sTREM2 is a plasma biomarker for human NASH and promotes hepatocyte lipid accumulation.

BACKGROUND: Pathogenetic mechanisms of the progression of NAFL to advanced NASH coupled with potential noninvasive biomarkers and novel therapeutic targets are active areas of investigation. The recent finding that increased plasma levels of a protein shed by myeloid cells -soluble Triggering Receptor Expressed on Myeloid cells 2 (sTREM2) -may be a biomarker for NASH has received much interest. We aimed to test sTREM2 as a biomarker for human NASH and investigate the role of sTREM2 in the pathogenesis of NASH. METHODS: We conducted studies in both humans (comparing patients with NASH vs. NAFL) and in mice (comparing different mouse models of NASH) involving measurements of TREM2 gene and protein expression levels in the liver as well as circulating sTREM2 levels in plasma. We investigated the pathogenetic role of sTREM2 in hepatic steatosis using primary hepatocytes and bone marrow derived macrophages. RESULTS: RNA sequencing analysis of livers from patients with NASH or NAFL as well as livers from 2 mouse models of NASH revealed elevated TREM2 expression in patients/mice with NASH as compared with NAFL. Plasma levels of sTREM2 were significantly higher in a well-characterized cohort of patients with biopsy-proven NASH versus NAFL (area under receiver-operating curve 0.807). Mechanistic studies revealed that cocultures of primary hepatocytes and macrophages with an impaired ability to shed sTREM2 resulted in reduced hepatocyte lipid droplet formation on palmitate stimulation, an effect that was counteracted by the addition of exogenous sTREM2 chimeric protein. Conversely, exogenous sTREM2 chimeric protein increased lipid droplet formation, triglyceride content, and expression of the lipid transporter CD36 in hepatocytes. Furthermore, inhibition of CD36 markedly attenuated sTREM2-induced lipid droplet formation in mouse primary hepatocytes. CONCLUSIONS: Elevated levels of sTREM2 due to TREM2 shedding may directly contribute to the pathogenesis of NAFLD by promoting hepatocyte lipid accumulation, as well as serving as a biomarker for distinguishing patients with NASH versus NAFL. Further investigation of sTREM2 as a clinically useful diagnostic biomarker and of the therapeutic effects of targeting sTREM2 in NASH is warranted.

Sexually Dimorphic Relationships Among Saa3 (Serum Amyloid A3), Inflammation, and Cholesterol Metabolism Modulate Atherosclerosis in Mice.

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Adipocyte-Derived Versican and Macrophage-Derived Biglycan Control Adipose Tissue Inflammation in Obesity.

Obesity is characterized by adipose tissue inflammation. Because proteoglycans regulate inflammation, here we investigate their role in adipose tissue inflammation in obesity. We find that adipose tissue versican and biglycan increase in obesity. Versican is produced mainly by adipocytes and biglycan by adipose tissue macrophages. Both proteoglycans are also present in adipose tissue from obese human subjects undergoing gastric bypass surgery. Deletion of adipocyte-specific versican or macrophage-specific biglycan in mice reduces macrophage accumulation and chemokine and cytokine expression, although only adipocyte-specific versican deletion leads to sustained improvement in glucose tolerance. Macrophage-derived biglycan activates inflammatory genes in adipocytes. Versican expression increases in cultured adipocytes exposed to excess glucose, and adipocyte-conditioned medium stimulates inflammation in resident peritoneal macrophages, in part because of a versican breakdown product, versikine. These findings provide insights into the role of adipocyte- and macrophage-derived proteoglycans in adipose tissue inflammation in obesity.

Aromatase deficiency in hematopoietic cells improves glucose tolerance in male mice through skeletal muscle-specific effects.

Estrogens are important for maintaining metabolic health in males. However, the key sources of local estrogen production for regulating energy metabolism have not been fully defined. Immune cells exhibit aromatase activity and are resident in metabolic tissues. To determine the relative contribution of immune cell-derived estrogens for metabolic health in males, C57BL6/J mice underwent bone marrow transplant with marrow from either wild-type (WT(WT)) or aromatase-deficient (WT(ArKO)) donors. Body weight, body composition, and glucose and insulin tolerance were assessed over 24 weeks with mice maintained on a regular chow diet. No differences were found in insulin sensitivity between groups, but WT(ArKO) mice were more glucose tolerant than WT(WT) mice 20 weeks after transplant, suggestive of enhanced glucose disposal (AUCglucose 6061±3349 in WT(WT) mice versus 3406±1367 in WT(ArKO) mice, p = 0.01). Consistent with this, skeletal muscle from WT(ArKO) mice showed higher expression of the mitochondrial genes Ppargc1a (p = 0.03) and Nrf1 (p = 0.01), as well as glucose transporter type 4 (GLUT4, Scl2a4; p = 0.02). Skeletal muscle from WT(ArKO) mice had a lower concentration of 17ß-estradiol (5489±2189 pg/gm in WT(WT) mice versus 3836±2160 pg/gm in WT(ArKO) mice, p = 0.08) but higher expression of estrogen receptor-α (ERα, Esr1), raising the possibility that aromatase deficiency in immune cells led to a compensatory increase in ERα signaling. No differences between groups were found with regard to body weight, adiposity, or gene expression within adipose tissue or liver. Immune cells are a key source of local 17ß-estradiol production and contribute to metabolic regulation in males, particularly within skeletal muscle. The respective intracrine and paracrine roles of immune cell-derived estrogens require further delineation, as do the pathways that regulate aromatase activity in immune cells specifically within metabolic tissues.

10,12 Conjugated Linoleic Acid-Driven Weight Loss Is Protective against Atherosclerosis in Mice and Is Associated with Alternative Macrophage Enrichment in Perivascular Adipose Tissue.

The dietary fatty acid 10,12 conjugated linoleic acid (10,12 CLA) promotes weight loss by increasing fat oxidation, but its effects on atherosclerosis are less clear. We recently showed that weight loss induced by 10,12 CLA in an atherosclerosis-susceptible mouse model with characteristics similar to human metabolic syndrome is accompanied by accumulation of alternatively activated macrophages within subcutaneous adipose tissue. The objective of this study was to evaluate whether 10,12 CLA-mediated weight loss was associated with an atheroprotective phenotype. Male low-density lipoprotein receptor deficient (Ldlr-/-) mice were made obese with 12 weeks of a high-fat, high-sucrose diet feeding (HFHS: 36% fat, 36% sucrose, 0.15% added cholesterol), then either continued on the HFHS diet with or without caloric restriction (CR), or switched to a diet with 1% of the lard replaced by either 9,11 CLA or 10,12 CLA for 8 weeks. Atherosclerosis and lipid levels were quantified at sacrifice. Weight loss in mice following 10,12 CLA supplementation or CR as a weight-matched control group had improved cholesterol and triglyceride levels, yet only the 10,12 CLA-treated mice had improved en face and aortic sinus atherosclerosis. 10,12 CLA-supplemented mice had increased lesion macrophage content, with enrichment of surrounding perivascular adipose tissue (PVAT) alternative macrophages, which may contribute to the anti-atherosclerotic effect of 10,12 CLA.

Androgen receptor deficiency in monocytes/macrophages does not alter adiposity or glucose homeostasis in male mice.

Androgen deprivation in men leads to increased adiposity, but the mechanisms underlying androgen regulation of fat mass have not been fully defined. Androgen receptor (AR) is expressed in monocytes/macrophages, which are resident in key metabolic tissues and influence energy metabolism in surrounding cells. Male mice bearing a cell-specific knockout of the AR in monocytes/macrophages (M-ARKO) were generated to determine whether selective loss of androgen signaling in these cells would lead to altered body composition. Wild-type (WT) and M-ARKO mice (12-22 weeks of age, n = 12 per group) were maintained on a regular chow diet for 8 weeks and then switched to a high-fat diet for 8 additional weeks. At baseline and on both the regular chow and high-fat diets, no differences in lean mass or fat mass were observed between groups. Consistent with the absence of differential body weight or adiposity, no differences in food intake (3.0 ± 0.5 g per day for WT mice vs 2.8 ± 0.4 g per day for M-ARKO mice) or total energy expenditure (0.6 ± 0.1 Kcal h-1 for WT mice vs 0.5 ± 0.1 Kcal h-1 for M-ARKO mice) were evident between groups during high-fat feeding. Liver weight was greater in M-ARKO than that in WT mice (1.5 ± 0.1 g vs 1.3 ± 0.0 g, respectively, P = 0.02). Finally, M-ARKO mice did not exhibit impairments in glucose tolerance or insulin sensitivity relative to WT mice at any study time point. In aggregate, these findings suggest that AR signaling specifically in monocytes/macrophages does not contribute to the regulation of systemic energy balance, adiposity, or insulin sensitivity in male mice.

Adipocyte-Specific Deficiency of NADPH Oxidase 4 Delays the Onset of Insulin Resistance and Attenuates Adipose Tissue Inflammation in Obesity.

OBJECTIVE: Obesity is associated with insulin resistance and adipose tissue inflammation. Reactive oxygen species (ROS) increase in adipose tissue during the development of obesity. We previously showed that in response to excess nutrients like glucose and palmitate, adipocytes generated ROS via NADPH oxidase (NOX) 4, the major adipocyte isoform, instead of using mitochondrial oxidation. However, the role of NOX4-derived ROS in the development of whole body insulin resistance, adipocyte inflammation, and recruitment of macrophages to adipose tissue during the development of obesity is unknown. APPROACH AND RESULTS: In this study, control C57BL/6 mice and mice in which NOX4 has been deleted specifically in adipocytes were fed a high-fat, high-sucrose diet. During the development of obesity in control mice, adipocyte NOX4 and pentose phosphate pathway activity were transiently increased. Primary adipocytes differentiated from mice with adipocytes deficient in NOX4 showed resistance against high glucose or palmitate-induced adipocyte inflammation. Mice with adipocytes deficient in NOX4 showed a delayed onset of insulin resistance during the development of obesity, with an initial reduction in adipose tissue inflammation that normalized with prolonged high-fat, high-sucrose feeding. CONCLUSIONS: These findings imply that NOX4-derived ROS may play a role in the onset of insulin resistance and adipose tissue inflammation. As such, therapeutics targeting NOX4-mediated ROS production could be effective in preventing obesity-associated conditions, such as insulin resistance.

Cutting Edge: BAFF Overexpression Reduces Atherosclerosis via TACI-Dependent B Cell Activation.

Patients with systemic lupus erythematosus exhibit accelerated atherosclerosis, a chronic inflammatory disease of the arterial wall. The impact of B cells in atherosclerosis is controversial, with both protective and pathogenic roles described. For example, natural IgM binding conserved oxidized lipid epitopes protect against atherosclerosis, whereas anti-oxidized low-density lipoprotein (oxLDL) IgG likely promotes disease. Because BAFF promotes B cell class-switch recombination and humoral autoimmunity, we hypothesized that excess BAFF would accelerate atherosclerosis. In contrast, BAFF overexpression markedly reduced hypercholesterolemia and atherosclerosis in hyperlipidemic mice. BAFF-mediated atheroprotection required B cells and was associated with increased protective anti-oxLDL IgM. Surprisingly, high-titer anti-oxLDL IgM production and reduced atherosclerosis was dependent on the BAFF family receptor transmembrane activator and CAML interactor. In summary, we identified a novel role for B cell-specific, BAFF-dependent transmembrane activator and CAML interactor signals in atherosclerosis pathogenesis, of particular relevance to the use of BAFF-targeted therapies in systemic lupus erythematosus.

Polybrominated diphenyl ether congener, BDE-47, impairs insulin sensitivity in mice with liver-specific Pten deficiency.

BACKGROUND: The potential health effects of polybrominated diphenyl ethers (PBDEs) that are widely used as flame-retardants in consumer products have been attributed, in part, to their endocrine disrupting properties. The purpose of this study is to examine the in vivo effects of an early exposure to PBDEs on the development of insulin resistance in mice. RESULTS: The metabolic consequences of BDE-47 in mice with varying insulin sensitivities secondary to liver-specific activation of Akt (Pten (fl/fl);Alb (Cre)) and mTORC1 (Tsc1 (fl/fl);Alb (Cre)) as well as wild-type littermates, were studied. BDE-47, a dominant congener of PBDE, was given daily (1 mg/kg/day) for six weeks by oral gavage in young mice following weaning. At the end of the exposure, there were no significant differences in total body, liver, or white adipose tissue weights between the BDE-47-treated vs. DMSO-treated mice for each respective genotype. Metabolic studies revealed significant impairment in insulin sensitivity in the BDE-47-treated Pten (fl/fl);Alb (Cre) mice, but not in wild-type or Tsc1 (fl/fl);Alb (Cre) mice. This was not accompanied by significant alterations in plasma insulin levels or hepatic triglyceride accumulation in the Pten (fl/fl);Alb (Cre) mice. The mean plasma BDE-47 level in the wild-type mice was 11.7 ± 2.9 ng/g (wet weight). CONCLUSIONS: Our findings indicate that BDE-47 exposure during the early post-natal period induces a mild disturbance in glucose metabolism in susceptible mice with increased baseline insulin sensitivity. These results suggest an interaction between BDE-47 and genetic factors that regulate insulin signaling, which may result in long-term consequences.

Increased levels of invariant natural killer T lymphocytes worsen metabolic abnormalities and atherosclerosis in obese mice.

Obesity is a chronic inflammatory state characterized by infiltration of adipose tissue by immune cell populations, including T lymphocytes. Natural killer T (NKT) cells, a specialized lymphocyte subset recognizing lipid antigens, can be pro- or anti-inflammatory. Their role in adipose inflammation continues to be inconclusive and contradictory. In obesity, the infiltration of tissues by invariant NKT (iNKT) cells is decreased. We therefore hypothesized that an excess iNKT cell complement might improve metabolic abnormalities in obesity. Vα14 transgenic (Vα14tg) mice, with increased iNKT cell numbers, on a LDL receptor-deficient (Ldlr(-/-)) background and control Ldlr(-/-) mice were placed on an obesogenic diet for 16 weeks. Vα14tg.Ldlr(-/-) mice gained 25% more weight and had increased adiposity than littermate controls. Transgenic mice also developed greater dyslipidemia, hyperinsulinemia, insulin resistance, and hepatic triglyceride accumulation. Increased macrophage Mac2 immunostaining and proinflammatory macrophage gene expression suggested worsened adipose inflammation. Concurrently, these mice had increased atherosclerotic lesion area and aortic inflammation. Thus, increasing the complement of iNKT cells surprisingly exacerbated the metabolic, inflammatory, and atherosclerotic features of obesity. These findings suggest that the reduction of iNKT cells normally observed in obesity may represent a physiological attempt to compensate for this inflammatory condition.

T cell activation inhibitors reduce CD8+ T cell and pro-inflammatory macrophage accumulation in adipose tissue of obese mice.

Adipose tissue inflammation and specifically, pro-inflammatory macrophages are believed to contribute to insulin resistance (IR) in obesity in humans and animal models. Recent studies have invoked T cells in the recruitment of pro-inflammatory macrophages and the development of IR. To test the role of the T cell response in adipose tissue of mice fed an obesogenic diet, we used two agents (CTLA-4 Ig and anti-CD40L antibody) that block co-stimulation, which is essential for full T cell activation. C57BL/6 mice were fed an obesogenic diet for 16 weeks, and concomitantly either treated with CTLA-4 Ig, anti-CD40L antibody or an IgG control (300 µg/week). The treatments altered the immune cell composition of adipose tissue in obese mice. Treated mice demonstrated a marked reduction in pro-inflammatory adipose tissue macrophages and activated CD8+ T cells. Mice treated with anti-CD40L exhibited reduced weight gain, which was accompanied by a trend toward improved IR. CTLA-4 Ig treatment, however, was not associated with improved IR. These data suggest that the presence of pro-inflammatory T cells and macrophages can be altered with co-stimulatory inhibitors, but may not be a significant contributor to the whole body IR phenotype.

Inhibition of intestinal cholesterol absorption decreases atherosclerosis but not adipose tissue inflammation.

Adipose tissue inflammation is associated with insulin resistance and increased cardiovascular disease risk in obesity. We previously showed that addition of cholesterol to a diet rich in saturated fat and refined carbohydrate significantly worsens dyslipidemia, insulin resistance, adipose tissue macrophage accumulation, systemic inflammation, and atherosclerosis in LDL receptor-deficient (Ldlr(-/-)) mice. To test whether inhibition of intestinal cholesterol absorption would improve metabolic abnormalities and adipose tissue inflammation in obesity, we administered ezetimibe, a dietary and endogenous cholesterol absorption inhibitor, to Ldlr(-/-) mice fed chow or high-fat, high-sucrose (HFHS) diets without or with 0.15% cholesterol (HFHS+C). Ezetimibe blunted weight gain and markedly reduced plasma lipids in the HFHS+C group. Ezetimibe had no effect on glucose homeostasis or visceral adipose tissue macrophage gene expression in the HFHS+C fed mice, although circulating inflammatory markers serum amyloid A (SSA) and serum amyloid P (SSP) levels decreased. Nevertheless, ezetimibe treatment led to a striking (>85%) reduction in atherosclerotic lesion area with reduced lesion lipid and macrophage content in the HFHS+C group. Thus, in the presence of dietary cholesterol, ezetimibe did not improve adipose tissue inflammation in obese Ldlr(-/-) mice, but it led to a major reduction in atherosclerotic lesions associated with improved plasma lipids and lipoproteins.

Dietary cholesterol worsens adipose tissue macrophage accumulation and atherosclerosis in obese LDL receptor-deficient mice.

OBJECTIVE: Chronic systemic inflammation accompanies obesity and predicts development of cardiovascular disease. Dietary cholesterol has been shown to increase inflammation and atherosclerosis in LDL receptor-deficient (LDLR(-/-)) mice. This study was undertaken to determine whether dietary cholesterol and obesity have additive effects on inflammation and atherosclerosis. METHODS AND RESULTS: LDLR(-/-) mice were fed chow, high-fat, high-carbohydrate (diabetogenic) diets without (DD) or with added cholesterol (DDC) for 24 weeks. Effects on adipose tissue, inflammatory markers, and atherosclerosis were studied. Despite similar weight gain between DD and DDC groups, addition of dietary cholesterol increased insulin resistance relative to DD. Adipocyte hypertrophy, macrophage accumulation, and local inflammation were observed in intraabdominal adipose tissue in DD and DDC, but were significantly higher in the DDC group. Circulating levels of the inflammatory protein serum amyloid A (SAA) were 4.4-fold higher in DD animals and 15-fold higher in DDC animals than controls, suggesting chronic systemic inflammation. Hepatic SAA mRNA levels were similarly elevated. Atherosclerosis was increased in the DD-fed animals and further increased in the DDC group. CONCLUSIONS: Obesity-induced macrophage accumulation in adipose tissue is exacerbated by dietary cholesterol. These local inflammatory changes in adipose tissue are associated with insulin resistance, systemic inflammation, and increased atherosclerosis in this mouse model.