Proteomic Profiling of Hepatic Endoplasmic Reticulum-associated Proteins in an Animal Model of Insulin Resistance and Metabolic Dyslipidemia

Abstract

Hepatic insulin resistance and lipoprotein overproduction are common features of the metabolic syndrome and insulin-resistant states. A fructose-fed, insulin-resistant hamster model was recently developed to investigate mechanisms linking the development of hepatic insulin resistance and overproduction of atherogenic lipoproteins. Here we report a systematic analysis of protein expression profiles in the endoplasmic reticulum (ER) fractions isolated from livers of fructose-fed hamsters with the intention of identifying new candidate proteins involved in hepatic complications of insulin resistance and lipoprotein dysregulation. We have profiled hepatic ER-associated proteins from chow-fed (control) and fructose-fed (insulin-resistant) hamsters using two-dimensional gel electrophoresis and mass spectrometry. A total of 26 large scale two-dimensional gels of hepatic ER were used to identify 34 differentially expressed hepatic ER protein spots observed to be at least 2-fold differentially expressed with fructose feeding and the onset of insulin resistance. Differentially expressed proteins were identified by matrix-assisted laser desorption ionization-quadrupole time of flight (MALDI-Q-TOF), MALDI-TOF-postsource decay, and database mining using ProteinProspector MS-fit and MS-tag or the PROWL ProFound search engine using a focused rodent or mammalian search. Hepatic ER proteins ER60, ERp46, ERp29, glutamate dehydrogenase, and TAP1 were shown to be more than 2-fold downregulated, whereas ␣-glucosidase, P-glycoprotein, fibrinogen, protein disulfide isomerase, GRP94, and apolipoprotein E were all found to be up-regulated in the hepatic ER of the fructose-fed hamster. Seven isoforms of ER60 in the hepatic ER were all shown to be downregulated at least 2-fold in hepatocytes from fructosefed/insulin-resistant hamsters. Implications of the differential expression of positively identified protein factors in the development of hepatic insulin resistance and lipoprotein abnormalities are discussed.

Proteomic screening of glucose-responsive and glucose non-responsive MIN-6 beta cells reveals differential expression of proteins involved in protein folding, secretion and oxidative stress

“…The expression of ERp29 reaches its highest levels in secretory cells; seems to play an important role in protein folding; consists of two domains of which the N-terminal domain of ERp29 resembles the thioredoxin module of PDI [26]; and has been shown to associate with GRP78 and GRP94 [27]. Recently, Morand et al [11] profiled hepatic ER-associated proteins from control and fructose-fed (insulin-resistant) hamsters using 2-DE and MS and found ERp29 to be 4.5-fold down-regulated in fructose-fed hamster livers. In our study, we found ERp29 to be significantly decreased in MIN-6(H) cells compared to MIN-6(L) cells that maintain their GSIS.…”

“…Many chaperones are heat shock proteins, that is, proteins expressed in response to elevated temperatures or other cellular stresses, and include 78-kDa glucose-regulated protein (GRP78), GRP58, GRP94, protein disulphide isomerase (PDI), calreticulin, 29-kDa ER protein (ERp29). Altered expression levels of several proteins including GRP78, GRP94 and PDI have been shown to be associated with the pathophysiology of diabetes [6][7][8][9][10][11].…”

Insulin resistance contributes to multidrug resistance in HepG2 cells via activation of the PERK signaling pathway and upregulation of Bcl-2 and P-gp

“…P-gp protein also functions as an ER chaperone protein to participate in the ER stress response by directly or indirectly transporting unfolded or misfolded protein and it could also provide protection for cells through inhibition of caspase-dependent cell apoptosis (17,18). In the present study, the expression levels of the P-gp protein were studied in the HepG2 cells at 6, 12, 24, 48 and 72 h post high-dose insulin exposure.…”

“…It is known that IR-induced hyperinsulinemia promotes the expression of P-gp, resulting in anticancer drug resistance in tumor cells (16). In addition, P-gp also plays protective roles as a chaperone for the transport of unfolded proteins during the ER stress reaction (17,18). During the process of IR induction in the HepG2 cells, the expression of MDR protein P-gp was significantly upregulated.…”

The endoplasmic reticulum stress response is associated with insulin resistance-mediated drug resistance in HepG2 cells

“…The expression of mdr1 in cells can be modified by several factors including stress signals and the metabolic and nutritional state [22][23]. P-gp can also play a role in the UPR, either directly by transporting misfolded proteins or indirectly by interacting with chaperones, conferring resistance to UPR-stimulated caspase-dependent apoptosis [23][24]. This suggests that MDR in tumors maybe related to the glucose abnormalities, ER stress, and the upregulation of mdr1.…”