Gelatinase A (MMP-2) promotes murine adipogenesis.

BACKGROUND: Expansion of adipose tissue is dependent on adipogenesis, angiogenesis and extracellular matrix remodeling. A functional role in these processes was suggested for the gelatinase subfamily of the matrix metalloproteinases. Here, we have evaluated a potential role of gelatinase A (MMP-2) in adipogenesis. METHODS: Murine embryonic fibroblasts (MEF) were derived from wild-type or MMP-2 deficient mice. Genetic manipulation of Mmp2 (shRNA-knockdown or overexpression) was performed in 3T3-F442A preadipocytes. Cell cultures were subjected to an adipogenic medium. As an in vivo model for de novo adipogenesis, 3T3-F442A preadipocytes with or without knockdown were injected subcutaneously in Nude BALB/c mice kept on high fat diet. RESULTS: Mmp2 deficient MEF, as compared to controls, showed significantly impaired differentiation into mature adipocytes, as demonstrated by 90% reduced intracellular lipid content and reduced expression of pro-adipogenic markers. Moreover, selective Mmp2 knockdown in 3T3-F442A preadipocytes resulted in significantly reduced differentiation. In contrast, overexpression of Mmp2 resulted in markedly enhanced differentiation. In de novo formed fat pads resulting from preadipocytes with Mmp2 knockdown expression of aP2, Ppar-γ and adiponectin was significantly lower, and collagen was more preserved. The fat pad weights as well as size and density of adipocytes or blood vessels were, however, not significantly different from controls. CONCLUSION: Our data directly support a functional role of MMP-2 in adipogenesis in vitro, and suggest a potential role in in vivo adipogenesis. GENERAL SIGNIFICANCE: Selective modulation of MMP-2 levels affects adipogenesis.

ADAMTS5 deficiency protects against non-alcoholic steatohepatitis in obesity.

BACKGROUND & AIMS: Increased prevalence of obesity is paralleled by an increase in non-alcoholic steatohepatitis (NASH). We previously found that the expression of ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) is enhanced in expanding adipose tissue. However, no information is available on a potential role in liver pathology. We studied the effect of ADAMTS5 deficiency on NASH in mice. METHODS: Wild-type (Adamts5+/+ ) and deficient (Adamts5-/- ) mice were kept on a standard- or high-fat diet (HFD) for 15 weeks. Alternatively, steatohepatitis was induced with methionine/choline-deficient (MCD) diet. RESULTS: HFD feeding resulted in comparable body weights for both genotypes, but Adamts5-/- mice had approximately 40% lower liver weight (P = 0.0004). In the Adamts5-/- mice, the HFD as well as the MCD diet consistently induced less NASH with less fibrosis. The deteriorating effect of ADAMTS5 on the liver during diet-induced obesity may be due, at least in part, to proteolytic cleavage of the matrix components syndecan-1 and versican, thereby enhancing hepatic triglyceride clearance from the circulation. Plasma lipid levels were elevated in obese Adamts5-/- mice. There was no clear effect of ADAMTS5 deficiency on glycaemia or glucose tolerance, whereas insulin sensitivity was somewhat improved. Furthermore, Adamts5-/- mice were protected from hepatic mitochondrial dysfunction, as indicated by increased mitochondrial respiratory chain complex activity, higher ATP levels and higher expression of antioxidant enzymes. CONCLUSIONS: Absence of ADAMTS5 preserves liver integrity in a diet-induced obesity model. Selective targeting of ADAMTS5 could provide a new therapeutic strategy for treatment/prevention of NASH.

Elastin fragmentation in atherosclerotic mice leads to intraplaque neovascularization, plaque rupture, myocardial infarction, stroke, and sudden death.

AIMS: There is a need for animal models of plaque rupture. We previously reported that elastin fragmentation, due to a mutation (C1039G(+/-)) in the fibrillin-1 (Fbn1) gene, promotes atherogenesis and a highly unstable plaque phenotype in apolipoprotein E deficient (ApoE(-/-)) mice on a Western-type diet (WD). Here, we investigated whether plaque rupture occurred in ApoE(-/-)Fbn1(C1039G+/-) mice and was associated with myocardial infarction, stroke, and sudden death. METHODS AND RESULTS: Female ApoE(-/-)Fbn1(C1039G+/-) and ApoE(-/-) mice were fed a WD for up to 35 weeks. Compared to ApoE(-/-) mice, plaques of ApoE(-/-)Fbn1(C1039G+/-) mice showed a threefold increase in necrotic core size, augmented T-cell infiltration, a decreased collagen I content (70 ± 10%), extensive neovascularization, intraplaque haemorrhage, and a significant increase in matrix metalloproteinase-2, -9, -12, and -13 expression or activity. Plaque rupture was observed in 70% of ascending aortas and in 50% of brachiocephalic arteries of ApoE(-/-)Fbn1(C1039G+/-) mice. In ApoE(-/-) mice, plaque rupture was not seen in ascending aortas and only in 10% of brachiocephalic arteries. Seventy percent of ApoE(-/-)Fbn1(C1039G+/-) mice died suddenly, whereas all ApoE(-/-) mice survived. ApoE(-/-)Fbn1(C1039G+/-) mice showed coronary plaques and myocardial infarction (75% of mice). Furthermore, they displayed head tilt, disorientation, and motor disturbances (66% of cases), disturbed cerebral blood flow (73% of cases; MR angiograms) and brain hypoxia (64% of cases), indicative of stroke. CONCLUSIONS: Elastin fragmentation plays a key role in plaque destabilization and rupture. ApoE(-/-)Fbn1(C1039G+/-) mice represent a unique model of acute plaque rupture with human-like complications.

Thrombin Generation by Fetoscopic Trauma to the Fetal Membranes: An in vivo and in vitro Study.

OBJECTIVE: We first aimed to investigate in vivo thrombin generation induced by fetoscopy, and second we used term membrane explants for measurement of thrombin generation, thrombin receptor location and induction of selected matrix metalloproteinases (MMPs) in tissue culture. MATERIALS AND METHODS: In vivo study (37 cases): samples of amniotic fluid were taken at the beginning and end of fetoscopy (mean gestational age 26.7 weeks) and analyzed by ELISA for thrombin-antithrombin complexes. In vitro study: fetal membranes were put in culture and punctured for measurement of thrombin generation by calibrated automated thrombography and ELISA. Induction of MMP-9 and MMP-2 was analyzed by zymography. PAR-1 was localized by immunohistochemistry. RESULTS: No significant increase in thrombin-antithrombin was measured in amniotic fluid obtained during fetoscopy. In vitro, thrombin generation induced by needle trauma of membrane cultures is correlated to the amount of plasma. Activity of MMP-9 but not MMP-2 was elevated in cultured membranes but could not be inhibited by a thrombin inhibitor. On histology, the thrombin receptor PAR-1 was located in the chorion and decidua, but not in the amnion. DISCUSSION: Despite the influence of thrombin on punctured fetal membranes in vitro, the role of thrombin in iatrogenic preterm premature rupture of membranes is questionable.

Differential effects of a gelatinase inhibitor on adipocyte differentiation and adipose tissue development.

(1) A potential role for the gelatinases in adipocyte differentiation in vitro and adipose tissue development in vivo was investigated using the gelatinase inhibitor tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4]oxadiazol-5-yl)-benzenesulphonylamino]-butyric acid). (2) Differentiation of murine 3T3-F442A preadipocytes (12 days after reaching confluence) into mature adipocytes in vitro was promoted in the presence of tolylsam (10-100 µmol/L). (3) De novo development of fat tissue in nude mice injected with preadipocytes and kept on a high-fat diet was significantly impaired following treatment with tolylsam (100 mg/kg per day for 4 weeks). (4) Adipose tissue development in matrix metalloproteinase (MMP)-2 deficient mice, kept on a high-fat diet, was significantly impaired following administration of tolylsam (100 mg/kg per day for 15 weeks). This was associated with markedly enhanced metabolic rate. (5) Treatment of MMP-2-deficient mice with tolylsam (100 mg/kg per day, 15 weeks) was associated with the preservation of collagen and a reduction in blood vessel size in adipose tissues in vivo. (6) Furthermore, plasma levels of triglycerides and free fatty acids were reduced by tolylsam treatment of MMP-2-deficient mice (100 mg/kg per day, 15 weeks), whereas nutrient adsorption in the intestine was not affected. (7) The results of the present study indicate that tolylsam promotes preadipocyte differentiation in vitro, but impairs adipose tissue development in vivo.

Does butein affect adipogenesis?

Butein is a plant flavonoid chalcone, with presumed anti-adipogenic properties. It was reported to impair preadipocyte differentiation, limit adipose tissue (AT) development and enhance white AT browning in rodents. In this study, we investigated the hypothesis that these effects of butein may occur via reduction of ADAMTS5 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 5) expression. Murine 3T3-L1 or 3T3-F442A preadipocytes were differentiated into mature adipocytes in the presence of butein or vehicle. At regular time intervals RNA was collected for gene expression studies. Male hemizygous mice for Tg(Ucp1-luc2,-tdTomato)1Kajim (ThermoMouse) were exposed to butein or vehicle, after which ATs were analyzed for Adamts5 and uncoupling protein-1 (Ucp-1) mRNA level changes. During preadipocyte differentiation, butein (25 - 50 mM) did not affect Adamts5 or Ucp-1 expression. Oil Red O analysis and monitoring of differentiation markers failed to demonstrate effects of butein on the differentiation extent. Furthermore, butein administration to the ThermoMouse (10 or 20 mg/kg, 4 days) or to the C57BL6/Rj mice (20 mg/kg, 4 weeks) did not enhance Adamts5 or Ucp-1 expression. Thus, we could not demonstrate marked effects of butein on the preadipocyte differentiation extent or AT development and browning, nor on Adamts5 or Ucp-1 gene expression during these processes.

Functional role of ADAMTS5 in adiposity and metabolic health.

Previous studies with gene-deficient mice (ADAMTS5-P) revealed that ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs, member 5) plays a functional role in adiposity and metabolic health. To confirm these observations, we have performed similar studies with an independently generated strain of ADAMTS5 deficient mice (ADAMTS5-J). Upon cold exposure as well as after high-fat diet feeding (diet-induced obesity or DIO model), these knockout (KO) mice developed less subcutaneous and gonadal white adipose tissue (WAT) as compared to their wild-type (WT) littermates (reduction was more pronounced in ADAMTS5-P mice). Enhanced browning of WAT, as monitored by expression of UCP-1 was seen in the ADAMTS5-J KO mice upon cold exposure but not in the DIO model (seen in both conditions with the ADAMTS5-P mice). Brown adipose tissue (BAT) mass was not different between KO and WT ADAMTS5-J mice, either upon cold exposure or in the DIO model (in contrast to the enhanced BAT mass with the ADAMTS5-P mice). Energy expenditure and thermogenesis were not significantly different between KO and WT ADAMTS5-J mice (in contrast to somewhat enhanced levels in ADAMTS5-P mice). Insulin sensitivity was improved in the ADAMTS5-J KO mice, and they were protected against non-alcoholic steatohepatitis in the DIO model (as the ADAMTS5-P mice). These data are thus similar for both strains of KO mice, confirming specificity of the phenotype, but some quantitative and qualitative differences are also observed.

ADAMTS13 deficiency promotes microthrombosis in a murine model of diet-induced liver steatosis.

ADAMTS13 cleaves ultralarge multimeric von Willebrand Factor (VWF), thereby preventing formation of platelet-rich microthrombi. ADAMTS13 is mainly produced by hepatic stellate cells, and numerous studies have suggested a functional role of ADAMTS13 in the pathogenesis of liver diseases. The aim of our study was to investigate a potential role of ADAMTS13 in formation of hepatic microthrombi and development of non-alcoholic steatohepatitis (NASH), and furthermore to evaluate whether plasmin can compensate for the absence of ADAMTS13 in removal of thrombi. Therefore, we used a model of high-fat diet-induced steatosis in Adamts13 deficient (Adamts13-/-) and wild-type (WT) control mice. Microthrombi were more abundant in the liver of obese Adamts13-/- as compared to obese WT or to lean Adamts13-/- mice. Obese Adamts13-/- mice displayed lower platelet counts and higher prevalence of ultra-large VWF multimers. Hepatic plasmin-α2-antiplasmin complex levels were comparable for obese WT and Adamts13-/- mice and were lower for lean Adamts13-/- than WT mice, not supporting marked activation of the fibrinolytic system. High fat diet feeding, as compared to normal chow, resulted in enhanced liver triglyceride levels for both genotypes (p < 0.0001) and steatosis (p < 0.0001 for WT mice, p = 0.002 for Adamts13-/- mice) without differences between the genotypes. Expression of markers of inflammation, oxidative stress, steatosis and fibrosis was affected by diet, but not by genotype. Thus, our data confirm that obesity promotes NASH, but do not support a detrimental role of ADAMTS13 in its development. However, Adamts13 deficiency in obese mice promotes hepatic microthrombosis, whereas a compensatory role of plasmin in removal of microthrombi in the absence of ADAMTS13 could not be demonstrated.

Loss of ADAMTS5 enhances brown adipose tissue mass and promotes browning of white adipose tissue via CREB signaling.

OBJECTIVE: A potential strategy to treat obesity - and the associated metabolic consequences - is to increase energy expenditure. This could be achieved by stimulating thermogenesis through activation of brown adipose tissue (BAT) and/or the induction of browning of white adipose tissue (WAT). Over the last years, it has become clear that several metalloproteinases play an important role in adipocyte biology. Here, we investigated the potential role of ADAMTS5. METHODS: Mice deficient in ADAMTS5 (Adamts5-/-) and wild-type (Adamts5+/+) littermates were kept on a standard of Western-type diet for 15 weeks. Energy expenditure and heat production was followed by indirect calorimetry. To activate thermogenesis, mice were treated with the ß3-adrenergic receptor (ß3-AR) agonist CL-316,243 or alternatively, exposed to cold for 2 weeks. RESULTS: Compared to Adamts5+/+ mice, Adamts5-/- mice have significantly more interscapular BAT and marked browning of their subcutaneous (SC) WAT. Thermogenic pathway analysis indicated, in the absence of ADAMTS5, enhanced ß3-AR signaling via activation of the cAMP response element-binding protein (CREB). Additional ß3-AR stimulation with CL-316,243 promoted browning of WAT in Adamts5+/+ mice but had no additive effect in Adamts5-/- mice. However, cold exposure induced more pronounced browning of WAT in Adamts5-/- mice. CONCLUSIONS: These data indicate that ADAMTS5 plays a functional role in development of BAT and browning of WAT. Hence, selective targeting of ADAMTS5 could provide a novel therapeutic strategy for treatment/prevention of obesity and metabolic diseases.

ADAMTS5 promotes murine adipogenesis and visceral adipose tissue expansion.

Enhanced expression of the aggrecanase ADAMTS5 (A Disintegrin And Metalloproteinase with Thrombospondin type 1 motifs; member 5) has been observed in adipose tissue (AT) of obese rodents. Here, we have investigated the role of ADAMTS5 in adipogenesis, AT expansion and associated angiogenesis. In vitro differentiation of precursor cells into mature adipocytes was studied using murine embryonic fibroblasts (MEF) derived from wild-type (Adamts5(+/+)) and ADAMTS5 deficient (Adamts5(-/-)) mice, or 3T3-F442A preadipocytes with stable gene silencing of Adamts5. De novo adipogenesis was monitored by injection of 3T3-F442A cells with or without Adamts5 knockdown in Nude mice. Furthermore, Adamts5(+/+)and Adamts5(-/-) mice were kept on a high-fat diet (HFD) to monitor AT development. Adamts5(-/-) MEF, as well as 3T3-F442A preadipocytes with Adamts5 knockdown, showed significantly reduced differentiation as compared to control cells. In mice, de novo formed fat pads arising from 3T3-F442A cells with Adamts5 knockdown were significantly smaller as compared to controls. After 15 or 25 weeks on HFD, total body weight and subcutaneous AT weight were similar for Adamts5(+/+) and Adamts5(-/-) mice, but visceral/gonadal fat mass was significantly lower for Adamts5(-/-) mice. These data were confirmed by magnetic resonance imaging. In addition, the blood vessel density in adipose tissue was higher for Adamts5(-/-) mice kept on HFD. In conclusion, our data support the concept that ADAMTS5 promotes adipogenesis in vitro and in vivo, as well as development of visceral AT and associated angiogenesis in mice kept on HFD.

The Anti-Adipogenic Potential of COUP-TFII Is Mediated by Downregulation of the Notch Target Gene Hey1.

BACKGROUND: Chicken ovalbumin upstream promoter transcription factor II (COUP-TFII) belongs to the steroid/thyroid hormone receptor superfamily and may contribute to the pathogenesis of obesity. It has not conclusively been established, however, whether its role is pro- or anti-adipogenic. METHODS AND RESULTS: Gene silencing of Coup-tfII in 3T3-F442A preadipocytes resulted in enhanced differentiation into mature adipocytes. This was associated with upregulation of the Notch signaling target gene Hey1. A functional role of Hey1 was confirmed by gene silencing in 3T3-F442A preadipocytes, resulting in impaired differentiation. In vivo, de novo fat pad formation in NUDE mice was significantly stimulated following injection of preadipocytes with Coup-tfII gene silencing, but impaired with Hey1 gene silencing. Moreover, expression of Coup-tfII was lower and that of Hey1 higher in isolated adipocytes of obese as compared to lean adipose tissue. CONCLUSIONS: These in vitro and in vivo data support an anti-adipogenic role of COUP-TFII via downregulating the Notch signaling target gene Hey1.

The Gustatory Signaling Pathway and Bitter Taste Receptors Affect the Development of Obesity and Adipocyte Metabolism in Mice.

Intestinal chemosensory signaling pathways involving the gustatory G-protein, gustducin, and bitter taste receptors (TAS2R) have been implicated in gut hormone release. Alterations in gut hormone profiles may contribute to the success of bariatric surgery. This study investigated the involvement of the gustatory signaling pathway in the development of diet-induced obesity and the therapeutic potential of targeting TAS2Rs to induce body weight loss. α-gustducin-deficient (α-gust-/-) mice became less obese than wild type (WT) mice when fed a high-fat diet (HFD). White adipose tissue (WAT) mass was lower in α-gust-/- mice due to increased heat production as a result of increases in brown adipose tissue (BAT) thermogenic activity, involving increased protein expression of uncoupling protein 1. Intra-gastric treatment of obese WT and α-gust-/- mice with the bitter agonists denatonium benzoate (DB) or quinine (Q) during 4 weeks resulted in an α-gustducin-dependent decrease in body weight gain associated with a decrease in food intake (DB), but not involving major changes in gut peptide release. Both WAT and 3T3-F442A pre-adipocytes express TAS2Rs. Treatment of pre-adipocytes with DB or Q decreased differentiation into mature adipocytes. In conclusion, interfering with the gustatory signaling pathway protects against the development of HFD-induced obesity presumably through promoting BAT activity. Intra-gastric bitter treatment inhibits weight gain, possibly by directly affecting adipocyte metabolism.

Gelatinase B (MMP-9) gene silencing does not affect murine preadipocyte differentiation.

Growth of adipose tissue involves differentiation of preadipocytes into mature lipid-containing adipocytes. The matrix metalloproteinases (MMPs) are known regulators of adipose tissue biology, and previous studies suggested a key role for gelatinase B (MMP-9) in adipogenesis. In the present study we have evaluated a potential functional role of MMP-9 by performing gene silencing in 3T3-F442A preadipocytes. At the end of a 12-day differentiation period, no significant differences were observed between MMP-9 knockdown cells and the control cells with respect to intracytoplasmatic lipid content, or expression of the adipogenic markers aP2, PPARγ, Lpl, and adiponectin, or of the preadipocyte marker Pref-1. Thus, in vitro differentiation of 3T3-F442A preadipocytes into mature adipocytes is not significantly affected by the loss of MMP-9.