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F G Hamel

Publications and source records attributed to F G Hamel.

52 records · Page 3Linked to original sources
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↗
PubMed1988

Hepatic metabolism of insulin.

The liver plays a major role in the metabolism of insulin, but the precise cellular mechanisms, the enzymes involved, and the products generated have only recently become clarified. The initial step in insulin degradation by the liver is binding to a cell membrane receptor, following which some insulin is degraded and the products released into the incubation medium, whereas some insulin is internalized and degraded intracellularly. Recently, it has been demonstrated that the degradation of insulin by hepatocytes produces products identical to those generated by the enzyme insulin protease. With both enzyme and intact hepatocytes, two A-chain cleavages and four major and three minor B-chain cleavages occur in intact insulin. It has also been demonstrated that internalized insulin is degraded in early endosomes, primarily by cleavages in the B chain and occurring prior to acidification of the endosome and thus prior to dissociation of insulin from its receptor. The initial cleavages in the B chain of insulin occur in the same sites as are cleaved by insulin protease, supporting a role for this enzyme, both in the extracellular and intracellular metabolism of insulin. These findings also indicate that lysosomes probably play a minor or secondary role for hepatic insulin metabolism.

Animals↗
PubMed1988

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.

Adipose Tissue↗
PubMed1988

Isolation of insulin degradation products from endosomes derived from intact rat liver.

Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease.

Animals↗
PubMed1988

Degradation products of insulin generated by hepatocytes and by insulin protease.

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1988

Insulin-like effects of vanadate in isolated rat adipocytes.

Vanadate has been shown to have a number of insulin-like effects in various cells, including isolated rat adipocytes. In the present study we compared the activities of vanadate and insulin in isolated fat cells using a number of different assays of insulin-like activity. Both insulin and vanadate stimulated [2-3H]glucose incorporation into fat cell lipid in a dose-dependent manner, but the maximal effect of vanadate was markedly greater than that of insulin. At 10(-2) M vanadate the effect was 3-4 times as great as the maximal effect of insulin. This effect was dependent on specific glucose transport. Combinations of insulin and vanadate were not more effective than vanadate alone. Vanadate also produced antilipolysis with an effect somewhat greater than that of insulin. Using [U-14C]glucose both vanadate and insulin stimulated 14CO2 production and [14C]glucose incorporation into lipid, and again the effect of vanadate was greater than that of insulin. Vanadate had a greater effect on 14CO2 production than on [14C]glucose incorporation into lipid. When [1-14C]glucose was used vanadate again had a significantly greater effect on 14CO2 production than did insulin, but when [6-14C]glucose was used the effects of vanadate and insulin were equal. These results demonstrate that vanadate has insulin-like effects in isolated fat cells, but it selectively stimulates certain pathways to a greater extent than does insulin. The greater effect of vanadate than insulin appears to be primarily on the pentose phosphate shunt, suggesting that this agent may be useful for examination of this intracellular pathway in fat cells.

Adipose Tissue↗
PubMed1988

High performance liquid chromatographic analysis of insulin degradation products from a cultured kidney cell line.

The kidney is a major site for insulin removal and degradation, but the subcellular processes and enzymes involved have not been established. We have examined this process by analyzing insulin degradation products by HPLC. Monoiodoinsulin specifically labeled on either the A14 or B26 tyrosine residue was incubated with a cultured kidney epithelial cell line, and both intracellular and extracellular products were examined on HPLC. The products were then compared with products of known structure generated by hepatocytes and the enzyme insulin protease. Intracellular and extracellular products were different, suggesting two different degradative pathways, as previously shown in liver. The extracellular degradation products eluted from HPLC both before and after sulfitolysis similarly with hepatocyte products and products generated by insulin protease. The intracellular products also eluted identically with hepatocyte products. Based on comparisons with identified products, the kidney cell generates two fragments from the A chain of intact insulin, one with a cleavage at A13-A14 and the other at A14-A15. The B chain of intact insulin is cleaved in a number of different sites, resulting in peptides that elute identically with B chain peptides cleaved at B9-B10, B13-B14, B16-B17, B24-B25, and B25-B26. These similarities with hepatocytes and insulin protease suggest that liver and kidney have similar mechanisms for insulin degradation and that insulin protease or a very similar enzyme is involved in both tissues.

Animals↗
PubMed1987

Identification of A chain cleavage sites in intact insulin produced by insulin protease and isolated hepatocytes.

The degradation of insulin by the enzyme insulin protease and by isolated hepatocytes results in proteolytic cleavages in both the A and B chains of intact insulin. Previous studies have shown that one of the A chain cleavages is between A13 leucine and A14 tyrosine and that a second cleavage occurs carboxyl to the A14 residue. In the present study we have used insulin specifically iodinated on the A19 tyrosine and examined the A chain cleavages by the enzyme and by hepatocytes. Insulin degradation products were purified by HPLC and sequenced by automated Edman degradation. Only two A chain cleavage sites were identified, one the previously reported A13-A14 and the other between A14 tyrosine and A15 glutamine. These data thus identify the second A chain cleavage site and further support the role of insulin protease in hepatic metabolism of insulin.

Amino Acid Sequence↗
PubMed1987

HPLC analysis of insulin degradation products from isolated hepatocytes. Effects of inhibitors suggest intracellular and extracellular pathways.

Isolated rat hepatocytes were incubated with A14-[125I]monoiodotyrosyl insulin for 30 min, and labeled material was extracted from the cells and incubation media. The medium and the cell extract were chromatographed on a Sephadex G-50 column, and radioactivity eluting in the position of intact insulin was concentrated and analyzed on HPLC. The HPLC analysis of the cell extract showed two major products eluting from the column at 19 and 23 min, whereas medium extracts showed one prominent product eluting at 14 min. Inclusion of chloroquine in the incubation blocked the formation of cellular products at 19 and 23 min and caused the accumulation of a product eluting at 41 min while not affecting the media products. After sulfitolysis all cellular products contained an intact A-chain. Dansylcadaverine increased media products and altered the cell-extracted product pattern such that it had a major peak at 14 min, similar to media. These results suggest that two pathways for insulin degradation exist within hepatocytes. The extracellular process forms products that are essentially unchanged by chloroquine and dansylcadaverine. The intracellular process is altered by chloroquine and apparently inhibited by dansylcadaverine.

Animals↗
PubMed1986

High performance liquid chromatographic analysis of insulin degradation by rat skeletal muscle insulin protease.

The degradation of [125I]iodoinsulin (A14) by insulin protease (EC 3.4.22.11) was studied using HPLC. A reverse phase HPLC method is presented which allows the separation and quantitation of insulin degradation products. After incubation of [125I]iodoinsulin (A14) with insulin protease, there was an initial rapid loss of radioactivity from the [125I] iodoinsulin (A14) peak, which was quantitatively accounted for by the appearance of radioactivity in 11 different peaks, but was not accompanied by a proportional increase in the solubility of the sample in trichloroacetic acid. Two of the peaks showed appreciable accumulation before the others, and all but the first-eluted peak plateaued by 20 min. After 20 min of incubation, the amount of radioactivity present as the first-eluted peak, solubility in trichloroacetic acid, and insulin loss continued to increase at a steady, but slowed, rate. The order of appearance suggests that insulin protease acts on insulin in an ordered sequence of steps to generate a number of intermediates that are precipitable by trichloroacetic acid, but are subsequently degraded to material that is soluble in trichloroacetic acid. Sulfitolysis of 5 major peaks and subsequent HPLC analysis of the fragments showed none of the peaks to possess intact A chains. Peptide sequencing of 2 of the peaks indicates that the A-chain is cleaved in at least 2 positions, one beyond the 14th position, and one between the 13th and 14th amino acids (leucine and tyrosine).

Animals↗
PubMed1985

Evidence that bacitracin alters intracellular insulin metabolism in isolated rat hepatocytes.

The effect of bacitracin on intracellular insulin degradation was investigated using an isolated rat hepatocyte preparation in which essentially all insulin degradation was due to cell-mediated processes. Bacitracin produced a concentration-dependent decrease in the degradation of insulin to products soluble in trichloroacetic acid, with a half-maximal effect at approximately 0.5 mM. These results were confirmed by analysis of extracted cell-bound radioactivity by Sephadex G-50 molecular sieve chromatography. Radioactive material eluting in the position of intact insulin from the G-50 column was further analyzed by reversed-phase, high-performance liquid chromatography. In addition to intact insulin, two peaks of radioactive material less hydrophobic than insulin were evident. Incubation of cells in the presence of 0.5 mM bacitracin significantly (P less than 0.05) altered the distribution of radioactivity in these two peaks. These results indicate that bacitracin significantly affects hepatocyte insulin metabolism and suggest that the continued use of bacitracin in studies of hepatocyte-insulin interaction should be avoided.

Animals↗
PubMed1983

Changes in renal phospholipid fatty acids in diabetes mellitus: correlation with changes in adenylate cyclase activity.

Male Sprague-Dawley rats made diabetic with alloxan (37.5 mg/kg) or streptozotocin (65 mg/kg) were killed after 3-6 weeks of disease; renal tissues were studied for phospholipid content and for fatty acid composition of the phospholipids. No consistent change was noted in total phospholipid content nor in the proportion of various phospholipids in diabetics. However, diabetic animals showed a consistent reduction of arachidonic acid content in phosphatidylcholine (PC) and phosphatidylethanolamine in whole renal cortex, plasma membranes purified from renal cortex, and in isolated glomeruli. Associated with the fall in arachidonic acid was a rise in linoleic acid in the samples studied. Insulin therapy returned the fatty acid profiles to normal. These results are similar to patterns observed in other diabetic tissues and suggest that diabetes is associated with generalized changes in cell membranes. That these structural changes may have functional significance is suggested by demonstrated alterations in the temperature-dependence of adenylate cyclase in renal plasma membranes of diabetic animals. Adenylate cyclase is thought to be intimately associated with PC in plasma membranes, a phospholipid showing significant changes in fatty acid content in diabetes (unsaturation index 165 +/- 2 for normals, 147 +/- 5 for diabetics). Na+,K+-ATPase which is thought to be primarily associated in vivo with phosphatidylinositol (PI), shows no change in apparent energy of activation in diabetes. The fatty acid content of PI is minimally altered in diabetes, and the unsaturation index is unchanged.

Adenylyl Cyclases↗
PubMed1983

Effect of gentamicin treatment on adenylate cyclase and Na+, K+-ATPase activities in renal tissues of rats.

Gentamicin (20 mg/kg) treatment of male rats reduced Na+,K+-ATPase activity by 32% in renal cortical plasma membranes. In contrast, adenylate cyclase stimulation by isoproterenol or a guanyl nucleotide or both was enhanced by as much as twofold in glomeruli and in plasma membranes of gentamicin-treated rats. These effects of gentamicin are suggested to be related to the changes in renal phospholipid metabolism produced by the drug.

Adenylyl Cyclases↗
PubMed1978

Independence of proline chemotaxis and transport in Bacillus subtilis.

Values of KI for nine proline analogs as inhibitors of proline chemotaxis and of proline transport were determined. Two of them inhibited transport at substantially lower concentrations than chemotaxis; two at substantially higher concentrations. Moreover, mutants, believed to be in the component that binds proline, were isolated that showed a shift of KM for transport to higher concentrations, one as much as 40-fold. However, chemotaxis was virtually unaffected. Therefore, unlike galactose chemotaxis and transport in Escherichia coli, which share the galactose-binding protein, proline chemotaxis and transport in Bacillus subtilis are independent.

Amino Acids↗
PubMedDate not supplied

The relationship between insulin and vanadium metabolism in insulin target tissues.

Vanadium (V) is an orally effective treatment for diabetes, but relatively little is known about the mechanisms controlling its normal metabolism nor the long term pharmacokinetics of oral administration. We have examined the accumulation of V in various organs from rats fed liquid diet for up to 18 days, containing no additional V, 1.6, 80, or 160 mumole/kg/day as either sodium orthovanadate (SOV) or vanadyl sulfate (VS). V content was assayed using a sensitive neutron activation analysis method. The organs of the nonsupplemented animals contained widely varying concentrations (ng of V/g dry tissue weight) with brain < fat < blood < heart < muscle < lung < liver < testes < spleen < kidney. All organs accumulated V in a dose dependent manner. Not all organs showed steady state amount of V at 18 days, so additional rats were fed SOV or VS, switched to control diet, and assayed at 0, 4 and 8 days. From this data we calculated organ half lives of V. Insulin sensitive tissue tissues, such as liver and fat, had shorter half-lives than tissues that are relatively less insulin sensitive, such as spleen, brain and testes. SOV and VS fed rats showed similar patterns, but VS had somewhat shorter t1/2's. Additional studies of old and young rats fed control diet for 45 days show accumulation of V in spleen and testes. These results indicate that vanadium metabolism varies widely among different organs, and that insulin, either directly or indirectly has effects on the retention of vanadium. This may have impact on the therapeutic use of vanadium in Type I diabetics with no insulin, or Type II patients who may be relatively hyperinsulinemic.

Animals↗