10,12-Conjugated linoleic acid supplementation improves HDL composition and function in mice.

Obesity is associated with inflammation, insulin resistance, and type 2 diabetes, which are major risk factors for CVD. One dietary component of ruminant animal foods, 10,12-conjugated linoleic acid (10,12 CLA), has been shown to promote weight loss in humans. Previous work has shown that 10,12 CLA is atheroprotective in mice by a mechanism that may be distinct from its weight loss effects, but this exact mechanism is unclear. To investigate this, we evaluated HDL composition and function in obese LDL receptor (Ldlr-/-) mice that were losing weight because of 10,12 CLA supplementation or caloric restriction (CR; weight-matched control group) and in an obese control group consuming a high-fat high-sucrose diet. We show that 10,12 CLA-HDL exerted a stronger anti-inflammatory effect than CR- or high-fat high-sucrose-HDL in cultured adipocytes. Furthermore, the 10,12 CLA-HDL particle (HDL-P) concentration was higher, attributed to more medium- and large-sized HDL-Ps. Passive cholesterol efflux capacity of 10,12 CLA-HDL was elevated, as was expression of HDL receptor scavenger receptor class B type 1 in the aortic arch. Murine macrophages treated with 10,12 CLA in vitro exhibited increased expression of cholesterol transporters Abca1 and Abcg1, suggesting increased cholesterol efflux potential of these cells. Finally, proteomics analysis revealed elevated Apoa1 content in 10,12 CLA-HDL-Ps, consistent with a higher particle concentration, and particles were also enriched with alpha-1-antitrypsin, an emerging anti-inflammatory and antiatherosclerotic HDL-associated protein. We conclude that 10,12 CLA may therefore exert its atheroprotective effects by increasing HDL-P concentration, HDL anti-inflammatory potential, and promoting beneficial effects on cholesterol efflux.

Comparison between genetic and pharmaceutical disruption of Ldlr expression for the development of atherosclerosis.

Antisense oligonucleotides (ASOs) against Ldl receptor (Ldlr-ASO) represent a promising strategy to promote hypercholesterolemic atherosclerosis in animal models without the need for complex breeding strategies. Here, we sought to characterize and contrast atherosclerosis in mice given Ldlr-ASO with those bearing genetic Ldlr deficiency. To promote atherosclerosis, male and female C57Bl6/J mice were either given weekly injections of Ldlr-ASO (5 mg/kg once per week) or genetically deficient in Ldlr (Ldlr-/-). Mice consumed either standard rodent chow or a diet high in saturated fat and sucrose with 0.15% added cholesterol for 16 weeks. While both models of Ldlr deficiency promoted hypercholesterolemia, Ldlr-/- mice exhibited nearly 2-fold higher cholesterol levels than Ldlr-ASO mice, reflected by increased VLDL and LDL levels. Consistent with this, the en face atherosclerotic lesion area was 3-fold and 3.6-fold greater in male and female mice with genetic Ldlr deficiency, respectively, as compared with the modest atherosclerosis observed following Ldlr-ASO treatment. Aortic sinus lesion sizes, fibrosis, smooth muscle actin, and necrotic core areas were also larger in Ldlr-/- mice, suggesting a more advanced phenotype. Despite a more modest effect on hypercholesterolemia, Ldlr-ASO induced greater hepatic inflammatory gene expression, macrophage accumulation, and histological lobular inflammation than was observed in Ldlr-/- mice. We conclude Ldlr-ASO is a promising tool for the generation of complex rodent models with which to study atherosclerosis but does not promote comparable levels of hypercholesterolemia or atherosclerosis as Ldlr-/- mice and increases hepatic inflammation. Thus, genetic Ldlr deficiency may be a superior model, depending on the proposed use.

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

[Figure: see text].

Obese Mice Losing Weight Due to trans-10,cis-12 Conjugated Linoleic Acid Supplementation or Food Restriction Harbor Distinct Gut Microbiota.

Background: trans-10,cis-12 Conjugated linoleic acid (t10,c12-CLA) is a dietary supplement that promotes weight loss by increasing fat oxidation and energy expenditure. We previously reported that in the absence of t10,c12-CLA, mice forced to lose equivalent body weight by food restriction (FR) do not exhibit increases in fat oxidation or energy expenditure but have improved glucose metabolism, consistent with FR as a metabolically healthy weight-loss method. Objective: Because diet is a primary determinant of gut bacterial populations, we hypothesized that the disparate metabolic effects accompanying weight loss from t10,c12-CLA or FR could be related to altered intestinal microbiota. Methods: Ten-week-old male LDL receptor-deficient (Ldlr-/-) mice were fed a high-fat, high-sucrose diet (HFHS; 36% lard fat, 36.2% sucrose + 0.15% cholesterol) for 12 wk (baseline), then switched to the HFHS diet alone (obese control), HFHS + 1% c9,t11-CLA (obese fatty acid control), HFHS + 1% t10,c12-CLA (weight-loss-inducing fatty acid), or HFHS + FR (weight-loss control group with 75-85% ad libitum HFHS food intake) for a further 8 wk. Fecal microbial content, short-chain fatty acids (butyrate, acetate), tissue CLA concentrations, and intestinal nutrient transporter expression were quantified. Results: Mice fed t10,c12-CLA or assigned to FR lost 14.5% of baseline body weight. t10,c12-CLA-fed mice had elevated concentrations of fecal butyrate (2-fold) and plasma acetate (1.5-fold) compared with HFHS-fed controls. Fecal α diversity decreased by 7.6-14% in all groups. Butyrivibrio and Roseburia, butyrate-producing microbes, were enriched over time by t10,c12-CLA. By comparing with each control group, we also identified bacterial genera significantly enriched in the t10,c12-CLA recipients, including Lactobacillus, Actinobacteria, and the newly identified Ileibacterium valens of the Allobaculum genus, whereas other taxa were enriched by FR, including Clostridiales and Bacteroides. Conclusion: Modalities resulting in equivalent weight loss but with divergent metabolic effects are associated with compositional differences in the mouse intestinal microbiota.

Chronic oral rapamycin decreases adiposity, hepatic triglycerides and insulin resistance in male mice fed a diet high in sucrose and saturated fat.

NEW FINDINGS: What is the central question of this study? Whether chronic oral rapamycin promotes beneficial effects on glucose/lipid metabolism and energy balance when administered to mice with an obesogenic diet rich in saturated fat and sucrose has not been explored. What is the main finding and its importance? Chronic oral rapamycin reduces body weight and fat gain, improves insulin sensitivity and reduces hepatic steatosis when administered to mice with a high-fat, high-sucrose diet. In addition, we make the new observation that there appear to be tissue-specific effects of rapamycin. Although rapamycin appears to impart its effects mainly on visceral adipose tissue, its effects on insulin sensitivity are mediated by subcutaneous adipose tissue. ABSTRACT: Excess adiposity is commonly associated with insulin resistance, which can increase the risk of cardiovascular disease. However, the exact molecular mechanisms by which obesity results in insulin resistance are yet to be understood clearly. The intracellular nutrient-sensing protein, mechanistic target of rapamycin (mTOR), is a crucial signalling component in the development of obesity-associated insulin resistance. Given that increased tissue activation of mTOR complex-1 (mTORC1) occurs in obesity, diabetes and ageing, we hypothesized that pharmacological inhibition of mTORC1 would improve metabolic dysregulation in diet-induced obesity. We administered continuous rapamycin, a specific mTORC1 inhibitor, orally to C57BL/6J mice concurrently with a high-fat, high-sucrose (HFHS) diet for 20 weeks. The control group received placebo microcapsules. Rapamycin-treated mice showed significantly reduced weight gain and adiposity (33.6 ± 4.9 versus 40.4 ± 3.0% body fat, P < 0.001, n = 8 mice per group), despite increased or equivalent food intake compared with the placebo group. The rapamycin-fed mice also demonstrated reduced plasma glucose (252 ± 57 versus 297 ± 67 mg dl-1 , P < 0.001) and improved insulin sensitivity during insulin and glucose tolerance testing. Rapamycin-treated mice also had lower plasma triglycerides (48 ± 13 versus 67 ± 11 mg/dL, P < 0.01) and hepatic triglyceride content (89 ± 15 versus 110 ± 19 mg/g liver, P < 0.05) compared with the placebo group. A moderately low dose of rapamycin decreased adiposity and improved the metabolic profile in a model of diet-induced obesity. These data suggest that low-grade chronic mTORC1 inhibition might be a potential strategy for anti-obesity therapies.

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.

Deficiency of Invariant Natural Killer T Cells Does Not Protect Against Obesity but Exacerbates Atherosclerosis in Ldlr-/- Mice.

Obesity is a chronic inflammatory state characterized by altered levels of adipose tissue immune cell populations. Natural killer T (NKT) cells are CD1d restricted lymphocyte subsets that recognize lipid antigens whose level decreases in obese adipose tissue. However, studies in mice with deficiency or increased levels of NKT cells have yielded contradictory results, so the exact role of these cells in obesity and adipose tissue inflammation is not yet established. We previously showed that Ldlr-/- mice with excess invariant NKT (iNKT) cells demonstrate significant weight gain, adiposity, metabolic abnormalities, and atherosclerosis. The current study evaluates the effects of NKT cell deficiency on obesity, associated metabolic changes, and atherosclerosis in Jα18-/-Ldlr-/- (lacking iNKT cells) and Cd1d-/-Ldlr-/- (lacking invariant and type II NKT cells) mice, and control mice were fed an obesogenic diet (high fat, sucrose, cholesterol) for 16 weeks. Contrary to expectations, Ja18-/-Ldlr-/- mice gained significantly more weight than Ldlr-/- or Cd1d-/-Ldlr-/- mice, developed hypertriglyceridemia, and had worsened adipose tissue inflammation. All the mice developed insulin resistance and hepatic triglyceride accumulation. Ja18-/-Ldlr-/- mice also had increased atherosclerotic lesion area. Our findings suggest that iNKT cells exacerbates the metabolic, inflammatory, and atherosclerotic features of diet-induced obesity. Further work is required to unravel the paradox of an apparently similar effect of iNKT cell surplus and depletion on obesity.

Apolipoprotein AI and high-density lipoprotein have anti-inflammatory effects on adipocytes via cholesterol transporters: ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptor B-1.

RATIONALE: Macrophage accumulation in adipose tissue associates with insulin resistance and increased cardiovascular disease risk. We previously have shown that generation of reactive oxygen species and monocyte chemotactic factors after exposure of adipocytes to saturated fatty acids, such as palmitate, occurs via translocation of NADPH oxidase 4 into lipid rafts (LRs). The anti-inflammatory effects of apolipoprotein AI (apoAI) and high-density lipoprotein (HDL) on macrophages and endothelial cells seem to occur via cholesterol depletion of LRs. However, little is known concerning anti-inflammatory effects of HDL and apoAI on adipocytes. OBJECTIVE: To determine whether apoAI and HDL inhibit inflammation in adipocytes and adipose tissue, and whether this is dependent on LRs. METHODS AND RESULTS: In 3T3L-1 adipocytes, apoAI, HDL, and methyl-ß-cyclodextrin inhibited chemotactic factor expression. ApoAI and HDL also disrupted LRs, reduced plasma membrane cholesterol content, inhibited NADPH oxidase 4 translocation into LRs, and reduced palmitate-induced reactive oxygen species generation and monocyte chemotactic factor expression. Silencing ATP-binding cassette A-1 abrogated the effect of apoAI, but not HDL, whereas silencing ATP-binding cassette G-1 or scavenger receptor B-1 abrogated the effect of HDL but not apoAI. In vivo, apoAI transgenic mice fed a high-fat, high-sucrose, cholesterol-containing diet showed reduced chemotactic factor and proinflammatory cytokine expression and reduced macrophage accumulation in adipose tissue. CONCLUSIONS: ApoAI and HDL have anti-inflammatory effects in adipocytes and adipose tissue similar to their effects in other cell types. These effects are consistent with disruption and removal of cholesterol from LRs, which are regulated by cholesterol transporters, such as ATP-binding cassette A-1, ATP-binding cassette G-1, and scavenger receptor B-1.

The role of intestinal microbiota in physiologic and body compositional changes that accompany CLA-mediated weight loss in obese mice.

OBJECTIVE: Obesity continues to be a major problem, despite known treatment strategies such as lifestyle modifications, pharmaceuticals, and surgical options, necessitating the development of novel weight loss approaches. The naturally occurring fatty acid, 10,12 conjugated linoleic acid (10,12 CLA), promotes weight loss by increasing fat oxidation and browning of white adipose tissue, leading to increased energy expenditure in obese mice. Coincident with weight loss, 10,12 CLA also alters the murine gut microbiota by enriching for microbes that produce short chain fatty acids (SCFAs), with concurrent elevations in fecal butyrate and plasma acetate. METHODS: To determine if the observed microbiota changes are required for 10,12 CLA-mediated weight loss, adult male mice with diet-induced obesity were given broad-spectrum antibiotics (ABX) to perturb the microbiota prior to and during 10,12 CLA-mediated weight loss. Conversely, to determine whether gut microbes were sufficient to induce weight loss, conventionally-raised and germ-free mice were transplanted with cecal contents from mice that had undergone weight loss by 10,12 CLA supplementation. RESULTS: While body weight was minimally modulated by ABX-mediated perturbation of gut bacterial populations, adult male mice given ABX were more resistant to the increased energy expenditure and fat loss that are induced by 10,12 CLA supplementation. Transplanting cecal contents from donor mice losing weight due to oral 10,12 CLA consumption into conventional or germ-free mice led to improved glucose metabolism with increased butyrate production. CONCLUSIONS: These data suggest a critical role for the microbiota in diet-modulated changes in energy balance and glucose metabolism, and distinguish the metabolic effects of orally delivered 10,12 CLA from cecal transplantation of the resulting microbiota.

10E,12Z-conjugated linoleic acid impairs adipocyte triglyceride storage by enhancing fatty acid oxidation, lipolysis, and mitochondrial reactive oxygen species.

Conjugated linoleic acid (CLA) is a naturally occurring dietary trans fatty acid found in food from ruminant sources. One specific CLA isomer, 10E,12Z-CLA, has been associated with health benefits, such as reduced adiposity, while simultaneously promoting deleterious effects, such as systemic inflammation, insulin resistance, and dyslipidemia. The precise mechanisms by which 10E,12Z-CLA exerts these effects remain unknown. Despite potential health consequences, CLA continues to be advertised as a natural weight loss supplement, warranting further studies on its effects on lipid metabolism. We hypothesized that 10E,12Z-CLA impairs lipid storage in adipose tissue by altering the lipid metabolism of white adipocytes. We demonstrate that 10E,12Z-CLA reduced triglyceride storage due to enhanced fatty acid oxidation and lipolysis, coupled with diminished glucose uptake and utilization in cultured adipocytes. This switch to lipid utilization was accompanied by a potent proinflammatory response, including the generation of cytokines, monocyte chemotactic factors, and mitochondrial superoxide. Disrupting fatty acid oxidation restored glucose utilization and attenuated the inflammatory response to 10E,12Z-CLA, suggesting that fatty acid oxidation is critical in promoting this phenotype. With further investigation into the biochemical pathways involved in adipocyte responses to 10E,12Z-CLA, we can discern more information about its safety and efficacy in promoting weight loss.

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.

Toll-like receptor 4 deficiency decreases atherosclerosis but does not protect against inflammation in obese low-density lipoprotein receptor-deficient mice.

OBJECTIVE: Obesity is associated with insulin resistance, chronic low-grade inflammation, and atherosclerosis. Toll-like receptor 4 (TLR4) participates in the cross talk between inflammation and insulin resistance, being activated by both lipopolysaccharide and saturated fatty acids. The present study was undertaken to determine whether TLR4 deficiency has a protective role in inflammation, insulin resistance, and atherosclerosis induced by a diabetogenic diet. METHODS AND RESULTS: TLR4 and low-density lipoprotein (LDL) receptor double knockout mice and LDL receptor-deficient mice were fed either a normal chow or a diabetogenic diet for 24 weeks. TLR4 and LDL receptor double knockout mice fed a diabetogenic diet showed improved plasma cholesterol and triglyceride levels but developed obesity, hyperinsulinemia, and glucose intolerance equivalent to obese LDL receptor-deficient mice. Adipocyte hypertrophy, macrophage accumulation, and local inflammation were not attenuated in intraabdominal adipose tissue in TLR4 and LDL receptor double knockout mice. However, TLR4 deficiency led to markedly decreased atherosclerosis in obese TLR4 and LDL receptor double knockout mice. Compensatory upregulation of TLR2 expression was observed both in obese TLR4-deficient mice and in palmitate-treated TLR4-silenced 3T3-L1 adipocytes. CONCLUSIONS: TLR4 deficiency decreases atherosclerosis without affecting obesity-induced inflammation and insulin resistance in LDL receptor-deficient mice. Alternative pathways may be responsible for adipose tissue macrophage infiltration and insulin resistance that occurs in obesity.

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.

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.

Serum amyloid A facilitates the binding of high-density lipoprotein from mice injected with lipopolysaccharide to vascular proteoglycans.

OBJECTIVE: Levels of serum amyloid A (SAA), an acute-phase protein carried on high-density lipoprotein (HDL), increase in inflammatory states and are associated with increased risk of cardiovascular disease. HDL colocalizes with vascular proteoglycans in atherosclerotic lesions. However, its major apolipoprotein, apolipoprotein A-I, has no proteoglycan-binding domains. Therefore, we investigated whether SAA, which has proteoglycan-binding domains, plays a role in HDL retention by proteoglycans. METHODS AND RESULTS: HDL from control mice and mice deficient in both SAA1.1 and SAA2.1 (SAA knockout mice) injected with bacterial lipopolysaccharide (LPS) was studied. SAA mRNA expression in the liver and plasma levels of SAA increased dramatically in C57BL/6 mice after LPS administration, although HDL cholesterol did not change. Fast protein liquid chromatography analysis showed most of the SAA to be in HDL. Mass spectrometric analysis indicated that HDL from LPS-injected control mice had high levels of SAA1.1/2.1 and reduced levels of apolipoprotein A-I. HDL from LPS-injected control mice demonstrated high-affinity binding to biglycan relative to normal mouse HDL. In contrast, HDL from LPS-injected SAA knockout mice showed very little binding to biglycan, consistent with SAA facilitating the binding of HDL to vascular proteoglycans. CONCLUSION: SAA enrichment of HDL under inflammatory conditions plays an important role in the binding of HDL to vascular proteoglycans.

Aging Increases Susceptibility to Develop Cardiac Hypertrophy following High Sugar Consumption.

Aging and poor diet are independent risk factors for heart disease, but the impact of high-sucrose (HS) consumption in the aging heart is understudied. Aging leads to impairments in mitochondrial function that result in muscle dysfunction (e.g., cardiac remodeling and sarcopenia). We tested whether HS diet (60%kcal sucrose) would accelerate muscle dysfunction in 24-month-old male CB6F1 mice. By week 1 on HS diet, mice developed significant cardiac hypertrophy compared to age-matched chow-fed controls. The increased weight of the heart persisted throughout the 4-week treatment, while body weight and strength declined more rapidly than controls. We then tested whether HS diet could worsen cardiac dysfunction in old mice and if the mitochondrial-targeted drug, elamipretide (ELAM), could prevent the diet-induced effect. Old and young mice were treated with either ELAM or saline as a control for 2 weeks, and provided with HS diet or chow on the last week. As demonstrated in the previous experiment, old mice had age-related cardiac hypertrophy that worsened after one week on HS and was prevented by ELAM treatment, while the HS diet had no detectable effect on hypertrophy in the young mice. As expected, mitochondrial respiration and reactive oxygen species (ROS) production were altered by age, but were not significantly affected by HS diet or ELAM. Our findings highlight the vulnerability of the aged heart to HS diet that can be prevented by systemic targeting of the mitochondria with ELAM.

Dietary cholesterol exacerbates hepatic steatosis and inflammation in obese LDL receptor-deficient mice.

Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of the metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver disease. Mechanisms that underlie this progression remain poorly understood, partly due to lack of good animal models that resemble human NASH. We previously showed that several metabolic syndrome features that develop in LDL receptor-deficient (LDLR-/-) mice fed a diabetogenic diet are worsened by dietary cholesterol. To test whether dietary cholesterol can alter the hepatic phenotype in the metabolic syndrome, we fed LDLR-/- mice a high-fat, high-carbohydrate diabetogenic diet (DD) without or with added cholesterol (DDC). Both groups of mice developed obesity and insulin resistance. Hyperinsulinemia, dyslipidemia, hepatic triglyceride, and alanine aminotransferase (ALT) elevations were greater with DDC. Livers of DD-fed mice showed histological changes resembling NAFLD, including steatosis and modest fibrotic changes; however, DDC-fed animals developed micro- and macrovesicular steatosis, inflammatory cell foci, and fibrosis resembling human NASH. Dietary cholesterol also exacerbated hepatic macrophage infiltration, apoptosis, and oxidative stress. Thus, LDLR-/- mice fed diabetogenic diets may be useful models for studying human NASH. Dietary cholesterol appears to confer a second "hit" that results in a distinct hepatic phenotype characterized by increased inflammation and oxidative stress.

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.

Serum amyloid A-containing HDL binds adipocyte-derived versican and macrophage-derived biglycan, reducing its antiinflammatory properties.

The ability of HDL to inhibit inflammation in adipocytes and adipose tissue is reduced when HDL contains serum amyloid A (SAA) that is trapped by proteoglycans at the adipocyte surface. Because we recently found that the major extracellular matrix proteoglycan produced by hypertrophic adipocytes is versican, whereas activated adipose tissue macrophages produce mainly biglycan, we further investigated the role of proteoglycans in determining the antiinflammatory properties of HDL. The distributions of versican, biglycan, apolipoprotein A1 (the major apolipoprotein of HDL), and SAA were similar in adipose tissue from obese mice and obese human subjects. Colocalization of SAA-enriched HDL with versican and biglycan at the cell surface of adipocyte and peritoneal macrophages, respectively, was blocked by silencing these proteoglycans, which also restored the antiinflammatory property of SAA-enriched HDL despite the presence of SAA. Similar to adipocytes, normal HDL exerted its antiinflammatory function in macrophages by reducing lipid rafts, reactive oxygen species generation, and translocation of Toll-like receptor 4 and NADPH oxidase 2 into lipid rafts, effects that were not observed with SAA-enriched HDL. These findings imply that SAA present in HDL can be trapped by adipocyte-derived versican and macrophage-derived biglycan, thereby blunting HDL's antiinflammatory properties.