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.     

Effect of down-regulation and return of insulin receptors on glycogen synthesis in cultured rat hepatocytes

The dependence of the regulation of insulin receptors by insulin on the time hepatocytes were maintained in culture and the relationship between the return of down-regulated receptors and glycogen synthesis from labelled glucose were investigated in primary cultures of adult rat hepatocytes. Insulin receptor numbers, but not ligand affinity, decreased significantly within the first 24 h of culture, even in the absence of insulin, and then returned to the immediate 'post-attachment' level during 24-48 h. Therefore, down-regulation of insulin receptors by 10 nmol/l insulin was only minor during the 1st day in culture, but amounted to 50% of control levels after the 2nd day, whereas the rate of insulin degradation remained unaltered throughout the entire period of culture. When down-regulated monolayers were switched to insulin-free medium, receptors returned to control levels within 5-10 h. The reduced basal rate of glycogenesis as well as insulin-sensitivity and insulin responsiveness of this metabolic pathway also gradually increased to control levels. However, the time-dependent receptor return was dissociated from the increase in insulin-sensitivity, emphasising the importance of postbinding events. Since the changes both in basal rates and in insulin responsiveness of glycogenesis during the period of receptor return were inversely related to differences in the actual glycogen content between control and down-regulated cells, cellular glycogen content might participate in the regulation of glycogenesis as a 'feedback inhibitor'.     

Aberrant regulation of the metabolism of the insulin receptor in Swarm rat chondrosarcoma chondrocytes.

Treatment of Swarm rat chondrosarcoma chondrocytes for 3 days in media containing either non-recombinant pig or recombinant human insulin (1 micrograms/ml) increased the rate of proteoglycan synthesis approximately 6-fold compared with cells cultured in the absence of insulin. The concentrations of human and pig insulin that stimulated the cells to double their rate of proteoglycan synthesis were approximately 1 ng/ml and approximately 2 ng/ml respectively. Because physiological concentrations of insulin do not influence proteoglycan synthesis in non-transformed chondrocytes, the findings indicated a possible abnormality in the insulin-dependent regulation of the insulin receptor in these tumour cells. Like most cells, chondrosarcoma chondrocytes down-regulated their insulin receptors when incubated with insulin for 30 min. However, the number of plasma-membrane and intracellular insulin receptors did not decrease when the chondrocytes were exposed to insulin chronically for 4 days. Chondrocytes were cultured in media containing 2H-, 13C- and 15N-labelled amino acids, and the heavy-isotope density-shift method was used to investigate both the rate of degradation and the rate of synthesis of the insulin receptor. Although the rate of synthesis of the receptor was slightly faster in the insulin-treated cultures, as assessed by a slightly faster rate of appearance of the 'heavy' receptor, the rate of degradation of the receptor was slower in the insulin-treated cultures. The half-lives for the 'light' receptors were approx. 18 h and 10 h for chondrocytes cultured in insulin-containing and insulin-free media respectively. These studies in vitro indicate that the apparent up-regulation of insulin receptors that occurs in this transformed cell upon long-term exposure to insulin is primarily the result of a decreased rate of receptor degradation.     

Insulin and prolactin synergistically stimulate beta-casein messenger ribonucleic acid translation by cytoplasmic polyadenylation

Previous studies have shown that the synthesis and stability of milk protein mRNAs are regulated by lactogenic hormones. We demonstrate here in cultured mouse mammary epithelial cells (CID 9) that insulin plus prolactin also synergistically increases the rate of milk protein mRNA translation. Insulin alone stimulates synthesis of both milk and nonmilk proteins, whereas prolactin alone has no effect, but insulin plus prolactin selectively stimulate synthesis of milk proteins more than insulin alone. The increase in beta-casein mRNA translation is also reflected in a shift to larger polysomes, indicating an effect on translational initiation. Inhibitors of the phosphatidylinositol 3-kinase, mammalian target of rapamycin, and MAPK pathways block insulin-stimulated total protein and beta-casein synthesis but not the synergistic stimulation. Conversely, cordycepin abolishes synergistic stimulation of protein synthesis without affecting insulin-stimulated translation. The poly(A) tract of beta-casein mRNA progressively increases from approximately 20 to about 200 A residues over 30 min of treatment with insulin plus prolactin. The 3'-untranslated region of beta-casein mRNA containing an unaltered cytoplasmic polyadenylation element is sufficient for the translational enhancement and mRNA-specific polyadenylation, based on transient transfection of cells with a reporter construct. Insulin and prolactin stimulate cytoplasmic polyadenylation element binding protein phosphorylation with no increase of cytoplasmic poly(A) polymerase activity.     

Effect of insulin on ultrastructure and glycogenesis in primary cultures of adult rat hepatocytes

Insulin in the presence of high concentrations of glucose has a beneficial trophic effect on the development of primary cultures of hepatocytes. Compared to the situation observed in hormone-free control cultures, the flattening of the reaggregated hepatocytes is enhanced, and the reconstituted cell trabeculae are enlarged and tend to form a confluent monolayer after 3 days; the survival time is prolonged from 3 to 5 or 6 days. Ultrastructural modifications are also initiated by insulin; numerous glycogen particles appear after 24 h, in between the cisternae of the proliferated smooth endoplasmic reticulum. After 48 h, large amounts of glycogen are stored, and numerous polysomes are present. A small number of cells showed an increased synthesis of lipid droplets in the lumen of the smooth endoplasmic reticulum and form liposomes at the same time. After 72 h, cytolysomes filled with glycogen develop, simulating glycogenosis type II. Simultaneously, microtubules and microfilaments, closely related to numerous polysomes, appear in cytoplasmic extensions constituting undulating membranes. The biochemical data demonstrate that, in the absence of insulin, a high concentration of glucose stimulates glycogenesis and hinders glycogenolysis. This effect of glucose on polysaccharide synthesis is progressively lost. The addition of insulin to the culture induces after 48 and 72 h, a three- to fivefold increase of the glucose incorporation into glycogen, as compared to the controls. The presence of insulin is required to maintain the hepatocyte's capacity to store glycogen. Glycogen synthetase is converted into its active form under the influence of glucose. Insulin increases the rate of activation.     

Temperature-induced production of recombinant human insulin in high-cell density cultures of recombinant Escherichia coli

The construction of expression vectors encoding either the human insulin A- or B-chains fused to a synthetic peptide and the temperature-induced expression of the recombinant genes in Escherichia coli are reported. Using this two-chain approach we also describe the separate isolation of the insulin A- and B-chains from inclusion bodies and their subsequent assembly into native human insulin. The production of the insulin fusion proteins were carried out in high-cell density fed-batch cultures using a synthetic medium with glucose as sole carbon and energy source. The expression of the recombinant genes by temperature-shift in high-cell density cultures of recombinant E. coli resulted in product yields of grams per litre of culture broth, e.g. 4.5 g of insulin B-chain fusion protein per litre of culture broth. This translates into an expression yield of about 800 mg of the insulin B-chain per litre of culture. Under similar cultivation conditions the expression yield of the insulin A-chain corresponds to approximately 600 mg per litre of culture. The metabolic burden imposed on the recombinant cells during temperature-induced production of insulin fusion proteins in high-cell density cultures is reflected in an increased respiratory activity and a reduction of the biomass yield coefficient with respect to glucose.     

Use of hepatocytes in primary culture for biochemical studies on liver functions

Summary Rat parenchymal hepatocytes isolated with collagenase were cultured as monolayers in Williams medium E supplemented with calf serum. Freshly isolated cells showed very low activities of various liver functions, and they had to be cultured for 6-24 h to allow recovery of these functions. Insulin and dexamethasone greatly increased cell viability in primary culture. After culture for 24 h, these cells showed various liver functions as seen in vivo and responded well to various added hormones and amino acids. The concentrations of amino acids in the medium regulated synthesis of serum proteins and insulin stimulated lipogenesis, which in turn regulated synthesis of lipoproteins. Insulin also stimulated glycogen synthesis and the stimulation was parallel with the number of insulin receptors. Glucagon stimulated glycogenolysis and its stimulation involved the function of the cytoskeleton. Glucagon and dexamethasone induced various enzymes of amino acid catabolism, such as tryptophan oxygenase, tyrosine aminotransferase and serine dehydratase. These inductions were inhibited by insulin or catecholamine. The effect of catecholamine was due to its -adrenergic action. The -action of isoproterenol was low in freshly isolated cells, but increased during culture of the cells. Acquirement of hormonal responses during neonatal development can be studied in this culture system. Mature hepatocytes in culture are usually quiescent, but when insulin and epidermal growth factor were added, DNA synthesis by the cells increased markedly and they showed density-dependent growth. In this culture system, serum could be omitted for 2 days when the dishes were coated with fibronectin without appreciable change of functions, but serum was needed for longer culture of the cells. A factor that increased cell survival was found in serum and in pituitary gland.These results show that hepatocytes in primary culture are a simple and useful system for studies of liver functions in vitro and related works were also reviewed.     

Chemically defined medium supporting cardiomyocyte differentiation of human embryonic stem cells

Many applications of human embryonic stem cells (hESCs) will require fully defined growth and differentiation conditions including media devoid of fetal calf serum. To identify factors that control lineage differentiation we have analyzed a serum-free (SF) medium conditioned by the cell line END2, which efficiently induces hESCs to form cardiomyocytes. Firstly, we noted that insulin, a commonly used medium supplement, acted as a potent inhibitor of cardiomyogenesis in multiple hESC lines and was rapidly cleared by medium conditioning. In the presence of insulin or IGF-1, which also suppressed cardiomyocyte differentiation, the PI3/Akt pathway was activated in undifferentiated hESC, suggesting that insulin/IGF-1 effects were mediated by this signaling cascade. Time course analysis and quantitative RT-PCR revealed impaired expression of endoderm and mesoderm markers in the presence of insulin, particularly if added during early stages of hESC differentiation. Relatively high levels of the neural ectoderm marker Sox1 were expressed under these conditions. Secondly, comparative gene expression showed that two key enzymes in the prostaglandin I2 (PGI2) synthesis pathway were highly up-regulated in END2 cells compared with a related, but non-cardiogenic, cell line. Biochemical analysis confirmed 6-10-fold higher PGI2 levels in END2 cell-conditioned medium (END2-CM) vs. controls. Optimized concentrations of PGI2 in a fully synthetic, insulin-free medium resulted in a cardiogenic activity equivalent to END2-CM. Addition of the p38 mitogen-activated protein kinase-inhibitor SB203580, which we have shown previously to enhance hESC cardiomyogenesis, to these insulin-free and serum-free conditions resulted in a cardiomyocyte content of >10% in differentiated cultures without any preselection. This study represents a significant step toward developing scalable production for cardiomyocytes from hESC using clinically compliant reagents compatible with Good Manufacturing Practice.     

THE INFLUENCE OF GROWTH-INHIBITING AND GROWTH-PROMOTING MEDIUM CONDITIONS ON CRYSTALLIN ACCUMULATION IN TRANSDIFFERENTIATING CULTURES OF EMBRYONIC CHICK NEURAL RETINA

The effects of media containing undialysed serum (controls) or dialysed serum with or without ascorbic acid, were compared during the second half of the 41-day culture period in embryonic chick neural retina cultures, which had all been grown in control medium prior to 19 days. Conditions permitting greatest culture growth (controls) showed earlier and more extensive development of lentoids, greater accumulation of total crystallin and a higher proportion of δ relative to α+β crystallins. Conditions allowing least culture growth (dialysed serum) gave converse results throughout. Thus changes in culture growth rate apparently affect δ crystallin production more than α or β crystallin production. Insulin promotes growth in neural retina cultures, whether present throughout the culture period (in this case 31 days), or only from 18 days onwards. The frequency and survival of putative neuronal cell aggregates are both increased by insulin during the first 18 days of culture. Delta crystallin production during subsequent transdifferentiation is selectively promoted by insulin when present throughout, but this effect is largely obviated when insulin is present only from 18 days onwards. This anomaly could arise through percursor cell selection during the earlier phases of culture, since it is possible that some (not all) lentoids may be derived from aggregates of neuronal-like cells in neural retina cultures. Thus precursor cell selection as well as culture growth rate may influence the pattern of crystallin production during transdifferentiation.     

Differentiation of newborn rat preadipocytes in culture: effects of insulin and dexamethasone

A preadipocyte cell population isolated from the inguinal tissue of 3-day-old rats converts at confluence into mature adipocytes when cultured with insulin (10(-9) M). Insulin is necessary only from Day 4 postplating. If the addition of insulin is further delayed, the proportion of cells which will undergo adipose conversion decreases. A loss of the differentiation competence is also observed when the cells are allowed to proliferate (seeding at a low density in a serum containing medium). A preexposure of the primary cells to dexamethasone during the insulin-insensitive period (Days 0-4) accelerates the subsequent "insulin-dependent" adipose conversion. In order to produce its effect, dexamethasone needs only to be present for 4 h on Day 2 postplating. The effect of dexamethasone is probably due neither to inhibition of cell proliferation nor to induction of the cell content of insulin receptors. The evolution of G3PDH enzyme activity as well as of G3PDH protein and mRNA was used as an indicator of the differentiation process. The enzyme accumulates to a low extent during culture in the absence of insulin. When insulin is present, the enzyme level is dramatically increased (maximum on Day 11). Dexamethasone pretreatment (Days 0-4, or 4 h on Day 2) accelerated the G3PDH enzyme activity increase as well as protein and mRNA accumulation. This was also true in cells maintained in insulin-free medium; however, in this case, the increase in the enzyme activity was limited to the first 8 days of culture and full differentiation did not take place. We conclude that: (1) the rat preadipocytes are committed to differentiate, requiring insulin as a sufficient physiological stimulus; (2) the differentiation program is progressively lost after greater than 4 days of culture without insulin and more rapidly if the cells are allowed to undergo divisions; and (3) dexamethasone accelerates the insulin-dependent adipose conversion but alone does not ensure the complete differentiation process.     

Regulatory relation between insulin receptor and its functional responses in primary cultured hepatocytes of adult rats

The relation between changes of insulin receptor and various metabolic responses were studied in adult rat hepatocytes in primary culture. In cells cultured for 3 h without insulin, the number of high affinity sites and the dissociation constant (Kd) of insulin receptor, determined from a Scatchard plot, were 1.05 x 10(5) sites/cell and 1.5 x 10(-9) M, respectively. The receptor number increased 2-fold, but the Kd value remained constant during 2-days culture in insulin-free medium (up-regulation). Addition of dexamethasone (Dex), growth hormone, glucagon or triiodothyronine did not change the number of insulin receptors or the Kd value. In contrast, 1-day culture in insulin (1 x 10(-7) M) medium decreased the receptor number by half (down-regulation) without change of the Kd value. Short-term responses of glycogenesis, amino acid transport and lipogenesis by insulin increased as the receptor number increased. In these cases, the sensitivity to insulin (Ka: half dose for the maximum response) did not change in cells with different receptor numbers, but the maximum response changed. These results show that hepatocytes, unlike adipocytes, do not have spare receptors of insulin. During down-regulation, the receptor number decreased by only half, but the insulin responses were lost almost completely. The receptor number returned to the normal level after culture in insulin-free medium for 12 h, but recovery of the responses took longer, suggesting that for the insulin response not only change of receptor number, but also other regulatory mechanisms for post-receptor processes, such as desensitization, are involved.     

Long-term maintenance of high rates of very-low-density-lipoprotein secretion in hepatocyte cultures. A model for studying the direct effects of insulin and insulin deficiency in vitro.

High rates of hepatic cellular triacylglycerol synthesis and very-low-density-lipoprotein (VLDL) triacylglycerol output were maintained in vitro for at least 3 days when hepatocytes were cultured in a medium lacking insulin but supplemented with 1 microM-dexamethasone, 10 mM-lactate, 1 mM-pyruvate and 0.75 mM-oleate (supplemented medium). Under these conditions VLDL output remained constant, whereas cell triacyglycerol content increased 10-fold over 3 days, suggesting that the secretory process was saturated. Insulin, present during the first 24 h period, enhanced the storage of cellular triacylglycerol by inhibiting the secretion of VLDL. This stored triacyglycerol was subsequently released into the medium as VLDL if insulin was removed. With the supplemented medium the increased rate of VLDL secretion after insulin removal exceeded that observed under 'saturating' conditions, suggesting that pre-treatment with insulin enhanced the capacity for VLDL secretion. In contrast with the short-term (24 h) effects of insulin, longer-term exposure (greater than 48 h) to insulin enhanced the secretion of VLDL compared with insulin-untreated cultures. Under these conditions, insulin increased the net rates of triacylglycerol synthesis. The results suggest that insulin affects the secretion of VLDL triacylglycerol by two distinct and opposing mechanisms: first, by direct inhibition of secretion; second by increasing triacylglycerol synthesis, which stimulates secretion. The net effect at any time depends upon the relative importance of each of these processes.     

Effect of insulin on murine megakaryocytopoiesis in a liquid culture system

To examine the influence of insulin on megakaryocytopoiesis, we tested its effect on murine bone marrow cultures in a liquid culture system. In the presence of pokeweed mitogen-stimulated spleen cell conditioned medium in culture, insulin markedly enhanced megakaryocyte colony formation and increased the number and size of free megakaryocytes seen after 7 days. Many of the cells in cultures with insulin, however, were classified as immature, since they had a basophilic cytoplasm, a low cytoplasmic/nuclear ratio and low acetylcholinesterase activity. It is suggested that insulin potentiates murine marrow megakaryocytopoiesis in vitro, but that this is not accompanied by differentiation of the cells from the immature to mature state.     

Comparative studies on the regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) and sex hormone-binding globulin (SHBG) production by insulin and insulin-like growth factors in human hepatoma cells

Production of insulin-like growth factor-binding protein-1 (IGFBP-1) by the liver is efficiently inhibited by insulin both in vivo and in vitro. Consequently, serum IGFBP-1 concentration reflects insulin bioactivity in portal vein. Sex hormone-binding globulin (SHBG) is another insulin-regulated liver-derived protein that has appeared promising in detecting individuals with portal hyperinsulinemia. We compared the regulation of IGFBP-1 and SHBG production by insulin and insulin-like growth factors (IGF-I and IGF-II) in human hepatoma cell cultures. Insulin equipotently inhibited IGFBP-1 and SHBG production, with maximal decrease in culture medium concentrations being about 35% for both proteins during 48 h of culture in serum-free medium. IGF-I and IGF-II also inhibited the IGFBP-1 and SHBG levels. We conclude that IGFBP-1 and SHBG are equally sensitive to ambient insulin concentrations in human hepatoma cell cultures, and the production of both proteins is also attenuated by the IGFs.     

Expression of differentiated function by hepatocytes in primary culture: variable effects of glucagon and insulin on gluconeogenesis during cell growth

Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchyma liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenesis activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity. The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.     

Expression of differentiated function by hepatocytes in primary culture: Variable effects of glucagon and insulin on gluconeogenesis during cell growth

Abstract. Hormonal effects on gluconeogenesis from lactate were studied during the growth cycle of adult rat parenchymal liver cells using a primary monolayer culture system previously described [25]. Basal and glucagon-stimulated gluconeogenic ability were found to decline rapidly during log phase, insulin-stimulated growth. A progressive recovery of gluconeogenic activity was observed after cell division subsided. Rates of lactate-gluconeogenesis were found also to decline in the absence of prior insulin exposure. This decline was not as rapid as the loss observed in cells cultured with insulin. However, in insulin-deficient cultures gluconeogenesis was completely abolished after 12 days and did not reelevate with further incubation unless cells were washed and exposed to glucagon. Decreasing growth rates of insulin-supplemented cultures by decreasing serum concentrations resulted in comparatively higher gluconeogenic activity.
The results presented here are consistent with previous observations of hepatic parenchymal expression of 'differentiated function' during cellular growth phases in culture (i.e., differentiated functions are generally lost during rapid growth and regained as cells become quiescent). The present study, however, presents unexpected effects of insulin on the apparent growth-state dependent gluconeogenic recovery. Our data imply that although insulin has long been known to inhibit gluconeogenesis, its presence in culture may facilitate long-term basal maintenance of gluconeogenic enzyme activity. Insulin also functions as a growth factor whose initial mitogenic effect correlates with decreased gluconeogenic function. These changes show no simple or predictive correlation with cyclic nucleotide metabolism.