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.     

Epidermal growth factor counteracts the glycogenic effect of insulin in parenchymal hepatocyte cultures.

Rat parenchymal hepatocytes in monolayer culture were used to study the metabolic effects of epidermal growth factor (EGF) and insulin on ketogenesis, gluconeogenesis and glycogen metabolism. EGF, unlike insulin, did not inhibit ketogenesis from palmitate or gluconeogenesis from pyruvate in hepatocyte cultures. It also had no effect on these pathways in the presence of insulin. In contrast, EGF potently counteracted the stimulation of [14C]pyruvate incorporation into glycogen by insulin, and also glycogen deposition from both gluconeogenic precursors and glucose. The EGF concentration causing half-maximal effect was about 0.1 nM. The anti-glycogenic effect of EGF was observed after both long-term (24 h) and short-term (1 h) exposure to EGF, and was more marked in the presence of insulin than in its absence. EGF did not displace bound insulin, suggesting that it neither competes for the insulin receptor nor affects the affinity of the receptor for insulin. EGF did not alter cellular cyclic AMP; and inhibition of cyclic AMP phosphodiesterase activity did not prevent the anti-glycogenic effect of EGF. In liver-derived dividing epithelial cells, Hep-G2 cells and fibroblasts, which have no capacity for gluconeogenesis, EGF did not counteract the stimulatory effect of insulin on [14C]glucose incorporation into glycogen, and in the epithelial cells EGF increased [14C]glucose incorporation into glycogen. The counter-effect of EGF on the glycogenic action of insulin in parenchymal hepatocytes may be due to a direct effect on glycogen metabolism or to an interaction with the post-receptor events in insulin action.     

Fuel metabolism in fasted newborn rabbits

Newborn rabbits delivered by Caesarean section at term were fasted for 72 h at 36 degrees C. Despite the abrupt interruption of maternal supply of energy substrates, glycaemia remains stable for 4 h after birth. This can be related to glucose production via rapid liver glycogenolysis; however, indirect evidence suggests that gluconeogenesis could also contribute to glucose production during this period. There is a selective decrease in the concentrations of gluconeogenic substrates and a suitable hormonal environment for gluconeogenesis as decreased insulin and increased glucagon concentration just after birth. The relative hypoglycaemia which develops after 6 h of life (2.6 mM at 72 h), despite high blood concentrations of non-esterified fatty acids and ketone bodies is not due to a deficient gluconeogenesis per se, as injection of gluconeogenic substrates to 72 h fasted newborns produces a three-fold increase in plasma glucose concentration. It is suggested that this relative hypoglycaemia is secondary to limited gluconeogenic substrate availability in the form of low circulting concentrations of gluconeogenic amino acids.     

Rapid effects of insulin and glucose on the hepatic incorporation of gluconeogenic substrates into glyceride glycerol and glycogen

1. The hepatic utilization of gluconeogenic substrates was investigated shortly after portal infusion of either insulin or glucose in fasted rats. 2. After 20 min of insulin infusion blood glucose concentration decreased. However, neither glucose generation from precursors such as alanine or pyruvate nor their incorporation into fatty acids was modified. Under these conditions, insulin rapidly increased the incorporation of gluconeogenic substrates into the hepatic glyceride glycerol fraction. Insulin treatment led to a decrease in substrate incorporation into liver glycogen. 3. After 20 min of portal glucose infusion both plasma insulin and glucose concentrations increased and the incorporation of pyruvate into hepatic glyceride glycerol and into glycogen was also stimulated. 4. A close relationship was observed between blood glucose concentrations and the level of incorporation of gluconeogenic substrates into liver glycogen. 5. In conclusion, during fasting insulin stimulates the incorporation of gluconeogenic substrates into the glycerol moiety of hepatic glycerides, which may be the preferential mechanism through which fatty acid esterification is accomplished during refeeding. This effect of insulin is rapid and detected even before other classical modifications induced by the hormone such as gluconeogenesis inhibition or lipogenesis activation. Furthermore, the effect is not related to insulin-induced hypoglycemia since glucose infusion mimics insulin action on glyceride glycerol synthesis.     

Regulation of phosphoenolpyruvate carboxykinase and pyruvate kinase in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources and the role of mitochondrial function on gluconeogenesis

Phosphoenolpyruvate carboxykinase (PEPCKase) and pyruvate kinase (PKase) were measured in Saccharomyces cerevisiae grown in the presence of glycolytic and gluconeogenic carbon sources. The PEPCKase activity was highest in ethanol-grown cells. However, high PEPCKase activity was also observed in cells grown in 1% glucose, especially as compared with the activity of sucrose-, maltose-, or galactose-grown cells. Activity was first detected after 12 h when glucose was exhausted from the growth medium. The PKase activity was very high in glucose-grown cells; considerable activity was also present in ethanol- and pyruvate-grown cells. The absolute requirement of respiration for gluconeogenesis was demonstrated by the absence or significantly low levels of PEPCKase and fructose-1,6-bisphosphatase activities observed in respiratory deficient mutants, as well as in wild-type S. cerevisiae cells grown in the presence of glucose and antimycin A or chloramphenicol. Obligate glycolytic and gluconeogenic enzymes were present simultaneously only in stationary phase cells, but not in exponential phase cells; hence futile cycling could not occur in log phase cells regardless of the presence of carbon source in the growth medium.     

Increase of L-serine dehydratase activity under gluconeogenic conditions in adult-rat hepatocytes cultured on collagen gel/nylon mesh.

Hormonal regulation of L-serine dehydratase [L-serine hydro-lyase (deaminating), EC 4.2.1.13] was studied in primary cultures of adult-rat hepatocytes. The hepatocytes were isolated by collagenase perfusion and maintained in culture on collagen-gel/nylon-mesh substrata. L-Serine dehydratase activity was measured with [14C]threonine as substrate. The enzyme activity in hepatocytes of normal adult rats was low and declined rapidly in culture in L-15 medium containing 0.1 micro M-insulin and even more in the presence of glucose. L-Serine dehydratase activity in hepatocytes of rats with streptozotocin-induced diabetes was initially 20-fold higher than that of normal rats, but fell rapidly to a low value by 4 days in culture. Hormonal regulation of the enzyme activity was manifested by treatment of the cultured hepatocytes with insulin (0.1 micro M), glucagon (0.3 micro M), dexamethasone (10 micro M) and combinations of these hormones. Either glucagon or dexamethasone in the absence of insulin enhanced the activity of L-serine dehydratase, but failed to do so in the presence of insulin. Treatment with both hormones resulted in a 2-3-fold increase in enzyme activity in culture on days 3 and 4. Under conditions in which the enzyme activity was enhanced, glucose production by the cultured hepatocytes was concomitantly increased. Glucose production resulted in part from gluconeogenesis from pyruvate and not entirely from glycogenolysis. The gluconeogenic conditions of culture resulted in a decrease in cellular lipids in the cultured hepatocytes, as evidenced by ultrastructural studies.     

Changes in DNA polymerase activity associated with cell fusion in cultures of embryonic muscle

In cultures of differentiating chicken embryo muscle cells there is a steep decline in DNA polymerase activity which closely parallels the time of rapid cell fusion and the formation of multinucleated myotubes. The DNA polymerase activity remaining in the cultures is almost completely associated with single unfused cells. Cell fusion does not require a confluent culture and fusion capability appears to be severely reduced in the remaining single cells following an approximately ten hour time period during which the majority of fusion takes place. A model is presented to explain the observed kinetics of cell growth and cell fusion in vitro.     

Hormonal regulation of key gluconeogenic enzymes and glucose release in cultured hepatocytes: effects of dexamethasone and gastrointestinal hormones on glucagon action

Hormonal regulation of key gluconeogenic enzymes and glucose release by glucagon, dexamethasone, secretin and somatostatin was evaluated in maintenance cultured rat hepatocytes. (i) Phosphoenolpyruvate (PEP)-carboxykinase activity declined rapidly during the first 24 h in serum- and hormone-free culture with a further slight decay during the following 2 days. Dexamethasone and glucagon independently increased PEP-carboxykinase and acted synergistically when added in combination. Glucose-6-phosphatase activity declining linearly during hormone-free culture was stimulated by glucagon. Dexamethasone itself was without significant effects but completely abolished glucagon action. Fructose-1,6-diphosphatase was maintained at its initial level during the first day under control conditions and declined thereafter. Neither glucagon nor dexamethasone affected total activity or substrate (fructose-1,6-diphosphate) affinity of this enzyme. In short-term experiments on cells cultured under control conditions, protein synthesis-dependent stimulation of PEP-carboxykinase by glucagon and the permissive action of dexamethasone was demonstrated. Glucose-6-phosphatase and fructose-1,6-diphosphatase were not altered by hormones within this period. (ii) Stimulation by glucagon of gluconeogenesis was independent of its action on PEP-carboxykinase. Dexamethasone inhibited glycogenolysis but maintained glucose release at control levels probably by stimulation of gluconeogenesis. When added in combination, the glycogen-preserving action of dexamethasone acutely reduced the glucose release in response to glucagon. Glucagon sensitivity remained unchanged. (iii) The gastrointestinal hormones secretin and somatostatin were ineffective in modulating basal or glucagon-stimulated glucose release and gluconeogenic key enzymes. They are therefore unlikely to play a physiological role in hepatic glucose metabolism.     

Recombinant protein synthesis and plasmid instability in continuous cultures of Escherichia coli JM103 harboring a high copy number plasmid

Escherichia coli JM103[pUC8] was employed as a model to investigate the behavior of a recombinant microbial system harboring a plasmid at high copy numbers. Experiments with batch and continuous cultures of recombinant and plasmid-free cells were conducted in a well-controlled bio-reactor. In batch experiments, plasmid copy number varied typically from an average of 500 during the exponential growth phase to as high as 1250 during the stationary phase. While the segregational plasmid instability was negligible in batch experiments, severe segregational instability occurred in continuous experiments conducted over a range of dilution rates, resulting in complete loss of plasmid-bearing cells from the continuous cultures within few residence times after transition to continuous operation. The profound differences in the specific growth rates and mass yields of the plasmid-free and plasmid-bearing cells resulting from the extra metabolic burden on the plasmid-bearing cells mainly due to excessive plasmid DNA content was the major cause for the plasmid instability. Plasmid multirnerization was detected in batch and continuous cultures and was found to have significant influence on the effective copy number and was partially responsible for the severe segregational instability in continuous cultures. A quasi-steady state representative of plasmid-bearing cells was established in the initial portion of each continuous culture experiment. Due to the profound growth rate differential between the two types of cells, transients of considerable duration were observed in each continuous culture experiment (initiated with a pure culture of plasmid bearing cells) following the slow accumulation of plasmid-free cells near the end of the quasi-steady state. Significant variations in various culture parameters (including a rapid decline in the plasmid-bearing fraction of the total cell population) occurred during this period, leading ultimately to a steady state for a culture dominated entirely by plasmid-free cells. In continuous cultures, plasmid copy number during the quasi-steady states increased with decreasing dilution rate from 50 (at 0.409 h(-1)) to 941 (at 0.233 h(-1)). Production of the plasmid-encoded protein (beta-lactamase) in these experiments was maximized at an intermediate dilution rate, corresponding to an optimum copy number of about 450. A similar optimum copy number was observed in batch cultures. Significant excretion of beta-lactamase was observed at both low and high dilution rates.     

Insulin effect on in vivo gluconeogenesis from [3-14C]pyruvate in the starved rat

During a 24 hr fast rats received 4 subcutaneous injections of insulin, and 15 min after the last injection they were given an intravenous pulse of [3-14C]pyruvate. The amount of [14C]glucose in blood 2 min after the tracer did not differ between insulin treated and control animals, whereas at 5 and 10 min values were significantly lower in the former group. At 10 min after the tracer, liver [14C]glycogen specific activity and [14C]fatty acid amount were higher in the insulin treated animals than in controls while plasma concentration of gluconeogenic amino acids was lower in the first group. Similar changes but less pronounced and more retarded were found in 24 hr fasted rats given only one insulin dose 15 min before the [3-14C]pyruvate pulse. Results indicate that gluconeogenesis from pyruvate is not directly modified by insulin treatment. Effects found at 5 and/or 10 min after the tracer and reported effects after prolonged insulin treatments may be caused by one or all of the following possibilities: enhanced utilization of the new-formed glucose, reduced availability of gluconeogenic substrates, and counteracting action on gluconeogenic hormones.     

PPAR-gamma-independent inhibitory effect of rosiglitazone on glucose synthesis in primary cultured rabbit kidney-cortex tubules

Therapeutic effect of rosiglitazone has been reported to result from an improvement of insulin sensitivity and inhibition of glucose synthesis. As the latter process occurs in both liver and kidney cortex the aim of this study was to elucidate the rosiglitazone action on glucose formation in both tissues. Primary cultured cells of both liver and kidney cortex grown in defined medium were use throughout. To identify the mechanism responsible for drug-induced changes, intracellular gluconeogenic intermediates and enzyme activities were determined. In contrast to hepatocytes, the administration of a 10 micromol/L concentration of rosiglitazone to renal tubules resulted in about a 70% decrease in the rate of gluconeogenesis, accompanied by an approximately 75% decrease in alanine utilization and a 35% increase in lactate synthesis. The effect of rosiglitazone was not abolished by GW9662, the PPAR-gamma irreversible antagonist, indicating that this action is not dependent on PPAR-gamma activation. In view of rosiglitazone-induced changes in gluconeogenic intermediates and a diminished incorporation of 14CO2 into pyruvate, it is likely that the drug causes a decline in flux through pyruvate carboxylase and (or) phosphoenolpyruvate carboxykinase. It is likely that the hypoglycemic action of rosiglitazone is PPAR-gamma independent and results mainly from its inhibitory effects on renal gluconeogenesis.     

Molecular signaling mechanisms of renal gluconeogenesis in nondiabetic and diabetic conditions

The kidneys are as involved as the liver in gluconeogenesis which can significantly contribute to hyperglycemia in the diabetic condition. Substantial evidence has demonstrated the overexpression of rate-limiting gluconeogenic enzymes, especially phosphoenolpyruvate carboxykinase and glucose 6 phosphatase, and the accelerated glucose release both in the isolated proximal tubular cells and in the kidneys of diabetic animal models and diabetic patients. The aim of this review is to provide an insight into the mechanisms that accelerate renal gluconeogenesis in the diabetic conditions and the therapeutic approaches that could affect this process in the kidney. Increase in gluconeogenic substrates, reduced insulin concentration or insulin resistance, downregulation of insulin receptors and insulin signaling, oxidative stress, and inappropriate activation of the renin–angiotensin system are likely to participate in enhancing renal gluconeogenesis in the diabetic milieu. Several studies have suggested that controlling glucose metabolism at the renal level favors effective overall glycemic control in both type 1 and type 2 diabetes. Therefore, renal gluconeogenesis may be a promising target for effective glycemic control as a therapeutic strategy in diabetes.     

Dissection of the insulin-sensitizing effect of liver X receptor ligands

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Treatment of insulin-resistant mice with synthetic LXR ligands enhances glucose tolerance, inducing changes in gene expression expected to decrease hepatic gluconeogenesis (via indirect suppression of gluconeogenic enzymes) and increase peripheral glucose disposal (via direct up-regulation of glut4 in fat). To evaluate the relative contribution of each of these effects on whole-body insulin sensitivity, we performed hyperinsulinemic-euglycemic clamps in high-fat-fed insulin-resistant rats treated with an LXR agonist or a peroxisome proliferator-activated receptor gamma ligand. Both groups showed significant improvement in insulin action. Interestingly, rats treated with LXR ligand had lower body weight and smaller fat cells than controls. Insulin-stimulated suppression of the rate of glucose appearance (Ra) was pronounced in LXR-treated rats, but treatment failed to enhance peripheral glucose uptake (R'g), despite increased expression of glut4 in epididymal fat. To ascertain whether LXR ligands suppress hepatic gluconeogenesis directly, mice lacking LXRalpha (the primary isotype in liver) were treated with LXR ligand, and gluconeogenic gene expression was assessed. LXR activation decreased expression of gluconeogenic genes in wild-type and LXRbeta null mice, but failed to do so in animals lacking LXRalpha. Our observations indicate that despite inducing suggestive gene expression changes in adipose tissue in this model of diet-induced insulin resistance, the antidiabetic effect of LXR ligands is primarily due to effects in the liver that appear to require LXRalpha. These findings have important implications for clinical development of LXR agonists as insulin sensitizers.     

Growth and function of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium.

The properties of primary rabbit kidney proximal tubule cells in glucose-free serum-free medium have been examined. Primary rabbit kidney proximal tubule cells were observed to grow at the same rate, 1.0 doublings/day, both in glucose-free and in glucose-supplemented medium. Growth in glucose-free medium was dependent upon the presence of an additional nutritional supplement, such as glutamine, pyruvate, palmitate, lactate, or beta hydroxybutyrate. Lactate, pyruvate, and glutamate are utilized for renal gluconeogenesis in vivo. The growth of the primary rabbit kidney proximal tubule cells in glucose-free medium was also dependent upon the presence of the three growth supplements insulin, transferrin, and hydrocortisone. Insulin was growth stimulatory to the primary proximal tubule cells in glucose-free medium, although insulin causes a reduction in the phosphoenolpyruvate carboxykinase (PEPCK) activity in these cells. PEPCK is a key regulatory enzyme in the gluconeogenic pathway. In order to evaluate whether or not the primary cells have gluconeogenic capacity, their glucose content was determined. The cells contained 5 pmoles D-glucose/mg protein. However, no significant glucose was detected in the medium. Presumably, the primary cells were either utilizing or storing the glucose made by the gluconeogenic pathway. Consistent with this latter possibility, cellular glycogen levels were observed to increase with time in culture. The effect of glucose on the expression of the alpha I(IV) collagen and laminin B1 chain genes was examined. Northern analysis indicated that the level of alpha I(IV) collagen mRNA was significantly elevated in glucose containing, as compared with glucose deficient, medium. In contrast, laminin B1 chain mRNA levels were not significantly affected by the glucose content of the medium.     

In vivo control of gluconeogenesis in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp) mutant.

The rate of cycloheximide-resistant incorporation of carbon from [14C]alanine and [14C]acetate into polysaccharidic material was used to study gluconeogenic activity in wild-type Neurospora crassa and in the adenylate cyclase-deficient cr-1 (crisp-1) mutant. The wild-type efficiently utilized alanine and acetate as gluconeogenic substrates, whereas the mutant used acetate efficiently but was unable to use alanine. Cycloheximide-resistant 14C-incorporating activity was sensitive to carbon catabolite effects (repression and inactivation) in the two strains, which suggested that cyclic AMP metabolism was not involved in these regulatory responses. In the wild type, gluconeogenesis was induced by incubation of the cells in the absence of a carbon source. In contrast, cr-1 required supplementation with acetate. This finding suggested that induction of gluconeogenesis in N. crassa could be mediated by metabolites formed in carbon-starved cells. The cr-1 mutant seemed to be deficient in this process and to depend on an exogenous effector to induce gluconeogenesis. Incubation of cr-1 with cyclic AMP partially overcame the acetate requirement for induction of gluconeogenesis.     

Hormone-sensitive carbohydrate metabolism in rat hepatocyte-hepatoma hybrid cells.

Hepatocyte-hepatoma hybrid cells were obtained by fusion of hepatocytes from adult rats and Fao hepatoma cells in the presence of polyethylene glycol. These hybrids were called hepatocytoma cells. The preservation of liver-specific enzyme activities and metabolic functions was studied in the hybrid clone 1E3. 1) The proliferating hepatocytoma cells formed monolayers presenting morphological similarity to primary cultures of hepatocytes. 2) In contrast to Fao hepatoma cells, activities of all gluconeogenic key enzymes were preserved at normal or reduced levels. 3) Lactate-dependent glucose formation was maintained at a state reduced to 36% of the gluconeogenesis in hepatocytes; no glucose formation was detected in Fao hepatoma cells. 4) The activity of the liver-specific glucokinase was reduced in hepatocytoma cells, but it was still present in contrast to Fao cells. The liver-specific isoenzyme pyruvate kinase type L was replaced by the isoenzyme type M2. 5) Gluconeogenic and glycolytic enzyme activities were regulated in hepatocytoma cells by glucagon (0.1 microM) and by insulin (0.1 microM). 6) The genome of hepatocytoma cells and its expression were stable for at least one year, when spontaneously dedifferentiating cells were removed by recloning in hypoxanthine-aminopterine-thymidine (HAT) medium.