ABSTRACT:Accumulation of lipid within non-adipose tissues can induce inflammation by promoting macrophage infiltration and activation. Oxidized lipoproteins (oxLDL) have been known to induce cellular dysfunction in resident macrophages through pro-inflammatory and pro-apoptotic properties. However research into the pro-inflammatory and pro-apoptotic effects of oxLDL in alternatively activated (M2) macrophages has been relatively sparse. In this study, the proinflammatory and pro-apoptotic effects of oxLDL (at concentrations of 1 and 40µg/ml) were investigated in M2 macrophages. Incubation of M2 macrophage for 24 hours with 1 or 40µg/ml oxLDL induced pro-inflammatory molecules (MCP-1 and IL-6) production. Induction of cell death via apoptosis was observed after 24 hours incubation with 1 or 40µg/ml, but statistical significant induction was only observed with 40µg/ml oxLDL. Taking into consideration that M2 macrophages have been proposed as an appropriate target for type 2 diabetes, these findings may be of use in enhancing our knowledge of the adverse effects of oxLDL, particularly in the context of obesity, type 2 diabetes and the metabolic syndrome.

Peroxisome Proliferator-Activated Receptor gamma (PPARγ) is a ligand-activated transcription factor which belongs to the nuclear hormone superfamily and has multiple pharmacological ligands called Thiazolidinediones (TZDs). TZDs are a class of drugs used in the treatment of type 2 diabetic patients. Rosiglitazone is one such TZD, and is used clinically to treat type 2 diabetes. In this study, the effect of Rosiglitazone on cell growth and apoptosis in cultured monocytic monomac 6 (MM6) cells was investigated. Over a 14 day period, MM6 cells were cultured in vitro and treated with 1µM and 10µM Rosiglitazone. Cell viability and proliferation were evaluated by Haemocytometer cell count and MTS assay respectively. Turbidity due to cell density was assessed spectrophotometrically. Apoptosis was determined by Caspase-Glo 3/7 assay. Expression of the endoplasmic reticulum (ER) stress-inducible protein sarco-endoplasmic reticulum Ca 2+ ATPase-2b (SERCA2b) was determined by Western blot. Neither 1µM nor 10µM Rosiglitazone exerted statistically significant inhibitory effects on cell proliferation, turbidity due to cell density, or cell viability (p > 0.05 in all cases). In contrast, Rosiglitazone induced increased apoptosis, but a significant difference was only observed in 10µM-treated cells compared with control cells (3.04 ± 0.52 control; p < 0.05) while 1µM-treated cells showed a non-significant increase (1.50 ± 0.06 control; p > 0.05). Meanwhile the expression of SERCA2b was up-regulated significantly in cells treated for >4hrs (e.g 2.45 ± 0.06 control at 24 hrs; p < 0.05) with 10µM Rosiglitazone. It was concluded that high doses (10µM) of Rosiglitazone up-regulate SERCA2b expression and induce apoptosis of MM6 cells by activating an ER stress response via a PPARγ-independent mechanism. The therapeutic relevance of these observations is a matter for further investigations.

168ABSTRACT: Obesity and associated disorders such as Type-2 Diabetes (T2D) and atherosclerosis are associated with elevated levels of circulating oxidized low-density lipoprotein (oxLDL). High levels of oxLDL lead to cell dysfunction and apoptosis, a phenomenon known as lipotoxicity. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response (UPR), which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. In the present study the lipotoxic effect of oxLDL was investigated on a monocyte/macrophage cell lines. The results demonstrate that oxLDL could induce ER stress and activation of the UPR pathway in mnocyte/macrophage cell lines as evident of the activation/up-regulation of ER stress/UPR genes. Cholesterol does not seem to exert effects in intact cells in our experiments; in contrast oxLDL did induce ER stress and UPR. In microsomal fractions, cholesterol but not oxLDL inhibit the ER Ca 2+ -ATPase activity. Gene expression analysis showed that macrophages express high levels of the oxLDL scavenger receptor CD36, than monocytes and oxLDL induced macrophage apoptosis via caspase-3/7 activation. The observations that oxLDL can induce UPRs in macrophages, and that cholesterol inhibit ER Ca 2+ -ATPase activity, suggest that cholesterol may be the oxLDL component responsible for macrophage lipotoxic ER stress effects as seen in obesity. As disrupted cellular Ca 2+ homeostasis/ER stress may be linked to macrophage lipotoxicity this data may enhance our understanding of the diverse effects of oxLDL, particularly in the context of obesity, type 2 diabetes and metabolic syndrome.