Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat
2001; Elsevier BV; Volume: 42; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)31533-9
ISSN1539-7262
AutoresMarshall B. Elam, Henry G. Wilcox, Lauren M. Cagen, Xiong Deng, Rajendra Raghow, Poonam Kumar, Murray Heimberg, James C. Russell,
Tópico(s)Liver Disease Diagnosis and Treatment
ResumoThe corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c.—Elam, M. B., H. G. Wilcox, L. M. Cagen, X. Deng, R. Raghow, P. Kumar, M. Heimberg, and J. C. Russell. Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat. J. Lipid Res. 2001. 42: 2039–2048. The corpulent JCR:LA-cp rat (cp/cp) is a useful model for study of the metabolic consequences of obesity and hyperinsulinemia. To assess the effect of hyperinsulinemia on VLDL secretion in this model, we measured rates of secretion of VLDL in perfused livers derived from cp/cp rats and their lean littermates. Livers of cp/cp rats secreted significantly greater amounts of VLDL triglyceride and apolipoprotein, compared with lean littermates. The content of apoB, apoE, and apoCs in both perfusate and plasma VLDL was greater in the cp/cp rat, as was the apolipoprotein (apo)C, apoA-I, and apoA-IV content of plasma HDL. Triglyceride content was also greater in cp/cp livers, as was hepatic lipogenesis and expression of lipogenic enzymes and sterol regulatory element binding protein-1 (SREBP-1). Hepatic mRNAs for apoE, and apoA-I were higher in livers of cp/cp rats. In contrast, the steady state levels of apoC-II, apoC-III, and apoB mRNAs were unchanged. Thus, livers of obese hyperinsulinemic cp/cp JCR:LA-cp rats secrete a greater number of VLDL particles that are enriched in triglyceride, apoE, and apoC. Greater secretion of VLDL in the cp/cp rat in part results from higher endogenous fatty acid synthesis, which in turn may occur in response to increased expression of the lipogenic enzyme regulator SREBP-1c. —Elam, M. B., H. G. Wilcox, L. M. Cagen, X. Deng, R. Raghow, P. Kumar, M. Heimberg, and J. C. Russell. Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR:LA-cp rat. J. Lipid Res. 2001. 42: 2039–2048. The JCR:LA-corpulent rat, when homozygous for the corpulent (cp) gene (also known as f), demonstrates characteristics of hyperphagia, obesity, hyperlipidemia, and hyperinsulinemia with impaired glucose tolerance (1Amy R.M. Dolphin P.J. Pederson R.A. Russell J.C. Atherogenesis in two strains of obese rats: the fatty Zucker and LA/N-corpulent.Atherosclerosis. 1988; 69: 199-209Google Scholar, 2Russell J.C. Graham S. Hameed M. Abnormal insulin and glucose metabolism in the JCR:LA-corpulent rat.Metabolism. 1994; 43: 538-543Google Scholar, 3Dolphin P.J. Stewart B. Amy R.M. Russell J.C. Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat.Biochim. Biophys. Acta. 1987; 919: 140-148Google Scholar). The defect is transmitted as an autosomal recessive trait (4Yen T.T. Shaw W.N. Yu P-L. Genetics of obesity in Zucker rats and Koletsky rats.Heredity. 1977; 38: 373-377Google Scholar) and has been identified as a Tyr763Stop nonsense mutation in the leptin receptor upstream of the putative transmembrane domain (5Wu-Pheng X.S. Streamson C.C. Okada N. Liu S-M. Nicolson M. Leibel R.L. Phenotype of the obese Koletsky (f) rat due to Tyr763Stop mutation in the extracellular domain of the leptin receptor (Lepr): evidence for deficient plasma-to-CSF transport of leptin in both the Zucker and Koletsky obese rat.Diabetes. 1997; 46: 513-518Google Scholar). The cp mutation arose in Koletsky's hypertensive rat strain and was crossbred with both LA/N and SHR/N strains (6Hansen C.T. Two new congenic rat strains for nutrition and obesity research.Fed. Proc. 1983; 42 (Abstract): 573Google Scholar). Insulin resistance and hyperinsulinemia are more severe in the male JCR:LA-cp rat than in the female, and, unlike the fatty Zucker rat, the male JCR:LA-cp rat develops atherosclerotic vascular lesions (7Russell J.C. Graham S.E. Richardson M. Cardiovascular disease in the JCR:LA-cp rat.Mol. Cell Biochem. 1998; 188: 113-126Google Scholar). Development of cardiovascular disease in the male JCR:LA-cp rat correlates strongly with hyperinsulinemia (7Russell J.C. Graham S.E. Richardson M. Cardiovascular disease in the JCR:LA-cp rat.Mol. Cell Biochem. 1998; 188: 113-126Google Scholar). The JCR:LA-cp rat is therefore a useful animal model for the study of the pathophysiology and metabolic consequences of obesity and hyperinsulinemia. Hyperlipidemia, in particular higher plasma levels of triglyceride enriched VLDL, is a prominent feature of the corpulent JCR:LA-cp rat (cp/cp) JCR:LA-cp phenotype (3Dolphin P.J. Stewart B. Amy R.M. Russell J.C. Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat.Biochim. Biophys. Acta. 1987; 919: 140-148Google Scholar). Although the pathogenesis of hyperlipidemia in the cp/cp JCR:LA-cp rat is not completely delineated, studies using triton WR1339 in intact animals, and with primary hepatocyte cultures from cp/cp rats suggest that overproduction of VLDL may play a significant role (8Russell J.C. Koeslag D.G. Amy R.M. Dolphin P.J. Plasma lipid secretion and clearance in hyperlipidemic JCR:LA-corpulent rats.Arteriosclerosis. 1989; 9: 869-876Google Scholar, 9Vance J.E. Russell J.C. Hypersecretion of VLDL, but not HDL, by hepatocytes from the JCR:LA-corpulent rat.J. Lipid Res. 1990; 31: 1491-1501Google Scholar). Assessment of VLDL secretion in the JCR:LA-cp rat, which exhibits insulin-resistance and hyperinsulinemia, is of interest because previous studies using in vitro hepatocyte cultures demonstrate that prolonged exposure to high levels of insulin increases secretion of both VLDL triglyceride and apoB (10Thorngate F.E. Raghow R. Wilcox H.G. Werner C.S. Elam M.B. Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by dramatically altering apolipoprotein B mRNA editing.Proc. Nat. Acad. Sci. USA. 1994; 91: 5392-5396Google Scholar, 11Bjornsson O.G. Duerden J.M. Bartlett S.M. Sparks J.D. Sparks C.E. Gibbons G.F. The role of pancreatic hormones in the regulation of lipid storage, oxidation and secretion in primary cultures of rat hepatocytes. Short and long-term effects.Biochem. J. 1992; 281: 381-386Google Scholar). Recently, it has been demonstrated that induction of the key lipogenic enzymes, fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC) by insulin is mediated by the transcription factor sterol regulatory element binding protein-1c (SREBP-1c) (12Foretz M. Guichard C. Ferre P. Foufelle F. Sterol regulatory element binding protein-1c is a major mediator of insulin action on the hepatic expression of glucokinase and lipogenesis-related genes.PNAS. 1999; 96: 12737-12742Google Scholar, 13Foretz M. Pacot C. Dugail I. Lemarchand P. Guichard C. Liepvre X.L. Berthelierw-Lubrano C. Spiegelman B. Kim J.B. Ferre P. Foufelle F. ADD1/SREBP-1c is required in the activation of hepatic lipogenic gene expression by glucose.Mol. Cell. Biol. 1999; 19: 3760-3768Google Scholar, 14Fukuda H. Katsurada A. Iritani N. Nutritional and hormonal regulation of mRNA levels of lipogenic eznymes in primary cultures of rat hepatocytes.J. Biochem. 1992; 111: 25-30Google Scholar), also known as adipocyte differentiation and determination factor (ADD-I) (15Kim J.B. Spiegelman B.M. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism.Genes Dev. 1996; 10: 1096-1107Google Scholar). These in vitro observations suggest that up-regulation of endogenous lipid synthesis via regulation of SREBP-1c/ADD-I, FAS, and ACC may play a significant role in the ability of insulin to increase VLDL secretion via increased availability of fatty acid for triglyceride synthesis. Therefore, we examined rates of secretion of VLDL lipid and apolipoprotein by isolated perfused livers of cp/cp rats in order to assess the effect of hyperinsulinemia on the quantity and composition of secreted VLDL. We also measured the steady-state levels of mRNAs encoding apolipoprotein (apo)B, apoC-II, apoC-III, apoE, apoA-I, FAS, ACC, and ADD-I/SREBP-1 in the livers of cp/cp JCR:LA-cp rats to gain insight into the potential mechanisms by which VLDL secretion is altered in response to hyperinsulinemia. Male rats were bred at the University of Alberta, Edmonton, Canada, and were shipped to the University of Tennessee at 4–8 weeks of age and maintained on a chow diet (Picolab Rodent Diet 20, Lab Diet 5053, PMI Nutrition International Inc., Brentwood, MO) for several weeks, at which time obese animals attained a weight of 450–500 g, while that of the lean animals was 300–350 g. Obese JCR:LA-cp rats are homozygous for the defective leptin receptor gene (cp/cp), whereas lean animals are heterozygous for the cp gene (cp/+) or homozygous for the functional leptin receptor gene (+/+). Lean animals are phenotypically indistinguishable and are therefore designated as +/? (3Dolphin P.J. Stewart B. Amy R.M. Russell J.C. Serum lipids and lipoproteins in the atherosclerosis prone LA/N corpulent rat.Biochim. Biophys. Acta. 1987; 919: 140-148Google Scholar). Livers were surgically removed under pentobarbital anesthesia, and liver perfusions were conducted in a humidified perfusion apparatus with recycling perfusion medium consisting of Krebs-Heneseleit-bicarbonate buffer, pH 7.4, with 100 mg/dl glucose, as has been described previously in detail (16Fungwe T.V. Cagen L.M. Wilcox H.G. Heimberg M. Regulation of hepatic secretion of the very low density lipoproteins by dietary cholesterol.J. Lipid Res. 1992; 33: 179-191Google Scholar). Blood was obtained from the abdominal aorta and placed into tubes containing EDTA prior to removal of the liver. Plasma samples were obtained after a brief centrifugation and processed as described below. Washed outdated human erythrocytes were present in the perfusion medium to provide an initial hematocrit of 25%. The starting perfusion medium contained 0.69 ± 0.03 mM (n = 14) sodium oleate complexed with 3% lipid-free purified BSA. Additional sodium oleate complexed with BSA (6%) was supplied by constant infusion at a rate of 166 μmol/h. The fatty acid concentration achieved at steady state during perfusion did not differ for livers of cp/cp or +/? rats (0.44 ± 0.02 and 0.40 μ 0.03 mM (n = 14) at 1.5 and 3 h, respectively). For experiments in which rates of secretion of newly synthesized apolipoprotein was assessed, trace amounts of [4, 5-3H]l-leucine (25 μCi) were added to the perfusion medium as a pulse and then infused at a rate of 60 μCi/h as described previously (17Salam W.H. Wilcox H.G. Heimberg M.H. Effects of oleic acid on the biosynthesis of lipoprotein apolipoproteins and distribution into the very low density lipoprotein by the isolated prefused rat liver.Biochem. J. 1988; 25: 809-816Google Scholar). Livers were perfused for up to 3 h. Samples of perfusate were collected at 0, 1, 2, and 3 h. At the termination of the per fusion, the liver was quickly flushed with ice-cold saline. A small piece of liver (0.1 g) was immediately placed in RNAzol for extraction of RNA. After weighing and mincing the remaining portion of the liver, lipids were extracted from 1-g aliquots. Livers that had not been perfused were also analyzed for lipid and mRNA content. Perfusate samples and arterial blood samples were collected in EDTA and perfusate and plasma was obtained after brief centrifugation. All samples were analyzed immediately, as described below, or were stored at −70 °. Pefused livers have the capacity for re-uptake of secreted nascent VLDL (18Wilcox H.G. Heimberg M. Secretion and uptake of nascent hepatic very low density lipoprotein by perfused livers from fed and fasted rats.J. Lipid Res. 1987; 28: 351-360Google Scholar). Therefore, greater accumulation of VLDL in perfusate of cp/cp livers could occur as a result of decreased reuptake rather than greater secretion. Differences in VLDL re-uptake might occur as a result of differing characteristics of cp/cp versus +/? livers, or may be due to differences in composition of the VLDL itself. For this reason, we measured re-uptake of VLDL by perfused livers using a crossover design. Metabolically labeled nascent VLDL was prepared by per fusing normal Sprague-Dawley donor livers with [4, 5-3H]l-leucine (1–3 mCi/liver). Labeled nascent VLDL was introduced into the medium perfusing cp/cp and +/? livers and disappearance of labeled VLDL protein (trichloroacetic acid precipitable counts) was assessed during the first 60 min of perfusion. Conversely, metabolically labeled VLDL produced by +/? and cp/cp donor livers, respectively, was introduced into the perfusate of normal Sprague-Dawley livers. The uptake studies were carried out using oleatecontaining medium as described above. Plasma lipoproteins were isolated by sequential ultracentrifugation after density adjustments with sodium bromide at d = 1.006 (VLDL), d = 1.063 (IDL/LDL), and d = 1.21 (HDL). Chemical analyses of all major classes of lipids (triglyceride, phospholipid, cholesterol, free fatty acid, and cholesteryl ester) were conducted after thin layer chromatography of plasma and lipoprotein extracts. Protein analyses were carried out by the method of Lowry as modified by Markwell et al. (19Markwell M.A.L. Hass S.M. Bieber L.L. Tolbert N.E. A modification of the Lowry procedure to simplify proteindetermination in membrane and lipoprotein samples.Anal. Biochem. 1978; 87: 206-210Google Scholar). Apolipoproteins of the various lipoprotein fractions of plasma were separated on Laemmli gradient gels (4–22% polyacrylamide) and stained with Coomassie Brilliant Blue R-250. Densitometric tracing of the gels, using NIH Image 161 software, was used to evaluate the distribution of the apolipoproteins within the particular perfusate or plasma lipoprotein fraction. For experiments in which 4,5-3H-labeled leucine was infused, Coomassie blue-stained apolipoprotein bands from tritium-labeled VLDL perfusion samples were excised for measurement of radioactivity (17Salam W.H. Wilcox H.G. Heimberg M.H. Effects of oleic acid on the biosynthesis of lipoprotein apolipoproteins and distribution into the very low density lipoprotein by the isolated prefused rat liver.Biochem. J. 1988; 25: 809-816Google Scholar). Total RNA was extracted from liver samples using RNA Stat-60 (Tel-Test, Inc., Friendswood, TX) according to the manufacturer's instructions and quantitated by absorbance at 260 nm. Fifteen μg of total RNA was loaded per lane of a formaldehyde/0.8% agarose gel, electrophoresed in 1XMOPS buffer, blotted onto Nytran membranes (Schleicher and Schuell, Keane, NH), and UV cross-linked. Ribosomal RNA bands were visualized by staining with ethidium bromide prior to transfer. Blots were prehybridized for 3 h at 42 °C in 50% formamide, 5 × SSPE, 5 × Denhardt's solution (5Prime-3Prime, Boulder, CO), 7.5% dextran sulfate, 1.5% sodium dodecyl sulfate (SDS), and 100 μg/ml sheared salmon sperm DNA (5Prime-3Prime). The probe for measurement of rat apoB mRNA was a 518 bp cDNA amplified by RT-PCR from samples of rat hepatocyte total RNA, with forward and reverse primers corresponding to nucleotides +126 to +150 and +609 to +643, respectively. The PCR product is 518 bp within the coding region, but distant from the editing site and free of obvious hairpin loops. Probes were prepared for apoA-I, apoC-II, apoC-III, and apoE using primers based on published sequences of rat liver cDNA for the corresponding protein (20Matsumoto A. Aburatani H. Shibasaki Y. Kodama T. Takaku F. Itakura H. Cloning and regulation of rat apolipoprotein B mRNA.Biochem. Biophys. Res. Commun. 1987; 142: 92-99Google Scholar, 21McLean J.W. Fukazawa C. Taylor J.M. Rat apolipoprotein E mRNA. Cloning and sequencing of double-stranded cDNA.J. Biol. Chem. 1983; 258: 8993-9000Google Scholar, 22Andersson Y. Thelander L. Bengtsson-Olivecrona G. Rat apolipoprotein C–II lacks the conserved site for proteolytic cleavage of the pro-form.J. Lipid Res. 1991; 32: 1805-1809Google Scholar, 23Haddad I.A. Ordovas J.M. Fitzpatrick T. Karathanasis S.K. Linkage, evolution, and expression of the rat apolipoprotein A-I, C–III, and A-IV genes.J. Biol. Chem. 1986; 261: 13268-13277Google Scholar). cDNAs were labeled with 32P-dCTP using a random primer labeling kit (Stratagene, La Jolla, CA), and hybridized according to the manufacturer recommendations. mRNA transcripts were visualized by autoradiography using Kodak X-Omat AR film (Rochester, NY). A digital image of the developed film was created and the intensity of bands determined by densitometry (Alpha Innotech, San Leandro, CA). Plasmids containing cDNAs used for measurement of ACC-1, FAS, and ADD-I/SREBP-1c mRNA levels were generously provided by Dr. Ki-Han Kim (Purdue University, West Lafayette, IN; ACC-1); Dr. Stuart Smith (Children's Hospital Research Institute, Oakland, CA; FAS); and Dr. Bruce M. Spiegelman (Dana Farber Cancer Institute, Boston, MA; ADD-I/SREBP-1c). B-actin mRNA was measured using mouse B-actin DECAprobe (Ambion, Inc., Austin, TX). ACC-1, FAS, ADD-I/SREBP-1c, and B-actin mRNA were detected by overnight hybridization at 42 °C with the cDNA probes, 32P-labeled by the random primer method using a commercial kit following the manufacturer's directions (Invitrogen, Carlsbad, CA). Unbound probe was removed by washing with 2 × SSC + 0.1% SDS at room temperature and then twice with 0.1 × SSC + 0.1% SDS at 65 °C for 30 min each. Membranes were exposed to X-Omat AR film (Kodak); a digital image of the developed film was created and bands quantitated by densitometry (Alpha Innotech Corp., San Leandro, CA). Plasma insulin levels were determined using a Micromedic Insulin RIA kit (ICN Pharmaceuticals Inc., Costa Mesa, CA), and glucose was determined using a glucose oxidase kit (Sigma Chemical, St. Louis, MO). Hepatocytes were prepared from livers of +/? and cp/cp rats by collagenase perfusion as described previously (10Thorngate F.E. Raghow R. Wilcox H.G. Werner C.S. Elam M.B. Insulin promotes the biosynthesis and secretion of apolipoprotein B-48 by dramatically altering apolipoprotein B mRNA editing.Proc. Nat. Acad. Sci. USA. 1994; 91: 5392-5396Google Scholar). Cells were suspended in modified Williams E medium (GIBCO/BRL) containing 20% fetal bovine serum (Sigma, St. Louis, MO) and 5.5 mM glucose and were plated on 60 mm culture dishes coated with rat tail collagen (Collaborative Biochemical Products, Bedford, MA) at 3 × 106 cells per plate. After 4 h, nonadherent cells were removed and the plates washed with PBS before measurement of lipogenesis. Rates of lipogenesis were assessed after incubation of the washed heptocyte monolayer for 3 h in serum-free, 5.5 mM glucose Williams E medium and 1 mM [2-14C]acetate (6 μCi). Lipids were extracted from the cells and medium and separated by TLC (16Fungwe T.V. Cagen L.M. Wilcox H.G. Heimberg M. Regulation of hepatic secretion of the very low density lipoproteins by dietary cholesterol.J. Lipid Res. 1992; 33: 179-191Google Scholar). Radioactivity in bands corresponding to triglyceride, phospholipids, free fatty acids, and cholesteryl esters was measured by liquid scintillation spectrometry (Beckman Model LS5000). Significance of differences in variables of interest between cp/cp and +/? rats were determined by Student's t-test using a microcomputer statistical analysis software package (StatView for Power Macintosh, version 4.51, 1996, Abacus Concepts, Berkeley, CA) on a Macintosh G3 computer. Both total body weight and liver weight were greater in cp/cp rats than those observed in lean (+/?) controls (Table 1). Cp/cp rats also demonstrated higher nonfasting insulin and glucose levels. Total plasma triglyceride, cholesterol, and cholesteryl ester content were also higher in the cp/cp rat. Surprisingly, plasma fatty acid levels in cp/cp rats were comparable to those observed in +/? controls.TABLE 1.Characteristics of cp/cp and +/? JCR ratsLean (+/?) (n = 9)Corpulent (cp/cp) (n = 9)Triglyceride (μmol/dl)49.9 ± 5.1186.1 ± 25.2aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Phospholipid (μmol/dl)107.7 ± 5.9185.1 ± 9.9aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Cholesterol (μmol/dl)43.3 ± 2.766.1 ± 4.4aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Cholesteryl ester (μmol/dl)92.6 ± 4.8161.3 ± 11.4aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Free fatty acid (μmol/dl)41.9 ± 3.246.7 ± 4.6Glucose (mg/dl)210 ± 5 (8)257 ± 17 (8)aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Insulin (μIU)100 ± 11 (3)178 ± 13 (3)aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Body weight (g)377 ± 4538 ± 21aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Liver weight (g)10.7 ± 0.317.6 ± 1.0aP < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice.Results are means ± SEM and the number of individual observations are in parentheses.a P < 0.05 compared with +/? rats. The plasma was taken in the fed state at the time of sacrifice. Open table in a new tab Results are means ± SEM and the number of individual observations are in parentheses. Table 2 depicts the apolipoprotein levels observed in plasma VLDL, LDL/IDL, and HDL of cp/cp and +/?rats. VLDL apolipoproteins (B-48, B-100, Cs, and apoA-I) were 4- to 6-fold greater in plasma of cp/cp rats, as compared with +/? rats. Similarly, HDL apolipoproteins' (A-I, A-IV, E, and Cs) levels were approximately 2-fold greater in the plasma of cp/cp rats. In contrast, the apolipoprotein content of the LDL/IDL fraction was comparable in cp/cp and +/? rats. Total plasma apoB-48 levels were higher in cp/cp rats, as were total plasma apoC, E, A-I, and A-IV (Table 2).TABLE 2.Plasma apolipoproteins in +/? and cp/cp JCR:LA/N ratsApolipoproteinApoB-48ApoB-100ApoEApoCApoA-IApoA-IVVLDL-+/?0.24 ± 0.020.22 ± 0.020.11 ± 0.020.57 ± 0.180.05 ± 0.020.11 ± 0.10VLDL-cp/cp1.81 ± 0.42aP < 0.05 cp/cp versus +/?.1.07 ± 0.26aP < 0.05 cp/cp versus +/?.0.23 ± 0.103.60 ± 0.78aP < 0.05 cp/cp versus +/?.0.26 ± 0.12aP < 0.05 cp/cp versus +/?.0.34 ± 0.21IDL/LDL-+/?1.52 ± 0.523.27 ± 0.901.02 ± 0.213.38 ± 1.610.20 ± 0.080.17 ± 0.14IDL/LDL-cp/cp1.36 ± 0.083.19 ± 0.511.42 ± 0.574.42 ± 1.020.62 ± 0.250.41 ± 0.35HDL-+/?NDND2.93 ± 0.6914.2 ± 2.916.8 ± 3.963.94 ± 0.92HDL-cp/cpNDND6.38 ± 1.30aP < 0.05 cp/cp versus +/?.33.0 ± 3.2aP < 0.05 cp/cp versus +/?.29.5 ± 3.5aP < 0.05 cp/cp versus +/?.8.59 ± 1.11aP < 0.05 cp/cp versus +/?.Total-+/?1.76 ± 0.533.49 ± 0.904.06 ± 0.7418.2 ± 2.317.0 ± 3.94.19 ± 0.82Total-cp/cp3.17 ± 0.43aP < 0.05 cp/cp versus +/?.4.26 ± 0.698.02 ± 0.90aP < 0.05 cp/cp versus +/?.41.0 ± 3.8aP < 0.05 cp/cp versus +/?.30.4 ± 3.4aP < 0.05 cp/cp versus +/?.9.33 ± 1.20aP < 0.05 cp/cp versus +/?.Data are mg/dl of plasma apolipoproteins in the VLDL, IDL/LDL, and HDL fractions and the total of the three lipoprotein fractions for +/? (N = 4) and cp/cp (N = 4) JCR:LA-cp rats. Lipoproteins were isolated from the plasma of (non-fasted) +/? and cp/cp JCR:LA-cp rats by sequential ultracentrifugation at d = 1.006 (VLDL), d = 1.006–1.063 (IDL/LDL), and d = 1.063–1.21 (HDL). Apolipoproteins were separated on Laemmli gradient gels (4 to 22% polyacrylamide) and stained with Coomassie Brilliant Blue R-250, and the mass of each apolipopro-tein was estimated from densitometric scanning of the stained gels. Separation of apoC species was not sufficient using this methodology to allow separate quantitation of apoC-III and apoC-II. Total apolipoprotein represents the sum of the apolipoprotein in VLDL, IDL/LDL, and HDL fractions. ND = none detectable.a P < 0.05 cp/cp versus +/?. Open table in a new tab Data are mg/dl of plasma apolipoproteins in the VLDL, IDL/LDL, and HDL fractions and the total of the three lipoprotein fractions for +/? (N = 4) and cp/cp (N = 4) JCR:LA-cp rats. Lipoproteins were isolated from the plasma of (non-fasted) +/? and cp/cp JCR:LA-cp rats by sequential ultracentrifugation at d = 1.006 (VLDL), d = 1.006–1.063 (IDL/LDL), and d = 1.063–1.21 (HDL). Apolipoproteins were separated on Laemmli gradient gels (4 to 22% polyacrylamide) and stained with Coomassie Brilliant Blue R-250, and the mass of each apolipopro-tein was estimated from densitometric scanning of the stained gels. Separation of apoC species was not sufficient using this methodology to allow separate quantitation of apoC-III and apoC-II. Total apolipoprotein represents the sum of the apolipoprotein in VLDL, IDL/LDL, and HDL fractions. ND = none detectable. To determine whether greater accumulation of triglyceride-rich lipoproteins in the plasma of cp/cp rats resulted from increased hepatic secretion of VLDL, we assessed lipid and lipoprotein secretion by isolated perfused livers of cp/cp and +/? rats. When livers were perfused with a constant infusion of oleic acid, triglyceride secretion was linear over the 3-h perfusion period and was consistently greater in livers derived from cp/cp rats compared with +/? controls (data not shown). Livers of cp/cp rats secreted 3 times as much triglyceride (VLDL and total perfusate) per gram of liver during the perfusion than did livers of +/? rats (Table 3). Secretion of VLDL phospholipid and cholesterol (but not cholesteryl ester) by livers of cp/cp rats was also greater. Because lipid and apolipoprotein secretion rates are presented as amount secreted per gram liver, the absolute magnitude of the difference in lipid and apolipoprotein secretion by livers of +/? and cp/cp rats was even greater, as cp/cp livers were 60% larger than +/? livers. Therefore, whole liver triglyceride secretion rates were up to 5-fold greater in livers of cp/cp rats. Surprisingly, despite increased triglyceride secretion, total fatty acid uptake by livers of cp/cp rats was lower per gram liver than that of +/? livers (Table 3). Because cp/cp livers were larger, however, total fatty acid uptake per liver was comparable to that observed in +/? livers.TABLE 3.Secretion of perfusate and VLDL lipids by perfused livers of +/? and cp/cp JCR:LA-cp rats+/? (9)cp/cp (9)μmol/g liver/3hTriglycerideTotal1.11 ± 0.123.19 ± 0.35aP < 0.05 compared with +/? rats.VLDL0.85 ± 0.112.62 ± 0.31aP < 0.05 compared with +/? rats.PhospholipidTotal1.62 ± 0.241.25 ± 0.18VLDL0.27 ± 0.020.55 ± 0.06aP < 0.05 compared with +/? rats.CholesterolTotal0.24 ± 0.040.28 ± 0.05VLDL0.10 ± 0.010.22 ± 0.03aP < 0.05 compared with +/? rats.Cholesteryl esterTotal0.14 ± 0.030.13 ± 0.04VLDL0.06 ± 0.010.08 ± 0.02FFA uptake (μmol/gm/liver/hr)129 ± 8.882 ± 5.2aP < 0.05 compared with +/? rats.Results are means ± SEM and the number of individual observations are in parentheses.a P < 0.05 compared with +/? rats. Open table in a new tab Results are means ± SEM and the number of individual observations are in parentheses. Calculation of the core/surface ratio (TG + CE/C + PL) provided an indication of the relative differences in the average size of the VLDL produced by cp/cp versus +/? livers. The core-surface ratios of VLDL produced by cp/cp livers were larger than that of +/? rats (3.53 ± 0.40 versus2.44 ± 0.24 for cp/cp versus +/?). This reflects marked enrichment with triglyceride of VLDL secreted by cp/cp livers. We assessed the rates of secretion of VLDL apolipoproteins by perfused livers of cp/cp and +/? rats both by densitometry on stained SDS-PAGE gels (Fig. 1A) and by measuring the secretion of metabolically labeled ([3H]leucine) VLDL apolipoprotein (Fig. 1B). Approximately 85% of secreted apolipoprotein was associated with the VLDL as assessed by both methods (data not shown). Secretion of VLDL associated apolipoproteins was greater in cp/cp rat livers (Fig. 1A and 1B). Although incorporation of [3H]leucine into total hepatic and perfusate protein (TCA precipitable counts) was only somewhat higher in cp/cp livers compared with +/? livers (2.53 μCi/g liver versus 2.11 μCi/g respectively), secretion of labeled apolipoproteins was dramatically increased (Fig. 1B). This indicated a specific increase in incorporation of labeled amino acid into apolipoproteinsin the cp/cp livers. Because each VLDL particle has one apoB molecule, the rate of secretion of apoB serves as an index of the number of VLDL particles secreted. Therefore, in addition to increased triglyceride secretion, livers of cp/cp rats also secreted a larger number of VLDL particles. Enrichment of the VLDL fraction with other apolipoproteins (apoE, apoCs) was also observed (Fig. 1). Enrichment of VLDL with triglyceride in the cp/cp rat also resulted in altered distribution of apolipoproteins among the l
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