Immunomanipulation of Appetite and Body Temperature through the Functional Mimicry of Leptin

Objective: Although current obesity therapies produce some benefits, there is a need for new strategies to treat obesity. A novel proposal is the use of anti‐idiotypic antibodies as surrogate ligands or hormones. These anti‐idiotypic antibodies carry an internal motif that imitates or mimics an epitope in the antigen (i.e., hormone or ligand). Thus, anti‐idiotypic antibodies to several ligands may mimic them in transducing signals when binding to their receptors. Research Methods and Procedures: We developed an anti‐idiotypic polyclonal antibody against the region of a leptin monoclonal antibody that competitively binds leptin, mimicking the active site structure of leptin. To test whether our anti‐idiotype could also reproduce leptin functions, we examined food intake, body weight, and colonic temperature in male Wistar rats (n = 9) in response to intracerebroventricular administration of the leptin anti‐idiotype. Results: Our leptin anti‐idiotype induced a significant reduction in food intake coupled with an increase in body temperature comparable to that of leptin. That is, the intracerebroventricular administration of 8.0 μg of leptin anti‐idiotype or 5.0 μg leptin significantly increased colonic temperature (Δ 1.9 ± 0.11 °C and Δ1.7 ± 0.12 °C, respectively). In addition, both decreased 24‐hour food intake (?26.4 ± 2.4% and ?21.9 ± 2.2%) compared with the control. The gain in body weight was also decreased by acute administration of the anti‐idiotype (?1.4 ± 0.28%) and leptin (?1.1 ± 0.17%) vs. the phosphate‐buffered saline control (1.3 ± 0.15%). Discussion: These studies revealed that the leptin anti‐idiotype inhibited food intake and enhanced heat production, mimicking leptin's central actions.     

Effect of the Interleukin‐6 (−174) G/C Promoter Polymorphism on Adiponectin and Insulin Sensitivity

We investigated the relation among the interleukin (IL)‐6 (?174) G/C promoter polymorphism, adipose tissue gene expression of IL6, circulating adiponectin, and systemic insulin sensitivity. Eighty‐five Swedish male subjects who had participated in our previous prediabetic phenotype characterization study were genotyped for the IL6 (?174) G/C polymorphism. Subcutaneous adipose tissue gene expression of IL6 and adiponectin was measured in 44 subjects. The IL6 (?174) G allele carriers had higher fasting plasma insulin levels (C/C, 7.8 ± 1.1; G/C, 9.0 ± 0.6; G/G, 10.5 ± 1.0 mU/L) and higher homeostasis model assessment for insulin resistance (C/C, 1.6 ± 0.2; G/C, 1.9 ± 0.1; G/G, 2.2 ± 0.2) compared with subjects with the C/C genotype. The circulating adiponectin levels were lower in the G allele carriers (C/C, 7.93 ± 0.45; G/C, 7.05 ± 0.44; G/G, 7.02 ± 0.46 μg/mL), whereas the IL‐6 levels did not differ among the three genotypes. Adipose tissue IL6 gene expression was significantly higher in the G allele carriers compared with the subjects homozygous for the C allele (C/C, 0.29 ± 0.15; G/C, 0.84 ± 0.29; G/G, 0.62 ± 0.35). Our results suggest that IL6 (?174) G/C polymorphism is associated with insulin resistance and increased adipose tissue IL6 gene expression, which can impair adiponectin production.     

Upstream stimulatory factor‐2 mediates quercetin‐induced suppression of PAI‐1 gene expression in human endothelial cells

The polyphenol quercetin (Quer) represses expression of the cardiovascular disease risk factor plasminogen activator inhibitor‐1 (PAI‐1) in cultured endothelial cells (ECs). Transfection of PAI‐1 promoter‐luciferase reporter deletion constructs identified a 251‐bp fragment (nucleotides ?800 to ?549) responsive to Quer. Two E‐box motifs (CACGTG), at map positions ?691 (E‐box1) and ?575 (E‐box2), are platforms for occupancy by several members of the c‐MYC family of basic helix‐loop‐helix leucine zipper (bHLH‐LZ) proteins. Promoter truncation and electrophoretic mobility shift/supershift analyses identified upstream stimulatory factor (USF)‐1 and USF‐2 as E‐box1/E‐box2 binding factors. ECs co‐transfected with a 251 bp PAI‐1 promoter fragment containing the two E‐box motifs (p251/luc) and a USF‐2 expression vector (pUSF‐2/pcDNA) exhibited reduced luciferase activity versus p251/luc alone. Overexpression of USF‐2 decreased, while transfection of a dominant‐negative USF construct increased, EC growth consistent with the known anti‐proliferative properties of USF proteins. Quer‐induced decreases in PAI‐1 expression and reduced cell proliferation may contribute, at least in part, to the cardioprotective benefit associated with daily intake of polyphenols. J. Cell. Biochem. 111: 720–726, 2010. © 2010 Wiley‐Liss, Inc.     

Inhibition of LPS‐induced C/EBPδ by trichostatin a has a positive effect on LPS‐induced cyclooxygenase 2 expression in RAW264.7 cells

Cyclooxygenase 2 (COX‐2) is an important inflammatory factor. Previous studies have indicated that COX‐2 is induced with lipopolysaccharide (LPS) treatment. Here, we found that an inhibitor of histone deacetylase (HDAC), trichostatin A (TSA), cannot repress LPS‐induced COX‐2 but it increased the COX‐2 level in RAW264.7 cells. We found no significant difference in NF‐κB activation and ERK1/2 phosphorylation, but LPS‐induced C/EBPδ expression was completely abolished after TSA treatment of LPS‐treated cells. Interesting, reporter assay of C/EBPδ promoter revealed that Sp1‐binding site is important. Although there was no alteration in c‐Jun levels, but the phosphorylation of c‐Jun at its C‐terminus was increased dramatically. A DNA‐associated protein assay (DAPA) and chromatin immunoprecipitation assay (ChIP) indicated that c‐Jun was recruited via Sp1 to the promoter of C/EBPδ after LPS treatment; this recruitment of c‐Jun was repressed by TSA. C/EBPδ inhibition by TSA resulted in increased binding of C/EBPα and C/EBPβ to the COX‐2 promoter. Therefore, TSA has a positive effect on LPS‐induced COX‐2 since it decreases the C/EBPδ level by reducing c‐Jun recruitment by Sp1 to the C/EBPδ promoter, resulting in increased the recruitment of C/EBPα and C/EBPβ to the COX‐2 promoter. J. Cell. Biochem. 110: 1430–1438, 2010. © 2010 Wiley‐Liss, Inc.     

Statins potently reduce the cytokine‐mediated IL‐6 release in SMC/MNC cocultures

Inflammatory pathways are involved in the development of atherosclerosis. Interaction of vessel wall cells and invading monocytes by cytokines may trigger local inflammatory processes. 3‐Hydroxy‐3‐methylglutaryl coenzyme A reductase inhibitors (statins) are standard medications used in cardiovascular diseases. They are thought to have anti‐inflammatory capacities, in addition to their lipid‐lowering effects. We investigated the anti‐inflammatory effect of statins in the cytokine‐mediated‐interaction‐model of human vascular smooth muscle cells (SMC) and human mononuclear cells (MNC). In this atherosclerosis‐related inflammatory model LPS (lipopolysaccharide, endotoxin), as well as high mobility group box 1 stimulation resulted in synergistic (i.e. over‐additive) IL‐6 (interleukin‐6) production as measured in ELISA. Recombinant IL‐1, tumour necrosis factor‐α and IL‐6 mediated the synergistic IL‐6 production. The standard anti‐inflammatory drugs aspirin and indomethacin (Indo) reduced the synergistic IL‐6 production by 60%. Simvastatin, atorvastatin, fluvastatin or pravastatin reduced the IL‐6 production by 53%, 50%, 64% and 60%, respectively. The inhibition by the statins was dose dependent. Combination of statins with aspirin and/or Indo resulted in complete inhibition of the synergistic IL‐6 production. The same inhibitors blocked STAT3 phosphorylation, providing evidence for an autocrine role of IL‐6 in the synergism. MNC from volunteers after 5 day aspirin or simvastatin administration showed no decreased IL‐6 production, probably due to drug removal during MNC isolation. Taken together, the data show that anti‐inflammatory functions (here shown for statins) can be sensitively and reproducibly determined in this novel SMC/MNC coculture model. These data implicate that statins have the capacity to affect atherosclerosis by regulating cytokine‐mediated innate inflammatory pathways in the vessel wall.     

Lifestyle intervention and/or statins for the reduction of C‐reactive protein in type 2 diabetes: From the look AHEAD study

Objective:

Cardiovascular risk remains high despite statin use. Overweight/obese diabetic persons usually have normal/low LDL‐cholesterol but high C‐reactive protein (CRP) levels. We aimed to examine the effects of intensive lifestyle intervention for weight loss (ILI) on CRP levels in overweight/obese diabetic individuals by statin use.

Design and Methods:

Look AHEAD was a randomized trial in overweight/obese type 2 diabetic individuals testing whether ILI would reduce cardiovascular mortality, when compared to usual care. CRP changes in 1,431 participants with biomarker levels, who remained on or off statin treatment for 1 year, were evaluated.

Results:

The reduction in CRP levels with ILI at 1 year in men and women on statins was ?44.9 and ?42.3%, respectively, compared to ?13.7 and ?21.0% for those on statins and usual care (P < 0.0001). At 1 year, median CRP levels were: 1.8 mg L^?1 in participants randomized to ILI on statin therapy; 2.6 mg L^?1 for those on statins randomized to usual care and 2.9 mg L^?1 for participants not on statins but randomized to ILI. Weight loss was associated with 1‐year CRP reduction (P < 0.0001) in statin and nonstatin users.

Conclusions:

Our findings suggest that in overweight/obese diabetic persons, ILI and statin therapy may have substantial additive anti‐inflammatory benefits.

     

The Expression of Cyclin D1 during Adipogenesis in Pig Primary Stromal–Vascular Cultures

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal‐vascular (S‐V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S‐V cells using 8% sodium dodecyl sulfate‐polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti‐mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti‐CCAAT/enhancer binding protein (C/EBPα) and anti‐cyclin D1 together. Cyclin D1 expression was evaluated in 105‐day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S‐V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid‐filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.     

A novel lncRNA BADLNCR1 inhibits bovine adipogenesis by repressing GLRX5 expression

Adipogenesis is a complex cellular process, which needs a series of molecular events, including long non‐coding RNA (lncRNA). In the present study, a novel lncRNA named BADLNCR1 was identified as a regulator during bovine adipocyte differentiation, which plays an inhibitory role in lipid droplet formation and adipogenic marker gene expression. CHIPR‐seq data demonstrated a potential competitive binding motif between BADLNCR1 and sterol regulatory element‐binding proteins 1 and 2 (SREBP1/2). Dual‐luciferase reporter assay indicated target relationship between KLF2 and BADLNCR1. Moreover, after the induction of KLF2, the expression of adipogenic gene reduced, while the expression of BADLNCR1 increased. Real‐time quantitative PCR (qPCR) showed that BADLNCR1 negatively regulated mRNA expression of GLRX5 gene, a stimulator of genes that promoted formation of lipid droplets and expression of adipogenic genes. GLRX5 could partially reverse the effect of BADLNCR1 in bovine adipocyte differentiation. Dual‐luciferase reporter assay stated that BADLNCR1 significantly reduced the enhancement of C/EBPα on promoter activity of GLRX5 gene. Furthermore, CHIP‐PCR and CHIRP‐PCR confirmed the suppressing effect of BADLNCR1 on binding of C/EBPα to GLRX5 promoter. Collectively, this study revealed the molecular mechanisms underlying the negative regulation of BADLNCR1 in bovine adipogenic differentiation.     

The apolipoprotein A‐I mimetic peptide,D‐4F,restrains neointimal formation through heme oxygenase‐1 up‐regulation

D‐4F, an apolipoprotein A‐I (apoA‐I) mimetic peptide, possesses distinctly anti‐atherogenic effects. However, the biological functions and mechanisms of D‐4F on the hyperplasia of vascular smooth muscle cells (VSMCs) remain unclear. This study aimed to determine its roles in the proliferation and migration of VSMCs. In vitro, D‐4F inhibited VSMC proliferation and migration induced by ox‐LDL in a dose‐dependent manner. D‐4F up‐regulated heme oxygenase‐1 (HO‐1) expression in VSMCs, and the PI3K/Akt/AMP‐activated protein kinase (AMPK) pathway was involved in these processes. HO‐1 down‐regulation with siRNA or inhibition with zinc protoporphyrin (Znpp) impaired the protective effects of D‐4F on the oxidative stress and the proliferation and migration of VSMCs. Moreover, down‐regulation of ATP‐binding cassette transporter A1 (ABCA1) abolished the activation of Akt and AMPK, the up‐regulation of HO‐1 and the anti‐oxidative effects of D‐4F. In vivo, D‐4F restrained neointimal formation and oxidative stress of carotid arteries in balloon‐injured Sprague Dawley rats. And inhibition of HO‐1 with Znpp decreased the inhibitory effects of D‐4F on neointimal formation and ROS production in arteries. In conclusion, D‐4F inhibited VSMC proliferation and migration in vitro and neointimal formation in vivo through HO‐1 up‐regulation, which provided a novel prophylactic and therapeutic strategy for anti‐restenosis of arteries.     

Reduced C/EBPβ‐LIP translation improves metabolic health

Inactivation of target of rapamycin complex 1 (TORC1) signaling is considered important for the beneficial effects of caloric restriction (CR) on metabolism and health span. It is however not fully elucidated which cellular processes downstream of TORC1 are the main regulators of metabolic health. In this issue of EMBO Reports, Zidek et al 1 describe that inhibition of mammalian TORC1 (mTORC1) leads to decreased translation of CCAAT/enhancer‐binding protein β (C/EBPβ)‐liver inhibitory protein (LIP). Moreover, loss of C/EBPβ‐LIP in mice improves metabolic health, similar to the effects of CR. Zidek et al 1 thus report that reduced C/EBPβ‐LIP translation is a novel mTORC1‐regulated process that could play a major role in mediating the beneficial metabolic effects of caloric restriction.     

The methylation of C/EBP β gene promoter and regulated by GATA-2 protein

Mammalian genomes are punctuated by DNA sequences containing an atypically high frequency of CpG sites (CpG islands; CGIs) that are associated with the majority of annotated gene promoters. Methylated C bases of CpG sites inhibit the expression of downstream genes. During the differentiation of 3T3-L1 preadipocytes, the CCAAT/enhancer-binding protein (C/EBP) β gene plays an important role. We studied the CpG island methylation status of the C/EBP β promoter and its relationship with the GATA-2 protein. We used computer analysis to determine that the C/EBP β promoter sequence is rich in CGIs, and observed that two of seven methylated C bases were demethylated during the preadipocyte differentiation using bisulfite sequencing PCR (BSP). This corresponded with the onset of notable C/EBP β gene expression. Immunofluorescence and molecular docking showed that the GATA-2 protein binds the C/EBP β promoter in front of the first demethylated CpG site. We also found that expression of GATA-2 and C/EBP β proteins is negatively correlated. These results indicate that the methylated C bases in the C/EBP β promoter relate to expression of the C/EBP β gene, and that its demethylation is linked with GATA-2 protein association.