Inhibition of angiotensin-induced aortic aneurysm by metformin in apolipoprotein E-deficient mice.

OBJECTIVE: Metformin is associated with a reduced incidence and growth of abdominal aortic aneurysms (AAAs). The aim of the present study was to investigate the inhibitory effects of metformin on AAA development and possible underlying mechanisms in experimentally induced AAAs in mice, along with the possible synergistic effects of metformin and imatinib. METHODS: Angiotensin II was used to induce AAAs in apolipoprotein E knockout (ApoE -/- ) mice for 28 days. The mice were treated with metformin (n = 11), metformin combined with imatinib (n = 7), or vehicle (n = 12), starting 3 days before angiotensin II infusion. Ultrasound examination was used to analyze aneurysm formation. Cholesterol and blood pressure levels were measured at the start and end of the study. Gene array and quantitative polymerase chain reaction were used to analyze the changes in gene expression in the aorta. Wire myography was used to study vascular function. RESULTS: Metformin (n = 11) suppressed the formation and progression of AAAs by 50% compared with the vehicle controls (n = 12), with no further effects from imatinib (n = 7). Metformin reduced total cholesterol and mRNA expression of SPP1 (encoding osteopontin), MMP12, and the glycoprotein genes Gpnmb and Clec7a. Furthermore, metformin inhibited blood pressure increases and reduced vascular contractions, as determined by wire myography, and restored the anticontractile function of perivascular adipose tissue. CONCLUSION: Metformin inhibited aneurysm formation and progression and normalized vascular function in ApoE -/- mice with no additional effect of imatinib. This might be mediated by the protective effects on vascular endothelial function and perivascular adipose tissue via reduced expression of genes promoting inflammation, including SPP1, MMP12, Gpnmb, and Clec7a. CLINICAL RELEVANCE: Retrospective studies of the effects of metformin in patients with aneurysm have so far only been performed of those with type 2 diabetes. The present study shows that metformin has effects on nondiabetic mice and revealed the mechanistic effects mediated by the drug that could also be important to study as outcomes in humans. Future clinical trials using metformin are warranted in patients without diabetes with abdominal aortic aneurysms.

miR-10b promotes aortic aneurysm formation and aortic rupture in angiotensin II-induced ApoE-deficient mice.

Abdominal aortic aneurysm (AAA) is associated with increased plasma levels of microRNA (miR) -10b. 5 nmols of miR-10b or miR control was administrated to Apolipoprotein E-deficient mice three days prior implantation of osmotic mini-pumps containing angiotensin II, and for three additional times once a week, which increased expression of miR-10b in plasma. Animals receiving miR-10b had a mortality rate due to aortic rupture of 61% compared to 11% in the miR controls (p < 0.05). Further, miR- 10b resulted in an increased aneurysm formation and growth (p < 0.05), which was accompanied by increased elastin degradation, neutrophil and mast cell markers (p < 0.05). In conclusion, miR-10b is functionally affecting aneurysm development and rupture and not only a marker of AAA. More mechanistic studies are required to better understand miR-10b's role in AAA formation.

Role of Kallikrein 7 in Body Weight and Fat Mass Regulation.

Increased plasma and adipose tissue protease activity is observed in patients with type 2 diabetes and obesity. It has been proposed that specific proteases contribute to the link between obesity, adipose tissue inflammation and metabolic diseases. We have recently shown that ablation of the serine protease kallikrein-related peptidase 7 (Klk7) specifically in adipose tissue preserves systemic insulin sensitivity and protects mice from obesity-related AT inflammation. Here, we investigated whether whole body Klk7 knockout (Klk7-/-) mice develop a phenotype distinct from that caused by reduced Klk7 expression in adipose tissue. Compared to littermate controls, Klk7-/- mice gain less body weight and fat mass both under chow and high fat diet (HFD) feeding, are hyper-responsive to exogenous insulin and exhibit preserved adipose tissue function due to adipocyte hyperplasia and lower inflammation. Klk7-/- mice exhibit increased adipose tissue thermogenesis, which is not related to altered thyroid function. These data strengthen our recently proposed role of Klk7 in the regulation of body weight, energy metabolism, and obesity-associated adipose tissue dysfunction. The protective effects of Klk7 deficiency in obesity are likely linked to a significant limitation of adipocyte hypertrophy. In conclusion, our data indicate potential application of specific KLK7 inhibitors to regulate KLK7 activity in the development of obesity and counteract obesity-associated inflammation and metabolic diseases.

The Fabp4-Cre-Model is Insufficient to Study Hoxc9 Function in Adipose Tissue.

Developmental genes are important regulators of fat distribution and adipose tissue (AT) function. In humans, the expression of homeobox c9 (HOXC9) is significantly higher in subcutaneous compared to omental AT and correlates with body fat mass. To gain more mechanistic insights into the role of Hoxc9 in AT, we generated Fabp4-Cre-mediated Hoxc9 knockout mice (ATHoxc9-/-). Male and female ATHoxc9-/- mice were studied together with littermate controls both under chow diet (CD) and high-fat diet (HFD) conditions. Under HFD, only male ATHoxc9-/- mice gained less body weight and exhibited improved glucose tolerance. In both male and female mice, body weight, as well as the parameters of glucose metabolism and AT function were not significantly different between ATHoxc9-/- and littermate control CD fed mice. We found that crossing Hoxc9 floxed mice with Fabp4-Cre mice did not produce a biologically relevant ablation of Hoxc9 in AT. However, we hypothesized that even subtle reductions of the generally low AT Hoxc9 expression may cause the leaner and metabolically healthier phenotype of male HFD-challenged ATHoxc9-/- mice. Different models of in vitro adipogenesis revealed that Hoxc9 expression precedes the expression of Fabp4, suggesting that ablation of Hoxc9 expression in AT needs to be achieved by targeting earlier stages of AT development.

Nicotinamide Nucleotide Transhydrogenase (Nnt) is Related to Obesity in Mice.

The C57BL/6J (B6J) mouse strain has been widely used as a control strain for the study of metabolic diseases and diet induced obesity (DIO). B6J mice carry a spontaneous deletion mutation in the nicotinamide nucleotide transhydrogenase (Nnt) gene eliminating exons 7-11, resulting in expression of a truncated form of Nnt, an enzyme that pumps protons across the inner mitochondrial membrane. It has been proposed that this mutation in B6J mice is associated with epigonadal fat mass and altered sensitivity to diet induced obesity. To define the role of Nnt in the development of diet induced obesity, we generated first backcross (BC1) hybrids of wild type Nnt C57BL/6NTac and mutated Nnt C57BL/6JRj [(C57BL/6NTac×C57BL/6JRj)F1×C57BL/6NTac]. Body weight gain and specific fat-pad depot mass were measured in BC1 hybrids under high fat diet conditions. Both sexes of BC1 hybrids indicate that mice with Nnt wild type allele are highly sensitive to DIO and exhibit higher relative fat mass. In summary, our data indicate that the Nnt mutation in mice is associated with sensitivity to DIO and fat mass.

Altered IL-32 Signaling in Abdominal Aortic Aneurysm.

INTRODUCTION AND OBJECTIVE: Interleukin (IL)-32 is a pro-inflammatory cytokine not previously studied in relation to abdominal aortic aneurysm (AAA). The aim of this study was to elucidate the expression and localization of IL-32 in AAA. METHODS: Expression and localization of IL-32 in human aortic tissue was studied with immunohistochemical analysis and Western blot (AAA: n = 5; controls: n = 4). ELISA was used to measure IL-32 in human plasma samples (AAA: n = 140; controls: n = 37) and in media from cultured peripheral blood mononuclear cells (PBMCs) from 3 healthy donors. IL-32 mRNA in PBMCs, endothelial cells, aortic smooth muscle cells (SMCs), and aortic tissue samples of AAA (n = 16) and control aortas (n = 9) was measured with qPCR. RESULTS: IL-32 was predominantly expressed in SMCs and T-cell-rich areas. Highest mRNA expression was observed in the intima/media layer of the AAA. A weaker protein expression was detected in non-aneurysmal aortas. Expression of IL-32 was confirmed in isolated T cells, macrophages, endothelial cells, and SMCs, where expression was also inducible by cytokines such as interferon-γ. There was no difference in IL-32 expression in plasma between patients and controls. CONCLUSION: IL-32 signaling is altered locally in AAA and could potentially play an important role in aneurysm development. Further studies using animal models would be helpful to study its potential role in AAA disease.

Effects of osteoprotegerin/TNFRSF11B in two models of abdominal aortic aneurysms.

Osteoprotegerin (OPG), additionally termed tumor necrosis factor receptor superfamily member 11B, is produced by vascular smooth muscle cells (VSMCs) and endothelial cells in the vasculature, and its release may be modulated by pro­inflammatory cytokines, including interleukin­1ß and tumor necrosis factor­α. The present study investigated the effects of treatment with low­dose human recombinant OPG on abdominal aortic aneurysm (AAA) development in mice. Mice were treated with 1 µg human recombinant OPG four times (or vehicle) for 2 weeks prior to inducing AAA. A total of two different models for inducing AAA were used to investigate the hypothesis as to whether OPG is involved in key events of AAA development, using osmotic mini­pumps with angiotensin II in apolipoprotein­E (ApoE­/­) mice for 28 days or using periaortic application of CaCl2 on the aorta in C57Bl/6J mice for 14 days. OPG was continuously administered during the experimental period. Histological staining using Masson's trichrome, Verhoeff's van­Gieson and picro­sirius red, in addition to reverse transcription­quantitative polymerase chain reaction analysis of various markers, were used to analyze phenotypic alterations. Treatment with OPG had no inhibitory effect on AAA development in the angiotensin II model in ApoE­/­ mice, which developed suprarenal aneurysms, although it increased vessel wall thickness of the aorta and total collagen in C57Bl/6J mice using the CaCl2 model that induced infrarenal dilation of the aorta. Treatment with OPG did not inhibit aneurysm development and key events, including inflammation, extracellular matrix or VSMC remodeling, in aortas from OPG­treated mice with periaortic treatment with CaCl2. The results indicated that mice treated with low levels of human recombinant OPG may have a more stable aneurysmal phenotype due to compensatory production of collagen and increased vessel wall thickness of the aorta, potentially protecting the aneurysm from rupture. Further studies investigating rupture models of AAA in addition to using higher levels of OPG are require to verify this speculation. Furthermore, treatment with low levels of OPG in patients with AAA may represent a novel therapeutic strategy for the treatment of AAA as well as attenuate the adverse effects associated with the administration of normal and high dosages of OPG.

PDGF-C and PDGF-D signaling in vascular diseases and animal models.

Members of the platelet-derived growth factor (PDGF) family are well known to be involved in different pathological conditions. The cellular and molecular mechanisms induced by the PDGF signaling have been well studied. Nevertheless, there is much more to discover about their functions and some important questions to be answered. This review summarizes the known roles of two of the PDGFs, PDGF-C and PDGF-D, in vascular diseases. There are clear implications for these growth factors in several vascular diseases, such as atherosclerosis and stroke. The PDGF receptors are broadly expressed in the cardiovascular system in cells such as fibroblasts, smooth muscle cells and pericytes. Altered expression of the receptors and the ligands have been found in various cardiovascular diseases and current studies have shown important implications of PDGF-C and PDGF-D signaling in fibrosis, neovascularization, atherosclerosis and restenosis.

Ablation of kallikrein 7 (KLK7) in adipose tissue ameliorates metabolic consequences of high fat diet-induced obesity by counteracting adipose tissue inflammation in vivo.

Vaspin is an adipokine which improves glucose metabolism and insulin sensitivity in obesity. Kallikrein 7 (KLK7) is the first known protease target inhibited by vaspin and a potential target for the treatment of metabolic disorders. Here, we tested the hypothesis that inhibition of KLK7 in adipose tissue may beneficially affect glucose metabolism and adipose tissue function. Therefore, we have inactivated the Klk7 gene in adipose tissue using conditional gene-targeting strategies in mice. Klk7-deficient mice (ATKlk7 -/-) exhibited less weight gain, predominant expansion of subcutaneous adipose tissue and improved whole body insulin sensitivity under a high fat diet (HFD). ATKlk7 -/- mice displayed higher energy expenditure and food intake, most likely due to altered adipokine secretion including lower circulating leptin. Pro-inflammatory cytokine expression was significantly reduced in combination with an increased percentage of alternatively activated (anti-inflammatory) M2 macrophages in epigonadal adipose tissue of ATKlk7 -/-. Taken together, by attenuating adipose tissue inflammation, altering adipokine secretion and epigonadal adipose tissue expansion, Klk7 deficiency in adipose tissue partially ameliorates the adverse effects of HFD-induced obesity. In summary, we provide first evidence for a previously unrecognized role of KLK7 in adipose tissue with effects on whole body energy expenditure and insulin sensitivity.

Hedgehog signalling in myeloid cells impacts on body weight, adipose tissue inflammation and glucose metabolism.

AIMS/HYPOTHESIS: Recently, hedgehog (Hh) was identified as a crucial player in adipose tissue development and energy expenditure. Therefore, we tested whether Hh ligands are regulated in obesity. Further, we aimed at identifying potential target cells of Hh signalling and studied the functional impact of Hh signalling on adipose tissue inflammation and glucose metabolism. METHODS: Hh ligands and receptors were analysed in adipose tissue or serum from lean and obese mice as well as in humans. To study the impact on adipose tissue inflammation and glucose metabolism, Hh signalling was specifically blocked in myeloid cells using a conditional knockout approach (Lys-Smo -/-). RESULTS: Desert Hh (DHH) and Indian Hh (IHH) are local Hh ligands, whereas Sonic Hh is not expressed in adipose tissue from mice or humans. In mice, obesity leads to a preferential upregulation of Hh ligands (Dhh) and signalling components (Ptch1, Smo and Gli1) in subcutaneous adipose tissue. Further, adipose tissue macrophages are Hh target cells owing to the expression of Hh receptors, such as Patched1 and 2. Conditional knockout of Smo (which encodes Smoothened, a mandatory Hh signalling component) in myeloid cells increases body weight and adipose tissue inflammation and attenuates glucose tolerance, suggesting an anti-inflammatory effect of Hh signalling. In humans, adipose tissue expression of DHH and serum IHH decrease with obesity and type 2 diabetes, which might be explained by the intake of metformin. Interestingly, metformin reduced Dhh and Ihh expression in mouse adipose tissue explants. CONCLUSIONS/INTERPRETATION: Hh signalling in myeloid cells affects adipose tissue inflammation and glucose metabolism and may be a potential target to treat type 2 diabetes.

Thyroid hormone status defines brown adipose tissue activity and browning of white adipose tissues in mice.

The present study aimed to determine the effect of thyroid hormone dysfunction on brown adipose tissue activity and white adipose tissue browning in mice. Twenty randomized female C57BL/6NTac mice per treatment group housed at room temperature were rendered hypothyroid or hyperthyroid. In-vivo small animal 18F-FDG PET/MRI was performed to determine the effects of hypo- and hyperthyroidism on BAT mass and BAT activity. Ex-vivo14C-acetate loading assay and assessment of thermogenic gene and protein expression permitted analysis of oxidative and thermogenic capacities of WAT and BAT of eu-, hyper and hypothyroid mice. 18F-FDG PET/MRI revealed a lack of brown adipose tissue activity in hypothyroid mice, whereas hyperthyroid mice displayed increased BAT mass alongside enhanced 18F-FDG uptake. In white adipose tissue of both, hyper- and hypothyroid mice, we found a significant induction of thermogenic genes together with multilocular adipocytes expressing UCP1. Taken together, these results suggest that both the hyperthyroid and hypothyroid state stimulate WAT thermogenesis most likely as a consequence of enhanced adrenergic signaling or compensation for impaired BAT function, respectively.

Repin1 deficiency improves insulin sensitivity and glucose metabolism in db/db mice by reducing adipose tissue mass and inflammation.

Replication initiator 1 (Repin1) is a zinc finger protein playing a role in insulin sensitivity, body fat mass and lipid metabolism by regulating the expression key genes of glucose and lipid metabolism. Here, we tested the hypothesis that introgression of a Repin1 deletion into db/db mice improves glucose metabolism in vivo. We generated a whole body Repin1 deficient db/db double knockout mouse (Rep1(-/-)x db/db) and systematically characterized the consequences of Repin1 deficiency on insulin sensitivity, glucose and lipid metabolism parameters and fat mass. Hyperinsulinemic-euglycemic clamp studies revealed significantly improved insulin sensitivity in Rep1(-/-)x db/db mice, which are also characterized by lower HbA1c, lower body fat mass and reduced adipose tissue (AT) inflammation area. Our study provides evidence that loss of Repin1 in db/db mice improves insulin sensitivity and reduces chronic hyperglycemia most likely by reducing fat mass and AT inflammation.

Fat depot-specific expression of HOXC9 and HOXC10 may contribute to adverse fat distribution and related metabolic traits.

OBJECTIVE: Independent previous studies in both rodents and humans suggest a role of developmental genes in the origin of obesity and body fat distribution. Here, the hypothesis that human adipose tissue (AT) expression of the developmental genes homeobox transcription factors C9 (HOXC9) and C10 (HOXC10) is fat depot-specific and related to obesity-related traits was tested. METHODS: In 636 individuals, HOXC9 and HOXC10 mRNA expression was investigated in paired abdominal subcutaneous (SC) and omental AT samples in relation to a wide range of age, BMI, fat distribution, and metabolic parameters and in subfractions of isolated adipocytes and cells of the stromal vascular fraction (SVF). RESULTS: HOXC9 and HOXC10 mRNA expression is significantly higher in SC compared to omental AT. HOXC9 and HOXC10 mRNA expression significantly correlates with body fat mass, even after adjustment for age and gender. In smaller subgroups (depending on the availability of data), fat depot-related significant gender- and BMI-independent associations between HOXC9 and HOXC10 gene expression and parameters of glucose metabolism and AT biology were found (e.g., adipocyte size). CONCLUSIONS: Taken together, these data suggest that HOXC9 and HOXC10 may play an important role in the development of obesity, adverse fat distribution, and subsequent alterations in whole-body metabolism and AT function.

Di-(2-Ethylhexyl)-Phthalate (DEHP) Causes Impaired Adipocyte Function and Alters Serum Metabolites.

Di-(2-ethylhexyl)-phthalate (DEHP), an ubiquitous environmental contaminant, has been shown to cause adverse effects on glucose homeostasis and insulin sensitivity in epidemiological studies, but the underlying mechanisms are still unknown. We therefore tested the hypothesis that chronic DEHP exposure causes impaired insulin sensitivity, affects body weight, adipose tissue (AT) function and circulating metabolic parameters of obesity resistant 129S6 mice in vivo. An obesity-resistant mouse model was chosen to reduce a potential obesity bias of DEHP effects on metabolic parameters and AT function. The metabolic effects of 10-weeks exposure to DEHP were tested by insulin tolerance tests and quantitative assessment of 183 metabolites in mice. Furthermore, 3T3-L1 cells were cultured with DEHP for two days, differentiated into mature adipocytes in which the effects on insulin stimulated glucose and palmitate uptake, lipid content as well as on mRNA/protein expression of key adipocyte genes were investigated. We observed in female mice that DEHP treatment causes enhanced weight gain, fat mass, impaired insulin tolerance, changes in circulating adiponectin and adipose tissue Pparg, adiponectin and estrogen expression. Serum metabolomics indicated a general increase in phospholipid and carnitine concentrations. In vitro, DEHP treatment increases the proliferation rate and alters glucose uptake in adipocytes. Taken together, DEHP has significant effects on adipose tissue (AT) function and alters specific serum metabolites. Although, DEHP treatment led to significantly impaired insulin tolerance, it did not affect glucose tolerance, HOMA-IR, fasting glucose, insulin or triglyceride serum concentrations. This may suggest that DEHP treatment does not cause impaired glucose metabolism at the whole body level.

Tamoxifen affects glucose and lipid metabolism parameters, causes browning of subcutaneous adipose tissue and transient body composition changes in C57BL/6NTac mice.

Tamoxifen is a selective estrogen receptor (ER) modulator which is widely used to generate inducible conditional transgenic mouse models. Activation of ER signaling plays an important role in the regulation of adipose tissue (AT) metabolism. We therefore tested the hypothesis that tamoxifen administration causes changes in AT biology in vivo. 12 weeks old male C57BL/6NTac mice were treated with either tamoxifen (n = 18) or vehicle (n = 18) for 5 consecutive days. Tamoxifen treatment effects on body composition, energy homeostasis, parameters of AT biology, glucose and lipid metabolism were investigated up to an age of 18 weeks. We found that tamoxifen treatment causes: I) significantly increased HbA1c, triglyceride and free fatty acid serum concentrations (p < 0.01), II) browning of subcutaneous AT and increased UCP-1 expression, III) increased AT proliferation marker Ki67 mRNA expression, IV) changes in adipocyte size distribution, and V) transient body composition changes. Tamoxifen may induce changes in body composition, whole body glucose and lipid metabolism and has significant effects on AT biology, which need to be considered when using Tamoxifen as a tool to induce conditional transgenic mouse models. Our data further suggest that tamoxifen-treated wildtype mice should be characterized in parallel to experimental transgenic models to control for tamoxifen administration effects.

Identification of genetic loci associated with different responses to high-fat diet-induced obesity in C57BL/6N and C57BL/6J substrains.

We have recently demonstrated that C57BL/6NTac and C57BL/6JRj substrains are significantly different in their response to high-fat diet-induced obesity (DIO). The C57BL/6JRj substrain seems to be protected from DIO and genetic differences between C57BL/6J and C57BL/6N substrains at 11 single nucleotide polymorphism (SNP) loci have been identified. To define genetic variants as well as differences in parameters of glucose homeostasis and insulin sensitivity between C57BL/6NTac and C57BL/6JRj substrains that may explain the different response to DIO, we analyzed 208 first backcross (BC1) hybrids of C57BL/6NTac and C57BL/6JRj [(C57BL/6NTac × C57BL/6JRj)F1 × C57BL/6NTac] mice. Body weight, epigonadal and subcutaneous fat mass, circulating leptin, as well as parameters of glucose metabolism were measured after 10 wk of high-fat diet (HFD). Genetic profiling of BC1 hybrids were performed using TaqMan SNP genotyping assays. Furthermore, to assess whether SNP polymorphisms could affect mRNA level, we carried out gene expression analysis in murine liver samples. Human subcutaneous adipose tissue was used to verify murine data of SNAP29. We identified four sex-specific variants that are associated with the extent of HFD-induced weight gain and fat depot mass. BC1 hybrids carrying the combination of risk or beneficial alleles exhibit the phenotypical extremes of the parental strains. Murine and human SC expression analysis revealed Snap29 as strongest candidate. Our data indicate an important role of these loci in responsiveness to HFD-induced obesity and suggest genes of the synaptic vesicle release system such as Snap29 being involved in the regulation of high-fat DIO.