Polyhormonal regulation of avian and mammalian corticosteroidogenesis in vitro

1. The combined actions of ACTH, corticosterone and prolactin (PRL) in the acute regulation of corticosteroidogenesis were investigated using isolated adrenocortical cells from intact and hypophysectomized (hypox) rats (Rattus norvegicus) and from intact male domestic fowl (Gallus gallus domesticus). 2. Exogenous corticosterone suppressed to about 50% ACTH-induced corticosterone production of cells from either species. This suppression, in part, was due to corticosterone degradation. 3. oPRL, in the presence or absence of ACTH, raised corticosterone production of hypox rat cells, but not intact rat and domestic fowl cells. 4. In addition, oPRL counteracted the corticosterone-induced suppression of net ACTH-stimulated corticosterone production of hypox rat and intact domestic fowl cells, but not intact rat cells. 5. The potency of oPRL with domestic fowl cells was 4 times that with hypox rat cells. 6. Furthermore, in domestic fowl cells, the effect of oPRL was Ca2+-dependent.     

Role of the hepatic ABCA1 transporter in modulating intrahepatic cholesterol and plasma HDL cholesterol concentrations

The current model for reverse cholesterol transport proposes that HDL transports excess cholesterol derived primarily from peripheral cells to the liver for removal. However, recent studies in ABCA1 transgenic mice suggest that the liver itself may be a major source of HDL cholesterol (HDL-C). To directly investigate the hepatic contribution to plasma HDL-C levels, we generated an adenovirus (rABCA1-GFP-AdV) that targets expression of mouse ABCA1-GFP in vivo to the liver. Compared with mice injected with control AdV, infusion of rABCA1-GFP-AdV into C57Bl/6 mice resulted in increased expression of mouse ABCA1 mRNA and protein in the liver. ApoA-I-dependent cholesterol efflux was increased 2.6-fold in primary hepatocytes isolated 1 day after rABCA1-GFP-AdV infusion. Hepatic ABCA1 expression in C57Bl/6 mice (n = 15) raised baseline levels of TC, PL, FC, HDL-C, apoE, and apoA-I by 150-300% (P < 0.05 all). ABCA1 expression led to significant compensatory changes in expression of genes that increase hepatic cholesterol, including HMG-CoA reductase (3.5-fold), LDLr (2.1-fold), and LRP (5-fold) in the liver. These combined results demonstrate that ABCA1 plays a key role in hepatic cholesterol efflux, inducing pathways that modulate cholesterol homeostasis in the liver, and establish the liver as a major source of plasma HDL-C.     

Transfer of excess cholesterol from human red blood cells to plasma in vitro

This study examined the ability of plasma and plasma fractions from normolipidaemic subjects and plasma from a patient with homozygous familial high density lipoprotein deficiency (Tangier disease) to promote loss of excess cholesterol from red blood cells in vitro. Isolated high density lipoproteins were the most potent plasma fraction for removing excess cellular cholesterol. Lipoprotein-deficient plasma and human serum albumin, but not very low density lipoproteins and low density lipoproteins, also removed excess cholesterol from the red blood cells. The near absence of high density lipoproteins in plasma from the patient with Tangier disease did not result in an abnormally low rate of cholesterol loss from the enriched red blood cells. These results suggest that normal levels of high density lipoproteins are not vital for the removal of excess cholesterol from red blood cells by plasma.     

Role of seminal plasma in damage to turkey spermatozoa during in vitro storage

In vitro storage of turkey spermatozoa is performed without consideration of the potential role of seminal plasma on sperm functions. We report the effects of seminal plasma on membrane permeability, lipid metabolism, energy status, motility and fertility of turkey spermatozoa stored at 4 or 20 degrees C. Phospholipid content (1077 nmol/10(9) spz versus 1219 nmol/10(9) spz at 48 h) and membrane permeability of spermatozoa were significantly damaged by the presence of seminal plasma after 48 h of storage at 4 degrees C, whereas damage to ATP content and fertility occurred earlier damaged by this presence (fertility after 24h storage 51% with seminal plasma versus 71% without). At 20 degrees C, seminal plasma decreased the phospholipid content of spermatozoa in the first hour of storage (1326 nmol/10(9) spz versus 1636 nmol/10(9) spz). Twenty-four hours later, this effect was masked by intense lipid peroxidation. These results show that seminal plasma is deleterious to storage of turkey spermatozoa at 4 degrees C and is involved in phospholipid metabolism of spermatozoa. Lipid peroxidation could be responsible for the acceleration of the degradation of sperm phospholipids during storage at 20 degrees C. However, lipid peroxidation seems not to be active at 4 degrees C. In this case, we suggest that phospholipase activation may contribute to sperm degradation, especially in the presence of seminal plasma.     

Regulation of endoplasmic reticulum cholesterol by plasma membrane cholesterol

The abundance of cell cholesterol is governed by multiple regulatory proteins in the endoplasmic reticulum (ER) which, in turn, are under the control of the cholesterol in that organelle. But how does ER cholesterol reflect cell (mostly plasma membrane) cholesterol? We have systematically quantitated this relationship for the first time. We found that ER cholesterol in resting human fibroblasts comprised approximately 0.5% of the cell total. The ER pool rose by more than 10-fold in less than 1 h as cell cholesterol was increased by approximately 50% from below to above its physiological value. The curve describing the dependence of ER on plasma membrane cholesterol had a J shape. Its vertex was at the ambient level of cell cholesterol and thus could correspond to a threshold. A variety of class 2 amphiphiles (e.g., U18666A) rapidly reduced ER cholesterol but caused only minor alterations in the J-curve. In contrast, brief exposure of cells to the oxysterol, 25-hydroxycholesterol, elevated and linearized the J-curve, increasing ER cholesterol at all values of cell cholesterol. This finding can explain the rapid action of oxysterols on cholesterol homeostasis. Other functions have also been observed to depend acutely on the level of plasma membrane cholesterol near its physiological level, perhaps reflecting a cholesterol-dependent structural or organizational transition in the bilayer. Such a physical transition could serve as a set-point above which excess plasma membrane cholesterol is transported to the ER where it would signal regulatory proteins to down-regulate its further accumulation.     

Rapid labeling of lipoproteins in plasma with radioactive cholesterol. Application for measurement of plasma cholesterol esterification

In order to efficiently and rapidly label lipoproteins in plasma with [3H]cholesterol, micelles consisting of lysophosphatidylcholine (lysoPC) and [3H]cholesterol (molar ratio, 50:1) were prepared. When trace amounts of these micelles were injected into plasma, [3H]cholesterol rapidly equilibrated among the plasma lipoproteins, as compared to [3H]cholesterol from an albumin-stabilized emulsion. The distributions of both [3H]cholesterol and unlabeled free cholesterol in plasma lipoproteins were similar in labeled plasma samples. This method of labeling can be used for the measurement of cholesterol esterification, or lecithin:cholesterol acyltransferase activity, in small amounts (20-40 microliters) of plasma samples.     

Essential fatty acid deficiency and cholesterol esterification activity of plasma and liver in vitro and in vivo

The esterification of cholesterol in vitro and in vivo was studied in rats fed diets containing and deficient in essential fatty acids (EFA). EFA deficiency resulted in increased cholesterol esterification activity in vitro, and this appeared to reflect an increased plasma cholesterol esterification activity in vitro, and this appeared to reflect an increase in enzyme concentrations. There was a decrease in plasma lipid phosphorus concentrations during the period of cholesterol esterification. Certain aspect of the fatty acid compositions of plasma phospholipids and cholesterol esters were consistent with a role of phospholipid fatty acids in the transesterification reaction which has been demonstrated by Glomset et al. (18). There was heterogeneous labeling of different subclasses of cholesterol esters when cholesterol-4-C¹⁴ was used as the substrate, the more unsaturated fatty acid esters having the highest relative specific activities. EFA deficiency resulted in decreased cholesterol esterification activity by hepatocytic organelles. This activity was also reduced when organelles were exposed to hypotonic media, ultrasound, or taurocholate. Rats deficient in EFA had increased rates of esterification of cholesterol-4-C¹⁴ in plasma and increased rates of elimination of radiocholesterol from the plsama after intravenous injection of labeled lipoproteins. This was associated with higher concentrations of total radioactivity in the livers of the EFA deficient rats. The specific activities of individual subclasses of cholesterol esters of plasma and liver were also heterogeneous for several hours in these in vivo experiments regardless of the dietary regimen involved. The more unsaturated fatty acid esters had the highest initial specific activities. Although liver and plasma free cholesterol specific activities rapidly equilibrated, the specific activities of the cholesterol esters and individual subclasses of cholesterol esters of plasma were greater than the activities of corresponding fractions from liver. It was proposed that the differences in plasma and liver esterification activities induced by EFA deficiency were secondary to alterations in the stability of hepatocytic organelles.     

Origin and fate of rat plasma cholesterol in vivo. Modelling of cholesterol movements between plasma and organs

A cholesterol system model was developed in the rat following a single injection of red cells containing free (unesterified) [3H]cholesterol. The radioactivity of free and esterified cholesterol in the different parts of the system was measured during the 48 h following tracer introduction. The model consisted of seven compartments (red cell free cholesterol, plasma and liver free and esterified cholesterol, total cholesterol in the rapidly and slowly exchangeable carcass pools). The model was validated by the similarity between simulated and experimental values during the 48 h following tracer introduction. Both the fractional rate of cholesterol esterification in the plasma (0.44 h-1) and liver (0.01 h-1) and the fractional exchange rate of free cholesterol from the plasma towards the various organs (particularly 3 h-1 towards the liver for a total of 7 h-1) can be estimated with this model. The results show that cholesterol movements between the plasma and the different organs take place mainly through intense free cholesterol exchanges, resulting in a low net flux.     

Measurement of cholesterol gallstone growth in vitro

Methods to study growth of gallstones in the laboratory have not been reported. We here present such a method. Human cholesterol gallstones were harvested from patients with multiple nearly identical stones. The gallstones were washed and added to supersaturated model biles and the formation of cholesterol crystals and the increases in mass of human cholesterol gallstones were studied concurrently, over a period of weeks, using nephelometry and a microbalance, respectively. All stones incubated in model biles supersaturated with cholesterol increased in mass. Increases in the degree of supersaturation of cholesterol in the model biles resulted in increased growth of stones. The mass increases, the growth rates, and the spatial orientation of accreted crystalline cholesterol differed among various stone types. The kinetics and structures of stone growth were similar when the stones were incubated in supersaturated, native, human gallbladder biles. The structure of accreted cholesterol was the same as found on the surface of some human gallstones that were harvested during apparent active growth in situ. This simple method allows accurate measurements of stone growth in vitro, in patterns that mimic stone growth in vivo, and is useful for studies on the relationships of gallstone growth and the kinetics of cholesterol crystallization.