Role Of LOX-1in Death, Inflammation And Adipogenesis Of Preadipocytes Induced By Palmitic Acid And The Underlying Mechanisms

Part1The correlation between LOX-1and obesity in C57BL/6J obese miceAbstract[Background] Obesity is a common disease defined as a state of excess body fat accumulation which stems from energy imbalance. Obesity is strongly associated with a wide variety of adverse health effects such as diabetes mellitus, cardiovascular disease, renal disease characterized by microalbuminuria, fatty liver disease, hormonal disturbances and certain forms of cancer. Obesity per se is a state of chronic inflammation, as indicated by increased plasma concentrations of inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-6(IL-6), C-reactive protein (CRP).Lectin-like oxidized low-density lipoprotein (ox-LDL) receptor-1(LOX-1)is a lectin-like receptor for ox-LDL.LOX-1is expressed in highly vascularized tissues, such as placenta and lung, and also expressed in renal tissue and adipose tissue.As for cells types, LOX-1is expressed not only in endothelial cells, but also in adipocytes, macrophages, vascular smooth muscle cells and platelets.LOX-1may be a key factor in hypertension, diabetes mellitus and hyperlipidemia, the most important risk factors for atherosclerosis.There is mounting evidence that LOX-1plays critical roles in the development of atherosclerosis and related disorders, and is up-regulated in hypertensive, dyslipidemic, and diabetic animals and humans.Recently, the association of LOX-1with obesity has raised the attention of investigators.It has been noticed that plasma levels of sLOX-1in obese women are elevated compared to lean and overweight women. Notably, LOX-1expression was found to be increased in the adipose tissue of obese compared with lean mice.The expression levels of LOX-1in the adipose tissue are closely correlated with MCP-1,IL-6, and macrophage inflammatory protein-1α. However, the roles of LOX-1and the underlying mechanisms in the pathological process of obesity remain unclear. We therefore aimed to explore the roles of LOX-1in obesity.[Method]1.Obese mice model:C57BL/6J male mice(15-17g) were fed normal or high-fat diet for16weeks.2.The body weight, Lee’s index, BMI was measured and calculated. The serum triglyeride (TG), total cholesterol (TC), high densitv lipoprotein (HDL), low-density lipoprotein (LDL) and free fatty acids (FFA) were detected.3.The mRNA and protein expression of LOX-1in visceral adipose was detected by real-time PCR and immunohistochemisty.4.TNF-a, IL-6and MCP-1mRNA levels in visceral adipose were measured by real-time PCR.5. The adipogenesis key genes (C/EBP β,PPAR γ and aP-2) mRNA expression was detected by real-time PCR.6. TK and RIPK1mRNA levels was also measured by real-time PCR.[Results]1.The body weight, Lee’s index and BMI were increased in obese mice.2.The serum TG, TC,LDL and FFA were significantly higher in obese mice than control.3.The mRNA and protein expression of LOX-1in visceral adipose was up-regulated in obese mice.4. Inflammatory cytokines (TNF-a, IL-6and MCP-1)mRNA levels were up-regulated in obese mice.5.The adipogenesis key genes (C/EBP β, PPARy and aP-2) mRNA expression was up-regulated in obese mice,6. Necrosis related gene-RIPK1mRNA level was also up-regulated.[Conclusion]LOX-1is correlated with obesity, and it may mediate the cell death, inflammation and adipogenesis in visceral adipose. Part2LOX-1mediates apoptosis and necrosis in mouse3T3-L1preadipocytes induced by palmitic acidAbstract[Background] Obesity is associated with chronic inflammation and adipose tissue remodeling, characterized with macrophage accumulation, adipocytes and preadipocytes apoptosis. Apoptosis has also been noted in adipose tissue of obese mice and humans.It is documented that adipocyte death increases in obese (db/db) mice (30fold) and humans, and exhibits ultrastructural features of necrosis but not apoptosis. The committed preadipocytes number was significantly reduced in obese women(17%) compared to the lean women (30%).Moreover, the preadipocytes of obese women exhibited reduced differentiation and were more susceptible to apoptotic stimuli than preadipocytes isolated from adipose tissue of lean women. Meanwhile, preadipocytes susceptibility to cell death is routinely assessed in primary culture.Apoptosis, the other key preadipocyte fate is related to adipose tissue remodeling. However, a relative deficit in preadipocyte number or their capacity to differentiate into adipocytes is associated with the development of dysfunctional hypertrophied adipocytes, leading to low-grade inflammation and insulin resistance in obese humans.Elevated levels of free fatty acids in obesity may contribute to the development of atherosclerosis and insulin resistance. Apoptosis or necrosis is pertinent to adipose tissue remodeling and inflammation.Lectin-like oxidized LDL receptor (LOX-1)plays an important role in apoptosis via activation of Bax/Bcl-2pathway and reactive oxygen species (ROS) signal pathway. We have now investigated the role of LOX-1on palmitic acid induced preadipocytes death and the intracellular signaling pathways.[Method] Onset of apoptosis and necrosis is determined by characteristic morphological features, cell viability, cell proliferation assay as well as Hoechst33258and annexin V-PI staining assay. Then we detected the mRNA expression of TK and RIPK1and the protein expression of Bax/Bcl-2and cytochrome c in3T3-L1preadipocytes induced by different concentrations of palmitic acid. The LOX-1mRNA expression level by palmitic acid at different times was determined by real-time PCR.We next inhibited the expression of LOX-1by LOX-1-neutralizing antibody and LOX-1siRNA.Cell apoptosis and necrosis was measured by Annexin V-PI staining assay and the genes expression was also detected ditto. The generation of ROS was detected after inhibition of LOX-1.At last the effects of anti-oxidants on cell death were also determined to examine where the ROS derived from.[Results] We observed that exposure of3T3-L1preadipocytes to palmitic acid strongly stimulated the onset of apoptosis and necrosis, however, palmitic acid-induced apoptosis and necrosis was inhibited by anti-LOX-1antibody and by LOX-1siRNA transfection. Furthermore, the down-regulation of LOX-1significantly decreased palmitic acid-induced ROS generation. In addition, Pre-and co-treatment with MnTBAP, an inhibitor of mitochondrial oxidative stress, protected cells from palmitic acid-induced cell death.[Conclusion] The study indicated that palmitic acid induces death via LOX-1-dependent ROS generation in3T3-L1preadipocytes. Part3The role of LOX-1in palmitic acid-induced mouse3T3-L1preadipocytes inflammation and the underlying mechanismsAbstract[Background] It is well established that metabolic disorders, including dyslipidemias, obesity, and diabetes,are often accompanied by elevated serum concentrations of fatty acids.Because these diseases are also characterized by marked increases in serum concentrations of inflammatory mediators, including TNF-a, IL-6, CRP, they are commonly referred to as inflammatory states.Over the past few years, it has become increasingly clear that inflammation could represent a mechanism responsible for cardiovascular diseases and that increased circulating inflammation markers could be predictive of cardiovascular events, as recently suggested. Indeed, both IL-6and TNF-a can induce insulin resistance in adipose cells by inhibiting insulin signaling. Thus, the chronological appearance of these inflammatory molecules before the development of insulin resistance, as well as their known ability to promote insulin resistance and other complications of obesity, strongly suggests adipose tissue inflammation protagonist in the development of obesity-related complications.Clearly, several inflammatory mediators are implicated in the development of obesity and the mechanisms responsible for the development of the chronic diseases associated with obesity.LOX-1interplays with inflammatory signaling pathway, as it activates PKC,resulting in a sequence of signals including MAPKs, NF-κB and AP-1.The role of LOX-1in the inflammation of preadipocytes remains unclear. We therefore explored the roles of LOX-1in palmitic acid-induced inflammation model of preadipocytes.[Method] We detected mRNA expression and the levels in culture medium of TNF-a, IL-6, and MCP-1in3T3-L1preadipocytes induced by different concentrations of palmitic acid at different time points.The LOX-1mRNA and protein expression induced by palmitic acid at different times was determined by real-time PCR or Western-blot.We next inhibited the expression of LOX-1by LOX-1-neutralizing antibody or LOX-1siRNA. The mRNA expression and the levels in culture medium of TNF-α,IL-6,and MCP-1were also determined. In addition, we analyzed the phosphorylation of AMPK, the protein expression of PP2A, the nuclear translocation and intracellular level of NF-κB p65subunit.At last, we used AMPK activator or inhibitor and determined the expression of TNF-α, IL-6,and MCP-1,the nuclear translocation and intracellular level of NF-κB p65subunit, and the expression of LOX-1.[Results] We observed the exposure of3T3-L1preadipocytes to palmitic acid strongly stimulated the mRNA expression and the levels in culture medium of TNF-α,IL-6,and MCP-1.We also found the LOX-1expression was up-regulated.However, the increases in inflammatory cytokines were inhibited by anti-LOX-1antibody and by LOX-1siRNA transfection.But the phosphorylation of AMPK and the protein expression of PP2A induced by palmitic acid were not affected.The NF-κB was activated by palmitic acid (translocation to nuclear),and the activation was inhibited after inhibition of LOX-1. AMPK activation or inhibition analyses suggested that AMPK activation prevented the inflammation in preadipocytes and inhibited the activation of NF-κB and LOX-1expression, and AMPK inhibition was just the reverse.[Conclusion] LOX-1mediates the inflammation in preadipocytes induced by palmitic acid via NF-κB pathway. The phosphorylation of AMPK is activated by palmitic acid, which may be a compensatory mechanism responsible for attenuating palmitic acid-induced preadipocyte oxidative injury via inhibition of LOX-1/NF-κB pathway. Part4LOX-1regulates palmitic acid-induced adipogenesis in3T3-L1adipocytes and the underlying mechanismsAbstract[Background] Individuals with adult-onset obesity in general exhibit increased adipocyte size, whereas individuals with early-onset obesity have both adipocyte hypertrophy and hyperplasia. Adipocytes are the main constituent of adipose tissue and play a vital role in control of whole body metabolism. Their primary function is to regulate energy balance by storing TG in periods of energy excess.Besides the classical function, adipocytes secrete numerous lipid and protein factors. Adipogenesis is the key factor to adipocyte hypertrophy through producing more lipids.Adipogensis is the differentiation of preadipocytes into mature lipid laden, insulin-responsive adipocytes.C/EBP protein family and PPAR-y are the key transcription factors in adipogenesis.Most of this food contains high amounts of saturated fatty acids (SFA).Fatty acids can induce the differentiation of preadipocytes. Recently it is reported that LOX-1may play an important role in adipogenesis.LOX-1deletion suppresses adipogenesis genes of C/EBP-β and PPAR-y. We therefore explored the roles of LOX-1in adipogenesis of preadipocytes induced by palmitic acid.[Method] Palmitic acid was added to3T3-L1preadipocytes to induce differentiation, then Oil Red O staining and determination of intracellular triglyceride to detect the adipogenesis in the process of differentiation. Real-time PCR and Western-blot were applied to measure the expression of C/EBP-β,PPAR-y, aP-2and Pref-1mRNA and protein levels. Meanwhile, we detected the expression of LOX-1.Next LOX-1was inhibited by siRNA, we analyzed the adipogenesis and the expression of C/EBP-β,PPAR-γ, aP-2and Pref-1by the methods mentioned above.[Results] Palmitic acid significantly increased the adipogenesis in3T3-L1preadipocytes and up-regulated the key transcription of C/EBP-β and PPAR-γ. The mRNA expression of aP-2was increased and pref-1was gradually decreased. The expression of LOX-1was up-regulated. After LOX-1inhibition by siRNA, the differentiation and adipogenesis of preadipocytes induced by palmitic acid was inhibited. Likewise, the expression of C/EBP-β, PPAR-γ, and aP-2was inhibited, and pref-1mRNA was increased.[Conclusion] LOX-1mediates adipogenesis of preadipocytes induced by palmitic acid via the regulation of C/EBP-β/PPAR-γ/aP-2and Pref-1.