Obesity and more particularly, high levels of low density lipoprotein (LDL) particles, together with low levels of HDL particles, are important risk factors leading to the development of T2D and the metabolic syndrome 
. The purpose of this study was to evaluate the impact of prolonged exposure to high levels of LDL-cholesterol on Cx36 expression since low Cx36 levels have been associated with reduced β-cell function and survival 
. The characterization of the ApoE−/− mice, which display drastically increased levels of circulating cholesterol 
, revealed that high plasma LDL concentrations are associated with increased levels of ICER-1, and concomitant decreased levels of Cx36 in pancreatic islets. Using purified human LDL particles, we found that oxidized LDL (oxLDL) particles specifically drive the overexpression of the repressor ICER-1/ICER-1γ, which in turn binds to the Cx36 promoter, leading to down-regulation of Cx36 transcripts and protein expression levels. We also show that oxLDL particles are more toxic than native LDL (nLDL) particles because defective storage and metabolism. We further demonstrate that Cx36 overexpression partially protects β-cells against oxLDL-induced apoptosis.
We previously showed that mice fed a high fat diet express increased levels of ICER-1 and reduced levels of Cx36 
. These mice had elevated levels of circulating FFA, but they were also slightly hyperglycemic and hypercholesterolemic (total cholesterol) 
. Here, we evaluated specifically the effect of cholesterol on Cx36 expression. The four months-old ApoE−/− mice used for this study had typically increased levels of circulating total cholesterol and no change in glycemia 
. These mice also displayed slightly increased circulating levels of FFA (2 fold), and we cannot exclude that these FFAs contribute to Cx36 down-regulation in ApoE−/− mice as previously shown upon a prolonged high fat diet 
. Despite the dramatic hypercholesterolemia, ICER-1 and Cx36 expression were not considerably changed in those mice. This is in accordance with our in vitro
data showing that normal LDL particles are not deleterious to Cx36 expression in β-cells. Of note, Cx36 protein levels as assessed in situ
by immunostaining were far more decreased in ApoE−/− animals compared to WT than at the mRNA level. This suggests that either Cx36 protein is further destabilized in ApoE−/− or reveals a biased assessment of Cx36 mRNA expression in freshly isolated islets due to the many artifacts (exposure to RNase, reducing agents, non linear RNA loss during islet and mRNA isolation…) and sampling bias (less than 1/3 of the existing islets can be isolated in mice as per the most efficient isolation procedures) which hinder analysis of gene expression from isolated islets.
The ApoE−/− mice have raised concerns owning to the very high “non-physiological” plasma cholesterol levels and the “quality” of lipoproteins 
, as cholesterol is carried mostly in large VLDL and chylomicron remnants 
, whereas pro-atherogenic particles are thought to be mostly small-dense oxidized/glycated LDL particles 
. Yet, the ApoE−/− mice have been extensively used to study atherosclerotic lesions because they rapidly develop large plaque throughout the length of the arterial tree 
, suggesting that a significant fraction of the circulating lipoprotein have atherogenic properties. In addition, and in contrast with the ApoE−/− mice originally described 
, our ApoE−/− mice also had increased circulating levels of HDL particles, which have been shown to protect against the deleterious effects of oxLDL particles 
. Altogether those limitations may account for the seemingly little impact of hypercholesterolemia on ICER-1 and Cx36 expression in vivo.
This study demonstrates that normal human LDL particles have no deleterious effects on β-cell survival whereas oxidized LDL particles have pro-apoptotic effects at fairly physiological concentration (2 mM), supporting the hypothesis that oxLDL particles greatly contribute to β-cell dysfunction and death in the pathophysiology of T2D. This is particularly important as small dense modified LDL particles are early markers of T2D 
. The mechanisms responsible for the detrimental impact of oxLDL particles on β-cell function and survival are still poorly understood. The fact that saturated FFA and oxLDL both induce ICER-1 overexpression 
and Cx36 down-regulation (
and this study) strongly suggest that a similar mechanism is responsible for the effects of the lipids in both forms. The deleterious impact of saturated FFAs such as palmitate is due to accumulation of fatty AcylCoA entering non-oxidative toxic pathways of fatty acids metabolism, such as de novo ceramide formation which trigger oxidative stress in the mitochondria 
. Our data indicate that nLDL particles stimulate lipid β-oxidation as assessed through Acc1 overexpression. On the other hand, oxLDL particles had only a marginal impact on ACC1 expression. Blocking lipid metabolism using the CPT1 inhibitor etomoxir 
rendered native LDL particles toxic and aggravated the pro-apoptotic impact of oxLDL. Conversely, the serine palmitoyltransferase inhibitor myriocin, which blocks the synthesis of ceramide, partially prevented oxLDL-induced apoptosis, ICER-1 overexpression and Cx36 downregulation, suggesting that ceramide production is instrumental in oxLDL-induced apoptosis. Altogether those data suggest that the toxic effect of oxLDL may partly be due to a defect in metabolism, leading to oxidative stress, ICER-1 overexpression, Cx36 down-regulation and apoptosis. Oil red O staining data also revealed that both nLDL and oxLDL particles lead to the formation of lipid droplets in β-cells. However, nLDL formed abundant big regular round shaped lipid droplets whereas oxLDL formed fewer and smaller lipid droplets, suggesting that oxLDL are not as well stored in the form of lipid droplets as nLDL in β-cells. These observations are confirmed at the molecular levels by lower levels of the known marker of lipid droplets Plin1 
in cell exposed to oxLDL as compared to nLDL particles. Altogether these data suggest that nLDL are not toxic at this concentration (2 mM) because they are partly β-oxidized and partly stored in the form of neutral harmless lipid droplets. On the other hand, oxLDL particles are toxic due to both defective storage and β-oxidation, which may both result in accumulation of toxic lipid metabolites such as ceramides generating oxidative stress both in the form of ROS/RNS and superoxide anions, similarly to what has been shown with palmitate 
Our oxidation protocol leads to the formation of mildly oxidized LDL 
. Therefore the oxLDL preparation probably still contains partially oxidized or native nLDL particles. Whether oxLDL particles themselves or nLDL particles remaining in the oxLDL preparation are responsible for the intermediate effect of oxLDL on β-oxidation or lipid droplets formation remains to be determined.
Our data indicate that oxLDL stimulate the production of ROS/RNS and superoxide species. ROS/RNS production can be averted by the antioxidant N-Acetyl cysteine (NAC), whereas superoxide production is not prevented. NAC partially protected cells against oxLDL toxicity, indicating that the ROS/RNS species are involved in oxLDL-induced apoptosis. We previously demonstrated that oxLDL-induced oxidative stress leads to ICER-1 overexpression 
. Furthermore, blocking ICER-1 overexpression using a siRNA strategy protects β-cell against oxLDL-induced apoptosis 
. However the exact mechanisms underlying the pro-apoptotic impact of ICER-1 is poorly understood. We recently showed that Cx36 protects mice against β-cell apoptosis induced by streptozotocin or alloxan, two models of induced Type1 Diabetes (T1D) 
. Here, we observed that Cx36 knock-down or knock-out sensitized β-cells to oxLDL particles and that Cx36 overexpression partially protected β-cells from oxLDL-induced apoptosis. We further show that Cx36 knock-down aggravated the production of ROS/RNS, but not superoxide species, suggesting that Cx36 is able to alleviate (“dilute”) the oxidative stress upon oxLDL exposure. Thus intercellular communication may provide protection against pro-apoptotic stresses involved in the pathophysiology of T2D. Together with our previous studies showing that prolonged exposure to glucose and/or saturated FFA lead to ICER-1 overexpression and Cx36 down-regulation in β-cells 
, this study underscores the importance of this particular mechanism in the pathophysiology of T2D. We further demonstrated that HDL counteracts the effect of oxLDL on Cx36 expression. This is in accordance with our previous finding that HDL particles prevent oxLDL-induced ICER-1 overexpression 
and other studies showing that HDL particles are potent antioxidants with strong anti-apoptotic properties in a variety of models 
Further studies are required to identify other ICER-1 target genes with anti-apoptotic properties in β-cells. Given the prominent role of Cx36 mediated intercellular communication in β-cell function 
and survival 
, we suggest that oxLDL-induced Cx36 down-regulation contributes to oxidative stress induced upon prolonged exposure to oxLDL, which is involved in β-cells dysfunction and apoptosis.