Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal alpha-glucosidase

The levels of functional mRNA encoding glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) were examined in hepatocytes from fasted and fasted/carbohydrate-refed rats and in hepatocytes inoculated into primary culture. Functional G6PDH mRNA was assessed in a cell-free protein synthesis system in vitro. We observed that hepatocytes from fasted/carbohydrate-refed rats had a 12-fold higher level of mRNA than did hepatocytes from fasted rats. The possibility that the adrenal glucocorticoids and insulin were responsible for the increase in G6PDH mRNA in refed rats was examined by studying the effect of insulin and the synthetic glucocorticoid, dexamethasone, on the level of functional G6PDH mRNA in primary cultures of rat hepatocytes maintained in a chemically defined medium. Hepatocytes from fasted rats were inoculated into primary culture and maintained for 48 h either in the absence of hormones or in the presence of insulin alone, dexamethasone alone or both hormones together. We observed that dexamethasone alone caused a fourfold increase in G6PDH mRNA while insulin caused about a twofold increase. Both hormones together elicited an increase that was additive. A comparison of functional G6PDH mRNA levels with the effect of the hormones on G6PDH activity and relative rate of enzyme synthesis suggests that the glucocorticoid elevates the level of G6PDH mRNA within the cell without causing a concommitant increase in the rate of synthesis of the enzyme or the level of G6PDH activity. The results obtained with the primary cultures of hepatocytes indicate that insulin and the glucocorticoids are probably involved with the regulation of hepatic G6PDH mRNA. However, involvement of other hormones, such as thyroid hormone, seems likely since the induced levels of G6PDH mRNA in hepatocytes in culture was one-third of that observed in refed rats.     

The effect of ethanol, alone and in combination with the glucocorticoids and insulin, on glucose-6-phosphate dehydrogenase synthesis and mRNA in primary cultures of hepatocytes.

The hormonal regulation of the relative rate of synthesis and mRNA of glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of adult-rat liver parenchymal cells maintained in a chemically defined medium. Maintenance of hepatocytes from starved animals in a culture medium devoid of any hormones resulted in a 4-fold increase in the relative rate of G6PDH synthesis in 48 h. Parallel cultures treated with glucocorticoids alone exhibited a rate of G6PDH synthesis comparable with that in the control cultures, whereas insulin alone caused a 6.5-fold increase in the rate of synthesis in 48 h. However, if the cultures were treated with glucocorticoids and insulin simultaneously, a 13-fold increase in the rate of synthesis was observed. The effect of ethanol, alone and in combination with the hormones, on the relative rate of G6PDH synthesis was studied also. Ethanol alone caused an 8-fold increase in the rate of synthesis in 48 h, whereas the combination of ethanol, glucocorticoid and insulin caused a 25-fold increase. The amount of functional mRNA encoding G6PDH, as measured in a cell-free translation system, was compared with enzyme activity and relative rate of enzyme synthesis. The increases in G6PDH activity and relative rate of synthesis in primary cultures of hepatocytes treated with ethanol, alone and in combination with the glucocorticoids and insulin, were paralleled by comparable increases in G6PDH mRNA. The results of this study show that the glucocorticoids acted in a permissive manner to amplify the insulin stimulation of G6PDH synthesis and that insulin, glucocorticoids and ethanol interact to stimulate synthesis of G6PDH primarily by increasing the concentration of functional G6PDH mRNA.     

Ethanol modulation of the hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase activity in primary cultures of rat hepatocytes.

The hormonal and nutritional regulation of glucose 6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Inoculation of hepatocytes from starved rats into primary cultures resulted in a 4-5-fold increase in G6PDH activity in 48 h in the absence of hormones. Parallel cultures treated simultaneously with glucocorticoids and insulin exhibited a 12-15-fold increase during the same time. Glucocorticoids by themselves did not elevate G6PDH activity, whereas insulin alone significantly stimulated enzyme activity. Thus the glucocorticoids acted in a 'permissive' role to amplify the insulin stimulation of G6PDH. Elevated concentrations of glucose in the culture medium increased enzyme activity in both the control cultures and those treated with hormones. Ethanol was found to potentiate G6PDH activity in cultures treated with glucocorticoids and insulin. The effect of ethanol was time- and dose-dependent. These results establish that insulin, glucocorticoids, glucose and ethanol interact in some undefined manner to regulate hepatic G6PDH activity.     

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.     

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.     

Regulation of malic enzyme gene expression by nutrients,hormones, and growth factors in fetal hepatocyte primary cultures

The culture of fetal hepatocytes for 64 h in medium supplemented with 5 mM glucose, T3, insulin, and dexamethasone resulted in the coordinate precocious expression of malic enzyme mRNA, protein, and specific activity. T3 was the main inducer; meanwhile, insulin exerted a small synergistic effect when added with T3. Dexamethasone had a potentiation effect on the T3 response of malic enzyme mRNA expression regardless of the presence of insulin. This effect of dexamethasone on T3 response of malic enzyme mRNA expression was time (64 h) and glucose dependent. Glucagon, and to a greater degree dibutyryl-cAMP, repressed malic enzyme mRNA as well as protein expression by T3 and dexamethasone, in the absence of insulin. Glucose and other carbon sources such as lactate-pyruvate or dihydroxyacetone induced the abundance of malic enzyme mRNA in the absence of hormones. Insulin and T3 produced a high accumulation of malic enzyme mRNA in lactate-pyruvate medium, this effect being decreased by dexamethasone. EGF supressed the induction produced by T3 and dexamethasone on malic enzyme mRNA, while the expression of β-actin mRNA remained essentially unmodified. © 1993 Wiley-Liss, Inc.     

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.     

Nutritional and hormonal regulation of mRNA levels of lipogenic enzymes in primary cultures of rat hepatocytes.

The effects of nutrients and hormones on the mRNA levels of acetyl-CoA carboxylase, fatty acid synthase, malic enzyme, and glucose 6-phosphate dehydrogenase were examined in primary cultures of rat hepatocytes during the process of induction. The addition of both glucose and insulin to the culture medium markedly enhanced the lipogenic enzyme mRNA induction due to either of them, in 16 h. Fructose or glycerol proved to be an effective substitute for glucose, suggesting that glycolytic metabolites were involved in the mRNA induction. It is remarkable that mRNA induction of acetyl-CoA carboxylase was the most sensitive to glucose and also to insulin among the lipogenic enzymes. Polyunsaturated fatty acids markedly reduced the mRNA induction of lipogenic enzymes. Dexamethasone enhanced all the lipogenic enzyme mRNA induction by insulin. On the other hand, triiodothyronine addition greatly increased the mRNA concentrations of lipogenic enzymes, but dexamethasone decreased rather than increased the mRNA induction by triiodothyronine. The effects of insulin on the induction of the lipogenic enzyme mRNAs were similar, but those of triiodothyronine were not. Triiodothyronine markedly enhanced malic enzyme mRNA induction by insulin with dexamethasone, and tended to enhance the induction of the acetyl-CoA carboxylase and fatty acid synthase mRNAs, but not that of glucose 6-phosphate dehydrogenase mRNA. It appeared that insulin and triiodothyronine synergistically enhanced lipogenic enzyme mRNA induction by glucose, but the mechanisms were different.     

Pretranslational regulation of tyrosine aminotransferase and phosphoenolpyruvate carboxykinase (GTP) synthesis by glucagon and dexamethasone in adult rat hepatocytes.

The regulation of synthesis of the gluconeogenic cytosolic enzyme phosphoenolpyruvate carboxykinase (PEPCK) and of tyrosine aminotransferase (TAT) by glucagon and glucocorticoid hormones was studied in hepatocytes maintained in suspension culture for 7 h. Specific antibodies were used to measure relative rates of enzyme synthesis after pulse-labelling of the cells with [3H]leucine or [35S]methionine. Concomitantly, amounts of mRNA were quantified after translation in vitro in a reticulocyte lysate and specific immunoprecipitation of the proteins. Glucagon stimulated the rate of synthesis of PEPCK by 4-6-fold and that of TAT by 6-8-fold in 2h. In contrast, dexamethasone had little effect on PEPCK synthesis, whereas it increased TAT synthesis by 5-9-fold. When used in combination, the two hormones displayed additive effects on TAT synthesis, whereas the glucocorticoid hormone strongly potentiated stimulation of PEPCK synthesis by glucagon. In every instance, changes in rates of synthesis of the two enzymes were totally accounted for by increases in amounts of the corresponding functional mRNA, suggesting a pretranslational site of action for both glucagon and dexamethasone.     

Molecular characterization of a novel glucose-6-phosphate dehydrogenase from potato (Solanum tuberosum L.)

We describe a novel G6PD cDNA from potato. The deduced amino acid sequence shares 77% identity with the known chloroplast enzyme, but only 47% with the corresponding cytosolic G6PDH. The sequence comprises the two cysteine residues conserved in other redox-regulated chloroplast G6PDH and a transit peptide capable of directing a GFP fusion protein to chloroplasts, demonstrating that the cDNA codes for a second plastidic G6PD isoform. The mature part was expressed in E. coli. When synthesized with a C-terminal Strep tag, the enzyme retained G6PDH activity upon affinity purification. In the presence of reductively activated spinach thioredoxin, G6PDH activity decreased by about 50%. This protein-mediated activity loss was completely reversed by addition of oxidant. In contrast to the chloroplast enzyme (P1), the presence of reduced dithiothreitol alone destroyed the activity of the new G6PDH (P2), and incubation with GSH had no effect. The Km values determined for both substrates were significantly lower compared to those of P1. The high Vmax and Ki [NADPH] values indicate that the P2 enzyme is more active than P1 and less susceptible to feedback inhibition by its product NADPH. At the level of mRNA accumulation, differences between the two plastid-localized isoforms are most prominent in roots and growing tissues. Immunoblot analyses of isolated plastid preparations revealed that the two plastidic enzymes are present in both root and leaf tissue. The data obtained indicate that we have characterized a second plastidic G6PDH with distinct biochemical features.     

Melatonin enhances leptin expression by rat adipocytes in the presence of insulin

Leptin and melatonin play an important role in the regulation of body mass and energy balance. Both hormones show a circadian rhythm, with increasing values at night. In addition, melatonin receptors were recently described in adipocytes, where leptin is synthesized. Here, we investigated the influence of melatonin and its interaction with insulin and dexamethasone on leptin expression. Isolated rat adipocytes were incubated with melatonin (1 nM) alone or in combination with insulin (5 nM) and/or dexamethasone (7 nM) for 6 h. Melatonin or insulin alone did not affect leptin expression, but together they increased it by 120%. Dexamethasone increased leptin mRNA content (105%), and this effect was not enhanced by melatonin. Simultaneous treatment with the three hormones provoked a further increase in leptin release (250%) and leptin mRNA (100%). Melatonin prevented the forskolin-induced inhibition (95%) of leptin expression. In addition, melatonin's ability to stimulate leptin release (in the presence of insulin) was completely blocked by pertussis toxin and luzindole. To gain further insight into the molecular basis of melatonin and insulin synergism, the insulin-signaling pathway was investigated. Melatonin increased the insulin-induced insulin receptor-beta tyrosine phosphorylation, which led to an increased serine phosphorylation of the downstream convergent protein Akt. We concluded that melatonin interacts with insulin and upregulates insulin-stimulated leptin expression. These effects are caused by melatonin binding to the pertussis toxin-sensitive G(i) protein-coupled membrane receptor (MT1 subtype) and the cross talk with insulin, since insulin receptor and its convergent target Akt are coactivated by melatonin.     

Depot-specific regulation of the conversion of cortisone to cortisol in human adipose tissue

Objective: Our main objective was to compare the regulation of cortisol production within omental (Om) and abdominal subcutaneous (Abd sc) human adipose tissue. Methods and Procedures: Om and Abd sc adipose tissue were obtained at surgery from subjects with a wide range of BMI. Hydroxysteroid dehydrogenase (HSD) activity (³H‐cortisone and ³H‐cortisol interconversion) and expression were measured before and after organ culture with insulin and/or dexamethasone. Results: Type 1 HSD (HSD1) mRNA and reductase activity were mainly expressed within adipocytes and tightly correlated with adipocyte size within both depots. There was no depot difference in HSD1 expression or reductase activity, while cortisol inactivation and HSD2 mRNA expression (expressed in stromal cells) were higher in Om suggesting higher cortisol turnover in this depot. Culture with insulin decreased HSD reductase activity in both depots. Culture with dexamethasone plus insulin compared to insulin alone increased HSD reductase activity only in the Om depot. This depot‐specific increase in reductase activity could not be explained by an alteration in HSD1 mRNA or protein, which was paradoxically decreased. However, in Om only, hexose‐6‐phosphate dehydrogenase (H6PDH) mRNA levels were increased by culture with dexamethasone plus insulin compared to insulin alone, suggesting that higher nicotinamide adenine dinucleotide phosphate‐oxidase (NADPH) production within the endoplasmic reticulum (ER) contributed to the higher HSD reductase activity. Discussion: We conclude that in the presence of insulin, glucocorticoids cause a depot‐specific increase in the activation of cortisone within Om adipose tissue, and that this mechanism may contribute to adipocyte hypertrophy and visceral obesity.