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.

Advanced-age C57BL/6JRj mice do not develop obesity upon western-type diet exposure.

Obesity has become a global health-threat for every age group. It is well known that young mice (10-12 weeks of age) fed a western-type diet (WD) become obese and develop higher cholesterol levels and liver steatosis whereas insulin sensitivity is reduced. Less is known, however, about the effect of a WD on advanced-age mice. Therefore, 10 week-old (young) and 22 month-old (advanced-age), male C57BL/6JRj mice were kept on either a WD or a control diet (SFD) for 15 weeks. In contrast to young mice, advanced-age mice on WD did not show a higher body weight or adipose tissue (AT)-masses, suggesting a protection against diet-induced obesity. Furthermore, plasma adiponectin and leptin levels were not affected upon WD-feeding. A WD, however, did induce more hepatic lipid accumulation as well as increased hepatic expression of the macrophage marker F4/80, in advanced-age mice. There were no significant differences in mRNA levels of uncoupling protein-1 or F4/80 in brown AT (BAT) or of several intestinal integrity markers in colon suggesting that the protection against obesity is not due to excessive BAT or to impaired intestinal absorption of fat. Thus, advanced-age mice, in contrast to their younger counterparts, appeared to be protected against diet-induced obesity.

Deficiency of Bmal1 disrupts the diurnal rhythm of haemostasis.

BACKGROUND: Mice deficient in the circadian clock gene BMAL1 (Brain and Muscle ARNT-like protein-1) exhibit a hypercoagulable state and an enhanced arterial and venous thrombogenicity, which aggravates with age. We investigated the effect of BMAL1 deficiency in mice at a different age on the diurnal rhythm of factors involved in coagulation and fibrinolysis. MATERIALS AND METHODS: Hepatic, cardiac and brain tissues were isolated from 10- and 25-weeks-old Bmal1-deficient (BMAL1-/-) and wild-type (BMAL1+/+) mice at ZT2 and at ZT14 to analyze the mRNA expression level of genes involved in coagulation and fibrinolysis. RESULTS: Body weight and brain weight were significantly lower in all BMAL1-/- versus BMAL1+/+ mice. Bmal1 deficiency disturbed the diurnal rhythm of plasminogen activator inhibitor-1 (PAI-1) in liver and plasma, but not in cardiac or brain tissues. BMAL1+/+ livers showed diurnal fluctuations in factor (F)VII, FVII, protein S and anti-thrombin gene expression, which were not observed in BMAL1-/- tissues. Interestingly, tissue plasminogen activator (t-PA) expression was significantly upregulated in all BMAL1-/- versus BMAL1+/+ brains at both time points. Plasma t-PA-PAI-1 complex levels were however similar for all groups. CONCLUSION: Bmal1 deficiency affected the biphasic rhythm of coagulation and fibrinolysis factors predominantly in the liver. In the brain, Bmal1-dependent control of t-PA gene expression was documented for the first time.

Monitoring reperfused myocardial infarction with delayed left ventricular systolic dysfunction in rabbits by longitudinal imaging.

BACKGROUND: An experimental imaging platform for longitudinal monitoring and evaluation of cardiac morphology-function changes has been long desired. We sought to establish such a platform by using a rabbit model of reperfused myocardial infarction (MI) that develops chronic left ventricle systolic dysfunction (LVSD) within 7 weeks. METHODS: Fifty-five New Zeeland white (NZW) rabbits received sham-operated or 60-min left circumflex coronary artery (LCx) ligation followed by reperfusion. Cardiac magnetic resonance imaging (cMRI), transthoracic echocardiography (echo), and blood samples were collected at baseline, in acute (48 hours or 1 week) and chronic (7 weeks) stage subsequent to MI for in vivo assessment of infarct size, cardiac morphology, LV function, and myocardial enzymes. Seven weeks post MI, animals were sacrificed and heart tissues were processed for histopathological staining. RESULTS: The success rate of surgical operation was 87.27%. The animal mortality rates were 12.7% and 3.6% both in acute and chronic stage separately. Serum levels of the myocardial enzyme cardiac Troponin T (cTnT) were significantly increased in MI rabbits as compared with sham animals after 4 hours of operation (P<0.05). According to cardiac morphology and function changes, 4 groups could be distinguished: sham rabbits (n=12), and MI rabbits with no (MI_NO_LVSD; n=10), moderate (MI_M_LVSD; n=9) and severe (MI_S_LVSD; n=15) LVSD. No significant differences in cardiac function or wall thickening between sham and MI_NO_LVSD rabbits were observed at both stages using both cMRI and echo methods. cMRI data showed that MI_M_LVSD rabbits exhibited a reduction of ejection fraction (EF) and an increase in end-systolic volume (ESV) at the acute phase, while at the chronic stage these parameters did not change further. Moreover, in MI_S_LVSD animals, these observations were more striking at the acute stage followed by a further decline in EF and increase in ESV at the chronic stage. Lateral wall thickening determined by cMRI was significantly decreased in MI_M_LVSD versus MI_NO_LVSD animals at both stages (P<0.05). As for MI_S_LVSD versus MI_M_LVSD rabbits, the thickening of anterior, inferior and lateral walls was significantly more decreased at both stages (P<0.05). Echo confirmed the findings of cMRI. Furthermore, these in vivo outcomes including those from vivid cine cMRI could be supported by exactly matched ex vivo histomorphological evidences. CONCLUSIONS: Our findings indicate that chronic LVSD developed over time after surgery-induced MI in rabbits can be longitudinally evaluated using non-invasive imaging techniques and confirmed by the entire-heart-slice histomorphology. This experimental LVSD platform in rabbits may interest researchers in the field of experimental cardiology and help strengthen drug development and translational research for the management of cardiovascular diseases.

Adiposity and metabolic health in mice deficient in intestinal alkaline phosphatase.

Intestinal alkaline phosphatase 3 (AKP3) is an enzyme that was reported to play a role in lipid metabolism and to prevent high fat diet-induced metabolic syndrome in mice. To investigate a potential functional role of AKP3 in diet-induced adiposity and metabolic health, we have kept male and female wild-type or AKP3 deficient mice on a high fat diet for 15 weeks to induce obesity and compared those with mice kept on standard fat diet. Body weight as well as adipose tissue mass were statistically significantly higher upon high fat diet feeding for mice of both genders and genotypes. Female mice of either genotype kept on high fat diet gained less weight, resulting in smaller adipose tissue depots with smaller adipocytes. However, AKP3 deficiency had no significant effect on body weight gain or adipose tissue mass and did not affect adipocyte size or density. Gene expression analysis revealed no effect of the genotype on inflammatory parameters in adipose tissue, except for tumor necrosis factor alpha, which was higher in mesenteric adipose tissue of female obese mice. Plasma glucose and insulin levels were also not affected in obese AKP3 deficient mice. Overall, our data do not support a functional role of AKP3 in adipose tissue development, or insulin sensitivity.

Evaluation of cardiac arrhythmic risks using a rabbit model of left ventricular systolic dysfunction.

Patients with heart disease have a higher risk to develop cardiac arrhythmias, either spontaneously or drug-induced. In this study, we have used a rabbit model of myocardial infarction (MI) with severe left ventricular systolic dysfunction (LVSD) to study potential drug-induced cardiac risks with N-(piperidin-2-ylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide (flecainide). Upon ligation of the left circumflex arteries, male New Zealand White rabbits developed a large MI and moderate or severe LVSD 7 weeks after surgery, in comparison to SHAM-operated animals. Subsequently, animals were exposed to escalating doses of flecainide (0.25-4 mg/kg) or solvent. Electrocardiograms (ECG) were recorded before surgery, 1 and 7 weeks after surgery and continuously during the drug protocol. The ECG biomarker iCEB (index of Cardio-Electrophysiological Balance = QT/QRS ratio) was calculated. During the ECG recording at week 1 and week 7 post MI, rabbits had no spontaneous cardiac arrhythmias. When rabbits were exposed to escalating doses of flecainide, 2 out of 5 rabbits with MI and moderate LVSD versus 0 out of 5 solvent-treated rabbits developed arrhythmias, such as ventricular tachycardia/ventricular fibrillation. These were preceded by a marked decrease of iCEB just before the onset (from 4.09 to 2.42 and from 5.56 to 2.25, respectively). Furthermore, 1 out of 5 MI rabbits with moderate LVSD and 1 out of 7 MI rabbits with severe LVSD developed total atrioventricular block after flecainide infusion and died. This rabbit model of MI and severe LVSD may be useful for preclinical evaluation of drug (similar mechanism as flecainide)-induced arrhythmic risks, which might be predicted by iCEB.

Role of ADAMTS13 in diet-induced liver steatosis.

Previous studies, predominantly based on increased or decreased plasma levels, have reported conflicting data on a potential functional role of ADAMTS13 in the pathogenesis of liver diseases, including non­alcoholic steatohepatitis (NASH). The aim of the current study was to evaluate whether ADAMTS13 deficiency affects development of NASH. Therefore, male wild­type (WT) and Adamts13 deficient (Adamts13­/­) mice were kept on a steatosis­inducing diet devoid of methionine and choline (MCD) or a control diet (MCC) for 4 weeks. Induction of NASH did not affect plasma ADAMTS13 antigen levels of WT mice. MCD as compared with MCC feeding resulted in reduced body and liver weight with no differences between the genotypes. Plasma levels of the liver enzymes AST and ALT were significantly higher for MCD vs. MCC fed Adamts13­/­ and WT mice, however were not different between the genotypes. Liver triglyceride levels were also higher after MCD feeding, but were not different between WT and Adamts13­/­ mice. Adamts13­/­ mice on the two diets exhibited higher insulin sensitivity when compared with WT mice. On the MCC diet, the genotype did not show clear histological abnormalities in the liver, whereas severe steatosis and fibrosis were observed on MCD diet, however were comparable for both genotypes. This was supported by comparably enhanced hepatic expression in the two genotypes on MCD diet of the steatosis marker CD36 and of the fibrosis marker tissue inhibitor of metalloproteinase 1. Thus, the results of the current study do not support a functional role of ADAMTS13 in this murine model of NASH.

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 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 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.

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.

Age-associated pro-inflammatory adaptations of the mouse thoracic aorta.

Arterial ageing may be associated with a reduction in vasodilation due to increased reactive oxygen species (ROS) production, whereas endothelial cell activation induces procoagulant changes. However, little is known on the effect of ageing on expression of anticoagulant endothelial markers such as endothelial protein C receptor (EPCR). To study age-associated alterations in smooth muscle cell (SMC) and endothelial cell (EC) structure and function, the aorta was isolated from 10-week- and 12- and 24-month-old C57BL/6J mice and analysed for its expression of genes involved in senescence, oxidative stress production, coagulation and matrix remodelling. In addition, vasorelaxation experiments were performed using 10-week- and 24-month-old thoracic aortic ring segments in organ chamber baths. The media thickness of the thoracic aorta progressively increased with age, associated with hypertrophy of vascular SMCs. Basal nitric oxide production and sensitivity to acetylcholine-mediated vasodilation in thoracic aorta rings was reduced with age, whereas no significant differences in ROS production could be demonstrated. Gene expression of tissue factor, EPCR and von Willebrand factor was not affected by ageing of the aorta, whereas that of thrombomodulin was mildly reduced and that of xanthine dehydrogenase, NADPH oxidase 4, tumour necrosis factor-α and vascular cell adhesion molecule-1 significantly enhanced. In conclusion, a reduction in endothelial cell-mediated vasodilation in aged thoracic aortas of C57BL/6J mice was accompanied by a shift towards a pro-inflammatory state of the endothelium.

From mice to men--mouse models in obesity research: what can we learn?

Obesity has become a world-wide epidemic and is associated with diseases such as diabetes, dyslipidaemia, cardiovascular disease and certain types of cancers. Understanding the adipose tissue developmental process, involving adipogenesis, angiogenesis and extracellular matrix remodelling, is therefore crucial to reveal the pathobiology of obesity. Experimental mouse models are extensively used to gain new insights into these processes and to evaluate the role of new key players, in particular proteolytic system components, in adipose tissue development and obesity. In this paper, we will review available in vitro and in vivo murine models of obesity and discuss their value in understanding the mechanisms contributing to obesity.

Effect of vascular endothelial growth factor receptor 2 antagonism on adiposity in obese mice.

Development and maintenance of fat depots require angiogenesis, in which vascular endothelial growth factor (VEGF) and its receptors play a crucial role. We have evaluated the effect of blocking VEGF receptor 2 (VEGF-R2) with a MAB (DC101) on adipose tissue of mice with established obesity. Therefore, obese male wild-type C57B1/6 mice were treated with i.p. injection of DC101 (40 mg/kg body weight, twice weekly during 13 weeks) or of the control antibody 1C8. Treatment with DC101 resulted in a slightly lower body weight but had no effect on subcutaneous (SC) or gonadal (GON) white adipose tissue mass, as monitored by MRI. Histochemical analysis of isolated SC and GON fat pads did not reveal significant effects of DC101 treatment on adipocyte or blood vessel size or density. Plasma levels of the liver enzymes aspartate aminotransferase and alanine aminotransferase as well as liver triglyceride levels were significantly decreased following DC101 treatment. Plasma glucose levels were markedly lower upon DC101 treatment, whereas insulin and adiponectin levels were not affected. Furthermore, Akt phosphorylation in adipose tissues was not affected. Thus, in vivo VEGF-R2 blockade in mice with established nutritionally induced obesity did not significantly affect insulin signaling in adipose tissue or adiposity.

CD36 promotes adipocyte differentiation and adipogenesis.

BACKGROUND: CD36 is a membrane glycoprotein, contributing to the pathogenesis of metabolic disorders, like obesity, which has become a major health concern worldwide. METHODS: A potential functional role of the scavenger receptor CD36 was investigated in in vitro adipocyte differentiation and in vivo adipogenesis. RESULTS: During differentiation of 3T3-F442A preadipocytes into mature adipocytes, expression of CD36 was upregulated and CD36 gene silencing resulted in impaired differentiation, as monitored by Oil Red O staining and expression of adipogenic markers. De novo fat pad formation in NUDE mice following injection of preadipocytes was significantly reduced upon CD36 gene silencing as compared to control. This was associated with marked adipocyte hypotrophy and reduced adipose tissue adipocyte content. Macrophage infiltration in de novo fat tissues derived from preadipocytes with CD36 gene silencing was not significantly different from controls. Collagen content was significantly higher in de novo fat with CD36 gene silencing. In a nutritionally induced obesity model, total body weight as well as subcutaneous and gonadal adipose tissue mass were significantly lower in CD36 deficient mice as compared to wild-type littermates. GENERAL SIGNIFICANCE: Thus, our data support a functional role of CD36 in promoting adipogenesis in vitro as well as in vivo.

Matrix metalloproteinase inhibition affects adipose tissue mass in obese mice.

1. Because the development of adipose tissue involves remodelling of the extracellular matrix (ECM), which requires matrix metalloproteinase (MMP) activity, we examined whether MMP inhibitors may have the potential to affect adipose tissue mass in obese mice. 2. Administration of the relatively gelatinase-specific MMP inhibitor tolylsam ((R)-3-methyl-2-[4-(3-p-tolyl-[1,2,4]oxadiazol-5-yl)-benzenesulphonylamino]-butyric acid; 100 mg/kg per day) for 7 weeks to obese wild-type mice on a high-fat diet resulted in significantly lower bodyweight (P < 0.05), lower subcutaneous (SC) and gonadal (GON) adipose tissue mass (both P < 0.05) and smaller adipocytes in both SC (P < 0.005) and GON (P < 0.0005) adipose tissues. 3. Magnetic resonance imaging confirmed a lower total body fat content in tolylsam-treated mice (P < 0.0005). In addition, tolylsam treatment of wild-type mice was associated with a marked enhancement in metabolic rate. 4. Electron microscopy analysis of tissue sections at the end of the 7 week feeding period revealed significantly higher collagen accumulation in the ECM of SC adipose tissues of tolylsam-treated mice (P < 0.001). 5. Thus, the relatively gelatinase-specific MMP inhibitor tolylsam has the potential to affect fat tissue growth in obese mice.

Effect of premature aging on murine adipose tissue.

To evaluate the effect of aging on adipose tissue development, subcutaneous (SC) and gonadal (GON) white and peri-aortic brown adipose tissues were analyzed of 10 and 30 week old mice deficient in the clock gene Bmal1 (brain and muscle arnt like protein 1) (Bmal1(-/-)) and wild-type littermates (Bmal1(+/+)) kept on a standard fat diet. At both ages, daily food intake was significantly decreased for Bmal1(-/-) mice, associated with reduced hypothalamic expression of PPARα. Between 10 and 30 weeks of age, the total body weight of Bmal1(+/+) mice increased significantly, but that of Bmal1(-/-) mice did not change. Whereas for Bmal1(+/+) mice, both SC and GON fat mass increased with age, these decreased for Bmal1(-/-) mice. This was associated with increased adipocyte size with age for Bmal1(+/+) but not for Bmal1(-/-) mice. Adipose tissue related angiogenesis was not affected by genotype or aging. Peri-aortic brown adipose tissue mass in 30 week old Bmal1(-/-) mice was significantly reduced as compared to age-matched Bmal1(+/+) mice. Comparison of gene expression profiles in SC and GON adipose tissues of both genotypes revealed very marked effects of Bmal1 gene deletion in itself on PAI-1 (4- to 13-fold downregulation), whereas the associated effect of premature aging was striking for leptin (90- to 130-fold downregulation). Thus, premature aging in Bmal1(-/-) mice kept on normal chow was associated with reduced adiposity.

Maximal PAI-1 inhibition in vivo requires neutralizing antibodies that recognize and inhibit glycosylated PAI-1.

Plasminogen activator inhibitor-1 (PAI-1) regulates the activity of t-PA and u-PA and is an important inhibitor of the plasminogen activator system. Elevated PAI-1 levels have been implicated in the pathogenesis of several diseases. Prior to the evaluation of PAI-1 inhibitors in humans, there is a strong need to study the effect of PAI-1 inhibition in mouse models. In the current study, four monoclonal antibodies previously reported to inhibit recombinant PAI-1 in vitro, were evaluated in an LPS-induced endotoxemia model in mice. Both MA-33H1F7 and MA-MP2D2 exerted a strong PAI-1 inhibitory effect, whereas for MA-H4B3 and MA-124K1 no reduced PAI-1 activity was observed in vivo. Importantly, the lack of PAI-1 inhibition observed for MA-124K1 and MA-H4B3 in vivo corresponded with the absence of inhibition toward glycosylated mouse PAI-1 in vitro. Three potential N-glycosylation sites were predicted for mouse PAI-1 (i.e. N209, N265 and N329). Electrophoretic mobility analysis of glycosylation knock-out mutants before and after deglycosylation indicates the presence of glycan chains at position N265. These data demonstrate that an inhibitory effect toward glycosylated PAI-1 is a prerequisite for efficient PAI-1 inhibition in mice. Our data also suggest that PAI-1 inhibitors for use in humans must preferably be screened on glycosylated PAI-1 and not on recombinant non-glycosylated PAI-1.

Caloric restriction improves coagulation and inflammation profile in obese mice.

To evaluate associations between adiposity and coagulation or inflammation profile, obese wild-type C57Bl/6 mice were subjected to drastic caloric restriction by switching from a high fat diet to restricted normal chow. After 6 weeks, total body weights as well as subcutaneous and gonadal adipose tissue mass were markedly reduced, associated with adipocyte hypotrophy (all p<0.001). Weight reduction was associated with markedly reduced plasma levels of plasminogen activator inhibitor-1, Factor VII and Factor VIII. Reduced oxidative stress and inflammation following weight reduction is supported by significantly lower expression in adipose tissues of pro-inflammatory interleukin-6, higher expression of anti-oxidant catalase, superoxide dismutase 1 and glutathione peroxidase 1, and lower plasma levels of C-reactive protein. Furthermore, reduced levels of leptin and enhanced levels of adiponectin were observed, whereas cholesterol and triglyceride levels were reduced. The content of structurally intact collagen fibers was significantly higher in subcutaeneous and gonadal adipose tissues after caloric restriction. Thus, caloric restriction and drastic weight loss in obese mice is associated with improved plasma coagulation profile and with reduced oxidative stress and inflammation in the adipose tissues.