Chronic hyperglycemia causes oxidative stress, which contributes to damage in various tissues and cells, including pancreatic β-cells. The expression levels of antioxidant enzymes in the islet are ...low compared with other tissues, rendering the β-cell more susceptible to damage caused by hyperglycemia. The aim of this study was to investigate whether increasing levels of endogenous glutathione peroxidase-1 (GPx-1), specifically in β-cells, can protect them against the adverse effects of chronic hyperglycemia and assess mechanisms that may be involved. C57BLKS/J mice overexpressing the antioxidant enzyme GPx-1 only in pancreatic β-cells were generated. The biological effectiveness of the overexpressed GPx-1 transgene was documented when β-cells of transgenic mice were protected from streptozotocin. The transgene was then introgressed into the β-cells of db/db mice. Without use of hypoglycemic agents, hyperglycemia in db/db-GPx(+) mice was initially ameliorated compared with db/db-GPx(−) animals and then substantially reversed by 20 wk of age. β-Cell volume and insulin granulation and immunostaining were greater in db/db-GPx(+) animals compared with db/db-GPx(−) animals. Importantly, the loss of intranuclear musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) that was observed in nontransgenic db/db mice was prevented by GPx-1 overexpression, making this a likely mechanism for the improved glycemic control. These studies demonstrate that enhancement of intrinsic antioxidant defenses of the β-cell protects it against deterioration during hyperglycemia.
β-cell-specific GPx-1 overexpression in db/db mice protects the β-cell from oxidative stress and progression in dysfunction associated with chronic hyperglycemia.
In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids ...and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue.
TNF-α signals through two receptors, TNFR1 and TNFR2. Our goals were: 1) determine the role of TNFRs in obesity and metabolic disease and 2) investigate whether TNFRs contribute to the link between ...obesity and adipose tissue macrophage infiltration and polarization. R1−/−R2−/− (RKO) and wild-type (WT) mice were fed standard chow or a high-fat/high-sucrose diet (HFHS) over 14 wk. Body composition, food intake, and energy expenditure were measured. Oral glucose tolerance and insulin sensitivity tests assessed glucose homeostasis. Adipose tissue and systemic inflammatory status were evaluated by quantifying plasma adipokine levels and macrophage-specific gene expression in fat. RKO mice were heavier (10%) and fatter (18%) than WT controls at 4 wk of age and were 26% heavier and 50% fatter than WT after 14 wk of HFHS diet feeding. Age- and diet-adjusted 24-h oxygen consumption, activity, and respiratory exchange ratio were significantly reduced in RKO mice. Obese RKO mice were markedly insulin resistant, suggesting that intact TNFR signaling is not required for the effect of obesity to impair glucose metabolism. Adipose tissue from HFHS-fed RKO mice exhibited increased macrophage infiltration, but compared with WT mice, macrophage phenotypic markers featured a predominance of antiinflammatory M2 over proinflammatory M1 cells. TNFRs play a physiological role to limit body weight and adiposity by modestly increasing metabolic rate and fatty acid oxidation, and they are required for obesity-induced activation of adipose tissue macrophages. Despite these effects, TNFRs are not required for obesity-induced insulin resistance.
In humans and rodents, obesity is associated with low-grade systemic and tissue inflammation, yet ablation of cytokines and their receptors, such as the two TNF receptors studied herein, predispose mice to obesity and do not protect mice from insulin resistance.
Insulin receptor (IR) translocates to the nucleus, but its recruitment to gene loci has not been demonstrated. Here, we tested the hypothesis that IR and its downstream mitogenic transducers are ...corecruited to two prototypic insulin-inducible genes: early growth response 1 (egr-1), involved in mitogenic response, and glucokinase (Gck), encoding a key metabolic enzyme.
We used RNA and chromatin from insulin-treated rat hepatic tumor cell line expressing human insulin receptor (HTC-IR) and livers from lean and insulin-resistant ob/ob glucose-fed mice in quantitative RT-PCR and chromatin immunoprecipitation studies to determine gene expression levels and associated recruitment of RNA polymerase II (Pol II), insulin receptor, and cognate signaling proteins to gene loci, respectively.
Insulin-induced egr-1 mRNA in HTC-IR cells was associated with corecruitment of IR signaling cascade (IR, SOS, Grb2, B-Raf, MEK, and ERK) to this gene. Recruitment profiles of phosphorylated IR, B-Raf, MEK, and Erk along egr-1 transcribed region were similar to those of elongating Pol II. Glucose-feeding increased Gck mRNA expression in livers of lean but not ob/ob mice. In lean mice, there was glucose feeding-induced recruitment of IR and its transducers to Gck gene synchronized with elongating Pol II. In sharp contrast, in glucose-fed ob/ob mice, the Gck recruitment patterns of active MEK/Erk, IR, and Pol II were asynchronous.
IR and its signal transducers recruited to genes coupled to elongating Pol II may play a role in maintaining productive mRNA synthesis of target genes. These studies suggest a possibility that impaired Pol II processivity along genes bearing aberrant levels of IR/signal transducers is a previously unrecognized facet of insulin resistance.
Laminin-β2 (LAMB2) is a critical component of the glomerular basement membrane as content of LAMB2 in part determines glomerular barrier permeability. Previously, we reported that high concentrations ...of glucose reduce expression of this laminin subunit at the translational level. The present studies were undertaken to further define systems that control Lamb2 translation and the effect of high glucose on those systems. Complementary studies were performed using in vitro differentiation of cultured podocytes and mesangial cells exposed to normal and elevated concentrations of glucose, and tissues from control and diabetic rats. Together, these studies provide evidence for regulation of Lamb2 translation by IMP2, an RNA binding protein that targets Lamb2 mRNA to the actin cytoskeleton. Expression of Imp2 itself is regulated by the transcription factor HMGA2, which in turn is regulated by the microRNA let-7b. Elevated concentrations of glucose increase let-7b, which reduces HMGA2 expression, in turn reducing IMP2 and LAMB2. Correlative changes in kidney tissues from control and streptozotocin-induced diabetic rats suggest these control mechanisms are operative in vivo and may contribute to proteinuria in diabetic nephropathy. To our knowledge, this is the first time that translation of Lamb2 mRNA has been linked to the actin cytoskeleton, as well as to specific RNA-binding proteins. These translational control points may provide new targets for therapy in proteinuric disorders such as diabetic nephropathy where LAMB2 levels are reduced.
Superoxide, the reduced form of molecular oxygen, has been implicated in the genesis of vascular disease. One potential mechanism involves oxidation of low density lipoprotein into an atherogenic ...particle. A second involves reaction with nitric oxide to generate peroxynitrite, a highly oxidizing intermediate. A third involves regulation of signal transduction in artery wall cells. One well-characterized pathway for superoxide production resides in macrophages, the cellular hallmark of the early atherosclerotic lesion. Macrophages contain a membrane-bound NADPH oxidase that reduces oxygen to superoxide. In the current studies, we used mice that are deficient in the gp91-phox subunit of the NADPH oxidase–a model of chronic granulomatous disease (CGD)–to explore the role of superoxide in atherosclerotic vascular disease. Wild-type and CGD mice on the C57BL/6 background received a high-fat diet for 20 weeks to induce hypercholesterolemia. At the end of this period, the 2 strains of mice had comparable plasma lipid levels, and their atherosclerotic lesions were similar in size. We also crossed CGD mice with apolipoprotein E–deficient (apoE−/−) mice to generate spontaneously hypercholesterolemic animals that lacked functional NADPH oxidase. After 24 weeks, the CGD-apoE−/− animals had lower plasma cholesterol and triglyceride levels than did the apoE−/− animals, but there was no difference in the extent of atherosclerotic plaque. Our findings suggest that superoxide generated by the NADPH oxidase of phagocytes does not promote atherosclerosis in mice with either diet-induced or genetic forms of hypercholesterolemia.
Inflammatory processes are involved with all phases of atherosclerotic lesion growth. Tumor necrosis factor-α (TNFα) is an inflammatory cytokine that is thought to contribute to lesion development. ...Lymphotoxin-α (LTα) is also a proinflammatory cytokine with homology to TNFα. However, its presence or function in lesion development has not been investigated. To study the role of these molecules in atherosclerosis, the expression of these cytokines in atherosclerotic lesions was examined. The presence of both cytokines was observed within aortic sinus fatty streak lesions. To determine the function of these molecules in regulating lesion growth, mice deficient for TNFα or LTα were examined for induction of atherosclerosis. Surprisingly, loss of TNFα did not alter lesion development compared with wild-type mice. This brings doubt to the generally held concept that TNFα is a “proatherogenic cytokine.” However, LTα deficiency resulted in a 62% reduction in lesion size. This demonstrates an unexpected role for LTα in promoting lesion growth. The presence of LTα was observed in aortic sinus lesions suggesting a direct role of LTα in modulating lesion growth. To determine which receptor mediated these responses, diet-induced atherosclerosis in mice deficient for each of the TNF receptors, termed p55 and p75, was examined. Results demonstrated that loss of p55 resulted in increased lesion development, but loss of p75 did not alter lesion size. The disparity in results between ligand- and receptor-deficient mice suggests there are undefined members of the TNF ligand and receptor signaling pathway involved with regulating atherogenesis.
ABCA1 exports excess cholesterol from cells to apoA-I and is essential for HDL synthesis. Genetic studies have shown that ABCA1 protects against cardiovascular disease. We have previously shown that ...the interaction of apoA-I with ABCA1 activates signaling molecule Janus kinase 2 (JAK2), which optimizes the cholesterol efflux activity of ABCA1. ABCA1-mediated activation of JAK2 also activates signal transducer and activator of transcription 3 (STAT3), which significantly attenuates proinflammatory cytokine expression in macrophages. To determine the mechanisms of the anti-inflammatory effects of apoA-I/ABCA1 interaction, we identified two special ABCA1 mutants, one with normal STAT3-activating capacity but lacking cholesterol efflux ability and the other with normal cholesterol efflux ability but lacking STAT3-activating capacity. We showed that activation of STAT3 by the interaction of apoA-I/ABCA1 without cholesterol efflux could significantly decrease proinflammatory cytokine expression in macrophages. Mechanistic studies showed that the anti-inflammatory effect of the apoA-I/ABCA1/STAT3 pathway is suppressor of cytokine signaling 3 dependent. Moreover, we showed that apoA-I/ABCA1-mediated cholesterol efflux without STAT3 activation can also reduce proinflammatory cytokine expression in macrophages. These findings suggest that the interaction of apoA-I/ABCA1 activates cholesterol efflux and STAT3 branch pathways to synergistically suppress inflammation in macrophages.
Abstract Objective The tumor necrosis factor superfamily may exert cardioprotective or atherogenic effects, depending on the state of lesion progression. Tumor necrosis factor-α (TNF) induces ...macrophage ATP-binding cassette transporter A1 (ABCA1), a cardioprotective transmembrane protein that exports cellular cholesterol to apolipoprotein A-I. Here we examined the role of TNF receptors (TNFRs) in ABCA1 induction and tested the effects of lymphotoxin-α (LT), another TNF family member, on macrophage ABCA1 levels. Methods Primary macrophages taken from mice deficient in TNF receptors were used to determine ABCA1 expression and cholesterol efflux activity in response to treatment with exogenous TNF or LT. Results We studied TNFR2−/− and TNFR1−/− /R2−/− mice and found that both receptors are necessary for maximal induction of ABCA1 by TNF. Peritoneal macrophages from TNFR1−/− /R2−/− mice had no change in ABCA1 mRNA levels when treated with TNF while cells from TNFR2−/− mice had ABCA1 mRNA levels that were half that of wild-type (WT) cells. In contrast, incubating TNFR1−/− /R2−/− mice with LT increased ABCA1 by stabilizing the protein, which was not observed in WT mice and this was associated with downstream signaling through the LTβ receptor. Conclusion TNF requires both of its receptors to maximally induce ABCA1. Despite previous studies suggesting that LT has proatherogenic properties, we found that LT increases ABCA1 protein in TNFR1−/− /R2−/− but not WT macrophages and may supplement TNF in enhancing ABCA1-dependent cholesterol export from early atherosclerotic lesions.
Metabolic diseases such as obesity and atherosclerosis result from complex interactions between environmental factors and genetic variants. A panel of chromosome substitution strains (CSSs) was ...developed to characterize genetic and dietary factors contributing to metabolic diseases and other biological traits and biomedical conditions. Our goal here was to identify quantitative trait loci (QTLs) contributing to obesity, energy expenditure, and atherosclerosis. Parental strains C57BL/6 and A/J together with a panel of 21 CSSs derived from these progenitors were subjected to chronic feeding of rodent chow and atherosclerotic (females) or diabetogenic (males) test diets, and evaluated for a variety of metabolic phenotypes including several traits unique to this report, namely fat pad weights, energy balance, and atherosclerosis. A total of 297 QTLs across 35 traits were discovered, two of which provided significant protection from atherosclerosis, and several dozen QTLs modulated body weight, body composition, and circulating lipid levels in females and males. While several QTLs confirmed previous reports, most QTLs were novel. Finally, we applied the CSS quantitative genetic approach to energy balance, and identified three novel QTLs controlling energy expenditure and one QTL modulating food intake. Overall, we identified many new QTLs and phenotyped several novel traits in this mouse model of diet-induced metabolic diseases.
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