George W. Melchior

The role of cholesteryl ester transfer protein in primate apolipoprotein A-I metabolism. Insights from studies with transgenic mice.

To assess the effects of cholesteryl ester transfer protein (CETP) on the primate lipoprotein profile, a transgenic mouse expressing cynomolgus monkey CETP was developed. The C57BL/6 mouse was used, and four lines expressing the primate CETP were established. The level of CETP activity in the plasma of the transgenic mice ranged from values similar to those obtained for the monkey to levels approximately sixfold higher than that in the normal monkey. When all of the lines were taken into consideration, there was a strong (r = -0.81 or higher, p less than 0.01) negative correlation between plasma CETP activity and total plasma cholesterol, plasma apolipoprotein (apo) A-I levels, and plasma apo A-I to apo B ratio. There was a strong positive correlation (r = 0.77) between plasma CETP activity and plasma apo B levels. The size of the apo A-I-containing lipoproteins was significantly reduced in mice with high plasma CETP activity, and that reduction in size was due to the absence of the larger (HDL1 and HDL2) apo A-I-containing particles in the plasma. When the transgenic mice were fed a high-fat, high-cholesterol diet, the effects of the diet on lipoprotein profile were more prominent in the CETP transgenic mice than the controls. The CETP transgenic mice had, for example, substantially higher plasma cholesterol and plasma apo B levels (p less than 0.01), and the apo B-containing lipoproteins were generally larger than those in the nontransgenic C57BL/6 mice consuming the same diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular cloning, sequence, and expression of cynomolgus monkey cholesteryl ester transfer protein. Inverse correlation between hepatic cholesteryl ester transfer protein mRNA levels and plasma high density lipoprotein levels.

A cDNA clone containing the coding region for cynomolgus monkey cholesteryl ester transfer protein (CETP) was isolated by the polymerase chain reaction with primers based on the human CETP cDNA sequence and cDNA synthesized from liver poly (A+) RNA. Analysis of that cDNA indicated that the nucleotide and amino acid sequences of cynomolgus monkey CETP were greater than 95% homologous with the human sequences. A fragment of the cDNA was used to develop an internal-standard/RNAse protection assay that allowed precise quantification of CETP mRNA levels. Analysis of total RNA from various tissues with this assay revealed that the liver and thoracic aorta expressed high levels of CETP mRNA; the mesenteric fat, adrenal gland, spleen, and abdominal aorta had low but detectable levels of the mRNA; and the brain, kidney, intestine, and skeletal muscle had undetectable levels of that mRNA. When the monkeys were made hypercholesterolemic by a high-fat, high-cholesterol (HFHC) diet, hepatic levels of CETP mRNA increased from 1.6 +/- 0.4 pg/micrograms total RNA (mean +/- SEM) to 4.1 +/- 0.8 pg/micrograms (p less than 0.005); mesenteric fat CETP mRNA increased from 0.4 +/- 0.1 pg/micrograms total RNA to 5.3 +/- 2.2 pg/micrograms (p less than 0.05); and plasma CET activity increased approximately fourfold. The CETP mRNA levels in the thoracic and abdominal aortas were not significantly increased in monkeys fed the HFHC diet, even though those animals had gross atherosclerosis. The apoprotein E mRNA levels, however, were markedly increased in the aortas of monkeys with atherosclerosis, with the largest increase occurring in the abdominal aorta. Taken together, these data suggest that lipid deposition in the artery was not accompanied by increased expression of the CETP gene in that tissue. Statistical analysis showed that a strong, negative correlation existed between hepatic CETP mRNA levels and both high density lipoprotein cholesterol (r = -0.85, p less than 0.001) and apoprotein A-I (r = -0.84, p less than 0.001). These data suggest that HFHC diet-induced changes in high density lipoprotein metabolism may be linked to altered expression of a function CETP gene.

Lipoprotein profile characterization of the KKA(y) mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing agent pioglitazone.

The purpose of this study was to characterize the lipoprotein profile in the KKA(y) mouse, a rodent model of type II diabetes, before and after treatment with the insulin-sensitizing drug pioglitazone. Analysis of the plasma from untreated KKA(y) mice showed that they were severely hyperglycemic, severely hypertriglyceridemic, and moderately hypercholesterolemic. Agarose column chromatographic analysis showed that essentially all of the triglyceride eluted with very low density lipoprotein, and the majority of the cholesterol eluted with high density lipoprotein. Thus, both the very low density lipoprotein and high density lipoprotein levels were markedly elevated in KKA(y) mice. Analysis of the lipoproteins by agarose electrophoresis-immunoblotting showed that apoprotein A-I and apoprotein B had aberrant electrophoretic behavior, typical of apoproteins that have been modified by nonenzymatic glycosylation. Treatment of KKA(y) mice with pioglitazone for 8 days caused a marked reduction in blood glucose and plasma triglyceride concentrations but had no significant effect on plasma cholesterol concentration or distribution. The aberrant electrophoretic behavior of the apoproteins was corrected to normal by drug treatment. These data show that the KKAy mouse has a severe dyslipoproteinemia that is probably secondary to its insulin resistance, but that its lipoprotein profile differs significantly from that of the insulin-resistant human in that the majority of the plasma cholesterol is carried in high density lipoprotein, and those high density lipoprotein levels are very high.

Cryopreservation of cynomolgus monkey (Macaca fascicularis) hepatocytes for subsequent culture and protein synthesis studies.

SummaryA method is described for the preservation and subsequent recovery of hepatocytes obtained by collagenase perfusion of cynomolgus monkey (Macaca, fascicularis) livers. The fresh cells are suspended in fetal bovine serum containing 10% dimethylsulfoxide and, using a microprocessor-controlled, liquid nitrogen freezing chamber and a specific cooling protocol, processed in such a way that they can be stored in liquid nitrogen for several months and still restored to active culture. When the cryopreserved cells were established in culture they were found to actively synthesize and secrete both albumin and apolipoprotein A-I. That, taken together with morphologic evidence, was viewed as indication that the cells recovered in culture were in fact hepatocytes and not some other cell type from the monkey liver. The availability of this procedure for storing hepatocytes should contribute significantly to the efficient use of nonhuman primates as models with which to study hepatic metabolism.

Apo B metabolism in the cynomolgus monkey: evidence for post-transcriptional regulation

Previous studies have shown that hepatic apo B mRNA levels do not increase in animals fed high cholesterol diets, even though plasma apo B concentrations increase markedly. As a result, it has been suggested that the diet-induced increase in plasma apo B levels was due solely to an inhibited clearance of those lipoproteins. The present study was undertaken to test that hypothesis. Hepatic apo B mRNA levels were measured in liver biopsies taken from five male cynomolgus monkeys before and twice after, they began to consume a high cholesterol diet. The diet had no effect on hepatic apo B mRNA levels, even though it caused a 7-fold increase in the plasma apo B levels. However, measurements of the apo B secretion rate in eight separate monkeys (four chow-fed and four cholesterol-fed) by isotope dilution showed that apo B secretion by the liver was increased 4-fold in the cholesterol-fed monkeys. These data, taken together, indicate that apo B secretion is not regulated by the rate at which the apo B gene is transcribed, but at some point further along in the secretion pathway.

Remodeling of the HDL in NIDDM: a fundamental role for cholesteryl ester transfer protein

When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAymouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 ± 25 to 25 ± 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 ± 3 to 22 ± 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 ± 2 to 60 ± 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.When the Ay gene is expressed in KK mice, the yellow offspring (KKAy mice) become obese, insulin resistant, hyperglycemic, and severely hypertriglyceridemic, yet they maintain extraordinarily high plasma high-density lipoprotein (HDL) levels. Mice lack the ability to redistribute neutral lipids among circulating lipoproteins, a process catalyzed in humans by cholesteryl ester transfer protein (CETP). To test the hypothesis that it is the absence of CETP that allows these hypertriglyceridemic mice to maintain high plasma HDL levels, simian CETP was expressed in the KKAy mouse. The KKAy-CETP mice retained the principal characteristics of KKAy mice except that their plasma HDL levels were reduced (from 159 +/- 25 to 25 +/- 6 mg/dl) and their free apolipoprotein A-I concentrations increased (from 7 +/- 3 to 22 +/- 6 mg/dl). These changes appeared to result from a CETP-induced enrichment of the HDL with triglyceride (from 6 +/- 2 to 60 +/- 18 mol of triglyceride/mol of HDL), an alteration that renders HDL susceptible to destruction by lipases. These data support the premise that CETP-mediated remodeling of the HDL is responsible for the low levels of that lipoprotein that accompany hypertriglyceridemic non-insulin-dependent diabetes mellitus.

The primary structure of cynomolgus monkey apolipoprotein A-1 deduced from the cDNA sequence: comparison to the human sequence.

We have cloned and analyzed a cDNA containing the complete coding sequence for cynomolgus monkey apolipoprotein A-1 (apoA-1). This cDNA clone was found to share approx. 97% nucleotide sequence identity with the published human apoA-1 and encodes a protein of the same size as the human protein. Paired proline residues are present at positions 3 and 4 in the mature protein as has been reported for other primate species and the propeptide sequence is identical to the human propeptide. The amino acid content derived from the nucleotide sequence predicts a more basic protein than human apoA-1 and this was confirmed by isoelectric focusing analysis. In addition, we present evidence for two different transcriptional initiation sites for the cynomolgus monkey gene in contrast to only one for human.

Apolipoprotein A-I metabolism in cynomolgus monkey. Identification and characterization of beta-migrating pools.

Fresh plasma from control (C) and hypercholesterolemic (HC) cynomolgus monkeys was analyzed by agarose electrophoresis-immunoblotting with antibody to cynomolgus monkey apolipoprotein (apo) A-I. Two bands were evident on the autoradiogram: an alpha-migrating band (high density lipoprotein) and a beta-migrating band that comigrated exactly with cynomolgus monkey low density lipoprotein (LDL). The presence of beta-migrating apo A-I in the plasma of these monkeys was confirmed by Geon-Pevikon preparative electrophoresis, crossed immunoelectrophoresis, and isotope dilution studies in which radiolabeled apo A-I was found to equilibrate also with alpha- and beta-migrating pools of apo A-I in the plasma. Subfractionation of C and HC plasma by agarose column chromatography (Bio-Gel A-0.5M and A-15M) followed by agarose electrophoresis-immunoblotting indicated that the beta-migrating apo A-I in C was relatively homogeneous and eluted with proteins of Mr approximately 50 kD [apo A-I(50 kD)], whereas two beta-migrating fractions were identified in HC, one that eluted with the 50-kD proteins, and the other that eluted in the LDL Mr range [apo A-I(LDL)]. The apo A-I(LDL) was precipitated by antibody to cynomolgus monkey apo B. The apo A-I(50 kD) accounted for 5 +/- 1% (mean +/- SD) of the plasma apo A-I in C plasma, and 15 +/- 7% in HC plasma. No apo A-I(LDL) was detected in C plasma, but that fraction accounted for 9 +/- 7% of the apo A-I in HC plasma. These data establish the presence of multiple pools of apo A-I in the cynomolgus monkey, which must be taken into consideration in any comprehensive model of apo A-I metabolism in this species.

Purification, cloning and nucleotide sequence determination of cynomolgus monkey apolipoprotein C-11: comparison to the human sequence

We have purified apolipoprotein C-II (apo C-II) from cynomolgus monkey plasma, prepared antibody against it and used the antibody to isolate a cDNA containing the complete coding sequence for cynomolgus monkey apo C-11. Sequence analysis indicated that the monkey apo C-11 cDNA was 200 by longer than the human and the difference in size was all in the 5° untranslated region of the mRNA. This was confirmed by Northern analysis of human and monkey RNA. There was an open reading frame in the monkey apo C-11 cDNA sequence encoding a preprotein of 101 amino acids — identical in size to the human protein. The carboxyl terminal 44 amino acids of the protein were 100% homologous to the human apo C-11 amino acid sequence indicating evolutionary conservation of both structure and function. However, the amino terminal 35 amino acids of the protein were only 75% homologous and the amino terminal 19 amino acids were only 58% homologous to the human sequence. The amino acid sequence derived from the nucleotide sequence predicts a more basic protein than the human apo C-11 and this is confirmed by isoelectric focusing and immunoblotting.

Evidence That Cynomolgus Monkey Cholesteryl Ester Transfer Protein Has Two Neutral Lipid Binding Sites

Two inhibitors of cynomolgus monkey cholesteryl ester transfer protein were evaluated. One, a monoclonal antibody made against purified cynomolgus monkey cholesteryl ester transfer protein, was capable of severely inhibiting triglyceride transfer, but had a variable effect on cholesteryl ester transfer. At low antibody to antigen ratios, there was what appeared to be a stoichiometric inhibition of cholesteryl ester transfer, but at high antibody to antigen ratios the inhibition of cholesteryl ester transfer was completely relieved, even though triglyceride transfer remained blocked. Fab fragments of the antibody had no effect whatsoever on cholesteryl ester transfer, but were capable of completely blocking triglyceride transfer. The other inhibitor, 6-chloromecuric cholesterol, severely inhibited cholesteryl ester transfer with minimal inhibition of triglyceride transfer. When both inhibitors were added to the assay, both cholesteryl ester and triglyceride transfer were inhibited; an indication that the inhibitors did not compete for the same binding site on cholesteryl ester transfer protein. When the antibody was given subcutaneously to cynomolgus monkeys at a dose which inhibited triglyceride transfer in the plasma by more than 90%, there was no detectable effect on the high density lipoprotein (HDL) cholesterol level, but the HDL triglyceride levels decreased from 13 ± 2 to 1 ± 0 mol/mol of HDL (mean ± S.D.); an indication that the antibody uncoupled cholesteryl ester and triglyceride transfer in vivo. The 6-chloromecuric cholesterol could not be evaluated in vivo because it is a potent lecithin:cholesterol acyltransferase inhibitor. The fact that cholesteryl ester transfer can be inhibited without effect on triglyceride transfer and, conversely, that triglyceride transfer can be inhibited without effect on cholesteryl ester transfer indicates that these two lipids are not transferred by a single, non-discriminatory process.