured by fluorimetric assay in EpiWAT from LFD and HFD fed mice (n…