Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines. Intrinsic differences in growth characteristics and insulin response

Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant (P less than 0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10(-10)-10(-9) M. Slow cultures bound more [125I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Growth and differentiation of chicken embryo muscle cell cultures derived from fast- and slow-growing lines

Abstract. Primary myogenic cell cultures derived from 12-day embryos of genetically fast-growing chickens (fast cultures) and slow-growing chickens (slow cultures) were grown under identical conditions to examine differences in growth and differentiation at the cellular level. The two types of cultures exhibited significant ( P <0.01) differences in proliferation, protein accumulation, response to the addition of insulin to the culture medium and the amount of insulin bound per nucleus. The fast cultures exhibited a larger number of both total nuclei and fused nuclei at 48, 72 and 96 h in culture, accumulated more protein per nucleus at 24, 48 and 72 h in culture and demonstrated a greater response to the addition of insulin to the culture medium, as reflected by increased fusion rate and protein accumulation at 24 h in culture. Maximal response to insulin in both types of cultures was obtained at 24 h to added insulin concentrations of 10⁻¹⁰−10⁻⁹ M . Slow cultures bound more [¹²⁵I]-insulin than fast cultures at 24 h in culture. These experiments suggest that different muscle growth potentials in animals of the same species are at least partly due to intrinsic cellular differences in the myogenic cells that give rise to adult muscle tissue.     

Insulin concentration is critical in culturing human neural stem cells and neurons

Cell culture of human-derived neural stem cells (NSCs) is a useful tool that contributes to our understanding of human brain development and allows for the development of therapies for intractable human brain disorders. Human NSC (hNSC) cultures, however, are not commonly used, mainly because of difficulty with consistently maintaining the cells in a healthy state. In this study, we show that hNSC cultures, unlike NSCs of rodent origins, are extremely sensitive to insulin, an indispensable culture supplement, and that the previously reported difficulty in culturing hNSCs is likely because of a lack of understanding of this relationship. Like other neural cell cultures, insulin is required for hNSC growth, as withdrawal of insulin supplementation results in massive cell death and delayed cell growth. However, severe apoptotic cell death was also detected in insulin concentrations optimized to rodent NSC cultures. Thus, healthy hNSC cultures were only produced in a narrow range of relatively low insulin concentrations. Insulin-mediated cell death manifested not only in all human NSCs tested, regardless of origin, but also in differentiated human neurons. The underlying cell death mechanism at high insulin concentrations was similar to insulin resistance, where cells became less responsive to insulin, resulting in a reduction in the activation of the PI3K/Akt pathway critical to cell survival signaling.     

Hormonal regulation of amino acid transport and cAMP production in monolayer cultures of rat hepatocytes

The effects of insulin, glucagon or Dexamethasone (DEX) and of glucagon with insulin or DEX were examined on the uptake of 2-amino [1-¹⁴C]isobutyric acid (AIB) and N-Methyl-2-amino [1-¹⁴C]isobutyric acid (NMe AIB) in monolayer cultures of rat hepatocytes. Insulin and glucagon stimulated the uptake of both the amino acids and DEX inhibited it, showing that all three of these hormones regulate the A system (the sodium-dependent system that permits the transport of NMe AIB) for amino acid transport in these cultures. Experiments investigating the transport of aminocyclopentane-1-carboxylic acid, 1- [carboxyl-¹⁴C] in the presence of excess AIB or in the absence of sodium showed that insulin had no effect on the activity of the L system (the sodium-independent system that prefers leucine). Experiments on the uptake of AIB in the presence of excess NMe AIB showed insulin had no effect on the transport activity of the ASC system (the sodium-dependent system that does not transport NEe AIB). Insulin concentrations ranging from 0.1 nM to 100 nM did not antagonize the stimulatory effect of optimum or suboptimum concentrations of glucagon on the uptake of either AIB or NMe AIB. Similarly, glucagon did not antagonize the stimulatory effect of optimum or suboptimum concentrations of insulin on the uptake of both the amino acids. The combined effect of insulin and glucagon was additive on the rate as well as the cumulative uptake of both AIB and NMe AIB. DEX alone inhibited the transport of both AIB and NMe AIB by about 25%, while glucagon caused a 2–3-fold increase; however, the addition of glucagon to cultures containing DEX caused a 7–8-fold increase in the uptake of both AIB and NMe AIB when compared to cultures containing DEX alone. The effect of insulin on the levels of cAMP was also investigated. Insulin had no effect on the cAMP levels in cultures treated or untreated with optimum or suboptimum concentrations of glucagon.     

Microtubules and beta cell function: effect of colchicine on microtubules and insulin secretion in vitro by mouse beta cells

A monolayer culture system was developed to study the role of microtubules in insulin secretion. Cultured cells were obtained to study the role of microtubules in insulin secretion. Cultured cells were obtained by enzymatic digestion of pancreases from C57BL-KsJ mice 6-12 wk of age. On day 4 of culture, the medium was changed, control or treatment medium added, and frequent samples were removed for insulin assay. Microtubules and beta cells were identified by indirect immunofluorescence with monospecific antibodies to tubulin and insulin. An extensive microtubule network radiates from the perinuclear region of the beta cell to the plasma membrane. Although alterations in the calcium concentration of the medium did not affect the microtubule pattern, the absence of calcium or glucose in the medium inhibited insulin secretion (P less than 0.001). Optimum insulin release occurred at a calcium concentration of 2.5 mM. Colchicine, in concentrations of 10(-10) M, did not affect the microtubule immunofluorescent pattern, whereas concentrations of 1 and 5 x 10(-7) M decreased the number of microtubules, and microtubules could not be identified in cultures treated with 10(-6) M colchicine for 2 h. After a 2-h preincubation, the prolonged release of insulin at either 2.0 or 4.5 mg/ml of glucose was decreased by 10(-6) M colchicine (P less than 0.02). The immediate release of insulin was similar to that in control plates and occurred in cultures with no identifiable microtubules. Microtubules and insulin secretion were not altered by 10(-6) M lumicolchicine and prolonged insulin secretion recovered 24 h after removal of colchicine. These studies show that the microtubules facilitate sustained secretion of insulin but are not required for the immediate release of the hormone. Alterations in the extracellular calcium concentration which play an essential role in insulin secretion do not alter the microtubule pattern in the beta cell.     

Insulin regulates the expression of the insulin-like growth factor binding protein 2 mRNA in rat hepatocytes.

The goal of this study was to find out whether GH or insulin regulate the mRNA expression of the fetal binding protein of insulin-like growth factor (IGFBP-2). Primary hepatocytes from adult rats were used as a test system. IGFBP-2 mRNA was abundant in cells cultured in the absence of hormones and markedly reduced in cultures containing insulin. Addition of GH had no effect on IGFBP-2 mRNA levels although the cells are responsive to GH as demonstrated by a GH mediated elevation of IGF l mRNA levels. Half-maximal down-regulation of IGFBP-2 mRNA levels occurred at an insulin concentration of 1 to 2 x 10(-10) M. The finding that insulin is a potent negative regulator of hepatic IGFBP-2 mRNA levels suggests a physiologically important regulatory link between the two hormones insulin and IGF l.     

The effects of glucocorticoids on insulin-stimulated lipogenesis in primary cultures of rat hepatocytes.

We used primary cultures of rat hepatocytes to evaluate the effects of glucocorticoids on insulin-responsive hepatic lipogenesis. The data indicate that hepatocytes incubated for 20 h with dexamethasone (0.1 microM) alone are profoundly resistant to the ability of insulin to stimulate lipogenesis acutely. In contrast, primary cultures of hepatocytes incubated with dexamethasone plus insulin are hyper-responsive to the ability of insulin to stimulate lipogenesis chronically. This potentiation of insulin action by a glucocorticoid occurs at physiological concentrations of the two hormones. Exposure to dexamethasone plus insulin for more than 4 h is required for the two hormones to enhance insulin action either by overcoming the insulin resistance induced by dexamethasone alone or by stimulating insulin action induced by insulin alone. Despite the marked potentiation of insulin action, hepatocytes exposed to dexamethasone plus insulin are less sensitive to insulin, as demonstrated by a shift to the right in the dose-response curve for insulin-stimulated lipogenesis. The resistance of hepatocytes to the acute effects of insulin after exposure to dexamethasone alone and the potentiation of insulin action and decreased sensitivity to insulin after exposure to insulin plus dexamethasone are all mediated by post-insulin-binding events. These studies demonstrate potentiation of insulin action in the liver by physiological concentrations of glucocorticoids and may have physiological significance for the regulation of normal hepatic lipogenesis, for the hyperlipidaemia observed with the pharmacological use of glucocorticoids, and for disease states in man associated with hyperinsulinaemia and hypercortisolism.     

Postreceptor regulation of insulin action in primary cultures of rat hepatocytes by oral hypoglycemic agents: effects of linogliride and chlorpropamide

We have previously demonstrated the ability of the sulfonylurea tolazamide to potentiate insulin action in primary cultures of hepatocytes prepared from normal and streptozotocin-diabetic rats. To determine whether the pirogliride derivative linogliride, a non-sulfonylurea orally effective hypoglycemic agent, can potentiate insulin action, we evaluated the ability of linogliride to affect insulin-stimulated lipogenesis in primary cultures of hepatocytes prepared from normal rats. In addition, we also evaluated the ability of the sulfonylurea chlorpropamide to affect insulin-stimulated lipogenesis in the same in vitro system. The exposure of hepatocytes for 18 h to either linogliride (100 ug/ml) or chlorpropamide (175 ug/ml) resulted in dose-dependent (0.1 to 100 nM insulin) increases in insulin-stimulated lipogenesis, although the effects of chlorpropamide are approximately two times those of linogliride. This increase in insulin responsiveness was not associated with any change in insulin sensitivity (ED50) or insulin binding. The results provide evidence for an extra-pancreatic effect of linogliride and chlorpropamide in the liver and indicate that these structurally unrelated oral hypoglycemic agents enhance insulin responsiveness through postbinding mechanisms.     

Hormonal regulation of amino acid transport system N in primary cultures of rat hepatocytes

The transport of histidine and glutamine via system N in cultured hepatocytes was found to be subject to hormonal control. This long-term regulation showed the following characteristics. The transport capacity for histidine and glutamine (system N) increased slowly in response to the combination of dexamethasone and insulin to about 4-fold that of controls after 18-30 h. A similar time course was found for the stimulation of system N (2.5-fold) by dexamethasone and glucagon. In contrast the uptake of alpha-aminoisobutyric acid (system A) was rapidly stimulated 3-fold by dexamethasone and insulin and 5-fold by dexamethasone and glucagon within 3-6 h but decreased towards control rates after 24 h of cultivation in minimal essential medium. Dexamethasone, insulin and glucagon each stimulated glutamine uptake about 2-fold in cultures maintained in W/AB 77 medium, while the combination of dexamethasone with either glucagon or insulin resulted in a 3-4-fold increase. Dexamethasone was most effective at about 0.1 microM. Higher concentrations were less efficient. Insulin reached its optimal effect at concentrations above 1 microM. Kinetic analysis revealed that the increased capacity of glutamine transport in response to hormones was due to an increase in Vmax, while Km was essentially unchanged. The hormone-induced stimulation of system N was prevented by cycloheximide. The induced uptake of glutamine was inhibited by excess amounts of asparagine and histidine but not of alpha-methylaminoisobutyric acid or cysteine. These results clearly differentiate the hormonal regulation of system N from that of system A.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Regulation of insulin binding and stimulation of sugar transport in cultured human fibroblasts by sugar levels in the culture medium

Studies were carried out on cultures of human skin fibroblasts to explore the effects of culture medium glucose levels on insulin binding and action. Cell cultures in 5.55 mm glucose-containing medium depleted their medium glucose within 3 days, and at that time exhibited elevated deoxy-d-glucose (2-DG) transport (84% greater than control cultures fed 22.2 mm glucose) and failure of insulin to stimulate 2-DG transport (an insulin:control transport ratio of 1.02). There was also a significant negative correlation between basal 2-DG transport and insulin binding (r = ?0.621; n = 29; P < 0.01), while insulin binding exhibited a significant positive correlation with insulin action (r = 0.816; n = 12; P < 0.01). Glucose starvation of cultures for 18 h resulted in several changes: (i) a 49% decrease in specific ¹²⁵I-insulin binding due to a reduction in binding capacity; (ii) elevated basal 2-DG transport; and (iii) an absence of insulin stimulation of 2-DG transport. Exposure to increasing concentrations of glucose for 18 h led to a glucose concentration-dependent increase in specific insulin binding. Additionally, the various changes in the glucose-starved group were reversed after as little as 6 h of glucose refeeding. The results indicate that basal sugar transport, and insulin binding and action can be regulated by the amount of glucose in the medium.     

In vitro toxicity of alloxan for guinea pig B cells: comparison with rat B cells

Previous studies have shown that guinea pigs are resistant to the in vivo diabetogenic action of alloxan and that this resistance may be accompanied by a regeneration of B cells in the initial days following administration of the drug. In the studies reported here, we used the measurement of insulin and glucagon released over a 7-day culture period as indices of islet cell viability and examined effects of in vitro exposure to alloxan upon subsequent release of insulin and glucagon from guinea pig (alloxan-resistant) and rat (alloxan-sensitive) islet cell cultures. An alloxan dose-dependent decrease in subsequent insulin release was found. However, whereas the lowest concentration of the drug (1 mM) produced a significant depression in insulin release in rat islet cultures, with maximal depression occurring after exposure to 5 mM alloxan, insulin release from guinea pig cultures was not significantly depressed by 1 or 2 mM alloxan, and 5 mM alloxan treatment produced a submaximal depression. Furthermore, insulin release from guinea pig but not rat cultures increased transiently at between 6 and 18 hr during the first day following exposure to all doses of alloxan. Treatment with high doses of the drug (40 mM or greater) caused the same maximal chronic depression of insulin release for both species. In contrast, glucagon release from cultures of both species was not affected significantly following alloxan treatment. Thus, guinea pig B cells are more resistant than those of the rat to the action of alloxan, but this resistance can be overcome by employing high doses of the drug. Other factions unidentified by the present studies may also be involved in the failure of guinea pigs to develop diabetes following in vivo treatment with alloxan.     

Release of PAI-1 by human preadipocytes and adipocytes independent of insulin and IGF-1

The effect of insulin and insulin-like growth factor-1 (IGF-1) on plasminogen activator inhibitor-1 (PAI-1) release from primary cultures of human preadipocytes and adipocytes has been investigated. Initial experiments measuring basal PAI-1 release (ng/ml) indicated variability between individual cultures. Using a novel technique for adipocyte quantitation, additional experiments were performed to determine PAI-1 release per cell, indicating a significant reduction with differentiation. Insulin and IGF-1 over a range of concentrations had no effect on PAI-1 release, and RT-PCR of PAI-1 mRNA following treatment with insulin and IGF-1 also indicated similar expression between treatments. The cultures did exhibit insulin-stimulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and leptin synthesis following differentiation to the adipocyte phenotype. This is the first report of PAI-1 secretion by primary cultures of human preadipocytes and adipocytes, indicating PAI-1 release independent of insulin and IGF-1 and implicating other factors in the elevated plasma PAI-1 observed with insulin resistance.     

Differential Effects of Insulin on Choline Acetyltransferase and Glutamic Acid Decarboxylase Activities in Neuron-Rich Striatal Cultures

We studied the effects of insulin, nerve growth factor (NGF), and tetrodotoxin (TTX) on cellular metabolism and the activity of glutamic acid decarboxylase (GAD) and choline acetyltransferase (ChAT) in neuron-rich cultures prepared from embryonic day 15 rat striatum. Insulin (5 micrograms/ml) increased glucose utilization, protein synthesis, and GAD activity in cultures plated over a range of cell densities (2,800-8,400 cells/mm2). TTX reduced GAD activity; NGF had no effect on GAD activity. Insulin treatment reversibly reduced ChAT activity in cultures plated at densities of greater than 4,000 cells/mm2, and the extent of this reduction increased with increasing cell density. The number of acetylcholinesterase-positive neurons was not reduced by insulin, suggesting that insulin acts by down-regulating ChAT rather than by killing cholinergic neurons. Insulin-like growth factor-1 (IGF-1) reduced ChAT activity at concentrations 10-fold lower than insulin, suggesting that insulin's effect on ChAT may involve the IGF-1 receptor. NGF increased ChAT activity; TTX had no effect on ChAT activity. These results suggest that striatal cholinergic and GABAergic neurons are subject to differential trophic control.     

Hormonal regulation of amino acid transport and gluconeogenesis in primary cultures of adult rat liver parenchymal cells.

Amino acid transport was studied in primary cultures of parenchymal cells isolated from adult rat liver by a collagenase perfusion technique and maintained as a monolayer in a serum-free culture medium. These cells carried out gluconeogenesis from three carbon precursors (alanine, pyruvate, and lactate) in response to glucagon addition. Amino acid transport was assayed by measuring the uptake of the nonmetabolizable amino acid, alpha-aminoisobutyric acid (AIB). Addition of insulin or glucagon to culture rat liver parenchymal cells resulted in an increased influx of AIB transport. The glucocorticoid, dexamethasone, when added alone to cultures did not affect AIB transport. However, prior or simultaneous addition of dexamethasone to glucagon-treated cells caused a strong potentiation of the glucagon induction of AIB transport. Kinetic analysis of the effects of insulin and glucagon demonstrated that insulin increased the Vmax for transport without changing the Km while glucagon primarily decreased the Km for AIB transport. The effect of dexamethasone was to increase the Vmax of the low Km system.     

Assay of prolyl 4-hydroxylase by the chromatographic determination of [14C]succinic acid on ion-exchange minicolumns.

Hepatic glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) is subject to nutritional regulation. To assess the possible role of hormones in this regulation, the amounts of G6PDH mRNA were studied in primary cultures of rat hepatocytes treated with insulin and dexamethasone, alone or in combination. Relative concentrations of G6PDH mRNA were directly assessed by a dot-blot hybridization procedure with nick-translated cDNA probes. G6PDH sequence abundance increased when the cultures were treated with insulin or dexamethasone, but the G6PDH mRNA induced by dexamethasone was not expressed at the protein level as active enzyme. In cultures treated with insulin and dexamethasone in combination, enzyme activity and G6PDH sequence abundance were greater than those induced by insulin alone. Our results directly demonstrate that G6PDH mRNA amounts are modulated in liver by these two classes of hormones and can partially account for the dietary induction of the enzyme observed in vivo.     

Regulation by Insulin and Insulin-Like Growth Factor of 2-Deoxyglucose Uptake in Primary Ependymal Cell Cultures

Ependymal cells have been reported to express the facilitative glucose carriers GLUT1, GLUT2, and GLUT4, as well as glucokinase. They are therefore speculated to be part of the cerebral glucose sensing system and may also respond to insulin with alterations in their glucose uptake rate. A cell culture model was employed to study the functional status of ependymal insulin-regulated glucose uptake in vitro. Insulin increased the uptake of the model substrate 2-deoxyglucose (2-DG) dependent on the insulin concentration. This was due to a near doubling of the maximal 2-DG uptake rate. Insulin-like growth factor (IGF-1) was at least 10 times more potent than insulin in stimulating the rate of ependymal 2-DG uptake, suggesting that IGF-1, rather than insulin, is the physiological agonist regulating glucose transport in ependymal cells. The predominant glucose transporter in ependymal cell cultures was found to be GLUT1, which is apparently regulated by IGF-1 in ependymal cells.     

Hormonal regulation of DNA synthesis in primary cultures of adult rat hepatocytes : Action of glucagon

Primary cultures of adult rat hepatocytes, grown in modified minimal essential medium (Eagle's) containing 10% calf serum, could be induced into DNA replication by combinations of epidermal growth factor (EGF), insulin and glucagon. The three hormones acted synergistically, and cells began entering DNA synthesis 48 h after hormone addition. The ability of the hormones to stimulate DNA synthesis was enhanced by plating cells at high cell concentrations or by conditioned medium, and was diminished by daily medium change. The contribution of glucagon to DNA synthesis was replaced by cAMP plus 1-methyl, 3-isobutyl xanthine or by adrenergic agents. Evidence is presented which suggests that all three hormones are required on the first day of culture, and that EGF and insulin are also required after the first day. This appears to be a useful system for studies on the hormonal initiation of growth in quiescent cells.     

Characterization of primary rabbit kidney cultures that express proximal tubule functions in a hormonally defined medium

Primary cultures of rabbit-kidney epithelial cells derived from purified proximal tubules were maintained without fibroblast overgrowth in a hormone-supplemented serum-free medium (Medium RK-1). A hormone- deletion study indicated that the primary cultures derived from purified rabbit proximal tubules required all of the three supplements in Medium RK-1 (insulin, transferrin, and hydrocortisone) for optimal growth but did not grow in response to EGF and T3. In contrast, the epithelial cells in primary cultures derived from an unpurified preparation of rabbit kidney tubules and glomeruli grew in response to EGF and T3, as well as insulin, transferrin, and hydrocortisone. These observations suggest that kidney epithelial cells derived from different segments of the nephron grow differently in response to hormones and growth factors. Differentiated functions of the primary cultures derived from proximal tubules were examined. Multicellular domes were observed, indicative of transepithelial solute transport by the monolayers. The proximal tubule cultures also accumulated alpha- methylglucoside (alpha-MG) against a concentration gradient. However, little or no alpha-MG accumulation was observed in the absence of Na+. Metabolic inhibitor studies also indicated that alpha-MG uptake by the primaries is an energy-dependent process, and depends upon the activity of the Na+/K+ ATPase. Phlorizin at 0.1 mM significantly inhibited 1 mM alpha-MG uptake whereas 0.1 mM phloretin did not have a significant inhibitory effect. Similar observations have been made concerning the Na+-dependent sugar-transport system located on the lumenal side of the proximal tubule, whereas the Na+-independent sugar transporter on the peritubular side is more sensitive to inhibition by phloretin than phlorizin. The cultures also exhibited PTH-sensitive cyclic AMP synthesis and brush-border enzymes typical of proximal cells. However, the activities of the enzymes leucine aminopeptidase, alkaline phosphatase, and gamma-glutamyl-transpeptidase were lower in the cultures than in purified proximal-tubule preparations from which they are derived.