Regulation of Hepatocellular Fatty Acid Uptake in Mouse Models of Fatty Liver Disease with and without Functional Leptin Signaling: Roles of NfKB and SREBP-1C and the Effects of Spexin.

The processes causing increased hepatic triglycerides (TGs) in mouse models of hepatic steatosis (HS) due to high fat diet (HFD)-induced obesity (DIO), EtOH consumption, or obesity mutations (ob/ob, db/db) are uncertain. This report summarizes two studies. Study 1 focused on regulation by five transcription factors (TFs) (NfKb, Srebp-lc, AMPK, PPARα, PPARγ) of seven, much-studied hepatic long-chain fatty acid (LCFA) transporters (FABPpm, CD36, FATPl, FATP2, FATP4, FATP5, & Caveolin-1 [CAV-1]), and expression of genes for enzymes of LCFA synthesis (SCD-1, FASN) in mice with HS from various causes. Study 2 examined the effects of spexin, a novel adipokine, on obesity, type 2 diabetes mellitus (T2DM), and HS in these mice. Study 1 showed that: (1) processes underlying HS differed in mice with normal leptin signaling (DIO, EtoH-fed) versus those without it (ob/ob, db/db). Increased hepatocellular LCFA uptake was the principal cause of HS in the former, but increased hepatocellular LCFA synthesis predominated in the latter. (2) Expression of individual transporters was variable in the HS models studied, but strong correlations between TF expression and mean expression of four transporter genes across multiple HS models suggested regulatory interaction, and support the postulate that complexes of several different transporters mediate hepatic LCFA uptake. Study 2 indicated (1) that obese DIO mice often also have T2DM and/or nonalcoholic fatty liver disease (NAFLD); (2) confirmed that spexin treatment caused weight loss in DIO mice; (3) in DIO mice with T2DM, spexin also improved glucose tolerance, decreasing insulin resistance and HbAlc. Incubation with spexin directly inhibited LCFA uptake by hepatocytes isolated from DIO mice with HS/NAFLD by ≤70%. Spexin treatment in vivo for 4 weeks reduced hepatic lipids by 60%, and reduced serum alanine and aspartate aminotransferases. These studies in mice with DIO, T2DM, and HS/NAFLD suggest spexin may be an effective treatment for all three conditions.

Differences in adipocyte long chain fatty acid uptake in Osborne-Mendel and S5B/Pl rats in response to high-fat diets.

OBJECTIVE: To determine whether strain differences in adipocyte uptake of long chain fatty acids (LCFAs) contribute to differences in weight gain by Osborne-Mendel (OM) and S5B/Pl rats (S) fed a high-fat diet (HFD). SUBJECTS: Ninety-four adult (12-14-week old) male OM and S rats. MEASUREMENTS: Body weight; epididymal fat pad weight; adipocyte size, number, LCFA uptake kinetics; and plasma insulin and leptin during administration of HFD or chow diets (CDs). RESULTS: In both strains, rate of weight gain (RWG) was greater on an HFD than a CD; RWG on an HFD was greater, overall, in OM than S. A significant RWG increase occurred on days 1 and 2 in both strains. It was normalized in S by days 6-9 but persisted at least till day 14 in OM. RWGs were significantly correlated (P<0.001) with the V(max) for saturable adipocyte LCFA uptake (V(max)). In S, an increase in V(max) on day 1 returned to baseline by day 7 and was correlated with both plasma insulin and leptin levels throughout. In OM, a greater increase in V(max) was evident by day 2, and persisted for at least 14 days, during which both insulin and leptin levels remained elevated. Growth in epididymal fat pads on the HFD correlated with body weight, reflecting hypertrophy in OM and both hypertrophy and hyperplasia in S. CONCLUSIONS: (a) Changes in V(max) contribute significantly to changes in RWG on HFDs. (b) There are important strain differences in circulating insulin and leptin responses to an HFD. (c) Both insulin and leptin responses to an HFD are closely correlated with V(max) of adipocyte fatty acid uptake in S animals, but suggest early onset of insulin resistance in OM. Thus, differences in hormonal regulation of adipocyte LCFA uptake may underlie the different responses of OM and S to HFD.

Hepatocellular uptake of sulfobromophthalein and bilirubin is selectively inhibited by an antibody to the liver plasma membrane sulfobromophthalein/bilirubin binding protein.

To clarify sulfobromophthalein (BSP) and bilirubin uptake mechanisms, isolated rat hepatocytes were incubated with [35S]BSP. The initial uptake velocity (V0), determined from the first, linear portion of the cumulative uptake curve, was saturable (Michaelis constant [Km] = 6.2 +/- 0.5 microM; Vmax = 638 +/- 33 pmol X min-1 per 10(5) hepatocytes), maximal at 37 degrees C and pH 7.4, and competitively inhibited by bilirubin, but not by taurocholate, cholate, or oleate. Preloading with unlabeled BSP led to trans-stimulation of V0. Sodium substitution or pretreatment of hepatocytes with ouabain or metabolic inhibitors had no effect on V0; trypsin reduced V0 by 39% (P less than 0.001). A rabbit antiserum to the rat liver plasma membrane (LPM)-BSP/bilirubin binding protein selectively reduced V0 of 5 microM [35S]BSP and [14C]bilirubin by 41 and 42%, respectively (P less than 0.01); uptakes of [3H]oleate, [3H]cholate and [3H]taurocholate were not affected. Hence, the LPM-BSP/bilirubin binding protein plays a role in the carrier-mediated uptake of BSP and bilirubin by hepatocytes.

Hepatic organic anion uptake in the rat.

The hepatic uptake of bilirubin (BR), indocyanine green (ICG), and sulfobromophthalein (BSP) was studied in 350 anesthetized Sprague-Dawley rats by determining the initial plasma disappearance rate (V) of various doses of unlabeled ICG, or of tracer quantities of [3H]BR or [35S]BSP injected into the jugular vein simultaneously with varying amounts of unlabeled BR or BSP. Similar studies were also performed involving the simultaneous injection of potential inhibitors of hepatic uptake. The results indicate that: (a) hepatic uptake determined by direct tissue measurement could be accurately estimated from the plasma disappearance data; (b) saturation of hepatic uptake with increasing dose was readily demonstrated for each of these three organic anions, and in each instance a plot of V versus dose took the form of a rectangular hyperbola analyzable in terms of Michaelis-Menten kinetics; (c) for BR, the saturable uptake process showed a Vmax more than 100 times the normal net transfer rate from plasma to bile; (d) hepatic uptake of BR, BSP, and ICG showed relatively selective, mutually competitive inhibition; glycoholic acid did not inhibit hepatic uptake of any of these substances; and (e) "counter-transport" could be demonstrated for each of the three test substances. These data are compatible with the existence of a carrier-mediated transport process for hepatic uptake of each of these three organic anions and clarify the relationship of hepatic BR uptake to its overall transport from plasma to bile.

Identification, isolation, and partial characterization of a fatty acid binding protein from rat jejunal microvillous membranes.

The mechanisms by which FFA are absorbed by the gut are unclear. To examine these processes, binding of [14C]oleate to isolated rat jejunal microvillous membranes (MVM) was studied in vitro. When [14C]oleate alone or compounded with bovine serum albumin at various molar ratios was incubated with MVM aliquots, binding was time- and temperature-dependent, inhibitable by addition of excess cold oleate, and decreased by heat denaturation or trypsin digestion of the membranes. When [14C]oleate binding to heat denatured MVM, which increased continuously as a function of the free oleate concentration and was taken as a measure of nonspecific binding, was subtracted from total binding to native MVM, a curve suggestive of saturable specific binding was observed. In contrast to fatty acids, there was no specific binding of [14C]taurocholate or [35S]sulfobromophthalein to jejunal MVM. After MVM solubilization with 1% Triton X-100, affinity chromatography over oleate-agarose and elution with 7 M urea yielded a single 40,000-mol-wt protein. This Sudan Black/periodic acid-Schiff-stain-negative protein co-chromatographed on Sephadex G-100 with [14C]oleate, [14C]palmitate, [14C]arachidonate, and [14C]linoleate, but not with the [14C]oleate ester of cholesterol, [14C]phosphatidylcholine, [14C]taurocholate, or [35S]sulfobromophthalein. A rabbit antibody to the previously reported hepatic membrane fatty acid binding protein (FABP) gave a single line of immunologic identity between the FABPs of rat jejunum and rat liver membrane. It inhibited the binding of [14C]oleate to native MVM but not heat denatured MVM, and, in immunohistochemical studies, demonstrated the presence of the FABP in the apical and lateral portions of the brush border cells of the jejunum, but not on the luminal surface of esophagus or colon. These data are compatible with the hypothesis that a specific FABP plays a role in fatty acid absorption from the gut.

Studies of bilirubin kinetics in normal adults.

This report describes studies of bilirubin kinetics in 13 healthy young adults. The plasma content of unconjugated bilirubin-(14)C was determined at frequent intervals for 24-30 hr after the intravenous injection of a tracer dose of unconjugated isotopic bilirubin. Fecal and urinary radioactivity were measured for 7 days. During this time cumulative recovery averaged 96% of the injected dose. The plasma curves were processed by digital computer. For the 30 hr experimental period, a sum of three exponentials, with average half-times of 18, 81, and 578 min, was required to describe the data. Using the plasma curve integral method, the hepatic bilirubin clearance (47 +/-10 ml/min, mean +/-SD), the bilirubin production rate (3.8 +/-0.6 mg/kg per day), and the mean red blood cell life span (101 +/-13 days) were calculated directly from the parameters of this function. To gain further insight into the metabolism of unconjugated bilirubin, the data were also used to determine the parameters of a multicompartmental model. In the model proposed, plasma unconjugated bilirubin exchanges with two additional pools one of which is thought to represent extrahepatic extravascular, and the other intrahepatic unconjugated bilirubin. Bilirubin is eliminated from the system via the proposed intrahepatic pool. From the data and the model, pool sizes and exchange rates between compartments were calculated, and the liver: plasma concentration gradient estimated. These studies provide a detailed analysis of the kinetics of unconjugated bilirubin in a healthy normal population and are intended to serve as a reference point for studies of abnormal states.

At physiologic albumin/oleate concentrations oleate uptake by isolated hepatocytes, cardiac myocytes, and adipocytes is a saturable function of the unbound oleate concentration. Uptake kinetics are consistent with the conventional theory.

To reexamine the role of albumin in cellular uptake of long chain fatty acids, we measured [3H]oleate uptake by isolated hepatocytes, adipocytes, and cardiac myocytes from incubations containing oleate/albumin complexes at molar ratios from 0.01:1 to 2:1. For each ratio the uptake was studied over a wide range of albumin concentrations. In all three cell types and at any given oleate/albumin ratio, the uptake appeared saturable with increasing concentrations of oleate:albumin complexes despite the fact that the unbound oleate concentration for each molar ratio is essentially constant. However, the "Km" but not the "Vmax" of these pseudosaturation curves was influenced by substrate availability. At low albumin concentrations, uptake velocities did not correlate with unbound oleate concentrations. However, observed and expected uptake velocities coincided at albumin concentrations approaching physiologic levels and were a saturable function of the oleate/albumin ratios and the consequent unbound oleate concentrations employed. Hence, under the experimental conditions employed in this study using a variety of suspended cell types, oleate uptake kinetics were consistent with the conventional theory at physiologic concentrations of albumin.

Oleate uptake by cardiac myocytes is carrier mediated and involves a 40-kD plasma membrane fatty acid binding protein similar to that in liver, adipose tissue, and gut.

Uptake of [3H]oleate by canine or rat cardiac myocytes is saturable, displays the countertransport phenomenon, and is inhibited by phloretin and trypsin. Cardiac myocytes contain a basic (pI approximately 9.1) 40-kD plasma membrane fatty acid binding protein (FABPPM) analogous to those recently isolated from liver, adipose tissue, and gut, unrelated to the 12-14-kD cytosolic FABP in these same tissues. An antibody to rat liver FABPPM selectively inhibits specific uptake of [3H]oleate by rat heart myocytes at 37 degrees C, but has no influence on nonspecific [3H]oleate uptake at 4 degrees C or on specific uptake of [3H]glucose. Uptake of long-chain free fatty acids by cardiac muscle cells, liver, and adipose tissue and absorption by gut epithelial cells is a facilitated process mediated by identical or closely related plasma membrane FABPs.

Hepatic accumulation and intracellular binding of conjugated bilirubin.

After the intravenous injection of unconjugated [(3)H]bilirubin into normal Sprague-Dawley and Wistar R rats, radiolabeled bile pigments rapidly accumulated in the liver. By 1.5 min after injection, an average of 36% of the injected isotope was present in liver homogenates. Between 3 and 15 min, 37-64% of the total intrahepatic radiolabeled bilirubin was conjugated, as demonstrated by extraction of label into the polar phase of a solvent partition system. This indicates both rapid conjugation, and accumulation of conjugated bilirubin within the liver cell. Fluorometric determination of the dissociation constants of purified bilirubin and its mono- and diglucuronides for homogeneous preparations of two human and four rat glutathione S-transferases, including ligandin, revealed avid binding of all three bile pigments to this class of proteins. Hence, the observation that the intrahepatic bile pigment pool contains substantial amounts of conjugated bilirubin can be attributed to the high binding affinities observed. Thin-layer chromatographic analysis of the (3)H-pigments produced by p-iodoaniline diazotization of homogenates and cytosol demonstrated that the intrahepatic pool of conjugated bilirubin was almost exclusively monoglucuronide. Examination of radiolabeled bilirubin conjugates excreted in bile during the first 20 min after injection of [(3)H]bilirubin showed no preferential excretion of diglucuronide. These studies indicate that (a) both bilirubin and its monoglucuronide accumulate within the liver cell as ligands with the glutathione S-transferase; and (b) bilirubin diglucuronide does not significantly accumulate within the general intrahepatocellular pool of protein-bound bile pigments. The latter observation is compatible with the formation and excretion of bilirubin diglucuronide directly from the canalicular pool of the liver cell.

Quantitative studies of the delivery of hepatic-synthesized bilirubin to plasma utilizing -aminolevulinic acid-4- 14 C and bilirubin- 3 H in man.

After the simultaneous intravenous administration of unconjugated bilirubin-(3)H and delta-aminolevulinic acid-4-(14)C, the plasma disappearance curves of unconjugated bilirubin-(3)H and the plasma appearance curves of biosynthesized unconjugated bilirubin-(14)C have been defined in seven patients, three of whom had acute intermittent porphyria (AIP). The incorporation of (14)C into plasma unconjugated bilirubin, derived by an analysis which involves deconvolution of the two plasma curves, varied between 13.1 and 23.5% (mean 19.3%) of the injected dose in the nonporphyric patients and between 5.4 and 13.6% (mean 8.3%) of the injected dose in the porphyric patients. In five of the patients, the stercobilin-(14)C specific activity in a pooled specimen of feces was measured, enabling the following further values to be calculated: (a) the total (14)C radioactivity incorporated into bilirubin (21.0 and 25.3% [mean 23.2%] of the injected dose in two of the nonporphyric patients and between 8.5 and 25.3% [mean 14.2%] of the injected dose in the porphyric patients), and (b) the proportion of hepatic synthesized bilirubin delivered directly to plasma in the unconjugated form (between 0.520 and 0.904; mean for nonporphyric patients 0.712; mean for porphyric patients 0.614). The results demonstrate that a large proportion of bilirubin derived from hepatic hemes passes through the plasma in the unconjugated form before conjugation and secretion into bile.

Removing substances from blood by affinity chromatography. II. Removing bilirubin from the blood of jaundiced rats by hemoperfusion over albumin-conjugated agarose beads.

In vitro studies indicate that bilirubin and other albumin-bound substances can be efficiently removed from plasma by filtration over albumin-conjugated agarose beads. The effectiveness of this technique in vivo was investigated in rats by using a closed extracorporeal hemoperfusion system. Five Gunn rats whose endogenous bilirubin pool had been labeled with [(3)H]bilirubin and five Sprague Dawley rats with surgically created biliary obstruction were chosen as models of unconjugated and conjugated hyperbilirubinemia. Indocyanine green was injected into rats and its removal also studied. In the Gunn rats, 98% of the bilirubin was removed from plasma during the initial pass over the column as determined isotopically and chemically. Plasma bilirubin levels fell more than 70% from 8.2+/-1.6 mg/100 ml (mean+/-SD) to 2.6+/-0.5 mg/100 ml during a 1-h hemoperfusion. An average of 1,061 mug of bilirubin was recovered from the columns, representing 22.5+/-4.2% of the total exchangeable bilirubin pool and 96+/-36.4% of the plasma pool. Results were similar in the rats with biliary obstruction and in those given indocyanine green. Normal Sprague Dawley rats experience minimal changes in formed blood elements, electrolytes, and proteins as the result of hemoperfusion. When the total volume of the column did not exceed 51% of the estimated blood volume of the animal, the survival rate was 100% in 20 studies, and the procedure was without observable ill effect. Extrapolation of both in vitro and in vivo data to man suggests that extracorporeal hemoperfusion over albumin-agarose columns may be a practical means of assisting hepatic excretory function.

Physicochemical and immunohistological studies of a sulfobromophthalein- and bilirubin-binding protein from rat liver plasma membranes.

Affinity chromatography over bilirubinagarose and sulfobromophthalein (BSP)-agarose was used to isolate two proteins, with high affinities for bilirubin and BSP, respectively, from Triton X-100-solubilized rat liver plasma membranes. The protein eluted from either affinity column migrated as a single band of approximately 55,000 D on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and either protein cochromatographed with both [14C]bilirubin and [35S]BSP on Sephadex G-75. On gradient gels without reduction or SDS, or on Sephadex G-150, the native BSP-binding protein had an estimated molecular mass of approximately 100,000 D. After incubation with SDS, an additional Sephadex G-150 peak of molecular mass of 56,000 D was observed. Both, the 100,000- and 56,000-D G-150 peaks cochromatographed with [35S]BSP. The native protein had an isoelectric point of 3.5, stained with periodic acid-Schiff but not Sudan black, and contained 4 mol of sialic acid per mol of protein. A rabbit antibody to the BSP-binding protein gave a line of identity with both the BSP- and bilirubin-binding antigens, and inhibited the binding of [14C]bilirubin and [35S]BSP, but not [14C]oleate or [14C]taurocholate, to rat liver plasma membranes. Immunohistochemical studies revealed the presence of the antigen on all surface domains of rat hepatocytes, but not on other cell populations from normal rat liver. It was not found in other organs. These data are compatible with the hypothesis that a specific liver cell plasma membrane protein mediates the hepatocytic sequestration of bilirubin and BSP.

Removing substances from blood by affinity chromatography. I. Removing bilirubin and other albumin-bound substances from plasma and blood with albumin-conjugated agarose beads.

Substances such as bilirubin that bind tightly to plasma proteins cannot readily be removed from blood. We describe here the use of affinity chromatography as a new approach to the removal of proteinbound metabolites and toxins from blood. Agarose beads were coupled via cyanogen bromide to human serum albumin so as to contain 30-50 mg of albumin/g wet wt. Such beads, when exposed to plasma from a patient with congenital nonhemolytic jaundice labeled with [(14)C]-bilirubin, bound more than 150 mug bilirubin/g of beads. The binding was saturable, concentration-dependent, relatively independent of flow rate, and reversible by elution with plasma, albumin, or 50% (vol/vol) ethanol. The beads could be repeatedly reused without loss of efficiency after ethanol elution and long storage in the cold. Salicylate, cortisol, and taurocholate, which bind weakly to albumin, were retarded by the beads but eluted with neutral buffer. Thyroxine, taurolithocholate, chenodeoxycholate, and digitoxin bound tightly but were eluted with 50% ethanol. Digoxin did not bind at all. When whole blood was passed over agarose-albumin beads, bilirubin was removed, calcium and magnesium fell slightly, but red cells, white cells, platelets, clotting factors, and a variety of electrolytes and proteins were substantially unchanged. Agarose-albumin beads may be useful for removing protein-bound substances from the blood of patients with liver failure, intoxication with protein-bound drugs, or specific metabolic deficits. Furthermore, it may be possible to make useful adsorbents by attaching other proteins to agarose or other polymer beads.

Studies of albumin binding to rat liver plasma membranes. Implications for the albumin receptor hypothesis.

To characterize a previously proposed hepatocyte albumin receptor, we examined the binding of native and defatted 125I-labeled rat albumin to rat liver plasma membranes. After incubation for 30 min, binding was determined from the distribution of radioactivity between membrane pellet and supernatant following initial centrifugation (15000 X g for 15 min), and after repeated cycles of washing with buffer and re-centrifugation. 125I-labeled albumin recovered in the initial membrane pellet averaged only 4% of that incubated. Moreover, this albumin was only loosely associated with the membrane, as indicated by recovery in the pellet of under 0.5% of the counts after three washes. Binding of 125I-labeled albumin to the plasma membranes was no greater than to erythrocyte ghosts, was not inhibited by excess unlabeled albumin, and was not decreased by heat denaturation of the membranes, all suggestive of a lack of specific binding. Failure to observe albumin binding to the membranes was not due to a rapid dissociation rate or 'off-time', as incubations in the presence of sufficient ultraviolet light to promote covalent binding of ligands to receptors did not increase 125I counts bound to the membrane. Finally, affinity chromatography over albumin/agarose gel of solubilized membrane proteins provided no evidence of a membrane protein with a high affinity for albumin. These studies, therefore, do not support the hypothesis that liver cell plasma membranes contain a specific albumin receptor.

Binding of unconjugated and conjugated sulfobromophthalein to rat liver plasma membrane fractions in vitro.

As part of a study of the mechanism whereby organic anionic dyes such as sulfobromophthalein and bilirubin enter hepatocytes, the binding of [35S]sulfobromophthalein and of its glutathione conjugate to two rat liver plasma membrane fractions were studied in vitro. Both fractions reversibly and saturably bound conjugated and unconjugated sulfobromophthalein. Three classes of binding site were necessary to account for the observed sulfobromophthalein binding, their maximal binding capacities being 3.5 . 10(-11), 1.6 . 10(-7) and 5.4 . 10(-7) mol/mg membrane protein. The corresponding association constants were 5.5 . 10(7), 1.5 . 10(5) and 1.3 . 10(3) M-1. Binding of the glutathione conjugate could be accounted for by two classes of binding site only, their association constants being 2.0 . 10(8) and 1.9 . 10(3) M-1 and their maximal binding capacities 5.0 . 10(-11) and 2.2 . 10(-7) mol/mg protein, respectively. Conjugated and unconjugated sulfobromophthalein mutually competed for binding, KI being 7.8 . 10(-7) and 5.5 . 10(-5) M for conjugated and unconjugated sulfobromophthalein, respectively. Similarly, bilirubin and indocyanine green, but not taurocholate, competitively inhibited sulfobromophthalein binding. Treatment with trypsin and phospholipases reduced, while treatment with neuraminidase did not affect binding. Neither changes in pH nor substitution of other cations for Na+ in the incubation mixture significantly affected sulfobromophthalein binding. Heating the membranes increased binding. This was due to an increase in maximal binding capacity of the low-affinity, high-capacity sites. The description of saturable binding sites on hepatocellular surface membranes, the affinity of one of the sites exceeding the reported affinities for albumin and ligandin, supports the hypothesis that a membrane-located membrane carrier is responsible for hepatic uptake and biliary excretion of organic anionic dyes. Based on the studies with enzyme treatments, it is speculated that this carrier is a phospholipid-dependent, integral membrane protein.

The role of microfilaments and microtubules in taurocholate uptake by isolated rat liver cells.

The role of microfilaments and microtubules on bile salt transport was studied by investigating the influence of a microfilament and a microtubule inhibitor, cytochalasin B and colchicine, respectively, on taurocholate uptake by isolated hepatocytes in vitro. Hepatocytes were prepared by the enzyme perfusion method and [14C]taurocholate uptake velocity was determined by a filtration assay. Taurocholate uptake obeyed Michaelis-Menten kinetics, maximal uptake velocity and apparent half-saturation constants averaging 0.87 +/0 SD 0.05 nmol . s-1 . 10(-6) cells and 10.9 +/- 1.8 muM, respectively. Cytochalasin B (4.2-420 muM) inhibited taurocholate uptake in a competitive fashion; Ki being 33 +/- 7 muM. At concentrations above 100 muM the compound decreased 36Cl membrane potential and intracellular K+ concentration. Other parameters of cell viability were not affected by cytochalasin B. Colchicine (0.1-1.0 mM), by contrast, inhibited taurocholate uptake non-competitively, Ki being 0.47 +/- 0.07 mM. The inhibition brought about by colchicine was considerably smaller than that induced by cytochalasin B. None of the parameters of cell viability tested was affected by colchicine. These results suggest that microfilaments may be involved in the carrier-mediated hepatocellular transport of bile salts. This could, at least in part, account for cytochalasin B-induced cholestasis. The contribution of the microtubular system, if any, is less important quantitatively. The mechanisms whereby these two components of the cytoskeleton partake in bile salt transport remain to be elucidated.

The kinetics of sulfobromophthalein uptake by rat liver sinusoidal vesicles.

The kinetics of bromo[35S]sulfophthalein (35S-BSP) binding by and uptake across the hepatocyte sinusoidal membrane were investigated using isolated rat liver sinusoidal membrane vesicles containing K+ as the principal internal inorganic cation. Uptake of 35S-BSP into vesicles was found to be temperature dependent, with maximum uptake between 35 and 40 degrees C; only binding occurred at or below 15 degrees C. Uptake at 37 degrees C was saturable and resolvable by Eadee-Hofstee analysis into two components: one with high affinity (Km = 53.1 microM) but low capacity, and the second of low affinity (Km = 1150 microM) but high capacity. By pre- or post-incubation, respectively, with unlabelled BSP, trans-stimulation and counter transport of 35S-BSP could also be demonstrated in these vesicles. Uptake was inhibited competitively using 5 microM Rose bengal and 10 microM indocyanine green, and non-competitively using 10 microM DIDS. Taurocholate did not inhibit uptake, and actually enhanced transport at concentrations greater than or equal to 250 microM. Imposition of inwardly directed inorganic ion gradients resulted in the enhancement of 35S-BSP transport when chloride ions were part of this gradient, irrespective of the cation employed whereas there was no apparent cation effect. However, substitution of 10 mM Na+ for 10 mM K+ as the internal cation resulted in a significant increase in uptake in the presence of external K+ as compared to Na+ gradients. This effect was not observed when 10 mM Tris+ was employed as the internal cation. The kinetics of 35S-BSP uptake by isolated sinusoidal membrane vesicles are indicative of facilitated transport. While the observed inorganic ion effects suggest a possible electrogenic component, the driving forces for hepatic BSP uptake remain uncertain. Isolated sinusoidal membrane vesicles provide a useful technique for studying hepatic uptake processes independent of circulatory or subsequent cellular phenomena.

Therapeutic recommendations in polycythemia vera based on Polycythemia Vera Study Group protocols.

The PVSG was organized in 1967 to establish effective diagnostic criteria for polycythemia vera, to study the natural history of the disease and to define the optimal treatment. Although polycythemia vera and the other myeloproliferative diseases are relatively uncommon, the PVSG was able to accumulate well over 1,000 patients with these various disorders and to study them according to a total of 15 different protocols. PVSG-01, a long-term randomized controlled study of phlebotomy alone compared with the myelosuppressive agents, 32P or chlorambucil supplemented by phlebotomy, continues to receive follow-up data on 93% of surviving patients 18 years after initiation of the study. During its lifetime, PVSG has developed a widely accepted and highly effective set of criteria for the specific diagnosis of polycythemia vera as well as useful criteria for the diagnosis of essential thrombocythemia. It has gathered an enormous volume of data on the natural history of the myeloproliferative diseases and in particular on the nature of the prevalent complications, such as thrombotic events and hematologic and nonhematologic malignancies. With respect to the final question, the optimal treatment for polycythemia vera, it is apparent that the expectation of a single optimal therapy that would apply to all patients at all ages and stages of the disease was naive. Nevertheless considerable progress has been made. Moreover, the group has defined more precisely than ever before the nature of the complications of the disease and the association of the risks of specific complications with specific forms of therapy. It thus has made it possible to pose the next series of therapeutic questions that must be addressed in this disorder with a greater degree of sophistication than was previously possible.

Hyperbilirubinemia and cholestasis.

Although the morphologist continues to describe cholestasis on the basis of precipitated bile seen on light microscopic sections of the liver or dilated canaliculi with loss of microvilli seen by electron microscopy, the physiologist can distinguish clearly between hyperbilirubinemia and cholestasis. Both bilirubin and bile acids are specifically removed from sinusoidal plasma by the normal hepatocyte and appear in bile in high concentration. Bilirubin conjugation and excretion appear to be governed by hepatocellular mechanisms that are, for the most part, separate from the conjugation and excretion of bile acids. Disturbances in bilirubin transport are recognized by hyperbilirubinemia which represents a number of clinical syndromes that can be classified by the nature of the block in the transport system. Serum bile acids appear to remain normal in hyperbilirubinemic syndromes. By contrast, cholestatic syndromes are characterized by marked bile acidemia with normal to slightly elevated bilirubin levels. Severe cholestasis, because of the marked reduction in bile flow, can however, engender jaundice. Further exploration of these excretory pathways will provide interesting new insights on the numerous cholestatic and hyperbilirubinemic syndromes that occur in nature.

Effect of low-dose phenobarbital on hepatic microsomal UDP-glucuronyl transferase activity.

To determine whether hepatic microsomal enzyme induction occurs in rats following administration of phenobarbital at doses similar to those used in humans (0.5 to 7.5 mg/kg), UDP-glucuronyl transferase (UDPGT) and cytochrome P-450 activities were measured in liver homogenate and microsomal preparations from control rats and rats treated for 6 days with phenobarbital at 1 and 3 mg per kg per day. While no significant increases in liver weight and protein content of homogenate and microsomal preparations were observed with either dose of the drug, both UDPGT and P-450 activities were enhanced significantly following administration of phenobarbital at 3 mg per kg per day. The activity of P-450 was increased by approximately 30% and that of UDPGT by 15-24 and 45-66%, respectively, employing bilirubin and p-nitrophenol as the acceptor substrate. The extent of induction of bilirubin or p-nitrophenol UDPGT was similar when measured with "native" enzyme or with enzyme activated by UDP-N-acetyl glucosamine, digitonin or deoxycholate. These data suggest that the discordant effects of phenobarbital on UDPGT and cytochrome P-450 previously reported in humans and rats may not be attributable solely to differences in the drug doses employed.