Diabetes has been shown to accelerate atherosclerosis. However, it is currently unknown whether proatherogenic processes, such as the formation of oxidized LDL or foam cells, exacerbate the existing state of insulin resistance. In this study, we studied the effect of high doses of insulin and glucose on foam cell formation and cytokine production by macrophages. We found that incubation of MDM with oxidized types of LDL under hyperinsulinemic hyperglycemic conditions led to enhanced secretion of the inflammatory cytokines IL-lβ, IL-6 and IL-12, and the 5-lipoxygenase product 5-HETE. Our data also suggest that foam cell formation in the presence of pathophysiological concentrations of both insulin and glucose in vivo leads to the modification of the insulin signaling cascade.
Insulin is a key hormone regulating glucose and lipid metabolism [33, 34]. Upon binding insulin, the insulin receptor is activated and subsequently phosphorylates insulin-receptor substrates (IRS) and substrate protein Shc on tyrosine residues. The interaction of tyrosine phosphorylated IRS with the regulatory subunit(s) of phosphatidylinositol 3' kinase p85 activates the catalytic subunit(s) p110. The consequent production of phosphatydylinositol-3-phosphates leads to the phosphorylation and/or activation of downstream targets, including PKB/Akt . mmLDL-induced Akt phosphorylation has been previously described in mouse macrophages .
Our findings, specifically the increased expression of insulin receptor and IRS-2 genes in the presence of OxLDL and HIHG conditions, are consistent with increased insulin signaling. Indeed we found that Akt phosphorylation in human macrophages was upregulated (Figure 5A lower and 5B). It is possible that the effects of high insulin are mediated through the IGF-1 receptor , since we could mimic both an increase in INS-R expression (Figure 3) and LDL-induced Akt phosphorylation (Figure 5B) using high amounts of IGF-1. However, effects of high IGF-1 may also be mediated by the insulin receptors. Thus, it is still possible that our findings with oxidized LDL are mediated through the insulin and not the IGF-1 receptor.
Insulin affects the activity of genes that have insulin-responsive regions in their promoters. Glucose-6-phosphate dehydrogenase (G6PD) is one such example . The recently discovered Insulin-Induced genes (Insig) may be responsible for other insulin mediated effects [37, 38]. Insulin-Induced genes 1 and 2 (Insig-1 and Insig-2) encode proteins of the endoplasmic reticulum that block proteolytic activation of sterol regulatory element-binding proteins (SREBPs). SREBPs are transcription factors that activate the synthesis of cholesterol and fatty acids in the liver and other cells . Insig genes are down-regulated by insulin and the fall in Insig expression allows SREBPs to be processed, thereby allowing insulin to stimulate fatty acid synthesis . We detected significant down-regulation of Insig-1 gene expression by OxLDL, while mmLDL had a much weaker effect (Table 1). Thus, OxLDL may potentiate the chronic effect of insulin, behaving as an "insulin sensitizer". However, the overall effect of modified LDL on the insulin signaling cascade represents a sum of different effects, including the upregulation of INS-R and IRS2 genes, and the downregulation of Insig-1 and PIKSCD. The high correlation present between these four genes (Table 2) indicates that modified LDL may induce a "compensatory shift" in the expression of certain components of the insulin signaling cascade in macrophages cultivated under HIHG conditions.
mmLDL increased the expression of the insulin-regulated aminopeptidase protein (Figure 5A, upper row). IRAP is a member of the family of zinc-dependent membrane aminopeptidases. In fat and muscle cells IRAP localizes in an intracellular compartment under basal conditions and redistributes to the cell surface in response to insulin . Its precise role in insulin action remains unknown . The consequence of increased protein expression of IRAP in insulin signaling in foam cells has to be determined.
The products of 5-lipoxygenase, 5-hydro(pero)xyeicosatetraenoic acid, and the leukotrienes, especially leukotriene B4, may also be considered factors mediating the effects of foam cell formation on the insulin signaling cascade. 5-lipoxygenase is upregulated during foam cell formation , and its products are believed to play a significant role in inflammation [44, 45]. The level of 5-HETE in the cell supernatants was significantly upregulated by OxLDL and positively correlated with the presence of insulin and glucose in cultivation medium (Figure 6).
Insulin resistance and type 2 diabetes are associated with systemic inflammation, possibly through intermediates, such as cytokines and other factors [46, 47]. Circulating levels of inflammatory cytokines such as IL-6 and TNFα are increased in type 2 diabetes . Inflammatory cytokines can activate protein kinases that phosphorylate IRS on serine residues, leading to impaired insulin signaling . Despite some controversy regarding the potential role of IL-6 in insulin resistance [50–52], IL-6 has been shown to inhibit insulin signaling and insulin action in isolated hepatocytes . Additionally, IL-6 depletion selectively improves hepatic insulin action in obesity . Moreover, IL-6 leads to insulin resistance in vivo when chronically administered to mice at levels that are similar to those found in obese individuals . Hypersecretion of IL-6 and TNFα may exert major stimulatory effects on the synthesis of acute-phase proteins such as PAI-1, which is also related to insulin resistance . Elevated blood levels of IL-lβ and IL-6 increase the risk of type 2 diabetes , and lack of IL-lβ decreases the severity of atherosclerosis in ApoE-deficient mice on the C57B1/6 background , a model that develops insulin resistance when fed a Western diet . IL-12 is known to favor differentiation of naive T cells along the T-helper (Thl) pathway and play a significant role in atherogenesis . We found OxLDL significantly increased the biosynthesis of IL-lβ and IL-12 by macrophages incubated under HIHG conditions, while mmLDL significantly upregulated IL-6. Increased synthesis of these proinflammatory cytokines in turn can promote insulin resistance in other surrounding cells and tissues.
Dandona et al. noted that increased concentrations of TNFα and IL-6 in type 2 diabetes may modulate insulin action by suppressing insulin signal transduction and this may "shift" the effects of insulin to favor a more proinflammatory state . We hypothesize that oxidized forms of LDL can also "shift" or modulate insulin signaling, at least in macrophages, to a more proinflammatory state. Modified kinds of LDL may bring about effects similar to "selective insulin resistance"  or insulin resistance on the level of separate cells/tissues. This could play a role in pathogenesis of "total insulin resistance", thus exacerbating the development of type 2 diabetes. Liang et al. have recently observed that increased CD36 protein expression on mouse macrophages may be connected with defective insulin signaling in these cells as estimated by reduced expression and signaling of insulin receptors . Human macrophages treated with OxLDL in vitro indeed show increased surface expression of CD36 [61, 62]. Here we report that this change could be associated with an increase in mRNA for insulin receptor and modified insulin signaling. Thus, our results suggest that not exclusively "defective", but rather "modified" or "shifted" macrophage insulin signaling may cause a predisposition to foam cell formation and atherosclerosis in insulin-resistant states.
Our data suggest that the presence or formation of modified/oxidized/aggregated LDL in insulin-resistant patients, who have increased blood insulin and glucose, may exacerbate existing insulin resistance and contribute to the development and progression of type 2 diabetes. Although the treatment of human macrophages with both minimally oxidized LDL or extensively oxidized LDL leads to cholesterol accumulation and foam cell formation in vitro, the effects of mmLDL on the insulin signaling cascade in macrophages appear to be less profound or even opposite to those of OxLDL. Such OxLDL-specific effects suggest involvement of the scavenger receptors in a number of downstream events. Further investigations are necessary to evaluate the differential roles of scavenger receptors, including CD36, and CD14/Toll-like receptor 4 in mediating effects of OxLDL and mmLDL, respectively.