Targeting senescent cells alleviates obesity‐induced metabolic dysfunction

Adipose tissue inflammation and dysfunction are associated with obesity‐related insulin resistance and diabetes, but mechanisms underlying this relationship are unclear. Although senescent cells accumulate in adipose tissue of obese humans and rodents, a direct pathogenic role for these cells in the development of diabetes remains to be demonstrated. Here, we show that reducing senescent cell burden in obese mice, either by activating drug‐inducible “suicide” genes driven by the p16^Ink4a promoter or by treatment with senolytic agents, alleviates metabolic and adipose tissue dysfunction. These senolytic interventions improved glucose tolerance, enhanced insulin sensitivity, lowered circulating inflammatory mediators, and promoted adipogenesis in obese mice. Elimination of senescent cells also prevented the migration of transplanted monocytes into intra‐abdominal adipose tissue and reduced the number of macrophages in this tissue. In addition, microalbuminuria, renal podocyte function, and cardiac diastolic function improved with senolytic therapy. Our results implicate cellular senescence as a causal factor in obesity‐related inflammation and metabolic derangements and show that emerging senolytic agents hold promise for treating obesity‐related metabolic dysfunction and its complications.     

Dietary alpha‐ketoglutarate promotes beige adipogenesis and prevents obesity in middle‐aged mice

Aging usually involves the progressive development of certain illnesses, including diabetes and obesity. Due to incapacity to form new white adipocytes, adipose expansion in aged mice primarily depends on adipocyte hypertrophy, which induces metabolic dysfunction. On the other hand, brown adipose tissue burns fatty acids, preventing ectopic lipid accumulation and metabolic diseases. However, the capacity of brown/beige adipogenesis declines inevitably during the aging process. Previously, we reported that DNA demethylation in the Prdm16 promoter is required for beige adipogenesis. DNA methylation is mediated by ten–eleven family proteins (TET) using alpha‐ketoglutarate (AKG) as a cofactor. Here, we demonstrated that the circulatory AKG concentration was reduced in middle‐aged mice (10‐month‐old) compared with young mice (2‐month‐old). Through AKG administration replenishing the AKG pool, aged mice were associated with the lower body weight gain and fat mass, and improved glucose tolerance after challenged with high‐fat diet (HFD). These metabolic changes are accompanied by increased expression of brown adipose genes and proteins in inguinal adipose tissue. Cold‐induced brown/beige adipogenesis was impeded in HFD mice, whereas AKG rescued the impairment of beige adipocyte functionality in middle‐aged mice. Besides, AKG administration up‐regulated Prdm16 expression, which was correlated with an increase of DNA demethylation in the Prdm16 promoter. In summary, AKG supplementation promotes beige adipogenesis and alleviates HFD‐induced obesity in middle‐aged mice, which is associated with enhanced DNA demethylation of the Prdm16 gene.     

Increased Expression of DNA Methyltransferase 3a in Obese Adipose Tissue: Studies With Transgenic Mice

Epigenetic mechanisms are likely to be involved in the development of obesity. This study was designed to examine the role of a DNA methyltransferase (Dnmt3a), in obese adipose tissue. The gene expression of Dnmts was examined by quantitative real‐time PCR analysis. Transgenic mice overexpressing Dnmt3a in the adipose tissue driven by the aP2 promoter were created (Dnmt3a mice). DNA methylation of downregulated genes was examined using bisulfite DNA methylation analysis. Dnmt3a mice were fed a methyl‐supplemented or high‐fat diet, and subjected to body weight measurement and gene expression analysis of the adipose tissue. Expression of Dnmt3a was markedly upregulated in the adipose tissue of obese mice. The complementary DNA (cDNA) microarray analysis of Dnmt3a mice revealed a slight decrease in the gene expression of secreted frizzled‐related protein 1 (SFRP1) and marked increase in that of interferon responsive factor 9 (IRF9). In the SFRP1 promoter, DNA methylation was not markedly increased in Dnmt3a mice relative to wild‐type mice. In experiments with a high‐fat diet or methyl‐supplemented diet, body weight did not differ significantly with the genotypes. Gene expression levels of inflammatory cytokines such as tumor necrosis factor‐α (TNF‐α) and monocyte chemoattractant protein‐1 (MCP‐1) were higher in Dnmt3a mice than in wild‐type mice on a high‐fat diet. This study suggests that increased expression of Dnmt3a in the adipose tissue may contribute to obesity‐related inflammation. The data highlight the potential role of Dnmt3a in the adult tissue as well as in the developing embryo and cancer.     

Macrophage-colony stimulating factor in obese adipose tissue: studies with heterozygous op/+ mice

Objective: We examined the gene expression of macrophage‐colony stimulating factor (M‐CSF) in mice with diet‐induced obesity and in genetically obese mice. We also examined the effect of decreased M‐CSF signaling on the susceptibility to obesity and macrophage recruitment into the adipose tissue of mice. Research Methods and Procedures: The adipose tissue from mice with diet‐induced obesity, obese KKA^y mice, and ob/ob obese mice was used for RNA preparation. Production of M‐CSF and monocyte chemoattractant protein‐1 (MCP‐1) was examined by quantitative real‐time polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay. The op/+ heterozygous mice, with decreased functional M‐CSF expression, were placed on a high‐fat diet or crossed with KKA^y mice to study the susceptibility to obesity. The gene expression of macrophage markers in adipose tissue was examined. Results: The expression of M‐CSF was not significantly changed in mice on a high‐fat diet or in either type of genetic obesity (KKA^y or ob/ob mice). No change in the degree of obesity or macrophage‐related gene expression (F4/80, CD68, and MCP‐1) in the adipose tissue was observed in op/+ mice compared with +/+ control mice, which were either treated with a high‐fat diet or crossed with KKA^y mice. Discussion: This study demonstrated that there was no significant change in the expression of M‐CSF in the adipose tissue from obese mice and only a minor phenotypic change, such as macrophage infiltration, in the adipose tissue from op/+ mice, suggesting that M‐CSF does not play a major role in macrophage recruitment in the adipose tissue of obese mice.     

Elevated serum monocyte chemoattractant protein-4 and chronic inflammation in overweight subjects

Objective: Chronic inflammation observed in obesity has been reported to be implicated in the development of atherosclerosis. We screened candidate chemokines that link chronic inflammation and obesity. Research Methods and Procedures: Japanese overweight (n = 39, BMI 28.7 ± 0.65 kg/m²) and normal‐weight (n = 24, BMI 22.3 ± 0.45 kg/m²) subjects were enrolled. Using antibody‐based protein microarray, spot intensities of monocyte chemoattractant protein (MCP)‐4, eotaxin, and eotaxin‐2 correlated with anthropometric parameters. We further measured serum concentration of these chemokines and mRNA levels in adipose tissues obtained from volunteers. Results: Serum MCP‐4 levels showed positive correlation with BMI (r = 0.318, p = 0.014), waist (r = 0.316, p = 0.018), and waist‐to‐hip ratio (WHR) (r = 0.264, p = 0.049). Furthermore, MCP‐4 correlated with homeostasis model assessment of insulin resistance (r = 0.392, p = 0.002), high‐sensitivity C‐reactive protein (hsCRP) (r = 0.350, p = 0.006). In step‐wise multiple regression analyses, hsCRP independently correlated with MCP‐4 levels. The expression of MCP‐4 mRNA in visceral adipose tissue positively correlates with BMI. Serum eotaxin levels correlate with BMI (r = 0.262, p = 0.045) and WHR (r = 0.383, p = 0.003). Serum eotaxin‐2 levels correlated with BMI (r = 0.464, p < 0.001), waist (r = 0.333, p = 0.017), and WHR (r = 0.278, p = 0.048). However, eotaxin and eotaxin‐2 levels did not show significant correlation with hsCRP. Discussion: Serum levels of MCP‐4, eotaxin, and eotaxin‐2, which belong to CC chemokine family and share CC chemokine receptor 3, correlated with BMI. These chemokines, especially MCP‐4, may be critical molecules that link obesity and chronic inflammation.     

Galectin-3 stimulates preadipocyte proliferation and is up-regulated in growing adipose tissue

Objective: Some cytokines and mediators of inflammation can alter adiposity through their effects on adipocyte number. To probe the molecular basis of obesity, this study determined whether galectin‐3 was present in adipose tissue and investigated its effects on fat cell number. Research Methods and Procedures: In the first study, obesity‐prone C57BL/6J mice were fed with high‐fat (58%) diet. Epididymal fat pads were collected at Day 0, Day 60, and Day 120 after the start of high‐fat feeding. Results: Levels of adipocyte galectin‐3 protein, determined using Western blot analysis, increased as the mice became obese. Galectin‐3 mRNA and protein were then detected in human adipose tissue, primarily in the preadipocyte fraction. It was found that recombinant human galectin‐3 stimulated proliferation of primary cultured preadipocytes as well as DNA synthesis through lectin‐carbohydrate interaction. Discussion: Galectin‐3, which has been known to play a versatile role especially in immune cells, might play a role also in adipose tissue and be associated with the pathophysiology of obesity.     

Effect of peroxisome proliferator-activated receptor-alpha ligands in the interaction between adipocytes and macrophages in obese adipose tissue

Objective: This study was designed to examine the effect of peroxisome proliferator‐activated receptor‐α (PPAR‐α) ligands on the inflammatory changes induced by the interaction between adipocytes and macrophages in obese adipose tissue. Methods and Procedures: PPAR‐α ligands (Wy‐14,643 and fenofibrate) were added to 3T3‐L1 adipocytes, RAW264 macrophages, or co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages in vitro, and monocyte chemoattractant protein‐1 (MCP‐1) and tumor necrosis factor‐α (TNF‐α) mRNA expression and secretion were examined. PPAR‐α ligands were administered to genetically obese ob/ob mice for 2 weeks. Moreover, the effect of PPAR‐α ligands was also evaluated in the adipose tissue explants and peritoneal macrophages obtained from PPAR‐α‐deficient mice. Results: In the co‐culture of 3T3‐L1 adipocytes and RAW264 macrophages, PPAR‐α ligands reduced MCP‐1 and TNF‐α mRNA expression and secretion in vitro relative to vehicle‐treated group. The anti‐inflammatory effect of Wy‐14,643 was observed in adipocytes treated with macrophage‐conditioned media or mouse recombinant TNF‐α and in macrophages treated with adipocyte‐conditioned media or palmitate. Systemic administration of PPAR‐α ligands inhibited the inflammatory changes in adipose tissue from ob/ob mice. Wy‐14,643 also exerted an anti‐inflammatory effect in the adipose tissue explants but not in peritoneal macrophages obtained from PPAR‐α‐deficient mice. Discussion: This study provides evidence for the anti‐inflammatory effect of PPAR‐α ligands in the interaction between adipocytes and macrophages in obese adipose tissue, thereby improving the dysregulation of adipocytokine production and obesity‐related metabolic syndrome.     

Extensive changes in innate immune gene expression in obese Göttingen minipigs do not lead to changes in concentrations of circulating cytokines and acute phase proteins

The usefulness of Göttingen minipigs as models for obesity and obesity‐related pathologies is well established. The low‐grade inflammation associated with obesity involves a range of innate immune factors; however, to our knowledge, the impact of obesity on innate immune factor expression has not been studied in Göttingen minipigs. Therefore, we studied the expression of innate immune genes in liver and adipose tissues as well as serum concentrations of cytokines and acute phase proteins in obese vs. lean Göttingen minipigs. In the liver, of 35 investigated genes, the expression of nine was significantly different in obese pigs (three up‐regulated, six down‐regulated). Of 33 genes in adipose tissues, obesity was associated with changed expression of 12 genes in the visceral adipose tissue (VAT) (three up‐regulated), 11 in the abdominal retroperitoneal adipose tissue (RPAT) (seven of these up‐regulated) and eight in the subcutaneous adipose tissue (SAT) from the neck (five of which were up‐regulated). Obesity‐associated expression changes were observed for three genes in all adipose tissues, namely chemokine (C‐C motif) ligand 3‐like 1 (up‐regulated), CD200 molecule (down‐regulated) and interleukin 1 receptor antagonist (up‐regulated) with interleukin 1 receptor antagonist being the most highly regulated gene in both VAT and RPAT. Looking at patterns of expression across the three types of adipose tissues, obesity was associated with an increased number of acute phase proteins differentially expressed between adipose tissues and a decreased tissue‐specific expression of cytokines and chemokines. In contrast to obese humans, no changes in serum concentrations of haptoglobin, C‐reactive protein, serum amyloid A, tumor necrosis factor‐α and interleukin 6 were found in obese Göttingen minipigs.     

The Effect of the Interleukin‐1 Cytokine Family Members IL‐1F6 and IL‐1F8 on Adipocyte Differentiation

Obesity is characterized by chronic low‐grade inflammation originating from expanding adipose tissue. In the present study, we examined the adipogenic expression levels of IL‐1F6 and IL‐1F8, both members of the IL‐1 family of cytokines, and their effects on adipose tissue gene expression. Although IL‐1F6 is primarily present in adipose tissue resident macrophages and induced by inflammation, IL‐1F8 is absent. IL‐1F6, but not IL‐1F8, reduces adipocyte differentiation, as shown by a significant decrease in PPARγ gene expression. Finally, both IL‐1F6 and IL‐1F8 are able to induce inflammatory gene expression in mature adipocytes. In conclusion, we demonstrate for the first time that IL‐1F6 is present in adipose tissue and that IL‐1F6 and IL‐1F8 are involved in the regulation of adipose tissue gene expression. Importantly, IL‐1F6 inhibits PPARγ expression which may lead to reduced adipocyte differentiation suggesting metabolic effects of this cytokine.     

Inflammation Correlates With Markers of T‐Cell Subsets Including Regulatory T Cells in Adipose Tissue From Obese Patients

Accumulation of cytotoxic and T‐helper (T_h)1 cells together with a loss of regulatory T cells in gonadal adipose tissue was recently shown to contribute to obesity‐induced adipose tissue inflammation and insulin resistance in mice. Human data on T‐cell populations in obese adipose tissue and their potential functional relevance are very limited. We aimed to investigate abundance and proportion of T‐lymphocyte sub‐populations in human adipose tissue in obesity and potential correlations with anthropometric data, insulin resistance, and systemic and adipose tissue inflammation. Therefore, we analyzed expression of marker genes specific for pan‐T cells and T‐cell subsets in visceral and subcutaneous adipose tissue from highly obese patients (BMI >40 kg/m², n = 20) and lean to overweight control subjects matched for age and sex (BMI <30 kg/m²; n = 20). All T‐cell markers were significantly upregulated in obese adipose tissue and correlated with adipose tissue inflammation. Proportions of cytotoxic T cells and T_h1 cells were unchanged, whereas those of regulatory T cells and T_h2 were increased in visceral adipose tissue from obese compared to control subjects. Systemic and adipose tissue inflammation positively correlated with the visceral adipose abundance of cytotoxic T cells and T_h1 cells but also regulatory T cells within the obese group. Therefore, this study confirms a potential role of T cells in human obesity‐driven inflammation but does not support a loss of protective regulatory T cells to contribute to adipose tissue inflammation in obese patients as suggested by recent animal studies.     

The Human Visceral Fat Depot Has a Unique Inflammatory Profile

Obesity can be considered as a low‐grade inflammatory condition, strongly linked to adverse metabolic outcomes. Obesity‐associated adipose tissue inflammation is characterized by infiltration of macrophages and increased cytokine and chemokine production. The distribution of adipose tissue impacts the outcomes of obesity, with the accumulation of fat in visceral adipose tissue (VAT) and deep subcutaneous adipose tissue (SAT), but not superficial SAT, being linked to insulin resistance. We hypothesized that the inflammatory gene expression in deep SAT and VAT is higher than in superficial SAT. A total of 17 apparently healthy women (BMI: 29.3±5.5 kg/m²) were included in the study. Body fat (dual‐energy X‐ray absorptiometry) and distribution (computed tomography) were measured, and insulin sensitivity, blood lipids, and blood pressure were determined. Inflammation‐related differences in gene expression (real‐time PCR) from VAT, superficial and deep SAT biopsies were analyzed using univariate and multivariate data analyses. Using multivariate discrimination analysis, VAT appeared as a distinct depot in adipose tissue inflammation, while the SAT depots had a similar pattern, with respect to gene expression. A significantly elevated (P < 0.01) expression of the CC chemokine receptor 2 (CCR2) and macrophage migration inhibitory factor (MIF) in VAT contributed strongly to the discrimination. In conclusion, the human adipose tissue depots have unique inflammatory patterns, with CCR2 and MIF distinguishing between VAT and the SAT depots.     

Leukocyte Migration in Adipose Tissue of Mice Null for ICAM‐1 and Mac‐1 Adhesion Receptors

Objective: To determine whether the leukocyte adhesion receptors ICAM‐1 and Mac‐1, regulators of immune cell migration, have an intrinsic role within adipose tissue by 1) analyzing the expression of ICAM‐1 in adipose tissue, 2) identifying leukocyte populations within adipose tissue, and 3) determining whether ICAM‐1 and Mac‐1 mutant mice exhibit abnormal numbers of adipose tissue leukocytes. Research Methods and Procedures: Wild‐type, ICAM‐1^?/?, and Mac‐1^?/? mice were fed a long‐term high‐fat diet. ICAM‐1 expression was analyzed by Northern blot and immunohistochemistry. Leukocytes within adipose tissue were identified by immunohistochemistry and flow cytometry. Results: ICAM‐1 was expressed in adipose tissue and localized to the vascular endothelium. Macrophages and lymphocytes were prevalent within the stromal‐vascular cell fraction of adipose tissue, and gender‐specific differences were observed, with adipose tissue from female mice containing significantly more macrophages than tissue from male mice. Numbers of leukocytes in ICAM‐1^?/? and Mac‐1^?/? mice were not different from wild‐types, however, indicating that these adhesion receptors are not required for leukocyte migration into adipose tissue. Discussion: Our results documented leukocyte populations within adipose tissue, which may be involved in the development of heightened inflammation that is characteristic of obesity.     

Food-derived regulatory factors against obesity and metabolic syndrome

Abstract

Obesity is a key factor in metabolic syndrome. The study of metabolic syndrome focuses on the anti-weight gain properties of physiological mechanisms and food components. Abnormal energy metabolism is a major risk factor of metabolic syndrome. Chronic inflammation is a feature of obesity; cytokines from hypertrophied adipocytes cause inflammation in both adipose tissue and blood vessels, resulting in symptoms of metabolic syndrome. Tumor necrosis factor-α causes insulin resistance in adipocytes and regression of brown adipocytes, resulting in abnormal energy metabolism. Functional foods can serve as a strategy for prevention and treatment of obesity linked with metabolic processes in white and brown adipose tissues. Diet-induced thermogenesis caused by certain food components stimulates burning of stored fat within adipose tissues. A mechanistic understanding of dietary thermogenesis via the sympathetic nerve system will prove valuable for the development of precise strategies for the practical prevention of metabolic syndrome.     

Role of adiponectin and inflammation in insulin resistance of Mc3r and Mc4r knockout mice

Objective: To investigate the involvement of hypoadiponectinemia and inflammation in coupling obesity to insulin resistance in melanocortin‐3 receptor and melanocortin‐4 receptor knockout (KO) mice (Mc3/4rKO). Research Methods and Procedures: Sera and tissue were collected from 6‐month‐old Mc3rKO, Mc4rKO, and wild‐type C57BL6J litter mates maintained on low‐fat diet or exposed to high‐fat diet (HFD) for 1 or 3 months. Inflammation was assessed by both real‐time polymerase chain reaction analysis of macrophage‐specific gene expression and immunohistochemistry. Results: Mc4rKO exhibited hypoadiponectinemia, exacerbated by HFD and obesity, previously reported in murine models of obesity. Mc4r deficiency was also associated with high levels of macrophage infiltration of adipose tissue, again exacerbated by HFD. In contrast, Mc3rKO exhibited normal serum adiponectin levels, irrespective of diet or obesity, and a delayed inflammatory response to HFD relative to Mc4rKO. Discussion: Our findings suggest that severe insulin resistance of Mc4rKO fed a HFD, as reported in other models of obesity such as leptin‐deficient (Lep^ob/Lep^ob) and KK‐A^y mice, is linked to reduced serum adiponectin and high levels of inflammation in adipose tissue. Conversely, maintenance of normal serum adiponectin may be a factor in the relatively mild insulin‐resistant phenotype of severely obese Mc3rKO. Mc3rKO are, thus, a unique mouse model where obesity is not associated with reduced serum adiponectin levels. A delay in macrophage infiltration of adipose tissue of Mc3rKO during exposure to HFD may also be a factor contributing to the mild insulin resistance in this model.