Inhibition of apolipoprotein A-I expression by TNF-alpha in HepG2 cells: requirement for c-jun.

Tumor necrosis factor alpha (TNF α) signals in part through the mitogen activated protein (MAP) kinase c-jun-N-terminal kinase (JNK). Activation of JNK has been shown to promote insulin resistance and dyslipidemia, including reductions in plasma high-density lipoprotein (HDL) and apolipoprotein A-I (apo A-I). To examine how TNF α-mediated JNK activation inhibits hepatic apo A-I production, the effects of c-jun activation on apo A-I gene expression were examined in HepG2 cells. Apo A-I gene expression and promoter activity were measured by Northern and Western blotting and transient transfection. Transient transfection and siRNA were used to specifically over-express or knockout c-jun, c-jun-N-terminal kinase-1 and -2 (JNK1 and JNK2, respectively) and mitogen-activated protein kinase-4 (MKK4). TNF α-treatment of HepG2 cells induced rapid phosphorylation of c-jun on serine 63. In cells treated with phorbol-12-myristate-13-acetate (PMA), apo A-I gene promoter activity was inhibited and apo A-I mRNA content and apo A-I protein secretion decreased. Likewise, over-expression of JNK1 and JNK2 inhibited apo A-I promoter activity. Over-expression of constitutively active MKK4, an upstream protein kinase that directly activates JNK, also inhibited apo A-I promoter activity, while over-expression of a dominant-negative MKK4 de-repressed apo A-I promoter activity in TNF α-treated cells. Inhibition of c-jun synthesis using siRNA but not a control siRNA prevented TNF α-mediated inhibition of apo A-I. These results suggest that the MKK4/JNK/c-jun signaling pathway mediates TNF α-dependent inhibition of apo A-I synthesis.

The effect of glucosamine on Serum HDL cholesterol and apolipoprotein AI levels in people with diabetes.

OBJECTIVE: Dietary and nutritional supplements are modulators of HDL cholesterol levels and production of apolipoprotein (apo) AI. Previously, in vitro treatment of hepatocyte cell lines with glucosamine increased apoAI production by stabilization of apoAI mRNA. The hypothesis is that the neutraceutical glucosamine, when given in conventional doses (1,500 mg/day) may increase apoAI and HDL cholesterol levels in subjects with diabetes and low HDL cholesterol. RESEARCH DESIGN AND METHODS: Twelve subjects (three men and nine women) with type 1 (n = 2) and type 2 (n = 10) diabetes, aged 55 +/- 12 years (mean +/- SD), who had low HDL cholesterol (1.03 +/- 0.20 mmol/l), were randomly assigned to a double-blind, placebo-controlled, cross-over trial of 500 mg glucosamine or placebo orally three times daily for 2 weeks, followed by a 4-week washout phase and a 2-week cross-over to the alternate therapy. RESULTS: Fasting serum glucose, fructosamine, and total cholesterol remained stable during the drug and placebo phases. Glucosamine had no significant effect after therapy on serum levels of HDL cholesterol (from baseline of 1.02 +/- 0.15 to 1.05 +/- 0.16 mmol/l compared with placebo from 1.04 +/- 0.21 to 1.06 +/- 0.16 mmol/l) nor in changes in apoAI levels (from baseline of 147 +/- 15 to 140 +/- 126 mg/dl with glucosamine and from 146 +/- 25 to 142 +/- 17 mg/dl with placebo). CONCLUSIONS: These observations suggest that glucosamine at commonly consumed doses does not have significant effects on glycemic control, lipid profile, or levels of apoAI in diabetic subjects after 2 weeks of supplementation.

Severe insulin resistance associated with subcutaneous amyloid deposition.

A 59-year-old man with type 1 diabetes mellitus presented with severe resistance to subcutaneously injected insulin. Histological analysis of the injection sites, demonstrated foreign body type granulomas surrounding areas of amyloidosis. It is suggested that the granulomas were the source of an insulin-degrading enzyme (IDE) which simultaneously degraded amyloidogenic precursors into localized amyloid deposits. These findings may add insight into the role of insulin-degrading enzymes in the etiology of subcutaneous insulin resistance syndromes.

Statins prevent dextrose-induced endothelial barrier dysfunction, possibly through inhibition of superoxide formation.

Statins may have favorable effects on endothelial barrier function, possibly through reduction of oxidative stress and modulation of expression of vasoactive proteins. The permeability of human umbilical endothelial cells in culture to a group of fluorescein isothiocyanate dextrans of different molecular weights were studied under various experimental conditions. Superoxide anion production was measured with an ethidium bromide fluorescence method. Cellular endothelin 1 mRNA and endothelin 1 in culture media were measured with Northern blots and enzyme immunoassays, respectively. Rosuvastatin (10 nmol/l) normalized the 500 mg/dl dextrose-induced permeability changes. Superoxide anion production induced by 500 mg/dl dextrose was inhibited by therapeutic concentrations of rosuvastatin or simvastatin (10 nmol/l), whereas the increased levels of cellular endothelin 1 mRNA and endothelin 1 in culture media was inhibited by supratherapeutic concentrations of statins (> or =0.1 micromol/l). In conclusion, 1) endothelial cell barrier dysfunction occurs in cells treated with high concentrations of dextrose, 2) statin treatment of endothelial cells normalizes barrier permeability, and 3) the favorable effects of statins may be attributed to the inhibition of the dextrose-induced increase in superoxide anions, whereas inhibition of endothelin expression was observed only at supratherapeutic concentrations.

Effect of thyroid hormone responsive protein (THRP) expression on PC12 cell survival.

The thyroid hormone responsive protein (THRP) is a novel gene product that remains responsive to thyroid hormone in the cerebral cortex of adult rats. The biological effects of THRP are currently unknown. Since thyroid hormones (TH) are known to cause cell death in primary neuronal cultures, the effect of exogenous THRP expression on PC12 cell viability was investigated. Co-transfection of the THRP expression plasmid with the selectable marker pSV2neo resulted in a lower number of surviving PC12 cells compared to transfection with pSV2neo and the empty vector, pSVL. Similar results were observed when PC12 cells were transfected with the plasmid pCMV. SPORT beta-gal with and without pSVL-THRP. However, expression of exogenous THRP in the colonic epithelial cell line Caco-2 and the glial cell line U251 had no effect on cell viability. Coexpression of THRP with either the wild-type (WT)-c-Abl or a kinase-defective mutant c-Abl (K290R) did not alter the cell viability changes induced by THRP alone. Under these experimental conditions the predominant form of cell death was necrosis as evidenced by in situ analyses, such as terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) and staining with membrane permeating and non-permeating nuclear dyes, Hoechst 33342 and propidium iodide respectively. In addition cell cycle arrest induced by THRP was demonstrated by reduced (3)H-thymidine incorporation into cellular DNA. The number of PC12 cells treated with 10(-7) M of l-3, 5, 3'-triiodothyronine (T(3)) was significantly reduced after the fourth day of culture. Treatment of the cells with T(3 )resulted in a dose dependent induction of THRP mRNA. It is concluded that: (1). THRP expression induces PC12 cell death; (2). under these experimental conditions the form of cell death is predominantly necrosis although cell cycle arrest may also occur; (3). the effect of THRP on cell viability is not modulated by c-Abl tyrosine kinase; and (4). the effect of T(3 )treatment on PC12 cell survival may be mediated by THRP.