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R K Ockner

Publications and source records attributed to R K Ockner.

At least 37 records · Page 2Linked to original sources

The nucleotide sequence of rat liver fatty acid binding protein mRNA.

Rat liver fatty acid binding protein mRNA is present in both liver and intestinal epithelium. It is the most abundant mRNA in the small intestinal mucosa. We have determined the sequence of this mRNA from cloned cDNA. It consists of a 5'-nontranslated domain at least 39 nucleotides long, a coding region which specifies a 127-amino acid, 14,184-dalton polypeptide, and a 3'-nontranslated region 68 nucleotides long. An AAUAAA sequence begins 21 nucleotides before the 3'-terminal poly(A) tail. The derived protein sequence was verified by automated sequential Edman degradation of (i) the primary translation product of the mRNA and (ii) a cyanogen bromide peptide generated from the mature liver protein. Evidence is presented which suggests that this protein is targeted to the cytoplasm. The NH2 terminus of the primary translation product is acetylated in vitro. No cleavable signal peptide sequence or internal signal sequence equivalent was detectable in a co-translational cleavage assay. Cloned cDNA was used to establish that this mRNA was 12 times more abundant in intestinal epithelial than in liver RNA.

Amino Acid Sequence↗

Hepatic uptake of albumin-bound substances: albumin receptor concept.

The single-pass hepatic uptake of long-chain fatty acids and other substances bound tightly to albumin in plasma is surprisingly efficient. Recent kinetic studies for several of these substances suggest that uptake is mediated primarily by direct interaction of the albumin-ligand complex with the hepatocyte surface rather than by the small fraction of unbound ligand, as has been generally believed. Furthermore, 125I-albumin has been found to bind specifically, saturably, and reversibly to isolated hepatocytes, adipocytes, and erythrocytes. Although the nature and possible regulation of these binding sites remain to be fully elucidated, the putative albumin receptor may play an important role in the bidirectional transfer of many classes of endogenous and exogenous substances between albumin and cells.

Animals↗

Fatty acid binding protein. Isolation from rat liver, characterization, and immunochemical quantification.

Fatty acid-binding protein (FABP) was identified and isolated from rat liver cytosol by gel filtration, thin layer isoelectric focusing, and affinity chromatography. FABP (Mr 12,080 +/- 80) exists in several immunochemically identical forms differing in isoelectric pH, which may in part reflect differences in their respective complements of bound endogenous ligand. FABP-bound fatty acids accounted for 60% of total cytosolic long chain fatty acids but contained no detectable phospholipid; the substantial enrichment of FABP in 18:2 and 20:4 as compared with whole liver homogenate was not influenced by homogenization of tissue in EDTA. The amino acid composition of FABP suggests that it is closely related or identical with certain similar neutral and acidic cytosolic proteins reported from other laboratories. By quantitative radial immunodiffusion, FABP concentration in cytosol from livers of sexually mature female rats exceeded that from mature males (51.7 +/- 3.0 versus 39.8 +/- 4.0 micrograms/mg of protein, p less than 0.05), confirming earlier studies in which sex steroid effects on rates of fatty acid utilization were correlated with FABP concentration as determined by means of a binding assay. The abundance of FABP, its importance in the cytosolic binding of endogenous as well as exogenous fatty acids, and its demonstrated correlation with rates of hepatocyte fatty acid utilization provide additional evidence for its relationship to the cellular metabolism of long chain fatty acids.

Aging↗

Sex differences in hepatic uptake of long chain fatty acids in single-pass perfused rat liver.

The primary determinants of hepatic uptake of long chain fatty acids have been considered to be the plasma concentrations of fatty acid and albumin, with little or no intrinsic control by the hepatocyte itself. However, recent studies of liver cell suspensions have shown that in immature, adult, castrated, and hormone-treated rats, sex steroids exert striking effects on [(14)C]oleate uptake and utilization (which were significantly increased by estradiol and diminished by testosterone). To determine whether these observed sex differences in fatty acid uptake also were present in the intact liver, single-pass [(14)C]oleate uptake was measured in isolated perfused livers. Livers from sexually mature female and male rats were perfused single-pass with albumin-bound [(14)C]oleate in Krebs-Ringer bicarbonate buffer. Net uptake, calculated as the product of the transhepatic difference in (14)C-labeled fatty acid concentration and perfusate flow rate, reached a steady-state within 1 min and remained constant throughout the 10-min perfusion period. At 0.17 mM [(14)C]oleate and 0.15 mM albumin, extraction fraction and net uptake of [(14)C]oleate per gram liver were more than twice as great in females as in male livers (0.33 +/- 0.03 versus 0.15 +/- 0.02, P < 0.001; and 218 +/- 22 versus 101 +/- 15 nmol/g liver, P < 0.01, with parallel differences in [(14)C]oleate total utilization and incorporation into triglycerides. Significant differences in uptake also were observed at higher [(14)C]oleate concentrations (0.34 and 0.68 mM). Under all conditions, oxidation of [(14)C]oleate in female liver equaled or exceeded that in male liver, indicating that the increased incorporation into triglycerides and other glycerolipids was not simply the result of differences in the distribution of [(14)C]oleate among cellular metabolic pathways. These studies demonstrate that in the intact liver, as in isolated hepatocytes, there are profound sex differences in the uptake of long chain fatty acids. This difference may account in part for the observed sex steroid effects on hepatic triglyceride biosynthesis and VLDL production. The mechanism of these uptake differences remains to be determined.-Kushlan, M. C., J. L. Gollan, W-L. Ma, and R. K. Ockner. Sex differences in hepatic uptake of long chain fatty acids in single-pass perfused rat liver.

Animals↗

Sex steroid modulation of fatty acid utilization and fatty acid binding protein concentration in rat liver.

The mechanism by which sex steroids influence very low density hepatic lipoprotein triglyceride production has not been fully elucidated. In previous studies we showed that [(14)C]oleate utilization and incorporation into triglycerides were greater in hepatocyte suspensions from adult female rats than from males. The sex differences were not related to activities of the enzymes of triglyceride biosynthesis, whereas fatty acid binding protein (FABP) concentration in liver cytosol was greater in females. These findings suggested that sex differences in lipoprotein could reflect a sex steroid influence on the availability of fatty acids for hepatocellular triglyceride biosynthesis. In the present studies, sex steroid effects on hepatocyte [(14)C]oleate utilization and FABP concentration were investigated directly. Hepatocytes from immature (30-d-old) rats exhibited no sex differences in [(14)C]oleate utilization. With maturation, total [(14)C]oleate utilization and triglyceride biosynthesis increased moderately in female cells and decreased markedly in male cells; the profound sex differences in adults were maximal by age 60 d. Fatty acid oxidation was little affected. Rats were castrated at age 30 d, and received estradiol, testosterone, or no hormone until age 60 d, when hepatocyte [(14)C]oleate utilization was studied. Castration virtually eliminated maturational changes and blunted the sex differences in adults. Estradiol or testosterone largely reproduced the appropriate adult pattern of [(14)C]oleate utilization regardless of the genotypic sex of the treated animal. In immature females and males, total cytosolic FABP concentrations were similar. In 60-d-old animals, there was a striking correlation among all groups (females, males, castrates, and hormone-treated) between mean cytosolic FABP concentration on the one hand, and mean total [(14)C]oleate utilization (r = 0.91) and incorporation into triglycerides (r = 0.94) on the other. In 30-d-old animals rates of [(14)C]oleate utilization were greater, relative to FABP concentrations, than in 60-d-old animals. The sex differences that characterize fatty acid utilization in adult rat hepatocytes are not present in cells from immature animals, and reflect in part the influence of sex steroids. It remains to be determined whether the observed relationship of hepatic FABP concentration to [(14)C]oleate utilization in adult cells is causal or secondary to changes in cellular fatty acid uptake effected through another mechanism. In either case, modulation of triglyceride-rich lipoprotein production by six steroids appears to be mediated to a significant extent by their effects on hepatic fatty acid utilization.

Aging↗

Sex differences in long chain fatty acid utilization and fatty acid binding protein concentration in rat liver.

Female sex and estrogen administration are associated with increased hepatic production of triglyceride-rich lipoproteins; the basis for this has not been fully elucidated. Inasmuch as hepatic lipoprotein production is also influenced by FFA availability and triglyceride biosynthesis, we investigated sex differences in FFA utilization in rat hepatocyte suspensions and in the components of the triglyceride biosynthetic pathway. Isolated adult rat hepatocyte suspensions were incubated with albumin-bound [(14)C]oleate for up to 15 min. At physiological and low oleate concentrations, cells from females incorporated significantly more (14)C into glycerolipids, especially triglycerides, and into oxidation products than did male cells, per milligram cell protein. At 0.44 mM oleate, incorporation into triglycerides in female cells was approximately twice that in male cells. Comparable sex differences were observed in cells from fasted animals and when [(14)C]-glycerol incorporation was measured. At higher oleate concentrations, i.e., fatty acid:albumin mole ratios in excess of 2:1, these sex differences were no longer demonstrable, suggesting that maximal rates of fatty acid esterification and oxidation were similar in female and male cells. In female and male hepatic microsomes, specific activities of long chain acyl coenzyme A synthetase, phosphatidate phosphohydrolase, and diglyceride acyltransferase were similar, but glycerol-3-phosphate acyltransferase activity was slightly greater in females at certain substrate concentrations. Microsomal incorporation of [(14)C]oleate into total glycerolipids was not significantly greater in females. In further contrast to intact cells, microsomal incorporation of [(14)C]oleate into triglycerides, although significantly greater in female microsomes, accounted for only a small fraction of the fatty acid esterified.The binding affinity and stoichiometry of partially purified female hepatic fatty acid binding protein (FABP) were similar to those of male FABP. In contrast, the concentration of FABP, per milligram cytosolic protein, was 44% greater in female liver than in male, as indicated by measurement of [(14)C]oleate binding and of 280 nm OD in the FABP fraction of 105,000 g supernate after gel filtration chromatography. These experiments demonstrate profound sex differences in hepatocyte utilization of long chain fatty acids at concentrations within and below the physiological range, and suggest that these are attributable at least in part to corresponding differences in cytosolic FABP concentration. At higher FFA concentrations, sex differences in hepatocyte FFA utilization are virtually eliminated, suggesting that under these conditions, differences in FABP concentration are not rate determining. Sex differences in hepatic lipoprotein production may largely reflect these important differences in the initial stages of hepatocyte FFA utilization.

Albumins↗

Studies of relationship among bile flow, liver plasma membrane NaK-ATPase, and membrane microviscosity in the rat.

Liver plasma membrane (LPM) NaK-ATPase activity, LPM fluidity, and bile acid-independent flow (BAIF) were studied in rats pretreated with one of five experimental agents. Compared with controls, BAIF was increased 24.6% by thyroid hormone and 34.4% by phenobarbital, decreased by ethinyl estradiol, but unchanged by propylene glycol and cortisone acetate. Parallel to the observed changes in BAIF, NaK-ATPase activity also was increased by thyroid hormone (40.8%) and decreased by ethinyl estradiol (26.2%). In contrast, NaK-ATPase activity failed to increase after phenobarbital but did increase 36% after propylene glycol and 34.8% after cortisone acetate. Thus BAIF and NaK-ATPase activity did not always change in parallel. The NaK-ATPase K(m) for ATP was not affected by any of these agents.LPM fluidity, measured by fluorescence polarization using the probe 1,6-diphenyl-1,3,5-hexatriene, was found to be increased by propylene glycol, thyroid hormone, and cortisone acetate, decreased by ethinyl estradiol, and unaffected by phenobarbital. Thus in these cases, induced changes in LPM fluidity paralleled those in NaK-ATPase activity. In no case did Mg-ATPase or 5'-nucleotidase activities change in the same direction as NaK-ATPase, and the activity of neither of these enzymes correlated with LPM fluidity, thus indicating the selective nature of the changes in LPM enzyme activity caused by the agents. These findings indicate that LPM fluidity correlates with NaK-ATPase activity and may influence the activity of this enzyme. However, the nature of the role of LPM NaK-ATPase in bile secretion is uncertain and needs further study.

Animals↗

Validation of a recording spectrophotometric method for measurement of membrane-associated Mg- and NaK-ATPase activity.

Conventional methods for determination of membrane-associated Mg- and NaK-ATPase activity typically involve a timed incubation of enzyme with ATP followed by measurement of released Pi. They are time-consuming, require a large amount of enzyme protein, and show considerable variability induplicate samples. These problems can be largely eliminated with a coupled enzyme assay in which formation of ADP by ATPase is coupled to NADH oxidation with the intermediate enzymes PK and LDH and the intermediate substrate phosphoenolpyruvate present in excess. NADH oxidation is continuously recorded at 340 nm, and NaK-ATPase represents the ouabain suppressible fraction of total ATPase. This method allows accurate determinatin of initial reaction rates which vary linearly with respect to enzyme protein. Furthermore, it eliminates the need to measure Pi and prevents accumulation of inhibitory ADP. In over 100 preparations of LPM prepared from rats pretreated with agents known to alter ATPase activity or exposed in vitro to ATPase inhibitors. ATPase activity by the coupled-enzyme assay paralleled results obtained with the conventional method. Moreover, the coupled-enzyme assay required less membrane protein, showed less variability in duplicate samples, required half the time, and also yielded accurate values in brain, kidney, and heart tissue. This improved assay should find broad application in the study of membrane ATPase as they relate to a variety of cellular functions.

Adenosine Triphosphatases↗

Alterations in hepatic pericanalicular cytoplasm during enhanced bile secretory activity.

In an attempt to demonstrate the morphology of the bile secretory apparatus, male rats were restrained and maintained on an isocaloric diet with (experimental) and without (control) taurocholate, which was continuously infused via a duodenal cannula. This method of taurocholate administration promotes a 2-fold increase in the bile acid pool size and bile secretory rate and increases the transport maximum of taurocholate by approximately 50%. After 48 hours, the livers from both the control and experimental animals were perfusion-fixed and whole hepatocytes as well as pericanalicular cytoplasm (defined as a 1-micron. wide zone of cytoplasm adjacent to the bile canaliculus) in both centrolobular and periportal cells were subjected to a stereologic analysis. Although taurocholate infusion produced relatively few changes in the amounts of organelles or inclusionswithin hepatocytes, it caused highly significant increases in the amount ofGolgi-rich area, Golgi membranes, and the number of vesicles with diameters greater than 1000 A in the pericanalicular area of cytoplasm. In addition to these changes, which occurred in both central and periportal zones, decreases in the volume of lysosomes and the surface area of smooth surfaced endoplasmic reticulum were observed. These data provide new evidence that the "bile secretory apparatus" may encompass several hepatocellular components which include the Golgi complex and a vesicular transport system.

Animals↗

A quantitative analysis of hepatic ultrastructure in rats during enhanced bile secretion.

The ultrastructural changes in hepatocytes of rats subjected to selective biliary obstruction (SBO), wherein the biliary system draining approximately two-thirds of the liver is obstructed, were evaluated by quantitative electron microscopy or stereology. The remaining unobstructed portion of the organ compensates for this loss of bile secretion by functioning in a hypersecretory mode. This animal model permits the comparison of hepatocellular fine structure associated with the conditions of nonsecretion and hypersecretion of bile with that found in normal secreting sham-operated rats. Since recent evidence suggests the presence of lobular gradients in hepatic structure and function, both centrolobular and periportal hepatocytes were examined. The low incidence of Golgi membrane profiles in high magnification electron micrographs results in a low confidence level of sampling and, thus, necessitates the application of a novel parameter for estimating the amount of Golgi complex, i.e., the Golgi-rich area. For the most part, the lobular variation in hepatic fine structure in the sham-operated animals was similar to that described by Loud ('68). However, the periportal parenchyma contained approximately twice the volume of Golgi-rich area as the centrolobular tissue. The amount of cytoplasmic lipid increased significantly in the SBO unobstructed lobes, although there were few or no changes in the other intracellular organelles or inclusions except those related to the Golgi complex. The volume of Golgi-rich area increased significantly in the centrolobular tissue of the SBO unobstructed (hypersecretory) lobes to the extent that both intralobular zones contained similar amounts of this component. These data suggest that the Golgi complex is a dynamic unit which responds to changes in hepatocellular activity and may be involved in bile secretion.

Animals↗

Bile acid-induced increase in bile acid-independent flow and plasma membrane NaK-ATPase activity in rat liver.

Previous studies showed that in rats with obstruction of the bile ducts draining the median and left hepatic lobes, and in rats with normal bile ducts in which the bile acid pool size and secretion were augmented by 48-h intraduodenal infusion of taurocholate, bile acid flux through secreting hepatocytes was increased. Under these conditions, taurocholate transport maximum exhibited a time-dependent adaptation to increased secretory load.Unexpectedly, bile acid-independent canalicular flow in these experimental models also was found to be increased when measured at 48 h. Relative to controls, bile acid-independent flow per gram of nonobstructed liver was increased approximately threefold in selectively obstructed rats and 43% in bile acid-loaded rats with normal ducts. In rats infused with bile acids at similar rates for only 16 h, no increase was observed. Studies with [(14)C]erythritol suggested that the increased bile flow under these conditions was of canalicular origin.NaK-ATPase activity in canaliculi-enriched liver plasma membrane preparations from the nonobstructed lobes of selectively obstructed rats and from 48-h bile acid-loaded rats was increased by 47% and 52%, respectively, relative to controls, but was not increased in membranes from 16-h bile acid-loaded rats. Canalicular membrane 5'-nucleotidase and Mg ATPase also were increased. These studies show that augmented bile acid flux through secreting liver causes an adaptive increase in bile acid-"independent" flow and in the activity of canalicular membrane enzymes. The mechanism by which bile acids modulate this and previously reported aspects of bile secretion remains to be elucidated.

Adenosine Triphosphatases↗