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L Jarett

Publications and source records attributed to L Jarett.

At least 73 records · Page 4Linked to original sources
PubMed1987

Partial structure of an insulin-sensitive glycophospholipid.

The structure of a glycophospholipid, which has been involved in insulin action, has been investigated using H35 cells and rat liver membranes. The present evidence indicates that this molecule contains a phosphatidyl-chiro-inositol moiety, glycosidically linked to a non-N-acetylated glucosamine. In addition, the polar head group of the lipid contains galactose, probably four residues, and a total number of three phosphates.

Amino Acid Sequence↗
PubMed1987

Adenosine and oxytocin reverse antagonism of cyclic AMP elevating agents to insulin activation of adipocyte pyruvate dehydrogenase.

ACTH, isoprenaline, forskolin, and dibutyryl cyclic AMP prevented insulin from stimulating adipocyte pyruvate dehydrogenase in the presence of adenosine deaminase. Antagonism was reversed by N6-phenylisopropyladenosine as well as oxytocin. The stimulatory effects of insulin, adenosine and oxytocin on adipocyte pyruvate dehydrogenase appear to be through (a) mechanism(s) which is (are) similar or related.

Adenosine↗
PubMed1987

Nuclear translocation of the insulin receptor. A possible mediator of insulin's long term effects.

The translocation of occupied surface insulin receptors to the nuclei of isolated hepatocytes was studied using the biologically active photosensitive insulin derivative, B2(2-nitro-4-azidophenylacetyl)-des-PheB1-insulin (NAPA-DP-insulin). When hepatocytes were photolabeled at 4 degrees C, extensively washed, and then further incubated at 37 degrees C for 1 h, photolabeled insulin receptors, which were initially localized to the cell surface, accumulated in the subsequently isolated nuclei. When the isolated nuclei were solubilized and subjected to polyacrylamide gel electrophoresis and radioautography, labeled proteins with Mr identical to the cell surface insulin receptor were detected. Light microscopic radioautography of nuclei isolated from cells incubated for 1 ha at 37 degrees C demonstrated that 28% of these nuclei were specifically labeled with one or more grains. Electron microscopic radioautography of intact cultured hepatocytes, incubated 60 min at 37 degrees C, revealed that 26% of the thin-sectioned nuclei contained at least a single grain and 8.3% of the total cell-associated associated grains were located over the nuclei. Only 1.6% of grains were localized to lysosomes. In contrast, if photolabeled hepatocytes were incubated at 4 degrees C for up to 2 h, negligible accumulation of nuclear radioactivity was observed by polyacrylamide gel electrophoresis on light or electron microscopic radioautography. Conclusions are as follows. Occupied cell surface insulin receptors can internalize and translocate to the nucleus of intact hepatocytes by a time- and temperature-dependent mechanism. Accumulation and possible degradation of insulin receptors in lysosomes involves only a small percentage of the receptors internalized. Nuclear translocation of occupied cell surface insulin receptors may be a mechanism which mediates insulin's long term effects.

Affinity Labels↗
PubMed1987

Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells.

This study identifies and partially characterizes an insulin-sensitive glycophospholipid in H35 hepatoma cells. The incorporation of [3H]glucosamine into cell lipids was investigated. A major labeled lipid was purified by sequential thin layer chromatography using first an acid followed by a basic solvent system. After hydrochloric acid hydrolysis and sugar analysis by thin layer chromatography, 80% of the radioactivity in the purified lipid was found to comigrate with glucosamine. H35 cells were prelabeled with [3H]glucosamine for either 4 or 24 h and treated with insulin causing a dose-dependent stimulation of turnover of the glycophospholipid which was detected within 1 min. The purified glycolipid was cleaved by nitrous acid deamination indicating that the glucosamine C-1 was linked to the lipid moiety through a glycosidic bond. [14C]Ethanolamine, [3H]inositol, and [3H]sorbitol were not incorporated into the purified glycolipid. The incorporation of various fatty acids into this glycolipid was also studied. [3H]Palmitate was found to be preferentially incorporated while myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid were either not incorporated or incorporated less than 10% of palmitate. The purified glycolipid labeled with [3H]palmitate was cleaved by treatment with phospholipase A2 but was resistant to mild alkali hydrolysis suggesting the presence of a 1-hexadecyl,2-palmitoyl-glyceryl moiety in the purified lipid. Treatment of labeled glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus generated a compound migrating as 1-alkyl,2-acyl-glycerol and a polar head group with a size in the range from 800 to 3500. These findings coupled with the nitrous acid deamination demonstrate that glucosamine was covalently linked through a phosphodiester bond to the glyceryl moiety of the purified glycolipid. These findings suggest that insulin acts on this glycophospholipid by stimulating an insulin-sensitive phospholipase C. This unique glycophospholipid may play an important role in insulin action by serving as precursor of insulin-generated mediators.

Acetylation↗
PubMed1987

Insulin binding and processing by H4IIEC3 hepatoma cells: ultrastructural and biochemical evidence for a unique route of internalization and processing.

Biochemical and ultrastructural studies of insulin binding and cellular processing by cultured H4IIEC3 hepatoma cells were performed. Insulin binding and intracellular accumulation were rapid and after 30 min at 37 degrees C, 65% of the total cell-associated 125I-insulin was in an acid-stable compartment. Chloroquine had no significant effect on the amount of total cell-associated insulin or the percentage of insulin in the acid-stable compartment or cell-associated insulin degradation under those conditions, but after 60-min incubations, it slightly decreased the rate of dissociation of internalized hormone. Ultrastructural analysis revealed that monomeric ferritin-insulin (Fm-I) initially bound to single or paired receptors on microvilli. Within 5 min occupied insulin receptors microaggregated and migrated to the intervillous cell surface. During the next 5-10 min occupied receptors aggregated into large clusters on the plasma membrane. Large amounts of insulin were internalized by macropinocytosis and the majority of internalized Fm-I was found in phagosomes. Less than 10% of the membrane-bound insulin was associated with pinocytotic invaginations or coated pits and less than 5% of the total cell-associated insulin was found in lysosomes. Chloroquine had no detectable effect on the amount of Fm-I or its distribution among the intracellular organelles. These studies demonstrated that, compared to previous studies with rat adipocytes or 3T3-L1 adipocytes, insulin interalization and intracellular processing in this hepatoma cell were unique. These differences provide further evidence that insulin binding and processing may be controlled by cell-specific mechanisms and that substantial heterogeneity exists in pathways previously presumed to be similar for all cell types.

Animals↗
PubMed1987

Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events.

This ultrastructural study compared the endocytosis of a peptide hormone, ferritin-labeled insulin (Fm-I) or gold-labeled insulin (Au-I), and a non-hormonal ligand, gold-labeled alpha-2-macroglobulin-methylamine (Au-alpha 2MGMA), by rat adipocytes. Quantitative analysis of the cell surface showed that coated pits occupied 0.4% of the adipocyte surface. This was one fifth to one tenth of that which has been reported on fibroblasts and hepatocytes, cell types in which receptor-mediated endocytosis has been extensively studied. In contrast, uncoated micropinocytotic invaginations were quite numerous and occupied 13.1% of the adipocyte cell surface. The frequency of micropinocytotic invaginations, 13.8 per micron 2 of plasma membrane, was 7-12 times greater than has been reported on fibroblasts. Therefore, the ultrastructure of the endocytic apparatus on rat adipocytes was different from more commonly studied cell types. At 4 degrees C, Au-alpha 2MGMA concentrated within coated pits to a density that was 52 times greater than that on the uncoated plasma membrane. Au-alpha 2MGMA was excluded from micropinocytotic invaginations by more than 93%; this exclusion was unrelated to the size of the Au-alpha 2MGMA particle. In contrast, at 4 degrees C, Fm-I did not concentrate within coated pits and occupied micropinocytotic invaginations in a random manner. At 37 degrees C, coated pits accounted for all of the endocytosis of Au-alpha 2MGMA, proving that these structures were functional despite their atypically low density. In contrast, greater than 99% of the endocytosis of Fm-I or Au-I occurred through micropinocytotic invaginations. These results demonstrated for the first time by a comparative, quantitative, ultrastructural method that insulin and Au-alpha 2MGMA undergo endocytosis by dissimilar mechanisms on rat adipocytes. Dissimilarities in the endocytosis of insulin and Au-alpha 2MGMA may be related to the different biological roles of these two molecules.

Adipose Tissue↗
PubMed1987

Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: II. Comparison of intracellular events.

A previous ultrastructural study showed that gold-labeled insulin (Au-I) and the non-hormonal ligand gold-labeled alpha-2-macroglobulin-methylamine (Au-alpha 2MGMA) underwent endocytosis by dissimilar cell surface structures on rat adipocytes. The present ultrastructural study compared the intracellular routes taken by these two ligands in adipocytes. Intracellular Au-alpha 2MGMA was initially found within apparent coated vesicles but Au-I was not, consistent with the previous demonstration that Au-alpha 2MGMA underwent endocytosis by coated pits whereas Au-I was internalized by uncoated micropinocytotic invaginations. Early in the endocytic pathway, the two ligands were segregated within separate small vesicles and tubulovesicles. Au-alpha 2MGMA was concentrated in a small number of these structures whereas Au-I was sparsely distributed among a relatively large number. Subsequently, the two endocytic pathways converged as the ligands intermingled within pale multivesicular bodies and lysosome-like structures. Au-I was less efficiently transferred to lysosomes than Au-alpha 2MGMA since a greater proportion of intracellular Au-I remained associated with small vesicles and tubulovesicles. This study indicates that early intracellular events in the endocytic pathways of insulin and alpha 2MGMA are distinct. These findings are discussed in light of the fundamentally dissimilar biological roles of these two molecules and the possible involvement of the endocytic pathway in the insulin signaling mechanism.

Adipose Tissue↗
PubMed1987

Glucose transport and antilipolysis are differentially regulated by the polar head group of an insulin-sensitive glycophospholipid.

A glycophospholipid has been purified from rat liver membranes, which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid, which is a phosphooligosaccharide, was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. There was an "insulin-like" inhibitory effect of this phosphooligosaccharide on isoproterenol-stimulated lipolysis in adipocytes, whereas there was no effect on glucose oxidation under conditions that measure glucose transport. The antilipolytic effect of this phosphooligosaccharide was demonstrated in intact adipocytes. There was a linear correlation between the concentration of phosphooligosaccharide and its antilipolytic effect, the magnitude and time course of which were similar to that obtained with physiological concentrations of insulin. Submaximal concentrations of insulin and phosphooligosaccharide produced an additive antilipolytic effect. The antilipolytic effect of the phosphooligosaccharide was demonstrated only after release of this compound from the precursor glycophospholipid with phosphatidylinositol-specific phospholipase C, and the activity of the phosphooligosaccharide was sensitive to alkali. It is proposed that this phosphooligosaccharide plays a role in mediating certain insulin actions.

Adipose Tissue↗
PubMed1987

Ultrastructural evidence for the accumulation of insulin in nuclei of intact 3T3-L1 adipocytes by an insulin-receptor mediated process.

Monomeric ferritin-labeled insulin (Fm-Ins), a biologically active, electron-dense marker of occupied insulin receptors, was used to characterize the internalization of insulin in 3T3-L1 adipocytes. Fm-Ins bound specifically to insulin receptors and was internalized in a time- and temperature-dependent manner. Fm-Ins was found in cytoplasmic vesicles within 5-10 min at 37 degrees C and subsequently was observed in multivesicular bodies and lysosomes. In addition, small amounts of Fm-Ins were associated with nuclei after 30 min. The number of Fm-Ins particles observed in nuclei continued to increase in a time-dependent manner until at least 90 min. In the nucleus, several Fm-Ins particles usually were found in the same general location--near nuclear pores, associated with the periphery of the condensed chromatin. Addition of a 250-fold excess of unlabeled insulin or incubation at 15 degrees C reduced the number of Fm-Ins particles found in nuclei after 90 min by 99% or 92%, respectively. Nuclear accumulation of unlabeled ferritin was only 2% of that found with Fm-Ins after 90 min at 37 degrees C. Biochemical experiments utilizing 125I-labeled insulin and subcellular fractionation indicated that intact 3T3-L1 adipocytes internalized insulin rapidly and that approximately equal to 3% of the internalized ligand accumulated in nuclei after 1 hr. These data provide biochemical and high-resolution ultrastructural evidence that 3T3-L1 adipocytes accumulate potentially significant amounts of insulin in nuclei by an insulin receptor-mediated process. The transport of insulin or the insulin-receptor complex to nuclei in this cell or in others may be directly involved in the long-term biological effects of insulin--in particular, in the control of DNA and RNA synthesis.

Adipose Tissue↗
PubMed1986

The polar head group of a novel insulin-sensitive glycophospholipid mimics insulin action on phospholipid methyltransferase.

A phospholipid has been purified from rat liver membranes which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this phospholipid was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and purified through a C18 extraction column. Like insulin, the addition of this polar head group to isolated rat adipocytes inhibited the stimulatory effect of isoproterenol on phospholipid methyltransferase. The polar head group was also active on a subcellular fraction. The addition of the polar head group to microsomes isolated from isoproterenol-treated adipocytes produced a time-dependent inactivation of phospholipid methyltransferase, approaching basal activity. It is proposed that the effects of insulin on phospholipid methyltransferase may be mediated by this polar head group.

Adipose Tissue↗
PubMed1986

Insulin processing by cultured hepatocytes.

The chloroquine-sensitive and chloroquine-insensitive steps in insulin degradation by cultured hepatocytes have been explored by employing low temperature to retard processing. Under standard conditions (90 min association and 60 min dissociation) chloroquine inhibited insulin degradation at 15 degrees C but not at 37 degrees C. However, if the association and dissociation periods were short so that only early degradation was examined, marked inhibition of insulin degradation by chloroquine could also be observed at 37 degrees C. This inhibitory effect was observed only during the first 15 min, being masked by increased insulin degradation subsequently. An increase in slowly dissociable insulin, as well as a twofold increase in volume density of multivesicular bodies (MVB), occurred in the presence of chloroquine at both 37 and 15 degrees C. Rapid insulin processing from the slowly dissociable compartment at 37 degrees C masked chloroquine's effect on insulin processing under usual conditions at that temperature. At physiological temperature the chloroquine-sensitive step is not obligatory for insulin degradation by hepatocytes.

Animals↗
PubMed1986

Cell type-specific variability of bacitracin's effects on insulin binding and intracellular accumulation.

Bacitracin is known to inhibit proteolytic degradation of insulin and several other peptide hormones. Previous work with isolated rat adipocytes showed that bacitracin blocked insulin degradation by the plasma membrane and, even in the absence of detectable insulin degradation, bacitracin increased insulin binding by decreasing the rate of insulin dissociation. The present study examined the effects of bacitracin on insulin binding and degradation and on levels of intracellular insulin in a variety of cell types. Bacitracin inhibited insulin degradation in all cell types. Maximal inhibition varied from 70% (H4IIEC3 hepatoma cells) to 95% (rat adipocytes); concentrations giving half-maximal inhibition varied from 25 microM (3T3-A31 fibroblasts) to 250 microM (H4IIEC3). Dose-response curves showed three distinctive effects on insulin binding: dose-dependent stimulation (rat adipocytes), a biphasic curve with slight stimulation at low doses and inhibition at concentrations greater than 50 microM (human fibroblasts, H4IIEC3, and 3T3-L1 adipocytes), or dose-dependent inhibition of binding (3T3-L1 preadipocytes and 3T3-A31 fibroblasts). The intracellular accumulation of insulin rat adipocytes was not affected by bacitracin but was decreased in all other cell types. These data illustrated type-specific variability in the effects of bacitracin on insulin processing resulting from cellular heterogeneity either in processing insulin or in response to bacitracin, or both, and suggest that insulin binding studies performed in the presence of bacitracin can be biased.

Adipose Tissue↗
PubMed1986

Heterogeneous effects of inhibitors of receptor processing on insulin binding and intracellular accumulation in various cell types.

This study utilized uniform incubation conditions and demonstrated heterogeneity in the effects of various agents on 125-insulin binding and intracellular accumulation in five cell types. The cells used in this study included rat adipocytes, H4IIEC3 cultured hepatoma cells, normal human fibroblasts, and 3T3-L1 preadipocytes and adipocytes. Bacitracin increased insulin binding to rat adipocytes but inhibited binding to all other cells. Chloroquine increased total cell-associated insulin in all cells except H4IIEC3 hepatoma cells. Methylamine and dansylcadaverine increased or decreased binding depending on cell type. Similar heterogeneity was found in the intracellular accumulation of insulin. Under control conditions, intracellular insulin at steady state varied from 16 to 52% of the total cell-associated insulin. Bacitracin decreased intracellular accumulation of insulin in 3T3-L1 adipocytes, preadipocytes, and human fibroblasts but increased accumulation in rat adipocytes. Chloroquine increased insulin accumulation in all cell types except H411EC3 hepatoma cells. Both methylamine and dansylcadaverine increased intracellular insulin in rat adipocytes and decreased accumulation in human fibroblasts. These results provide additional evidence of heterogeneity in insulin and/or insulin receptor processing among different cell types.

Adipose Tissue↗
PubMed1985

Adenosine effects on glucose oxidation of adipocytes isolated from streptozotocin-diabetic rats.

Streptozotocin-induced diabetes did not impair the response of adipocytes to adenosine effects in glucose oxidation. The greatest effect of adenosine in potentiating the action of insulin was in the physiological concentration range of insulin (10-100 mu units/ml). The desensitization of cells by diabetes to the effects of insulin is therefore probably not related to the response of cells to adenosine.

Adenosine↗
PubMed1985

Adrenocorticotropic stimulation and insulin inhibition of adipocyte phospholipid methylation.

Treatment of isolated rat adipocytes with adrenocorticotropin (ACTH) caused a 1.5-fold increase in phospholipid methyltransferase activity within 5 min. This effect of ACTH was concentration-dependent with maximal activation at 2 milliunits/ml ACTH, and was reproduced by dibutyryl cyclic AMP. ACTH (2 milliunits/ml) caused an increase in the Vmax value of phospholipid methyltransferase without changing the Km for S-adenosyl-L-methionine. Insulin caused a concentration-dependent inhibition of both control and ACTH-stimulated phospholipid methyltransferase. Half-maximal inhibition by insulin was demonstrated with 5 microunits/ml insulin in control cells and with 25 microunits/ml insulin in ACTH-stimulated cells. The rapid and sensitive activation of adipocyte phospholipid methyltransferase by ACTH and inhibition by insulin are consistent with a role for this pathway in the hormonal response of the adipocyte.

Adipose Tissue↗