Christine K. Castle

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)

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.

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.

Apo A-I metabolism in cynomolgus monkeys: male-female differences.

Female cynomolgus monkeys have significantly higher plasma apo A-I concentrations than males (P = 0.04) and are able to maintain higher levels than the males even after consuming a high-cholesterol diet that severely depresses the apo A-I concentration in primates (P less than 0.05). The mechanism responsible for this difference was investigated by comparing apo A-I turnover (synthesis and catabolism) in males and females consuming monkey chow and in a separate group of males and females that had consumed the high-cholesterol diet for several weeks. The average length of time an apo A-I molecule remained in the plasma compartment of chow-fed monkeys was 2.62 days but decreased to 1.52 days (P less than 0.01) in animals fed the HC diet. There were no male-female differences in the residence times. The absolute turnover rate (mg/day) of apo A-I was not statistically affected by diet or sex; however, the females were substantially smaller than the males (3.8 vs. 4.8 kg; P less than 0.01) and their plasma volumes were significantly smaller than those of the males, even after correction for differences in body wt. (32.6 vs. 37.0 ml/kg, respectively; P less than 0.01). Taken together, the data indicate that females cynomolgus monkeys have higher apo A-I synthesis rates than males of comparable plasma volume (P = 0.03), which we would propose accounts for the higher plasma apo A-I concentrations evident in females.

Comparison of the lipid and apolipoprotein composition of skeletal muscle and peripheral lymph in control dogs and in dogs fed a high fat, high cholesterol, hypothyroid-inducing diet

Most studies of peripheral interstitial fluid lipoprotein composition have been made on interstitial fluid-derived from skin and connective tissue. We developed techniques which allowed simultaneous comparison of lymph (a model of interstitial fluid) from skeletal muscle and skin in control (C) and cholesterol-fed (CF) dogs. Lipoprotein fractions were separated by ultracentrifugation. Skeletal muscle interstitial fluid HDL concentrations were approximately twice those of skin. However, the concentration of VLDL-LDL particles was similar in both interstitial spaces. HDL particles from both microvascular beds showed evidence of extensive remodelling when compared to plasma HDL from the same animal. Relative to apo A-I, skeletal muscle HDL was enriched in free cholesterol and apo E (C and CF dogs) and apo A-IV (CF dogs). Skin-derived HDL was consistently enriched in free cholesterol, apo E and A-IV in both C and CF dogs. These studies indicate that similar remodeling of plasma HDL occurs in widely different tissues which together constitute approximately 70% of the total interstitial space. The relatively high concentration of plasma-derived and remodeled HDL within the interstitial space of skeletal muscle is consistent with that tissues importance in reverse cholesterol transport.