Michele Cavalera

Increased tumor necrosis factor α–converting enzyme activity induces insulin resistance and hepatosteatosis in mice

Tumor necrosis factor α–converting enzyme (TACE, also known as ADAM17) was recently involved in the pathogenesis of insulin resistance. We observed that TACE activity was significantly higher in livers of mice fed a high‐fat diet (HFD) for 1 month, and this activity was increased in liver > white adipose tissue > muscle after 5 months compared with chow control. In mouse hepatocytes, C2C12 myocytes, and 3T3F442A adipocytes, TACE activity was triggered by palmitic acid, lipolysaccharide, high glucose, and high insulin. TACE overexpression significantly impaired insulin‐dependent phosphorylation of AKT, GSK3, and FoxO1 in mouse hepatocytes. To test the role of TACE activation in vivo, we used tissue inhibitor of metalloproteinase 3 (Timp3) null mice, because Timp3 is the specific inhibitor of TACE and Timp3−/− mice have higher TACE activity compared with wild‐type (WT) mice. Timp3−/− mice fed a HFD for 5 months are glucose‐intolerant and insulin‐resistant; they showed macrovesicular steatosis and ballooning degeneration compared with WT mice, which presented only microvesicular steatosis. Shotgun proteomics analysis revealed that Timp3−/− liver showed a significant differential expression of 38 proteins, including lower levels of adenosine kinase, methionine adenosysltransferase I/III, and glycine N‐methyltransferase and higher levels of liver fatty acid‐binding protein 1. These changes in protein levels were also observed in hepatocytes infected with adenovirus encoding TACE. All these proteins play a role in fatty acid uptake, triglyceride synthesis, and methionine metabolism, providing a molecular explanation for the increased hepatosteatosis observed in Timp3−/− compared with WT mice. Conclusion: We have identified novel mechanisms, governed by the TACE–Timp3 interaction, involved in the determination of insulin resistance and liver steatosis during overfeeding in mice. (HEPATOLOGY 2009.)

Overexpression of Tissue Inhibitor of Metalloproteinase 3 in Macrophages Reduces Atherosclerosis in Low-Density Lipoprotein Receptor Knockout Mice

Objective— Tissue inhibitor of metalloproteinase 3 (TIMP3) is a stromal protein that inhibits the activity of proteases and receptors. TIMP3 is downregulated in metabolic and inflammatory disorders, such as type 2 diabetes mellitus and atherosclerosis, particularly in regions enriched with monocyte/macrophage cells. To investigate the role of TIMP3 in atherosclerosis, we generated a new mouse model in which Timp3 was overexpressed in the atherosclerotic plaque via a macrophage-specific promoter (MacT3). We elucidated any potential antiatherosclerotic effects of TIMP3, including regulation of monocyte/macrophage recruitment within atherosclerotic plaques, in MacT3 mice crossbred with low-density lipoprotein receptor knockout (LDLR−/−) mice. Methods and Results— MacT3/LDLR−/− mice had an improvement of atherosclerosis and metabolic parameters compared with LDLR−/−. En face aorta and aortic root examination of MacT3/LDLR−/− mice revealed smaller atherosclerotic plaques with features of stability, such as increased collagen content and decreased necrotic core formation. Atherosclerotic plaques in MacT3/LDLR−/− mice contained fewer T cells and macrophages. Furthermore, TIMP3 overexpression in macrophages resulted in reduced oxidative stress signals, as evidenced by lower lipid peroxidation, protein carbonylation, and nitration in atheromas. Conclusion— Our study confirmed that macrophage-specific overexpression of TIMP3 decreases the inflammatory content and the amplitude of atherosclerotic plaques in mice.

Loss of TIMP3 underlies diabetic nephropathy via FoxO1/STAT1 interplay.

ADAM17 and its inhibitor TIMP3 are involved in nephropathy, but their role in diabetic kidney disease (DKD) is unclear. Diabetic Timp3−/− mice showed increased albuminuria, increased membrane thickness and mesangial expansion. Microarray profiling uncovered a significant reduction of Foxo1 expression in diabetic Timp3−/− mice compared to WT, along with FoxO1 target genes involved in autophagy, while STAT1, a repressor of FoxO1 transcription, was increased. Re‐expression of Timp3 in Timp3−/− mesangial cells rescued the expression of Foxo1 and its targets, and decreased STAT1 expression to control levels; abolishing STAT1 expression led to a rescue of FoxO1, evoking a role of STAT1 in linking Timp3 deficiency to FoxO1. Studies on kidney biopsies from patients with diabetic nephropathy confirmed a significant reduction in TIMP3, FoxO1 and FoxO1 target genes involved in autophagy compared to controls, while STAT1 expression was strongly increased.

Regulation of TIMP3 in diabetic nephropathy: a role for microRNAs

Diabetic nephropathy (DN) is the major cause of chronic kidney disease in developed countries and contributes significantly to increased morbidity and mortality among diabetic patients. Morphologically, DN is characterized by tubulo-interstitial fibrosis, thickening of the glomerular basement membrane and mesangial expansion mainly due to accumulation of extracellular matrix (ECM). ECM turnover is regulated by metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) activities. In diabetic conditions, TIMP3 expression in kidney is strongly reduced, but the causes of this reduction are still unknown. The aim of this study was to elucidate at least one of these mechanisms which relies on differential expression of TIMP3-targeting microRNAs (miRs) in a hyperglycemic environment either in vitro (MES13 cell line) or in vivo (mouse kidney and human biopsies). Among the TIMP3-targeting miRs, miR-21 and miR-221 were significantly upregulated in kidneys from diabetic mice compared to control littermates, and in a mesangial cell line grown in high glucose conditions. In human samples, only miR-21 expression was increased in kidney biopsies from diabetic patients compared to healthy controls. The expression of miR-217, which targets TIMP3 indirectly through downregulation of SirT1, was also increased in diabetic kidney and MES13 cell line. In agreement with these result, SirT1 expression was reduced in mouse and human diabetic kidneys as well as in MES13 mesangial cell line. TIMP3 deficiency has recently emerged as a hallmark of DN in mouse and human. In this study, we demonstrated that this reduction is due, at least in part, to increased expression of certain TIMP3-targeting miRs in diabetic kidneys compared to healthy controls. Unveiling the post-transcriptional mechanisms responsible for TIMP3 downregulation in hyperglycemic conditions may orient toward the use of this protein as a possible therapeutic target in DN.

IL-21 is a major negative regulator of IRF4-dependent lipolysis affecting Tregs in adipose tissue and systemic insulin sensitivity.

Obesity elicits immune cell infiltration of adipose tissue provoking chronic low-grade inflammation. Regulatory T cells (Tregs) are specifically reduced in adipose tissue of obese animals. Since interleukin (IL)-21 plays an important role in inducing and maintaining immune-mediated chronic inflammatory processes and negatively regulates Treg differentiation/activity, we hypothesized that it could play a role in obesity-induced insulin resistance. We found IL-21 and IL-21R mRNA expression upregulated in adipose tissue of high-fat diet (HFD) wild-type (WT) mice and in stromal vascular fraction from human obese subjects in parallel to macrophage and inflammatory markers. Interestingly, a larger infiltration of Treg cells was seen in the adipose tissue of IL-21 knockout (KO) mice compared with WT animals fed both normal diet and HFD. In a context of diet-induced obesity, IL-21 KO mice, compared with WT animals, exhibited lower body weight, improved insulin sensitivity, and decreased adipose and hepatic inflammation. This metabolic phenotype is accompanied by a higher induction of interferon regulatory factor 4 (IRF4), a transcriptional regulator of fasting lipolysis in adipose tissue. Our data suggest that IL-21 exerts negative regulation on IRF4 and Treg activity, developing and maintaining adipose tissue inflammation in the obesity state.

Tissue inhibitor of metalloproteinase-3 regulates inflammation in human and mouse intestine.

BACKGROUND & AIMS Tissue inhibitor of metalloproteinases (TIMP)-3 is an inhibitor of matrix metalloproteinases, which regulates tissue inflammation, damage, and repair. We investigated the role of TIMP-3 in intestinal inflammation in human beings and mice. METHODS We used real-time polymerase chain reaction and flow cytometry to measure levels of TIMP-3 in intestine samples from patients with Crohns disease (CD) and those without (controls). We also analyzed TIMP-3 levels in lamina propria mononuclear cells (LPMCs) collected from biopsy samples of individuals with or without CD (controls) and then stimulated with transforming growth factor (TGF)-β1, as well as in biopsy samples collected from patients with CD and then incubated with a Smad7 anti-sense oligonucleotide (knock down). LPMCs and biopsy samples from patients with CD were cultured with exogenous TIMP-3 and levels of inflammatory cytokines were measured. We evaluated the susceptibility of wild-type, TIMP-3-knockout (TIMP-3-KO), and transgenic (TIMP-3-Tg) mice to induction of colitis with 2, 4, 6-trinitrobenzene-sulfonic-acid (TNBS), and the course of colitis in recombinase-activating gene-1-null mice after transfer of wild-type or TIMP-3-KO T cells. RESULTS Levels of TIMP-3 were reduced in intestine samples from patients with CD compared with controls. Incubation of control LPMCs with TGF-β1 up-regulated TIMP-3; knockdown of Smad7, an inhibitor of TGF-β1, in biopsy samples from patients with CD increased levels of TIMP-3. Exogenous TIMP-3 reduced levels of inflammatory cytokines in CD LPMCs and biopsy samples. TIMP-3-KO mice developed severe colitis after administration of TNBS, whereas TIMP-3-Tg mice were resistant to TNBS-induced colitis. Reconstitution of recombinase-activating gene-1-null mice with T cells from TIMP-3-KO mice increased the severity of colitis, compared with reconstitution with wild-type T cells. CONCLUSIONS TIMP-3 is down-regulated in inflamed intestine of patients with CD. Its expression is regulated by TGF-β1, and knock-down of Smad7 in intestinal tissues from patient with CD up-regulates TIMP-3. Loss or reduction of TIMP-3 in mice promotes development of colitis.

Loss of TIMP3 exacerbates atherosclerosis in ApoE null mice

BACKGROUND Tissue inhibitor of metalloproteinase 3 (TIMP3) is a stromal protein that inhibits the activity of various proteases and receptors. We have previously shown TIMP3 to be downregulated in metabolic and inflammatory disorders, such as type 2 diabetes mellitus. We have now generated an ApoE(-/-)Timp3(-/-) mouse model in which, through the use of genetics, metabolomics and in-vivo phenotypical analysis we investigated the role of TIMP3 in the development of atherosclerosis. METHODS AND RESULTS En face aorta analysis and aortic root examination showed that ApoE(-/-)Timp3(-/-) mice show increased atherosclerosis with increased infiltration of macrophages into the plaque. Serum concentration of MCP-1 were elevated in the serum of ApoE(-/-)Timp3(-/-) mice coupled with an expansion of the inflammatory (M1) Gr1+ macrophages, both in the circulation and within the aortic tissue. Targeted analysis of metabolites revealed a trend to reduced short chain acylcarnitines. CONCLUSIONS Our study shows that lack of TIMP3 increases inflammation and polarizes macrophages towards a more inflammatory phenotype resulting in increased atherosclerosis.

TIMP3 interplays with apelin to regulate cardiovascular metabolism in hypercholesterolemic mice.

Objective Tissue inhibitor of metalloproteinase 3 (TIMP3) is an extracellular matrix (ECM) bound protein, which has been shown to be downregulated in human subjects and experimental models with cardiometabolic disorders, including type 2 diabetes mellitus, hypertension and atherosclerosis. The aim of this study was to investigate the effects of TIMP3 on cardiac energy homeostasis during increased metabolic stress conditions. Methods ApoE−/−TIMP3−/− and ApoE−/− mice on a C57BL/6 background were subjected to telemetric ECG analysis and experimental myocardial infarction as models of cardiac stress induction. We used Western blot, qRT-PCR, histology, metabolomics, RNA-sequencing and in vivo phenotypical analysis to investigate the molecular mechanisms of altered cardiac energy metabolism. Results ApoE−/−TIMP3−/− revealed decreased lifespan. Telemetric ECG analysis showed increased arrhythmic episodes, and experimental myocardial infarction by left anterior descending artery (LAD) ligation resulted in increased peri-operative mortality together with increased scar formation, ventricular dilatation and a reduction of cardiac function after 4 weeks in the few survivors. Hearts of ApoE−/−TIMP3−/− exhibited accumulation of neutral lipids when fed a chow diet, which was exacerbated by a high fat, high cholesterol diet. Metabolomics analysis revealed an increase in circulating markers of oxidative stress with a reduction in long chain fatty acids. Using whole heart mRNA sequencing, we identified apelin as a putative modulator of these metabolic defects. Apelin is a regulator of fatty acid oxidation, and we found a reduction in the levels of enzymes involved in fatty acid oxidation in the left ventricle of ApoE−/−TIMP3−/− mice. Injection of apelin restored the hitherto identified metabolic defects of lipid oxidation. Conclusion TIMP3 regulates lipid metabolism as well as oxidative stress response via apelin. These findings therefore suggest that TIMP3 maintains metabolic flexibility in the heart, particularly during episodes of increased cardiac stress.