点击显示 收起
【摘要】 Chronic renal failure (CRF) is associated with hypertriglyceridemia and elevated plasma VLDL and IDL concentrations. These events can be due to either increased production or depressed catabolism of triglyceride-rich lipoproteins. Several studies have documented downregulation of lipoprotein lipase, hepatic triglyceride lipase, and the VLDL receptor, leading to depressed clearance and elevated plasma concentration of triglyceride-rich lipoproteins and their remnants in CRF. However, the effect of CRF on the triglyceride biosynthetic pathway has not been explored. Diglycerol acyltransferase (DGAT) is a microsomal enzyme that joins acyl-CoA to 1,2 diacylglycerol and, as such, constitutes the final step in triglyceride biosynthesis. Two distinct forms of DGAT (DGAT-1 and -2) have thus far been identified. The present study was undertaken to examine the effect of CRF on DGAT gene expression and activity in the liver, which is the source of endogenous triglycerides in the circulation. Male Sprague-Dawley rats were studied 8 wk after nephrectomy (CRF) or sham operation. DGAT-1 and DGAT-2 mRNA abundance and DGAT activity were quantified. The CRF group showed reduced creatinine clearance, elevated plasma triglycerides, and VLDL concentrations. This was accompanied by significant reductions in hepatic DGAT-2 mRNA abundance ( P < 0.01) and total DGAT activity ( P < 0.1), pointing to diminished hepatic triglyceride production capacity in CRF animals. In conclusion, CRF results in significant downregulation of hepatic DGAT gene expression and activity. Given the critical role of DGAT in triglyceride biosynthesis, the present study points to diminished, not increased, hepatic triglyceride synthetic capacity in CRF rats.
【关键词】 chronic kidney disease triglycerides atherogenesis dyslipidemia liver
CHRONIC RENAL FAILURE (CRF) is associated with profound quantitative and qualitative abnormalities of plasma lipoproteins. CRF-induced dyslipidemia is marked by hypertriglyceridemia, elevated concentration, impaired clearance, and triglyceride enrichment of apoB-containing lipoproteins, diminished apoA-1 concentration, relative reduction of high-density lipoprotein (HDL), and elevated lipoprotein (a) concentration ( 15 ). In addition to promoting atherosclerosis, dyslipidemia may accelerate the progression of renal disease, glomerulosclerosis, tubulointerstitial injury, and foam cell formation ( 7, 17 ).
In a series of earlier studies, we found marked downregulation of adipose tissue and skeletal muscle lipoprotein lipase and VLDL receptor in rats with CRF induced by nephrectomy ( 13, 22, 23, 26 ) and CRF rats with advanced spontaneous focal glomerulosclerosis ( 19 ). These findings, in part, account for hypertriglyceridemia, impaired clearance, triglyceride enrichment, and elevated concentration of apoB-containing lipoproteins, i.e., VLDL and chylomicrons. In addition, Klin et al. ( 11 ) and Sato et al. ( 18 ) have shown marked downregulation of hepatic triglyceride lipase in nephrectomized rats and CRF rats with advanced spontaneous glomerulosclerosis. This phenomenon can account for impaired clearance and accumulation of VLDL and chylomicron remnants in CRF. The impact of lipoprotein lipase deficiency is compounded by the lack of adequate supplies of apoC-II (lipoprotein lipase cofactor) and apoE (ligand for binding of VLDL and chylomicron to endothelium) in VLDL and chylomicrons, which normally receive these proteins from the cholesterol ester-rich HDL-2 particles. However, due to severe deficiency of LCAT ( 25 ), which is critical for HDL-mediated uptake of cholesterol from the peripheral tissues, maturation of cholesterol ester-poor HDL-3 to HDL-2 is greatly impaired in CRF. The reduction in HDL-2, which is the most efficient apoC-II and apoE donor, leads to reduced LPL activity and diminished VLDL and chylomicron binding to endothelium. These defects, in turn, diminish catalytic action of LPL.
Together, downregulation of LPL, hepatic lipase, and VLDL receptor and defective HDL maturation provide the basis for impaired clearance and catabolism of triglyceride-rich lipoproteins. However, due to lack of appropriate molecular tools, the triglyceride biosynthetic pathway had not been previously investigated. Recent identification of genes encoding diglycerol acyltransferase (DGAT), which catalyzes the final step in triglyceride biosynthesis ( 4, 5, 8, 20 ), has provided the opportunity to investigate regulation of triglyceride synthesis in various physiological and pathological states. Two distinct DGAT's have thus far been identified. DGAT-2 is expressed primarily in the liver, intestine, and white fat, whereas DGAT-1 is expressed in all tissues ( 4, 5 ). The present study was carried out to explore the effect of CRF on the hepatic triglyceride biosynthetic pathway by examining gene expression and activity of this enzyme in the liver. The liver was chosen because it is the primary source of endogenous triglyceride in the circulation. Accordingly, mRNA abundance of the two known DGAT's, i.e., DGAT-1 and DGAT-2, and microsomal DGAT activity were measured in rats 8 wk post- nephrectomy or sham operation.
METHODS
Animal models. Male Sprague-Dawley rats weighing 225-250 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). They were housed in a climate-controlled, light-regulated facility with a 12:12-h light-dark cycle. The animals were fed regular rat chow (Purina Mills, Brentwood, MO) and water ad libitum and randomly assigned to the CRF and control groups. Six animals were used for each group. The animals assigned to the CRF group were subjected to nephrectomy by surgical resection using a dorsal incision, as described previously ( 26 ). The animals assigned to the control group were subjected to a sham operation. All surgical procedures were carried out under general anesthesia (Nembutal 50 mg/kg ip), and strict hemostasis and aseptic techniques were observed. All animals were provided free access to food and water.
The animals were observed for 8 wk, at which point they were placed in metabolic cages for a 24-h urine collection. They were then killed by exsanguination via cardiac puncture between 9 and 11 AM. The liver was removed immediately, snap-frozen in liquid nitrogen, and stored at -70°C until processed. Serum cholesterol, triglyceride, LDL, VLDL, HDL, and creatinine concentrations and urinary protein and creatinine contents were determined as described previously ( 24 ). The protocol employed in this study was approved by the Institutional Committee for Care and Use of Animals at the University of California (Irvine, CA).
Isolation of liver microsomes. Four hundred milligrams of frozen liver were homogenized in ice-cold buffer A containing (in mM) 50 Tris·HCl, 250 sucrose, 1 EDTA, and 1 PMSF, pH 7.4. Microsomes were then prepared by differential centrifugation as described previously ( 14 ). The microsomal membranes were then resuspended in buffer A, divided into aliquots, and stored at -70°C until use.
Measurement of DGAT activity. The DGAT activity in the microsomal fraction was measured as described in our recent study ( 21 ). The assay is based on DGAT-catalyzed production of [ 14 C]trioleoylglycerol from [1- 14 C]oleoyl-CoA and 1,2-dioleoylglycerol. In brief, the reaction mixture used for total DGAT activity contained 175 mM Tris·HCl (pH 8.0), 8 mM MgCl 2, 5 mg/ml fatty acid-free BSA, 200 µM 1,2-dioleoylglycerol, 30 µl of [1- 14 C]oleoyl-CoA (50 µCi/ml, 56 mCi/mmol), and 50 µg of microsomal protein in a total volume of 200 µl. The reaction was initiated by addition of [1- 14 C]oleoyl-CoA and incubation of mixture for 30 min at 25°C. The reaction was terminated by adding 1.5 ml of a solution containing 2-propanol-heptane-water (80:20:2, vol/vol). The mixture was then vortexed briefly, 1 ml heptane and 0.5 ml water were added, and the mixutre was gently vortexed again and centrifuged at 1,000 g for 10 min. Thereafter, 1 ml of the top heptane phase was transferred to a new reaction tube to which 2 ml of alkaline ethanol solution (ethanol-0.5 N NaOH-water, 50:10:40, vol/vol) were added. The contents were then thoroughly mixed and centrifuged at 1,000 g for 10 min. A 650-µl aliquot of the top heptane phase containing [ 14 C]trioleoylglycerol was transferred to a scintillation vial to which 5 ml of Bio-Safe II scintillation fluid were added, and radioactivity was measured using a liquid scintillator.
RNA isolation and RT-PCR. RNA was isolated from frozen liver using TRIzol reagent (Invitrogen, Carlsbad, CA) and purified with a RNeasy kit (Qiagen, Valencia, CA). One microgram of total RNA from each sample was reverse transcribed to cDNA by using Superscript II RT (Invitrogen) with a mixture of oligo(dT) (200 ng/reaction) and random primers (200 ng/reaction) in a 20-µl volume at 45°C for 50 min. The reaction was stopped by heating at 90°C for 5 min.
Expression of DGAT-1 and DGAT-2 mRNAs was assessed by RT-PCR analysis using 18S as an internal control. The primer sequences are shown in Table 1. DGAT-1 and DGAT-2 primers were designed with the Primer3 program and purchased from Invitrogen. For 18S amplification, we used alternate 18S (Ambion, Austin, TX), which yielded a 324-bp product. In each PCR reaction, 18S ribosomal RNA was coamplified with the target cDNA (mRNA). The primers were tested for their compatibility with the alternate 18S primer. The cDNAs were amplified using standard PCR buffer, 0.2 mM dNTP, 1 µM specific primer set, 0.5 µM 18S primer/competimer mix, 2 mM MgCl 2, and 0.75 U of Taq DNA polymerase (Invitrogen) in 25 µl of total volume for 26 cycles. Each cycle consisted of 3 min of denaturation at 94°C, 45 s of annealing at 57°C, and 45 s of extension at 72°C. PCR products were separated on a 2.5% agarose gel with ethidium bromide by electrophoresis. Signal intensity was determined by laser-scanning densitometry. DGAT-1 and DGAT-2 mRNA values were normalized to their corresponding 18S measurements.
Table 1. Sense and antisense primers used for quantification of DGAT-1 and DGAT-2 mRNA abundance in the liver of study animals using RT-PCR
Measurement of diacylglycerol. Hepatic microsomal preparation was extracted by a modification of the method described by Ikeda et al. ( 9 ). Briefly, samples (125-µg protein equivalent) were extracted with 3 ml of chloroform/methanol (1:2, vol/vol) followed by addition of 0.8 ml of 1 M NaCl. The monophase was then mixed with 1 ml of chloroform and 1 ml of 1 M NaCl to separate phases. The mixture was centrifuged at 5,000 g for 2 min, and the lower chloroform phase was collected and analyzed for quantification of diacylglycerol (DAG) by the radioenzymatic assay using [ 32 P- ]ATP and an assay kit (DAG Biotrack assay reagent system, Amersham, Piscataway, NJ). 32 P-labeled phosphatidic acid was separated by liquid chromatography using Amersham Amprep minicolumns, and the eluates were analyzed by scintillation chromatography.
Data analysis. Student's t -test and regression analysis were used for statistical analysis of the data, which are presented as means ± SE. P values <0.05 were considered significant.
RESULTS
General data. Data are shown in Table 2. As expected, the CRF group showed a significant rise in serum creatinine concentration, a marked reduction in creatinine clearance, a moderate increase in urinary protein excretion, and a mild reduction of serum albumin level. This was associated with a fourfold rise in serum triglycerides and VLDL concentration, a twofold increase in free fatty acid, total cholesterol and LDL cholesterol concentrations, as well as a 50% increase in total cholesterol-to-HDL cholesterol ratio.
Table 2. Plasma concentrations of creatinine, albumin, triglycerides, free fatty acid, VLDL and LDL, total cholesterol-to-HDL-cholesterol ratio, urinary protein excretion, and creatinine clearance in rats with chronic renal failure and sham-operated control group
DGAT and DAG data. Representative and group data are shown in Figs. 1 - 3. The CRF group showed a significant reduction in liver tissue DGAT-2 mRNA abundance compared with that found in the control group. However, DGAT-1 mRNA abundance was unchanged in the liver of CRF animals. Downregulation of hepatic DGAT-2 gene expression was accompanied by a parallel reduction in DGAT enzymatic activity in the CRF animals. Hepatic microsomal concentration of DAG, the DGAT substrate, was higher in the CRF group than in the control animals. However, the difference did not attain statistical significance. A significant inverse correlation was found between hepatic microsomal DAG concentration and DGAT enzymatic activity ( r = 0.65, P 0.05).
Fig. 1. Representative RT-PCR and group data depicting hepatic tissue diglycerol acyltransferase (DGAT)-1 mRNA and 18S abundance in chronic renal failure (CRF) and sham-operated control (CTL) rats ( n = 6/group). P = not significant (NS).
Fig. 3. Hepatic tissue microsomal DGAT enzymatic activity ( A ) and diacylglycerol (DAG) concentration ( B ) in rats with CRF and CTL rats ( n = 6/group). * P < 0.05.
DISCUSSION
Fatty acids are a major fuel for energy production in muscle and long-term energy storage in adipose tissue. They are transported from the sites of absorption (intestine) and endogenous production (liver) as triglycerides by chylomicrons and VLDL particles, respectively. Accordingly, triglyceride biosynthesis is critical for delivery and metabolism of lipid fuel. The final step in triglyceride biosynthesis is catalized by DGAT, which is an endoplasmic reticulum membrane-associated enzyme. DGAT catalyzes production of triglycerides by covalently joining a long-chain fatty acyl-CoA to DAG ( 8 ). In most cells, DAG required for this reaction is primarily derived from the glycerol-phosphate pathway. This involves sequential acylation of glycerol phosphate at sn-1 and sn-2 positions followed by dephosphorylation of the resulting phosphatidate ( 12 ). In addition, in certain cells, including enterocytes and adipocytes, DAG is produced by monoglycerol acyltransferase ( 2, 6, 10 ) ( Fig. 4 ).
Fig. 4. Main pathways of triglyceride biosynthesis.
Although existence of DGAT activity has been known for some time ( 27 ) and the enzyme had been partially purified ( 1, 16 ), until recently the genes encoding DGAT were not identified. Cases et al. ( 4 ) were the first to identify mouse cDNA for DGAT, which was subsequently termed DGAT-1. Cells infected with the virus harboring the DGAT-1 gene were shown to produce a 498-amino acid, 47-kDa protein with exclusive substrate specificity for DAG and acyl-CoA. DGAT-1 is a member of the ACAT-DGAT gene family. It has 20% sequence homology with ACAT, which uses sterol as opposed to diglycerol as the acyl acceptor. The enzyme is expressed in all human and mouse tissues ( 4, 8 ). Although DGAT-1 is most abundant in the small intestine, it is not entirely essential for intestinal absorption of triglycerides or production of chylomicrons, as these processes are not severely impaired in DGAT-1-deficient mice ( 3 ). Moreover, while exhibiting reduced body fat, resistance to diet-induced obesity, and lactation defect, DGAT-1 knockout mice have normal plasma triglyceride concentration and abundant adipocyte triglyceride content ( 20 ). These observations pointed to the existence of other DGAT's and led to the identification of DGAT-2, which bears no sequence homology with DGAT-1. DGAT-2 is abundantly expressed in the liver and white fat but, unlike DGAT-1, is minimally expressed in the small intestine ( 5 ). However, its expression is upregulated in the intestine of DGAT-1-deficient mice ( 3 ). Based on its cDNA sequence, DGAT-2 is expected to encode a 388-amino acid protein with an approximate molecular mass of 44.5 kDa ( 5 ). Relative preservation of intestinal absorption in DGAT-1 knockout mice is due, in part, to compensatory upregulation of DGAT-2 and monoglycerol acyltransferase ( 3 ).
In an attempt to explore the effect of CRF on triglyceride biosynthesis, we studied the gene expression of DGAT-1 and DGAT-2 in the liver of CRF and sham-operated controls. While DGAT-1 mRNA abundance was unchanged, DGAT-2 mRNA abundance was significantly reduced in the liver of CRF animals. The reduction in hepatic tissue DGAT-2 mRNA abundance was coupled with a parallel reduction in total DGAT enzymatic activity in the CRF group. Because DGAT-1 expression was unchanged, the reduction of DGAT activity must be due to the observed downregulation of liver-specific DGAT-2 in our CRF animals. Reduction of hepatic DGAT-2 expression and DGAT activity in the CRF animals was accompanied by a mild elevation of microsomal DAG concentration. The latter observation is consistent with diminished hepatic DGAT activity in the CRF animals. These findings suggest that triglyceride biosynthetic capacity is significantly reduced in CRF liver. Accordingly, hypertriglyceridemia and triglyceride enrichment of apoB-containing lipoproteins in CRF appear to be due exclusively to its impaired clearance and catabolism, as opposed to increased endogenous triglyceride production. The underlying mechanism responsible for downregulation of hepatic DGAT-2 and DGAT activity in CRF is presently unknown. Nonetheless, this phenomenon may represent an adaptive response to the CRF-induced reduction of triglyceride catabolism/clearance.
In conclusion, CRF results in significant downregulation of liver DGAT-2 expression and DGAT activity, pointing to reduced hepatic triglyceride biosynthetic capacity.
Fig. 2. Representative RT-PCR and group data depicting hepatic tissue DGAT-2 mRNA and 18S abundance in CRF and CTL rats ( n = 6/group). * P < 0.05.
【参考文献】
Andersson M, Wettesten M, Boren J, Magnusson A, Sjoberg A, Rustaeus S, and Olofsson SO. Purification of diacylglycerol:acyltransferase from rat liver to near homogeneity. J Lipid Res 35: 535-545, 1994.
Bell RM and Coleman RA. Enzymes of glycerolipid synthesis in eukaryotes. Annu Rev Biochem 49: 459-487, 1980.
Buhman KK, Smith SJ, Stone SJ, Repa JJ, Wong JS, Knapp FF Jr, Burri BJ, Hamilton RL, Abumrad NA, and Farese RV Jr. DGAT1 is not essential for intestinal triacylglycerol absorption or chylomicron synthesis. J Biol Chem 277: 25474-25479, 2002.
Cases S, Smith SJ, Zheng YW, Myers HM, Lear SR, Sande E, Novak S, Collins C, Welch CB, Lusis AJ, Erickson SK, and Farese RV Jr. Identification of a gene encoding an acyl-CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis. Proc Natl Acad Sci USA 95: 13018-13023, 1998.
Cases S, Stone SJ, Zhou P, Yen E, Tow B, Lardizabal KD, Voelker T, and Farese RV Jr. Cloning of DGAT2: a second mammalian diacylglycerol acyltransferase, and related family members. J Biol Chem 276: 38870-38876, 2001.
Coleman RA. Diacylglycerol acyltransferase and monoacylglycerol acyltransferase from liver and intestine. Methods Enzymol 209: 98-104, 1992.
Eddy AA. Interstitial fibrosis in hypercholesterolemic rats: role of oxidation, matrix synthesis, and proteolytic cascades. Kidney Int 53: 1182-1189, 1998.
Farese RV Jr, Cases S, and Smith SJ. Triglyceride synthesis: insights from the cloning of diacylglycerol acyltransferase. Curr Opin Lipidol 11: 229-234, 2000.
Ikeda Y, Olsen GS, Ziv E, Hansen LL, Busch AK, Hansen BF, Shafrir E, and Mosthaf-Seedorf L. Cellular mechanism of nutritionally induced insulin resistance in Psammomys obesus : overexpression of protein kinase C in skeletal muscle precedes the onset of hyperinsulinemia and hyperglycemia. Diabetes 50: 584-592, 2001.
Jamdar SC and Cao WF. Properties of monoglycerol acyltransferase in rat adipocytes. Arch Biochem Biophys 296: 419-425, 1992.
Klin M, Smogorzewski M, Ni Z, Zhang G, and Massry SG. Abnormalities in hepatic lipase in chronic renal failure: role of excess parathyroid hormone. J Clin Invest 97: 2167-2173, 1996.
Lehner R and Kuksis A. Biosynthesis of triacylglycerols. Prog Lipid Res 35: 169-201, 1996.
Liang K, Oveisi F, and Vaziri ND. Role of secondary hyperparathyroidism in the genesis of hypertriglyceridemia and VLDL receptor deficiency in chronic renal failure. Kidney Int 53: 626-630, 1998.
Liang K and Vaziri ND. Upregulation of acyl-CoA:cholesterol acyltransferase (ACAT) in chronic renal failure. Am J Physiol Endocrinol Metab 283: E676-E681, 2002.
Mathur S, Devaraj S, and Jialal I. Accelerated atherosclerosis, dyslipidemia, and oxidative stress in end-stage renal disease. Curr Opin Nephrol Hypertens 11: 141-147, 2002.
Polokoff MA and Bell RM. Solubilization, partial purification and characterization of rat liver microsomal diacylglycerol acyltransferase. Biochim Biophys Acta 818: 129-142, 1980.
Sahadevan M and Kasiske BL. Hyperlipidemia in kidney disease: causes and consequences. Curr Opin Nephrol Hypertens 11: 323-329, 2002.
Sato T, Liang K, and Vaziri ND. Dietary protein restriction and oral absorbent, AST-120 ameliorates lipoprotein lipase, hepatic lipase and VLDL receptor deficiencies in rats with focal glomerulosclerosis. Kidney Int 64: 1780-1786, 2003.
Sato T, Liang K, and Vaziri ND. Downregulation of lipoprotein lipase and VLDL receptor in rats with focal glomerulosclerosis. Kidney Int 61: 157-162, 2002.
Smith SJ, Cases S, Jensen DR, Chen HC, Sande E, Tow B, Sanan DA, Raber J, Eckel RH, and Farese RV Jr. Obesity resistance and multiple mechanisms of triglyceride synthesis in mice lacking Dgat. Nat Genet 25: 87-90, 2000.
Vaziri ND, Kim CH, Phan D, Kim S, and Liang K. pregulation of hepatic acyl-CoA:diaclyglycerol acyltransferase (DGAT) expression in nephrotic syndrome. Kidney Int. In press.
Vaziri ND and Liang K. Downregulation of tissue lipoprotein lipase expression in experimental chronic renal failure. Kidney Int 50: 1928-1935, 1996.
Vaziri ND and Liang K. Downregulation of VLDL receptor expression in chronic experimental renal failure. Kidney Int 51: 913-919, 1997.
Vaziri ND, Liang K, and Parks JS. Acquired lecithin-cholesterol acyltransferase deficiency in nephrotic syndrome. Am J Physiol Renal Physiol 280: F823-F829, 2001.
Vaziri ND, Liang K, and Parks JS. Downregulation of hepatic lecithin: cholesterol acyltransferase gene expression in chronic renal failure. Kidney Int 59: 2192-2196, 2001.
Vaziri ND, Wang XQ, and Liang K. Secondary hyperparathyroidism downregulates lipoprotein lipase expression in chronic renal failure. Am J Physiol Renal Physiol 273: F925-F930, 1997.
Weiss SB, Kennedy EP, and Kiyasu JY. The enzymatic synthesis of triglycerides. J Biol Chem 235: 40-44, 1960.
Zhu D, Ganji SH, Kamanna VS, and Kashyap ML. Effect of gemfibrozil on apolipoprotein B secretion and diacylglycerol acyltransferase activity in human hepatoblastoma (HepG2) cells. Atherosclerosis 164: 221-228, 2002.
作者单位：Division of Nephrology and Hypertension, University of California, Irvine, California 92697