Continuous Presence Of Insulin Research Articles

Insulin-responsive Aminopeptidase Trafficking in 3T3-L1 Adipocytes

The insulin-responsive aminopeptidase (IRAP/VP165/gp160) was identified originally in GLUT4-containing vesicles and shown to translocate in response to insulin, much like the glucose transporter 4 (GLUT4). This study characterizes the trafficking and kinetics of IRAP in exocytosis, endocytosis, and recycling to the membrane in 3T3-L1 adipocytes. After exposure of 3T3-L1 adipocytes to insulin, IRAP translocated to the plasma membrane as assessed by either cell fractionation, surface biotinylation, or the plasma membrane sheet assay. The rate of exocytosis closely paralleled that of GLUT4. In the continuous presence of insulin, IRAP was endocytosed with a half-time of about 3-5 min. IRAP endocytosis is inhibited by cytosol acidification, a property of clathrin-mediated endocytosis, but not by the expression of a constitutively active Akt/PKB. Arrival in an LDM fraction derived via subcellular fractionation exhibited a slower time course than disappearance from the cell surface, suggesting additional endocytic intermediates. As assayed by membrane "sheets," GLUT4 and IRAP showed similar internalization rates that are wortmannin-insensitive and occur with a half-time of roughly 5 min. IRAP remaining on the cell surface 10 min following insulin removal was both biotin- and avidin-accessible, implying the absence of thin-necked invaginations. Finally, endocytosed IRAP quickly recycled back to the plasma membrane in a wortmannin-sensitive process. These results demonstrate rapid endocytosis and recycling of IRAP in the presence of insulin and trafficking that matches GLUT4 in rate.

Temporal Separation of Insulin-stimulated GLUT4/IRAP Vesicle Plasma Membrane Docking and Fusion in 3T3L1 Adipocytes

Examination of the time and temperature dependence of insulin-stimulated GLUT4/IRAP-containing vesicle trafficking demonstrated an approximate 7-fold increase in the half-time for plasma membrane translocation at 23 degrees C (t((1)/(2)) = approximately 30 min) compared with 37 degrees C (t((1)/(2)) = approximately 4 min) without a significant change in the extent of either GLUT4 or IRAP translocation. Localization of the endogenous GLUT4 and expressed GLUT4-enhanced green fluorescent protein fusion protein in intact 3T3L1 adipocytes demonstrated that at 23 degrees C there was a time-dependent accumulation of discrete GLUT4-containing vesicles adjacent to the inner face of the cell surface membrane but that was not contiguous and/or physically incorporated into the plasma membrane. Together, these data demonstrate that the temperature-dependent decrease in the rate of GLUT4 and IRAP translocation results from a reduction in GLUT4/IRAP-containing vesicle fusion and not trafficking or docking to the plasma membrane.

Divergent Insulin and Platelet-derived Growth Factor Regulation of Focal Adhesion Kinase (pp125FAK) Tyrosine Phosphorylation, and Rearrangement of Actin Stress Fibers

Insulin treatment of Chinese hamster ovary cells expressing high levels of the human insulin receptor resulted in the tyrosine dephosphorylation of the 125-kDa focal adhesion kinase (pp125FAK). The decrease in pp125FAK tyrosine phosphorylation paralleled a decrease in the cellular content of actin stress fibers, and these changes were independent of the extracellular matrix on which the cells were grown. The reduction in both pp125FAK tyrosine phosphorylation and actin stress fibers occurred in an insulin concentration-dependent manner, with significant effects at approximately 0.3 nM and a maximal effect at 3 nM. However, in the continuous presence of insulin, the decreases in the tyrosine phosphorylation state of pp125FAK and actin stress fiber content were transient. Maximal reduction of pp125FAK tyrosine phosphorylation was observed following 15 min of insulin treatment, with a return to unstimulated control levels by 60 min. Similarly, actin stress fiber content was maximally reduced by 15 min of insulin treatment and fully recovered by 60 min. In contrast to insulin, platelet-derived growth factor stimulation increased actin stress fiber content and enhanced pp125FAK tyrosine phosphorylation. These data demonstrate a novel signaling role for insulin in inducing the tyrosine dephosphorylation of pp125FAK and a concomitant reorganization of actin stress fibers, which underlies at least one aspect of signaling divergence between the insulin and platelet-derived growth factor receptor tyrosine kinases.

Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action.

The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismanose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos- 4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

Metformin blocks downregulation of cell surface GLUT4 caused by chronic insulin treatment of rat adipocytes.

Large decreases in insulin-responsive glucose transport occur in rat adipocytes maintained in culture for 24 h in the continuous presence of insulin. After 24 h in culture, an acute treatment with insulin increased 3-O-methyl-D-glucose transport by only approximately fivefold. In chronically insulin-treated cells, the transport activity was more severely reduced. The transport activity was only approximately twofold higher than in basal cells. To attribute changes in transport to alterations in cell surface transporters, we labeled the cell surface GLUT4 and GLUT1 transporters with the impermeant photoaffinity label 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy)-2- propylamine. Cell surface labeling was compared with the labeling obtained in digitonin-permeabilized cells where the normally impermeant reagent had access to the total cellular pool of transporters. Labeling showed that in basal cells the proportions of GLUT4 and GLUT1 at the cell surface were 20 and 22% of the total. After an acute treatment with insulin, the proportions of GLUT4 and GLUT1 at the cell surface were increased to 49 and 37% of the total, respectively. The chronic insulin treatment was associated with a very low proportion of GLUT4 (25% of the total) at the cell surface. The downregulation of GLUT4 observed after chronic insulin treatment was alleviated by metformin, and the proportion of GLUT4 at the cell surface was maintained at 60% of the total.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin and Glucose-dependent Regulation of the Glucose Transport System in the Rat L6 Skeletal Muscle Cell Line

Differentiated rat L6 skeletal muscle cell cultures maintained in glucose-deficient medium containing 25 mM xylose displayed a rapid, reversible, time- and concentration-dependent 3-5-fold increase in glucose transport activity. Glucose deprivation in the continuous presence of insulin (24 h) resulted in an overall 9-10-fold stimulation of glucose transport activity. In contrast, acute (30 min) and chronic (24 h) insulin treatment of L6 cells maintained in high glucose (25 mM)-containing medium resulted in a 1.5- and 4-fold induction of glucose transport activity, respectively. Acute glucose deprivation and/or insulin treatment had no significant effect on the total amount of glucose transporter protein, whereas the long-term insulin- and glucose-dependent regulation of glucose transport activity directly correlated with an increase in the cellular expression of the glucose transporter protein. In situ hybridization of the L6 cells demonstrated a 3-, 4-, and 6-fold increase in glucose transporter mRNA induced by glucose deprivation, insulin, and glucose deprivation plus insulin treatments, respectively. Similarly, Northern blot analysis of total RNA isolated from glucose-deprived, insulin, and glucose-deprived plus insulin-treated cells resulted in a 4-, 3-, and 9-fold induction of glucose transporter mRNA, respectively. The continuous presence of insulin in the medium, either in the presence or absence of glucose, resulted in a transient alteration of the glucose transporter mRNA. The relative amount of the glucose transporter mRNA was maximally increased at 6-12 h which subsequently returned to the basal steady-state level within 48 h. These data demonstrate a role for insulin and glucose in the overall regulation of glucose transporter gene expression which may account for the alteration of glucose transporter activity of muscle tissue observed in pathophysiological states such as type II diabetes mellitus.

Selective degradation of mRNA: the role of short-lived proteins in differential destabilization of insulin-induced creatine phosphokinase and myosin heavy chain mRNAs during rat skeletal muscle L6 cell differentiation.

This investigation concerns the combined effects of removal and readdition of insulin and inhibition of protein and RNA synthesis on the stability of insulin-induced mRNAs during and after differentiation of rat L6A1 myoblast cells in culture. Addition of insulin accompanying the withdrawal of the mitogenic stimulus of serum to myoblasts caused an 80-fold increase in creatine phosphokinase (CK) activity which was largely accounted for by a similar increase in the amount of CK mRNA. The latter was co-ordinately induced with myosin heavy chain (MHC) mRNA but not malic enzyme (ME) mRNA. Measurements of steady-state levels of mRNA showed that removal of insulin caused CK mRNA, but not MHC mRNA, to be rapidly degraded, the effect being reversed upon readdition of the hormone. Direct measurement of 3H-labeled CK, MHC and beta-actin mRNAs confirmed the selective stabilization and destabilization of CK mRNA by the hormone. Conditions were established for a time-window during which cycloheximide (Cx) produced a virtually total arrest of protein synthesis in myotubes that was reversible upon removal of the inhibitor. Under these conditions, Cx selectively prevented the degradation of CK mRNA in a reversible manner. Actinomycin D (Act D) also arrested the loss of this mRNA. Under the same conditions of mRNA stabilization during de-induction, a superinduction of CK mRNA, but not MHC mRNA, was observed if the two inhibitors were added during induction in the continuous presence of insulin. We conclude that a short-lived protein(s), encoded by a short-lived mRNA(s), selectively regulates the stability of reversibly inducible mRNA.

Synergistic action of thyroid hormone, insulin and hydrocortisone on astrocyte differentiation

We report here on the synergistic regulation of astrocyte development by 3 hormones: thyroid hormone (TH), insulin, and hydrocortisone (HC). Their effect, in a defined serum-free media, on astrocyte morphology, on glia fibrillary acidic protein (GFAP) immunostaining pattern, and on glutamine synthetase (GS) was investigated. TH transformed the flat, polygonal astrocytes into process-bearing cells. This effect was accentuated by insulin, which by itself had no effect on astrocyte morphology. The morphological transformations were accompanied by changes in the pattern of GFAP immunostaining which indicated a more organized and directed cytoskeleton arrangement in the TH-insulin treated cultures. Over 95% of the cells in the culture expressed GFAP. All 3 hormones regulated GS levels. TH increased GS levels by 50% and insulin raised its levels by 3-fold. While having no effect on astrocyte morphology, HC increased GS levels by 3.7-fold in both the hormone-free and insulin-supplemented medium. HC acted synergistically with insulin in its action on GS bringing about a 12-fold increase in the enzyme activity. In contrast, TH did not interact with insulin and was additive with HC in its action on GS. The continuous presence of insulin and TH was required to maintain their morphological and GS effect, suggesting that these hormones might not only be important for astrocyte differentiation, but later on for astrocyte function as well. Since astrocytes interact with and affect neurons and oligodendrocytes, the findings reported here might have bearing on the development and function of these other brain cells as well.

Insulin effects on protein synthesis and secretion in primary cultures of amphibian hepatocytes

The objective of the present study was to determine the effects of insulin on amphibian hepatocytes in primary culture. Hepatocytes were isolated from adult bullfrogs by collagenase perfusion and maintained as monolayers in serum-free medium. Cells cultured in the continuous presence of insulin exhibited a relatively constant rate of protein secretion over the first four to five days, whereas controls showed an almost three-fold decrease over the same time period. The decline in secreted proteins was equally represented in most exported proteins, except that serum albumin secretion showed twice as much of a decrease relative to the other proteins. The maintenance of protein secretion by insulin was the result of its effect on protein synthesis. The rate of protein synthesis was measured by the incorporation of (3H)-leucine into protein using culture medium containing 0.5 mM leucine, a condition where the specific radioactivity of leucyl-tRNA was shown to be equal to that of (3H)-leucine in the medium. Cultures maintained with insulin for 60 hours synthesized protein at two to three times the rate found in non-insulin treated controls whose rate of protein synthesis was first detectably decreased after nine hours of culture in the insulin-free medium. Sedimentation profiles of polyribosomes from hepatocytes maintained for 60 hours without insulin showed proportionately fewer ribosomes in large polysomes and more in monosomes and free ribosomal subunits than ribosomes from cells cultured with insulin. This result suggests that the decrease in protein synthesis found in the absence of insulin is due to a defect in initiation. Insulin does not exert its effect by regulating cellular levels of ATP; no change in ATP content was found in cells maintained with or without insulin. The results show that insulin maintains high levels of protein synthesis and secretion in amphibian hepatocytes. The hepatocytes in monlayer culture provide a system to study the molecular mechanisms involved in the translational control of protein synthesis by insulin.

An estrogen responsive primary amphibian liver cell culture system

Amphibian hepatocytes have been prepared in both high yield and purity using a collagenase perfusion technique. The isolated cells attach efficiently in serum-free medium to collagen-coated culture dishes and subsequently form monolayers. These cultures can be maintained in an appropriate medium for over one week with minimal cell loss. The nuclear labelling index of cells exposed to [ 3H]thymidine indicates a very low level of cell growth. Twenty-four hour exposure to dexamethasone induces tyrosine aminotransferase activity throughout the culture period. Monolayers incorporate [ 3H]leucine linearly into acid-insoluble material with approx. 40% of all synthesis devoted to secreted protein. Polyacrylamide gel electrophoresis of proteins in the presence of sodium dodecyl sulfate shows the majority of proteins present in whole serum are synthesized and secreted by the cultured hepatocytes. The absolute rate of protein secretion on the first day of culture is approx. 73 μg/day/mg cell protein which subsequently declines and plateaus at 30% of this level by the 4th–5th day of culture. However, when hepatocytes are cultured in the continued presence of insulin, the drop in protein secretion is completely inhibited. Cultures of hepatocytes isolated from female frogs and subsequently exposed to 17-B estradiol in culture, synthesize and secrete the egg-yolk protein precursor vitellogenin. The protein initially appears as a minor component in the medium 1–2 days after hormone addition. Its rate of synthesis, relative to other secreted proteins, increases with time so that it ultimately constitutes the majority of protein being exported after 6 days of treatment. Parallel with vitellogenin induction is an increase in rate of total protein secretion reaching a 2-fold increase at maximal stimulation. The results show that viable, monolayer cultures of amphibian hepatocytes can be prepared which retain the ability to respond directly to added estrogen by synthesizing vitellogenin.

Regulation of Lipid Synthesis in Cultured Animal Cells

Abstract Fatty acid and sterol biosynthesis were studied in several cell lines in culture. Two of these cell lines, L-M and NCTC 1469, demonstrated an 8.6- and 7.2-fold increase, respectively, in acetate incorporation into fatty acids and a 5.2- and 2.4-fold increase, respectively, in acetate incorporation into sterols within 24 hours after serum-containing medium was replaced with serum-free medium. Three other cell lines, Chang liver, HeLa, and NCTC 2544, demonstrated little change in fatty acid or sterol synthesis 48 hours after serum removal from the medium, but they demonstrated up to an 8-fold increase in fatty acid synthesis and a 3-fold increase in sterol synthesis 48 hours after serum removal when insulin was present in the serum-free medium. The increased fatty acid synthesis following transfer of L-M and NCTC 1469 cells to serum-free medium could be attributed to increased activities of both acetyl-coenzyme A carboxylase and fatty acid synthetase, whereas increased synthesis in Chang liver, HeLa, and NCTC 2544 cells was associated primarily with increased fatty acid synthetase activity. In Chang liver cells fatty acid synthetase activity did not change during the first 24 hours after replacement of serum-containing medium with serum-free medium. During the subsequent 24 hours activity increased approximately 160 % in the absence of insulin and approximately 450 % in the presence of insulin. Insulin at a concentration of 2 x 10-9 m produced 50 % of the maximal increase in synthetase activity, while proinsulin at 1.2 x 10-7 m was required for 50 % of maximal increase. The continued presence of insulin was necessary for maintenance of elevated fatty acid synthetase activity as demonstrated by the decrease observed when insulin was removed from the medium. Immunotitration experiments, using antiserum produced against pure fatty acid synthetase, indicated that the increased fatty acid synthetase activity observed after the removal of serum from the medium or the addition of insulin to serum-free medium was due to increased levels of enzyme in the cells. An increase in fatty acid synthetase synthesis presumably accounted for the increased levels of the enzyme since the effects of serum removal and insulin addition were blocked by cycloheximide, an inhibitor of protein synthesis. In Chang liver cells low levels of actinomycin D and camptothecin, inhibitors of RNA synthesis, prevented the induction of fatty acid synthetase by serum removal or insulin addition. A combination of theophylline and cyclic adenosine 3':5'-monophosphate also prevented induction.

The Uptake and Inactivation of Native Insulin by Isolated Fat Cells

Abstract With dilute suspensions of rat epididymal adipose tissue cells, glucose utilization was estimated by the conversion of glucose-14C to 14CO2 and 14C-labeled lipids. The maximal effect of insulin was observed with a glucose concentration of 1.25 mm or less. The same rate of glucose utilization was observed with and without insulin when the glucose concentration was 80 mm. Glucose utilization was stimulated with 1 microunit of insulin per ml, and insulin concentrations above 25 microunits per ml produced no further stimulation. The rate of glucose utilization was linear for 2 hours when the insulin concentration was 3 microunits per ml. With 5 microunits of insulin per ml, the glucose utilized in 2 hours did not increase when the volume of the incubation medium was doubled. The stimulatory effect of insulin was reversed by washing the cell suspension or by the addition of anti-insulin serum. These findings indicated that (a) a dilute suspension of fat cells did not inactivate insulin rapidly enough to lower the insulin concentration of the incubation medium significantly, and (b) the continued presence of insulin was required for a continuation of its biological effect. With a 30 times more concentrated suspension of fat cells, the change in immunoreactive insulin concentration of the incubation medium was measured. A rapid phase of insulin uptake occurred within the first minute, was linearly related to insulin concentration up to 10,000 microunits per ml, was not temperature-sensitive, and was abolished by a 30-sec exposure to 10-3 m maleimide. A subsequent phase of insulin uptake was slower, temperature-sensitive, and not abolished by maleimide. Cells previously incubated with insulin showed undiminished insulin uptake when reincubated in fresh medium. When insulin-treated cells were washed, no immunologically or biologically active insulin was released. It was concluded that (a) the rapid phase of insulin uptake may be the hormone-cell interaction which is necessary for the action of insulin to be manifest, and (b) a study of the factors which influence this process may lead to a better understanding of the mechanism of hormone action.

THE SOJOURN OF INSULIN IN THE BLOOD OF RABBITS AFTER THE ADMINISTRATION OF MASSIVE DOSES OF INSULIN1

THE INJECTION of insulin in a fasting animal causes a maximum decrease of the blood sugar within 2 hours, but the depression of the blood sugar is mani-fested for a longer period of time. This prolonged effect may be associated with the continued presence of insulin in the blood stream or to its persistent action in other tissues after it has disappeared from the circulation. Horsters (1) subscribes to the latter view. An answer to this problem may be obtained if the presence of insulin in the blood stream can be determined; obviously, when sufficiently large doses are injected intravenously into comparatively small laboratory animals, it may be shown that extracts made from the blood of such animals contain insulin.3 Each of 10 full-grown, unanesthetized rabbits received approximately 1000 U of insulin intravenously (50 mg. of insulin powder assaying about 20 U per mg.4 dissolved in 5 cc. of distilled water).