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 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 development of fatty liver is accelerated in transgenic mice expressing cynomolgus monkey cholesteryl ester transfer protein.

Expression of cynomolgus monkey cholesteryl ester transfer protein (CETP) in C57BL/6 mice has been shown to have a profound effect on the lipoprotein profile in those animals. The objective of this study was to examine the effect of CETP expression on the hepatic lipids of the CETP transgenic mice. The triglyceride, cholesterol and phospholipid composition of livers from 6- and 12-month-old transgenic mice were evaluated and compared with those of age-matched C57BL/6 mice. Statistical analysis indicated that fatty liver was more severe in CETP transgenic mice than C57BL/6 controls (p < 0.01); progressed with age (p < 0.01); and developed more rapidly in males than females (p < 0.01). The lipid that accumulated was triglyceride. These data indicate that CETP expression accelerates the development of fatty liver in the C57BL/6 mouse and raise the possibility that CETP may also contribute to the process of hepatic steatosis in man.

Apolipoprotein A-I metabolism in cholesteryl ester transfer protein transgenic mice. Insights into the mechanisms responsible for low plasma high density lipoprotein levels.

Expression of simian cholesteryl ester transfer protein (CETP) in C57BL/6 mice causes the animals' high density lipoprotein (HDL) levels to decrease. The purpose of these studies was to determine how CETP expression caused that reduction. Chemical analysis showed that the HDL of the CETP transgenic mice had about twice as much triglyceride and only about 60% as much cholesteryl ester as the HDL from the C57BL/6 mice. Both strains of mouse had high levels of a circulating lipase. When plasma from the mice was incubated at 37 degrees C for 5 h, the triglycerides in the HDL were hydrolyzed, and apoA-I was shed from the particle. However, apoA-I was shed from the CETP HDL more rapidly than it was shed from the C57BL/6 HDL. Because "free" apoA-I is rapidly cleared by the kidney, increased production of free apoA-I would be expected to shorten the average life span of apoA-I in the mouse. Kinetic analyses indicated that the life span of apoA-I was significantly reduced in the CETP transgenic mice. It was concluded that CETP expression enriched the core of the HDL with triglyceride, which rendered it vulnerable to lipolysis, causing apoA-I to be shed from the particle. That shortened the life span of apoA-I in the CETP mice, which led to lower plasma levels of the protein.

Effects of epidermal growth factor on apo B mRNA levels and apo B accumulation in the media of primate hepatocytes in culture.

EGF has been shown to augment albumin and apolipoprotein A-I secretion by cynomolgus monkey hepatocytes in primary culture without stimulating cell division. This study was undertaken to determine what effect EGF had on apo B secretion by those hepatocytes. The results indicate that EGF (3 nM final concentration) severely inhibits the rate at which apo B accumulates in the culture medium of primate hepatocytes. That effect was evident within 48 hours of treatment, and by 72 hours the rate that apo B accumulated was less than half that of cells treated with a hormone-free medium. However, the apo B mRNA levels in the EGF-treated cells were more than double those of hepatocytes given the hormone-free medium. These data indicate that EGF has a potent effect on the rate at which apo B accumulates in the culture medium of primate hepatocytes and that the effect is independent of apo B gene expression.

Arachidonic acid suppression of fatty acid synthase gene expression in cultured rat hepatocytes.

Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50-500 microM albumin-bound 20:1 (n-9) vs 20:4 (n-6). The induction of fatty acid synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of beta-actin mRNA was not suppressed by 20:4 (n-6). The expression of fatty acid synthase was actually stimulated 2-fold by 20:1 (n-9). It would appear that the in vivo inhibition of fatty acid synthase gene expression by dietary polyunsaturated fatty acids is a specific hepatocelluar event.

Suppression of rat hepatic fatty acid synthase and S14 gene transcription by dietary polyunsaturated fat.

The objective of this research was to determine whether dietary polyunsaturated fatty acids suppress hepatic fatty acid synthase (FAS) mRNA levels by altering FAS gene transcription. Male Sprague-Dawley rats were meal-fed for 10 d a high glucose diet supplemented with 20% digestible energy as menhaden oil or tripalmitin. The transcription rate for FAS was determined by nuclear run-on analysis in hepatic nuclei isolated from rats 2 h postmeal. The values for transcription rates of FAS and S14 (a putative lipogenic protein) in rats fed menhaden oil were only 6 and 21%, respectively, of the rates in rats fed the tripalmitin diet (p less than 0.02). Gene transcription for beta-actin and phosphoenolpyruvate carboxykinase did not differ between treatments. The reduction in hepatic FAS mRNA levels caused by dietary polyunsaturated fats appears to be caused primarily by an inhibition of FAS transcription. The control of transcription by polyunsaturated fats appears not to be mediated by cAMP because the transcription rate for phosphoenolpyruvate carboxykinase (whose gene is very sensitive to cAMP stimulation) was unaffected by the source of dietary fat.

Induction of adipose fatty acid binding protein (a-FABP) by insulin-like growth factor-1 (IGF-1) in 3T3-L1 preadipocytes.

Murine 3T3-L1 cells were cultured in the presence of fetal bovine serum (FBS) washed with an anion exchange resin and charcoal. Using the abundance of a-FABP and fatty acid synthase (FAS) as criteria of differentiation, IGF-1 was found to be 10-fold more potent than insulin as an inducer of preadipocyte differentiation. As little as 0.5 nM IGF-1 induced expression of FAS and a-FABP mRNAs whereas a minimum of 5 nM insulin was required. The data indicate IGF-1 specifically induces the expression of a-FABP in 3T3-L1 preadipocytes whereas the effect of insulin is likely via insulin's binding to the IGF-1 receptor.

Potency of polyunsaturated and saturated fats as short-term inhibitors of hepatic lipogenesis in rats.

Three dietary studies using male Sprague-Dawley rats conditioned to meal-eat a high glucose, fat-free diet and one in vitro study with isolated rat hepatocytes were designed to examine the hypothesis that polyunsaturated fats (i.e., safflower oil or linoleate) are more potent acute inhibitors of liver fatty acid synthesis than are saturated fats (i.e., beef tallow or palmitate). Fat in the first in vivo study was administered via intubation (1500 mg/rat) whereas in the second and third in vivo studies fat was added to the meal in amounts of 50, 100, 250 or 500 mg/g fat-free diet. When the rats were in a postprandial condition, significant suppression of hepatic lipogenesis required the meal to contain 38% of its energy as fat (i.e., 250 mg/g fat-free diet). At this level of fat, safflower oil was more inhibitory than beef tallow (p less than 0.05). The inhibition constant (Ki) for palmitate inhibition of fatty acid synthesis by isolated hepatocytes was fourfold greater than linoleate's Ki (fatty acid/albumin ratio of 1.4/1). When fat constituted 50% of the ingested energy, beef tallow was equivalent to safflower oil as an inhibitor of lipogenesis. Although a single meal containing 50 mg safflower oil/g fat-free diet did not decrease fatty acid synthesis, it effectively delayed the induction of lipogenesis during the first 30 min of the adaptive decrease in lipogenic enzymes attributed to polyunsaturated fats extends to short-term regulatory mechanisms.

Growth hormone acutely increases glucose output by hepatocytes isolated from hypophysectomized rats.

A series of experiments using isolated rat hepatocytes was carried out to establish rat liver cells in suspension as a physiological model for examining GH responses, and to determine whether acute recombinant bovine GH (rbGH) treatment of rat liver cells increased glucose output and/or suppressed fatty acid synthesis from lactate. Rat liver cells were isolated by collagenase perfusion and incubated in short-term (less than 60 min) suspension. The amount of insulin, glucagon or vasopressin required to elicit a half-maximal response was within the physiological range of the circulating hormone. When hepatocytes from normal rats were acutely (less than 60 min) treated with 0, 0.1, 10, 100 or 1000 nmol rbGH/l, rates of hepatocyte glucose output and fatty acid synthesis were unaltered. In addition, acute rbGH treatment (1000 nmol/l) did not alter hepatocyte responsiveness to insulin or vasopressin. However, acute rbGH treatment of hepatocytes isolated from hypophysectomized rats significantly (P less than 0.05) increased the rate of glucose output twofold and moderately (P less than 0.10) enhanced fatty acid synthesis. The accelerated rate of glucose production was not accompanied by an increase in the amount of glycogen phosphorylase-a. The observations with liver cells from hypophysectomized rats are not consistent with a GH receptor-transducing mechanism which is like that for glucagon (adenylate cyclase-linked) or insulin (tyrosine kinase-linked).

Modification of fatty acid composition of membrane phospholipid in hepatocyte monolayer with n-3, n-6 and n-9 fatty acids and its relationship to triacylglycerol production.

The objective of these studies with rat hepatocytes in primary culture was to establish that: (a) membrane phospholipids would become enriched with the specific fatty acid supplemented to the media and (b) hepatocyte monolayer triacylglycerol synthetic rates were dependent on the type of fatty acid enrichment of the membrane phospholipids. Hepatocytes cultured in the absence of media lipid developed a phospholipid fatty acid composition which is indicative of an essential fatty acid deficiency. The extensive rise in 18:1(n - 9) content indicated that delta 9-desaturase was active. The fatty acid composition of phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol in the microsomal- and mitochondrial-enriched fractions was highly dependent upon the type of fatty acid supplemented to the medium. Incorporation of fatty acids into phospholipids was rapid, and a new steady-state in fatty acid composition was achieved within approx. 36 h. Changes in the fatty acid composition of these hepatocyte phospholipid subclasses resulting from media supplementation with 18:2/20:4(n-6) or 20:5(n-3) were similar, but not identical, to changes which occurred in vivo as a result of consuming diets rich in 18:2(n-6) or 20:5(n-3). Hepatocyte lipogenesis was highly dependent upon the type of fatty acid supplemented to the medium. Prior conditioning with 16:0 increased triacylglycerol synthesis and secretion. Secretion of triacylglycerol was reduced by polyenoic fatty acid enrichment with 20:5(n-3) greater than 20:4/18:2(n-6). The suppression of triacylglycerol synthesis by 20:5(n-3) was due to an increased (P less than 0.05) diacylglycerol specific activity, which indicates that 20:5(n-3) suppression of hepatic triacylglycerol production may be caused in part by the inhibition of diacylglycerol acyltransferase.