Adipose Tissue Inflammation And Expression Of Lipid Droplet Associated Protein In Rat With Chronic Renal Failure

BackgroundDuring the past decade much knowledge has been gained on the metabolic and hormonal functions of adipose tissue. The adipose tissue organ, which can average from 20-30% of total body weight in adult humans, serve as a primary energy storage depot, as well as critical for maintaining proper body energy balance in higher animals. Prolonged fasting increases the cleavage triglycerides (TAG) and release of fatty acids (FA) and glycerol for provision of FA as an energy source, which is fat cell lipolysis. Obesity, cachexia, metabolicsyndrome and other condition in which lipid energy metabolism is important increase the rate of adipose tissue lipolysis and the release of FFA. High plasma FFA lead to increased import of FFAs into non-adipose tissues, particularly in the liver and skeletal muscles, is highly correlated with the development of insulin resistance and type 2 diabetes, which are important risk factors for arteriosclerosis and cardiovascular diseases.Chronic renal failure (CRF) is a kind of chronic infammation wasting disease, which is characterized by loss of adipose tissue and abnormal of lipid stored and mobilation. Many studies have shown that increased in vivo lipolysis is a key factor behind adipose cachexia in weight-losing cancer patients. Indeed, in privious study, we have proved that viscera and subcutaneous adipose tissue in CRF rat increase the rate of lipolysis, which include the increase of serum glycerol and the release of the glycerol in primary adipocyte or/and adipose tissue. When in stimulated condition, the rate of lipolysis in adipose tissue is significantly increase, which suggest that the response to hormone of CRF rat is more insensitive than sham rat. Although the increase of lipolysis in CRF maybe closely correlated with the development of insulin resistance and atherosclerosis, the mechanisms how to increase the lipolysis in CRF are not fully understood. In our study, HSL and ATGL activation are increased in viscera adipose tissue, even more, HSL and ATGL in subcutaneous adipose tissue are not only increasing the activity but also the high expression of the emzyse, which could explain increased lipolysis in CRF. ATGL and HSL is the key enzyme in lipolysis, which constitute 95% of the total lipoyme. The main role of ATGL seems to be to catalyse triglyceride to diglyceride. HSL catalyses the hydrolysis of triglycerides to diglycerides and FA. It can be phosphorylated on at least five serine residues, of which four are associated with lipolytic activation. Administration of a specific inhibitor of HSL diminishes the plasma level of FFAs in mice, rats and dogs, suggesting a greater role for HSL in basal lipolysis in vivo.Adipose tissue is no longer viewed as a passive reservoir of energy but has been shown to be a very active metabolic organ. Obesity is accompanied by an increased local infl ammatory response in adipose tissue, as measured by immunocompetent cell infi ltration, that in turn leads to increased production of proinflammatory factors such as interleukin-6(IL-6), tumor necrosis factor-a(TNF-a), monocyte chemoattractant protein-1 (MCP-1), and others. TNF-a, produced by adipocytes and by macrophages, stimulates lipolysis. TNF-a Binds its membrane receptor TNFR1, and activates the downstream MAP kinases p44/42 and JNK. Stimulation of lipolysis by TNF-a is slow prolonged and requires neither HSL nor ATGL. It confers a "dedifferentiated" expression pattern to adipocytes, decreasing the total perilipin level and increasing the fraction of perilipin that is phosphorylated. IL-6 is a multifunctional cytokine, adipose tissue is a major site of IL-6 secretion. Furthermore, plasma concentrations of IL-6 increase with obesity. Recent studies have shown that plasma IL-6 levels are closely related to adipocyte lipolysis. For example, in patients with type 2 diabetes, circulating IL-6 and FFA levels are significantly elevated, suggesting that IL-6 may act as an important inducer of lipolysis. In addition, after human breast adipocytes and 3T3-L1 adipocytes were cultured with IL-6 for 24 h, lipolysis showed a significant increase. Taken together, these studies demonstrated that high dose of IL-6 could stimulate lipolysis. Increased production of lipolytic factors from adipose tissue such as IL-6 and TNFa could explain increased lipolysis in cancer cachexia. Nuclear factor-KB (NF-κB), a primary regulator of inflammatory responses, plays a critical role in a variety of physiological and pathologic processes, has attracted interest as a new target for the treatment of inflammatory disease. Under basal conditions, NFκB exists in a complex with inhibitor of NFκB (IκB) in the cytoplasm. Upon activation of upstream inflammatory pathways, IκB is phosphorylated by the IκB kinase (IKK) complex, ubiquitinated and degraded, allowing NFκB to translocate into the nucleus and bind its target genes.Fat, mostly in the form of triglyceride, is stored in the lipid droplets in adipoc-ytes. Fat droplets, which constitute 95% of the total adipocyte volume, are compos-ed mainly of triglycerides. Recent analyses have revealed that a specific set of proteins called the PAT family, which are located on the lipid droplet surfaces, have central roles in lipid storage and breakdown. These proteins include PerilipinA, adipose differentiation-related protein (ADRP) and TIP47 (Tail-Interacting Protein of 47 kDa). These lipid droplets are coated with perilipins, the most abundant lipid droplet-associated proteins in adipocytes. PerilipinA is thought to function as a critical regulator of basal (nonstimulated) adipocyte lipolysis. the human immunodeficiency virus protease inhibitor, nelfinavir, are mediated by decreased perilipin protein content to increase the adipocite lipolytic action. The physiological role of perilipin as an inhib-itor of basal lipolytic rate was confirmed in perilipin knock-out mice, which exhibit a lean phenotype attributed to elevated basal lipolysis. Studies conducted in cell culture models and perilipin null mice reveal that perilipins play a dual role in the regulation of lipolysis in adipocytes. Under fed conditions, when circulating insulin promotes triacylglycerol storage in adipocytes, perilipin A forms a barrier at the surfaces of lipid droplets to restrict the access of cytosolic lipases to the lipid droplet. Once phosphorylated, however, by protein kinase A and extracellular regulated kinase1/2 (ERK1/2) upon stimulation by catecholamines, perilipin allows or even recruits lipases to access lipid droplets, and hence cause active lipolysis. PKA and ERK1/2 in viscera and subcutaneous adipose tissue are significantly activated. ADRP is another major PAT protein with a striking homology to perilipin in the N-terminus. Its expression is highly related to the amount of neutral lipid in the cell. Overexpression of ADRP has been associated with expansion of lipid droplet pools and increased cellular TAG mass in multiple studies. As was shown previously for perilipin, the effect of ADRP on cellular TAG and lipid droplets appears to have been due at least in part to reduced TAG catabolism. ADRP-null mice show a drastic reduction of LDs and lipid esters in the liver. So we can conclude that the downregulation of ADRP can increase the lipolysis activation. TIP47 shows significant similarity to ADRP both in amino acid sequence and three-dimen-sional structure. Knockdown of both TIP47 and ADRP was associated with lipid droplets that were much larger in size and fewer in number.Lipolysis is a potential therapeutic target for obesity, and other conditions in which lipid energy metabolism is important. Although, we have proved indeed that the lipolysis of adipose tissue in CRF rat is increased, the potential mechanism of how to result in this phenomenon is not understood. We therefore tested whether inflammation is associated with CRF and the expression of lipid droplet associated proteins in subnephrectomized (5/6Nx) rats, an established model of chronic renal failure, further suggesting how to regulate the signal pathway in adipose tissue inflammation. The aim is to investigate the other potential mechanism.Methods1. Animal model preparationsSD male rat were maintained on a 12/12-h light/dark cycle at 22℃and fed ad libitum a standard laboratory chow for a 1 week acclimation period.Subsequently, the animals were randomly assigned to two groups:sham (n=8), chronic renal failure (n=12), Surgical five-sixth nephrectomy (5/6Nx) and sham operations in rats were performed。Briefly, under pentobarbital anaesthesia, two-thirds of the left kidney was removed in the first stage of the procedure, and a week later the right kidney was totally excised (totally 5/6Nx). Control animals were sham-operated with only decapsulation of the kidney.2. The specimen collection and processingThe rats were killed by decapitation. Blood was allowed to clot, and the serum was stored frozen at-80℃until processed. The following WAT pads were rapidly isolated and completely dissected:intestine-related mesenteric WAT, perigonadal (epididymal), retroperitoneal cordons, and the subcutaneous inguinal fat pads. The samples were blotted and carefully cleaned of extraneous material (epididymis, pancreas, dermis), weighed, frozen in liquid nitrogen and kept at-80℃; this was done in the shortest time possible. The rest of subcutaneous WAT was later dissected and weighed.3. Blood sample and tissue were collected and used for biochemical measurements or histopathological studies. Plasma creatinine(Cr), BUN were determined by an auto biochemical analyzer. The adipose homogenate perilipinA, ADRP,TIP47,HNE,IKKa/pwere measured use western blotting. Plasma IL-1β,IL-6. TNF-aand MCP-1 were determined by an ELISA kit. The deposition of ED-1, MAC-1 in adipose tissue was detected by immunohistological staining. The infiltration of inflammatory cell was detected by hematoxylin-eosin staining. Real time-PCR was used to detect the present of cytokine and macrophages related proteins mRNA in the adipose tissue.Results1. The estabilish of the chronic renal failure modelBlood CR and BUN were higher significantly in chronic renal failure rats versus sham.2. The upregulation of cytokine TNF-α,IL-lβ,IL-6 and MCP-1 in blood were higher in chronic renal failure rats versus sham.3. The inflammation in adipose tissueThe infiltration of inflammation cell were higher in chronic renal failure rat versus sham as well as the expression of cytokine (TNFα,IL-1β,IL-6)mRNA were increased in chronic failure rat then sham.ED-1 and MAC-1 infiltration in adipose tissue:The results showed more recruitment of immunohistochemical ED-1 positive cells and MAC-1 positeve cells in renal failure rats than sham, plus the level of macrophage related proteins (ED-1,MAC-1,MIP-1α,PU.1 and MCP-1) mRNA were increased in chronic failure rat then sham.4. The expression of IKKα/pin adipose tissueThe active of signal pathway IKKα/βwere studied uesing western blotting, the totle IKKβprotein were increased in chronic renal failure rat than sham, AND the expression of p-IKKα/βwas significantly increased compared with that from sham rats. However, the totle protein IKKαwas not changed.5. Estimation of adipose tissue oxidative stress.To examine the activity of oxidative stess in chronic renal failure adipose tissue, we evaluated 4-HNE and H2O2 by western blotting and ELISA. The expression of 4-HNE and the release of H2O2 from chronic renal failure rats was significanyly increased compared with that from sham rats6. The expression of lipid droplet associated protein in adipose tissueWe investigate the level of perilipinA,ADRP and TIP47 in chronic renal failure use western blotting, the expression of lipid droplet associated protein were down-regulated in chronic renal failure.ConclusionsInflammative process and lipid droplet associated protein play an important role in the mechanism of chronic renal failue lipolysis. This research has confirmed oxidative stress and macrophages was infiltration in adipose tissue. We has studide IKKs signal pathway was included in the lipolysis.