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 down-regulated, whereas alpha-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 down-regulated 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.
Highlights
Proteomics involves the integration of a number of technologies with the aim of analyzing the complete complement of proteins expressed by a biological system in response to various stimuli and/or under different physiological or pathological conditions
Because a number of key signaling events as well as the pathways responsible for lipoprotein assembly occur in the endoplasmic reticulum (ER) compartment of hepatocytes, the current study focused on the identification of differentially expressed proteins in the ER fraction of hepatocytes derived from chow-fed and fructose-fed hamsters
This indicated that calnexin was present in both the heavier rough endoplasmic reticulum (RER) fractions, as well as the lighter smooth endoplasmic reticulum (SER) fractions, as described previously [10, 11]
Summary
Animal Surgery and Liver Perfusion—Surgery and liver perfusion were performed essentially as described previously [4, 8]. The pellet was resuspended in 54% Iodixanol (Optiprep, Axis Shield/Cedarlane, Hornby, ON, Canada) and subjected to ultracentrifugation at 350,000 ϫ g for 2:12 h at 4 °C using a V65.2-Ti vertical rotor After this spin, 20 fractions were unloaded from the top of the tube in 245-l volumes, and a protein assay was performed on each fraction. 200 g of protein (or up to 2 mg for preparative gels) was made up to 450 l with rehydration buffer (8 M urea, 0.5% w/v CHAPS, 15 mM dithiothreitol, 0.2%w/v Pharma Lyte 3–10) for the 24-cm Immobiline Drystrips (Amersham Biosciences). Based on difference map comparisons among numerous hepatic ER two-dimensional experiments, consistencies in differential protein expression between chow-fed and fructose-fed hamster hepatic ER were identified. Sequence data, obtained by MALDI-TOF-PSD, was subjected to database mining using the PSD-Sonar database
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