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R Rabkin

Publications and source records attributed to R Rabkin.

At least 19 recordsLinked to original sources
PubMed1992

Altered intestinal and renal brush border amino-oligopeptidase structure in diabetes and metabolic acidosis: normal and biobreed (BB) rats.

Amino-oligopeptidase (AOP, aminopeptidase N), a major glycoprotein hydrolase in intestinal and kidney brush border membranes, plays a crucial role in digesting peptide nutrients and salvaging filtered peptides. The molecular structure of rat intestinal and kidney AOP was compared for normal Wistar and congenitally diabetic BB Wistar (BBd) rats. Brush border membranes were isolated, solubilized with Triton X-100, and the AOP specifically immunoprecipitated with polyvalent rabbit antiserum and analyzed on 7% sodium dodecyl sulfate (SDS)-acrylamide electrophoresis. While the specific hydrolytic activity was maintained, BBd rats displayed an altered migration of AOP on SDS gels. Intestinal AOP migrated as a smaller species (130 kd) in the BBd than in the normal Wistar (135 to 140 kd). In some BBd rats, additional intestinal AOP species were observed (a 130- to 135-kd doublet or a 125-, 130-, or 135-kd triplet). Kidney AOP migrated as a broader band (125 to 140 kd) than intestine for all rat groups, probably due to carbohydrate chain heterogeneity, and was approximately 5 kd smaller in the BBd rat than in the normal Wistar. In contrast, no mass change was found in diabetes induced by streptozotocin (STZ). The altered intestinal AOP in the BBd rat was present when first inserted into the brush border membrane (6 hours after intraperitoneal [35S]methionine labeling), and hence was not due to nonenzymatic glycosylation (NEG). Abnormal intestinal and kidney AOP structure appeared in early diabetes, irrespective of high plasma glucose levels or ketoacidosis, and was reversed following evolution of the diabetes under prolonged (21 to 120 days) insulin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1992

Renal cortical endosomes participate in the degradation of insulin.

It has long been thought that metabolism of insulin by kidney proximal tubule cells takes place only in lysosomes. But previous studies with the perfused rat kidney and a proximal tubule-like cultured kidney cell line suggested that insulin degradation occurred in a nonlysosomal compartment. In this study we show that endosomes isolated from rat kidney cortex degrade insulin and that this process is ATP and pH dependent.

Adenosine Triphosphate↗
PubMed1992

Ammonium chloride increases kidney cell protein content.

Augmented renal ammoniagenesis and renal hypertrophy often occur together. Ammonia may increase cell protein content by modulating protein synthesis, protein degradation, or both. We conducted experiments to examine the effect of ammonium chloride on the synthesis and degradation of protein in cultured kidney cells. Quiescent opossum kidney cells were exposed to 20 mM NH4Cl for two days. Monolayers were then analyzed for cell number, protein content, protein synthesis ([14C]valine incorporation), protein degradation ([14C]valine release) and DNA synthesis ([3H]thymidine incorporation). Cell protein content was increased by 18% in NH4Cl-treated cells (190 +/- 6 pg/cell control vs. 225 +/- 7 pg/cell NH4Cl, p < 0.001). NH4Cl suppressed protein degradation (1.36 +/- 0.02%/h control vs. 1.12 +/- 0.04%/h NH4Cl, p < 0.001) but did not change protein synthesis, DNA synthesis, or cell number. Thus, reduced protein degradation accounts entirely for enhanced protein accumulation at 2 days in this in vitro model of kidney cell hypertrophy.

Ammonium Chloride↗
PubMed1991

Sucrase-alpha-dextrinase in diabetic BioBreed rats: reversible alteration of subunit structure.

BioBreed (BB) Wistar rats develop diabetes mellitus, which closely resembles the human disease, in 50% of progeny. Intestinal sucrase-alpha-dextrinase, a glycoprotein hydrolase of the enterocyte's brush border consisting of 140-kDa alpha-dextrinase and 125-kDa sucrase subunits, is essential for surface digestion of carbohydrate nutrients. Although its catalytic characteristics were found to be maintained in the diabetic state, the structure of the subunits, as compared with normal Wistar rats, was altered in the BB rat within 2 days of the onset of diabetes. Its capacity to react in a solid-phase immunoassay was reduced by 50%; when examined by 6% acrylamide electrophoresis, the sucrase subunit was increased in mass by 5 kDa and, in some BB rats, the dextrinase subunit was reduced by 5 kDa. Intact rats labeled intraintestinally with [35S]methionine displayed the alteration within 6 h of synthesis, indicating that nonenzymatic glycosylation could not account for the structural change. This mass change was not seen in streptozotocin-induced diabetes and was independent of the plasma glucose concentration or the degree of acidosis. Deglycosylation with peptide N-glycosidase indicated that the N-linked chains of the normal dextrinase subunit (11 kDa) have twice the mass of those in the BB rat (6 kDa) and that the sucrase subunit may have an increased mass of O-linked chains. Overall, these experiments point to changes in glycosylation as a mechanism of structural alteration in congenital diabetes. Despite persistence of the insulin-dependent diabetes, the subunit pattern eventually became indistinguishable from normal, but at differential rates (21 days and 35 days, respectively, for sucrase and dextrinase subunits).

Animals↗
PubMed1991

Amino acids regulate kidney cell protein breakdown.

Amino acids inhibit breakdown of long-lived intracellular proteins in some but not all tissues studied. Because no information is available relating to the effect of amino acids on kidney cell proteolysis, this study was conducted with cultured proximal-like opossum kidney (OK) cells and primary cultured rabbit proximal tubular cells in which long-lived cell proteins were labeled with carbon 14-labeled valine. These cultured cells were acutely deprived of amino acids; this was followed by a 57% to 66% increase in the proteolytic rate in OK cells and a 22% rate increase in the rabbit kidney cells. In cultured OK cells incubated in serum-free minimal essential medium containing 13 amino acids, proteolysis averaged 4.62% +/- 0.28%/2 hr and increased to 7.66% +/- 0.38%/2 hr when amino acids were deleted. Each amino acid was then added alone. Leucine, phenylalanine, and lysine had significant effects in inhibiting the deprivation response by 40%, 26%, and 22%, respectively. Leucine appears to inhibit proteolysis directly and not through its metabolites, since alpha-ketoisocaproate, the leucine transamination product, was without effect. Similarly, failure of tyrosine to inhibit proteolysis suggests a direct phenylalanine action. When leucine, phenylalanine, and lysine were simultaneously deleted from the incubation medium, the increase in proteolysis corresponded to 56% of the response after deletion of all amino acids. Thus to maximally affect proteolysis, amino acids, which on their own have little effect on protein breakdown, also appear to play a role. From this study we conclude that amino acids seem to play an important and direct role in the regulation of kidney epithelial cell protein breakdown.

Amino Acids↗
PubMed1990

Effect of bacitracin on retroendocytosis and degradation of insulin in cultured kidney epithelial cell line.

In an earlier study, we described the presence of a retroendocytotic pathway for insulin in a cultured kidney epithelial cell line. Derived from the opossum kidney (OK), these cells possess many features of proximal tubule epithelium, which is the major site of kidney insulin metabolism. We studied the interaction between the retroendocytotic and the degradative pathways with bacitracin as a pharmacological probe. Monolayers of OK cells were loaded with 125I-labeled insulin over 30 min, acid washed to remove membrane-bound insulin, then incubated in fresh medium for 60 min while the release of intracellular radioactivity was monitored. In experiments carried out in the presence of bacitracin (2 mM), there was a two-thirds increase in intracellular radioactivity at the end of the loading phase. Measurements made during the subsequent release phase showed that bacitracin reduced the release of degradation products. Thus, although controls released 72.1 +/- 8.1% of the internalized radioactivity as trichloroacetic acid (TCA)-soluble products, bacitracin-treated cells released 59.2 +/- 9.4% (P less than 0.02). In contrast, release of TCA-precipitable insulin increased from 15.2 +/- 4.6% in controls to 25.8 +/- 3.7% in bacitracin-treated cells (P less than 0.01). In separate experiments analyzed by gel-exclusion chromatography, 6.4 +/- 0.6% of radioactivity released from preloaded control cells into medium over 60 min was insulin sized compared to 29.7 +/- 1.4% in bacitracin-treated cells. High-performance liquid chromatography revealed that 61.5 +/- 3.5% of this insulin-sized material released from control cells preloaded with A14-insulin eluted as intact insulin and the remainder as unidentified intermediate degradation products.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Modulation of kidney cell protein degradation by insulin.

Insulin is an established regulator of intracellular proteolysis in several mammalian tissues but little is known about its role in the kidney. The present study was undertaken to determine whether insulin influences protein degradation in isolated rat renal proximal tubules and to investigate its mechanism of action in cultured proximal-like tubular epithelial cells from the opossum kidney. Long-lived protein degradation was determined from the release of carbon 14-labeled valine from previously labeled cellular protein under conditions designed to minimize label reutilization. In isolated tubules, the mean control rate of proteolysis was 2.18% per hour, indicating an appreciable turnover of cellular protein. Insulin (10(-6) mol/L) decreased the rate by 23%. In cultured kidney cells, the rate of protein degradation averaged 1.25% per hour in the presence of serum and 1.68% per hour in its absence, an increase of 34%. High insulin concentrations suppressed this acceleration completely, and physiologic levels inhibited it partially. No evidence was obtained to indicate that insulin action is mediated through stimulation of Na(+)-H+ antiport or through increased amino acid utilization. Ammonium chloride, however, strongly attenuated the serum deprivation response and the inhibitory effect of insulin. The exact mechanisms whereby insulin inhibits proteolysis is not known, but these findings are consistent with an inhibitory action of insulin on the lysosomal pathway.

Ammonium Chloride↗
PubMed1989

Retroendocytosis of insulin in a cultured kidney epithelial cell line.

It has been generally accepted that in renal tubular epithelium endocytosed proteohormones are transported to lysosomes where they undergo complete hydrolysis. En route, as endosomal pH falls, the proteohormone uncouples from the endocytosed membrane binding site, which recycles to the cell surface. However, studies in other tissues have uncovered alternate intracellular pathways for proteins. One such pathway is retroendocytosis (endocytosis then exocytosis). To determine whether a retroendocytotic pathway exists for insulin in renal epithelium, a study was carried out with confluent monolayers of a proximal-like opossum kidney cell line that exhibits receptor-mediated endocytosis of insulin. Cells were preloaded with 125I-labeled insulin (4 X 10(-10) M) for 30 min, surface-bound insulin was then removed by acid washing, and over the next 60 min the release of intracellular radioactivity into the medium was monitored. At 37 degrees C, control cells released on average 7-15% of the intracellular radioactivity as intact insulin [trichloroacetic acid (TCA)-precipitable radioactivity] and approximately 62% as TCA-soluble degradation products. In the presence of 0.1 mM chloroquine (an acidotropic agent) the release of intact insulin increased approximately twofold while degradation fell by nearly one-half. With Sephadex G-50 chromatography we found that the released radioactivity included insulin-size material that increased in the presence of chloroquine. High-performance liquid chromatography revealed that 53 (controls) and 81% (chloroquine treatment) of this latter material consisted of intact insulin. We conclude that, in addition to a major degradative pathway, cultured kidney epithelial cells exhibit a retroendocytotic pathway for insulin. Chloroquine inhibits degradation and appears to divert insulin from the degradative into the retroendocytotic pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Insulin degradation products from perfused rat kidney.

The kidney is a major site for insulin metabolism, but the enzymes involved and the products generated have not been established. To examine the products, we have perfused rat kidneys with insulin specifically iodinated on either the A14 or the B26 tyrosine. Labeled material from both the perfusate and kidney extract was examined by Sephadex G50 and high-performance liquid chromatography (HPLC). In perfusate from a filtering kidney, 22% of the insulin-sized material was not intact insulin on HPLC. With the nonfiltering kidney, 10.6% was not intact insulin. Labeled material from HPLC was sulfitolyzed and reinjected on HPLC. By use of 125I-iodo(A14)-insulin, almost all the degradation products contained an intact A-chain. By use of 125I-iodo(B26)-insulin, several different B-chain-cleaved products were obtained. The material extracted from the perfused kidney was different from perfusate products but similar to intracellular products from hepatocytes, suggesting that cellular metabolism by kidney and liver are similar. The major intracellular product had characteristics consistent with a cleavage between the B16 and B17 amino acids. This product and several of the perfusate products are also produced by insulin protease suggesting that this enzyme is involved in the degradation of insulin by kidney.

Animals↗
PubMed1989

Abnormal insulin metabolism by specific organs from rats with spontaneous hypertension.

Spontaneously hypertensive rats (SHR) have been shown to be both insulin resistant and hyperinsulinemic after oral glucose administration or infusion of exogenous insulin during an insulin suppression test. To determine if this hyperinsulinemia may be due to decreased removal of insulin, the metabolic clearance (k) of insulin was measured in isolated perfused liver, kidney, and hindlimb skeletal muscle from SHR and Wistar-Kyoto (WKY) control rats. The data indicate that the k for insulin removal by liver was similar in SHR and WKY rats, averaging 287 +/- 18 and 271 +/- 10 microliters.min-1.g-1 liver, respectively. In contrast, the k for insulin removal by hindlimbs from SHR was decreased 37% (P less than 0.001) compared with WKY rats (8.6 +/- 0.5 vs. 13.7 +/- 0.7 microliters.min-1.g-1 muscle), and this decrease was not accompanied by decreased binding of insulin to its receptor in plantaris muscle. Although the removal of insulin by glomerular filtration was similar in SHR and WKY rats (653 +/- 64 microliters/min vs. 665 +/- 90 microliters.min-1.kidney-1), total insulin removal by kidney was significantly lower (P less than 0.05) in SHR (710 +/- 78 microliters/min) compared with WKY rats (962 +/- 67 microliters/min), due to decreased peritubular clearance of insulin in SHR (56 +/- 73 vs. 297 +/- 59 microliters/min, P less than 0.05). These findings suggest that the decreased clearance of insulin in SHR rats was possibly not due to impaired hepatic removal of insulin but rather to decreased removal by skeletal muscle and kidneys.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Basolateral and apical binding, internalization, and degradation of insulin by cultured kidney epithelial cells.

In vivo, filtered insulin is absorbed and degraded in proximal tubules after binding to the apical membrane. Peritubular removal also occurs and involves basolateral receptor binding and degradation. Whether basolateral degradation proceeds within the cell or on the cell surface is unknown. Because of the difficulties in addressing this question in vivo, this study was carried out with a cultured opossum kidney epithelium cell line with proximal-like features and insulin receptors. Cells were grown in partitioned wells on polycarbonate filters and, when confluent, the monolayer effectively separated the culture well into apical and basolateral compartments. Apical and basolateral binding, internalization, and degradation were studied separately by incubating monolayers with 125I-insulin added to either the apical or basal compartment. At 37 degrees C insulin associated with either pole in a time-dependent manner. This interaction was specific, for it was competitively inhibited by cold insulin but not by unrelated peptides. Separation of surface-bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 92% of the radioactivity added to either side of the monolayer was surface-bound, whereas at 37 degrees C and after 1 h, 57% was surface-bound and 43% internalized. Affinity of apical and basolateral receptors were similar (1-2 nM), but basolateral receptor number was greater, for at high insulin concentrations (5 x 10(-8) M) basolateral membrane binding exceeded apical by fivefold (250 +/- 81 vs. 56 +/- 11 fm/10(6) cells). Degradation followed exposure to either pole of the cell.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenocorticotropic Hormone↗
PubMed1988

Internalization and catabolism of insulin by an established renal cell line.

Proximal renal tubules are a key site of insulin metabolism. To explore the kinetics and metabolic requirements of insulin internalization and catabolism, we used the opossum kidney cell line, which has proximal tubular-like features and possesses insulin-specific receptors. Internalization was determined by separating membrane-bound insulin from intracellular insulin by exposure to an acidified medium. Internalization of membrane-bound insulin was rapid, and half-maximal internalization occurred within 2.5 min. Degradation products did not accumulate in the cell but appeared in the medium after a delay of 5 min from the onset of internalization. In other experiments, addition of KCN (2 mM) or omission of glucose did not alter degradation, but KCN, combined with the omission of glucose, inhibited degradation by 64%. This was associated with a 240% increase in membrane-bound insulin and an 81% decrease in intracellular insulin. Accordingly, it appears that under these circumstances impaired degradation was a consequence of impaired internalization. In contrast, although 0.1 mM chloroquine, an endosomal-lysosomal inhibitor, also depressed degradation (by 57%), intracellular insulin increased fourfold, indicating failure of intracellular processing. We conclude that these cultured kidney cells rapidly internalize and degrade insulin and that internalization, a prerequisite for degradation, is dependent on energy that can be derived from anaerobic glycolysis or oxidative metabolism. Furthermore, the intracellular degradative processing of insulin by these cells involves a chloroquine-sensitive pathway.

Animals↗
PubMed1988

Differences in renal metabolism of insulin and cytochrome c.

Kidneys degrade small proteins such as cytochrome c (CYT c) by the classic lysosomal pathway. However, because alternate routes for the transport and degradation of protein hormones have been identified in other tissues, we set out to determine whether extralysosomal sites might participate in the renal degradation of insulin. First, we compared the effect of the lysosomal inhibitor NH4Cl on insulin and CYT c degradation by isolated perfused rat kidneys. After kidneys were loaded with radiolabeled proteins to allow for absorption and transport to lysosomes, degradation was measured in the presence or absence of inhibitors. Control kidneys degraded 45 +/- 1.5% of the trapped CYT c per hour, and this was inhibited 62 +/- 1.3% by NH4Cl. In contrast, 86 +/- 2.4% of the trapped insulin was degraded per hour, and this was inhibited 26 +/- 4% by NH4Cl. Next we followed the subcellular distribution of 125I-labeled insulin in kidneys exposed to 125I-labeled insulin in vivo or when isolated and perfused. Under both circumstances the distribution of insulin on a linear sucrose gradient differed from that of the lysosomal enzyme N-acetyl-beta-glucosaminidase. In contrast, [14CH3]CYT c, injected in vivo, distributed over a density similar to the lysosomal marker. Thus important differences exist between the renal metabolism of CYT c, which proceeds in lysosomes, and the renal metabolism of insulin. These include rate of degradation, sensitivity to NH4Cl, and subcellular sites of localization. Accordingly, we suggest that insulin degradation may occur, at least in part, in a different compartment from the classic lysosomal site of protein degradation.

Ammonium Chloride↗
PubMed1988

Insulin binding, internalization, and degradation by a cultured kidney cell line.

Proximal tubules are a key site of insulin metabolism, which is in part a receptor-mediated process. To explore the interaction between insulin and the kidney and to evaluate the role of receptors in insulin uptake and processing, a study was carried out with a cultured proximal-like opossum kidney (OK) cell line. 125I-insulin associated with confluent monolayers in a specific manner, and this interaction was competitively inhibited by insulin; unrelated peptides were relatively ineffective. Insulin degradation exhibited time and temperature dependency and up to a concentration of 5 X 10(-8) M was not saturable. Degradation exhibited partial hormone specificity. Separation of plasma membrane bound from internalized insulin was achieved by lowering extracellular pH. At 4 degrees C, 94% of cell-associated radioactivity was membrane bound, whereas at 37 degrees C, in the steady state, 33% was membrane bound and 67% was internalized. There was a significant correlation between membrane-bound insulin and the rate of degradation. These findings reveal that the binding and processing of insulin by the kidney cell line are compatible with the description of the uptake of filtered insulin by the proximal tubule in the intact kidney. Accordingly we conclude that this cell line provides a good model for studying renal epithelial uptake and metabolism of insulin.

Adrenocorticotropic Hormone↗
PubMed1988

Renal metabolism of calcitonin.

The kidneys account for approximately two-thirds of the metabolism of calcitonin, but relatively little is known regarding the details thereof. To further characterize this process, we examined the renal handling and metabolism of human calcitonin (hCT) by the isolated perfused rat kidney. We also studied the degradation of radiolabeled salmon calcitonin (sCT) by subcellular fractions prepared from isolated rabbit proximal tubules. The total renal (organ) clearance of immunoreactive hCT by the isolated kidney was 1.96 +/- 0.18 ml/min. This was independent of the perfusate total calcium concentration from 5.5 to 10.2 mg/dl. Total renal clearance exceeded the glomerular filtration rate (GFR, 0.68 +/- 0.05 ml/min), indicating filtration-independent removal. Urinary calcitonin clearance as a fraction of GFR averaged 2.6%. Gel filtration chromatography of medium from isolated kidneys perfused with 125I-labeled sCT showed the principal degradation products to be low molecular weight forms eluting with monoiodotyrosine. Intermediate size products were not detected. In the subcellular fractionation experiments, when carried out at pH 5.0, calcitonin hydrolysis exclusively followed the activities of the lysosomal enzyme N-acetyl-beta-glucosaminidase. Typically, at pH 7.5, 42% of total degradation occurred in the region of the brush-border enzyme alanyl aminopeptidase and 29% occurred in the region of the cytosolic enzyme phosphoglucomutase. Although 9% of the calcitonin-degrading activity was associated with basolateral membrane fractions, most of this activity could be accounted for by the presence of brush-border membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗