Glucose 6-phosphate stimulation of MgATP-dependent Ca2+ uptake by rat kidney microsomes

(1) The features of MgATP-dependent Ca2+ accumulation under stimulation with glucose 6-phosphate were studied in rat kidney microsomes. (2) Ca2+ accumulated in the presence of MgATP alone does not exceed approx. 2 nmol/mg protein. (3) Glucose 6-phosphate markedly stimulates Ca2+ accumulation, up to steady-state levels approx. 15-fold higher than in its absence. (4) The hydrolysis of glucose 6-phosphate by glucose-6-phosphatase is essential for the stimulation, as shown by inhibiting the glucose 6-phosphate hydrolysis with adequate concentrations of vanadate. Inorganic phosphate is accumulated in microsomal vesicles during glucose 6-phosphate-stimulated Ca2+ uptake in equimolar amounts with respects to Ca2+. (5) Increasing concentrations of glucose 6-phosphate result in increasing stimulations of Ca2+ uptake, until a maximal Ca2(+)-loading capacity of approx. 27 nmol/mg microsomal protein is reached. It is suggested that the enlargement of the kidney microsomal Ca2+ pool induced by glucose 6-phosphate (an important metabolite in kidney) might play a role in the regulation of Ca2+ homeostasis in kidney tubular cells.     

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.     

Liver glucose-6-phosphatase activity is modulated by physiological intracellular Ca2+ concentrations

The effect of varying concentrations of free Ca2+ on the formation of Pi from mannose-6-P or of Pi and [U-14C]glucose from [U-14C]glucose-6-P was investigated in isolated fasted rat hepatocytes made permeable by freezing and in liver microsomes. Free Ca2+ concentration was adjusted by the use of Ca-EGTA buffers. In permeabilized cells, glucose-6-phosphatase (EC 3.1.3.9) activity was inhibited up to 50% and in intact microsomes up to 70% by increasing free Ca2+ concentrations from 0.01 to 10 microM. The inhibition was reversible and competitive with respect to glucose-6-P. Treatment of microsomes with 0.4% deoxycholate exposed 90% of latent mannose-6-phosphatase activity which was insensitive to Ca2+. The results indicate that Ca2+ affects the glucose-6-P translocase rather than the phosphohydrolase component. It is concluded that the glucose-6-phosphatase system is modulated by changes in Ca2+ concentrations in the range of those occurring in the liver cell upon hormonal stimulation.     

MgATP-dependent glucose 6-phosphate-stimulated Ca2+ accumulation in liver microsomal fractions. Effects of inositol 1,4,5-trisphosphate and GTP

Ca2+ release triggered by inositol 1,4,5-trisphosphate (IP3) and/or GTP has been studied with rough and smooth microsomes isolated from rat liver. Microsomes were loaded with Ca2+ in the presence of MgATP and in the presence or in the absence of glucose 6-phosphate (glucose-6-P) which markedly stimulated the MgATP-dependent Ca2+ accumulation in rough and smooth microsomes (5- and 10-fold, respectively). Upon addition of IP3 (5 microM), rough and smooth microsomes rapidly release a part (not exceeding 20%) of the Ca2+ previously accumulated both in the absence and in the presence of glucose-6-P. Under the same experimental conditions, inositol 1,3,4,5-tetrakisphosphate was ineffective in triggering any Ca2+ release. Upon addition of GTP (10 microM) both the microsomal fractions progressively release the Ca2+ previously accumulated in the presence of glucose-6-P, when 3% polyethylene glycol was also present. In the absence of polyethylene glycol, GTP released Ca2+ from rough microsomes only, and GTP plus IP3 caused a Ca2+ release which was the sum of the Ca2+ releases caused by GTP and IP3 independently. Both IP3 and GTP, added to microsomes at the beginning of the glucose-6-P-stimulated Ca2+ uptake, reduced the Ca2+ accumulation into rough and smooth microsomes without modifying the initial rate (3 min) of Ca2+ uptake. Also in these conditions, the effects of GTP and IP3 were merely additive. These results indicate that both rough and smooth liver microsomes are responsive to IP3 and GTP with respect to Ca2+ release and that IP3 and GTP likely act independently.     

Role of a nonmitochondrial Ca2+ pool in the synergistic stimulation by cyclic AMP and vasopressin of Ca2+ uptake in isolated rat hepatocytes.

The subcellular distribution of 45Ca2+ accumulated by isolated rat hepatocytes exposed to dibutyryl cyclic AMP (dbcAMP) followed by vasopressin (Vp) was studied by means of a nondisruptive technique. When treated with dbcAMP followed by vasopressin, hepatocytes obtained from fed rats accumulated an amount of Ca2+ approximately fivefold higher than that attained under control conditions. Ca2+ released from the mitochondrial compartment by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) accounted for only a minor portion of the accumulated Ca2+. The largest portion was released by the Ca2+ ionophore A23187 and was attributable to a nonmitochondrial compartment. DbcAMP + Vp-treatment also caused a maximal stimulation of glucose production and a twofold increase in cellular glucose 6-phosphate levels. In hepatocytes obtained from fasted rats, dbcAMP + Vp-stimulated Ca2+ accumulation was lower, although with the same subcellular distribution, and was associated with a minimal glucose production. In the presence of gluconeogenetic substrates (lactate plus pyruvate) hepatocytes from fasted rats were comparable to cells isolated from fed animals. However, Ca2+ accumulation and glucose 6-phosphate production could be dissociated in the absence of dbcAMP, in the presence of lactate/pyruvate alone. Under this condition in fact Vp induced only a minimal accumulation of Ca2+ in hepatocytes isolated from fasted rats, although glucose production was markedly increased. Moreover, treatment of fed rat hepatocytes with 1 mM ATP caused a maximal activation of glycogenolysis, but only a moderate stimulation of cellular Ca2+ accumulation. In this case, sequestration of Ca2+ occurred mainly in the mitochondrial compartment. By contrast, the addition of ATP to dbcAMP-pretreated hepatocytes induced a large accumulation of Ca2+ in a nonmitochondrial pool. Additional experiments using the fluorescent Ca2+ indicator Fura-2 showed that dbcAMP pretreatment can enlarge and prolong the elevation of cytosolic free Ca2+ caused by Vp. A nonmitochondrial Ca2+ pool thus appears mainly responsible for the Ca2+ accumulation stimulated by dbcAMP and Vp in isolated hepatocytes, and cyclic AMP seems able to activate Ca2+ uptake in such a nonmitochondrial pool.     

Functional coupling of the calcium pump and glucose 6-phosphatase activity in liver microsomes: preliminary results

The addition of G-6-Pi to the incubation system for MgATP-dependent calcium transport in liver microsomes results in a marked stimulation of Ca2+ uptake. At physiological pH values (7.2-7.4), the G-6-Pi stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. In the system for the G-6-Pi-stimulated calcium uptake, G-6-Pi is actively hydrolyzed by the glucose 6-phosphatase activity of liver microsomes. Such an activity is not influenced by the concomitant calcium uptake. After the incubation of the system for the MgATP-dependent microsomal calcium transport in the presence of G-6-Pi, Pi and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose 6-phosphatase multicomponent system.     

MgATP-dependent accumulation of calcium ions and inorganic phosphate in a liver reticular pool.

1. MgATP-dependent Ca2+ uptake by rat liver microsomal preparations and permeabilized hepatocytes was measured in the presence or absence of Pi. 2. Monitoring of free Ca2+ in incubation systems with a Ca2+ electrode in the presence of Pi (2-7 mM) revealed a biphasic Ca2+ uptake, with the onset of a second, Pi-dependent, Ca2+ accumulation. 3. Increasing Pi concentrations (up to 10 mM) caused a progressive enlargement of 45Ca2(+)-loading capacity of microsomal fractions. 4. As a result of Pi stimulation of active Ca2+ uptake, [32P]Pi and 45Ca2+ were co-accumulated. 5. Experiments with permeabilized hepatocytes revealed that the amount of Ca2+ releasable by myo-inositol 1,4,5-trisphosphate is unaffected by Pi.     

Glucose 6-phosphate regulates Ca2+ steady state in endoplasmic reticulum of islets. A possible link in glucose-induced insulin secretion

Glucose stimulation of islets is coupled with the rapid intracellular release of myo-inositol 1,4,5-trisphosphate (IP3) and arachidonic acid which in turn mobilize Ca2+ stored in the endoplasmic reticulum (ER). The metabolism of glucose is required for insulin secretion although the link between glucose metabolism and the cellular events resulting in insulin release is unknown. In digitonin-permeabilized islets, glucose 6-phosphate (0.5-4 mM) increased significantly the ATP-dependent Ca2+ content of the ER at a free Ca2+ concentration of 1 microM. At 0.2 microM free Ca2+, glucose 6-phosphate (2-10 mM) had a smaller effect. Glucose, phosphate, mannose 6-phosphate, and fructose 1,6-diphosphate had no effect on the ATP-dependent Ca2+ content of the ER. Glucose 1-phosphate and fructose 6-phosphate also increased ATP-dependent Ca2+ content of the ER, presumably due to conversion to glucose 6-phosphate by islet phosphoglucomutase and phosphoglucoisomerase, respectively. The glucose 6-phosphate increase in the ATP-dependent Ca2+ content of the ER was shown to be mediated by glucose 6-phosphatase localized to the ER. Both arachidonic acid (10 microM) and the Ca2+ ionophore A23187 (2 microM) mobilized Ca2+ stored in the ER by glucose 6-phosphate. However, IP3-induced (10 microM) Ca2+ release from the ER was abolished in the presence of glucose 6-phosphate (0.5-10 mM). We propose that glucose 6-phosphate could provide a regulatory link between glucose metabolism and intracellular Ca2+ regulation by augmenting Ca2+ sequestered in the ER as well as attenuating IP3-induced Ca2+ release. Thus, glucose 6-phosphate would serve as an "off" signal leading to a decrease in intracellular Ca2+ when both the free Ca2+ and glucose 6-phosphate concentrations have increased following glucose stimulus.     

Reappraisal of the effect of Ca2+ on the activity of liver microsomal glucose-6-phosphatase

It has recently been reported that free Ca2+, a second hormonal messenger in the liver, can modulate the activity of liver glucose-6-phosphatase by inhibition (van de Werve, G. (1989) J. Biol. Chem. 264, 6033-6036) or activation (Yamagushi, M., Mori, S., and Suketa, Y. (1989) Chem. Pharm. Bull. (Tokyo) 37, 388-390). Such a controversial role for Ca2+ is reinvestigated by comparing the effect of the addition of free Ca2+ (10(-10) to 20.10(-3) M) under the form of CaCl2 or of Ca-EGTA buffers. We show that the glucose-6-phosphatase activity is: 1) increased in the presence of CaCl2 at concentrations higher than 10(-4) M and unaffected in the presence of CaCl2 at lower concentrations; 2) decreased in the presence of Ca-EGTA buffers yielding free Ca2+ concentrations higher than 10(-8) M; 3) the latter effect is not depending on free Ca2+ or free EGTA concentrations, but on Ca.EGTA complex concentration. In addition, these effects can be reproduced in the same concentration ranges by MgCl2 and Mg-EDTA buffers, respectively. It is concluded that a physiological role for free Ca2+ on the activity of liver glucose-6-phosphatase remains to be established.     

Glucose phosphorylation and dephosphorylation in chicken liver.

1. Glucokinase was absent from chicken liver and only the low Km hexokinases, inhibited by AMP, ADP but not ATP, were present. 2. The Km of chicken liver glucose-6-phosphatase for glucose-6-phosphate was reduced from 5.65 to 3.75 mM following starvation, and the enzyme was inhibited by glucose. 3. Starvation of chickens for 24 hr slightly lowered the hexokinase activity and doubled glucose-6-phosphatase activity; it did not change subcellular distribution of the enzymes. Oral glucose rapidly restored the activities to fed values. 4. It was concluded that glucose uptake into, and efflux from, chicken hepatocytes, was regulated by the activity and kinetic characteristics of glucose-6-phosphatase and by the glucose-6-phosphate concentration, and that the hexokinases had little regulatory function.     

Liver plasma membrane calcium transport. Evidence for a Na+-dependent Ca2+ flux

Plasma membrane vesicles isolated from rat liver exhibited an azide-insensitive Mg2+-ATP-dependent Ca2+ pump which accumulated Ca2+ at a rate of 5.1 +/- 0.5 nmol of calcium/mg of protein/min and reached a total accumulation of 33.2 +/- 2.6 nmol of calcium/mg of protein in 20 microM Ca2+ at 37 degrees C. Equiosmotic addition of 50 mM Na+ resulted in a loss of accumulated calcium. Measurement of Mg2+-ATP-dependent Ca2+ uptake in the presence of 50 mM Na+ revealed no effect of Na+ on the initial rate of Ca2+ uptake, but a decrease in the total accumulation. The half-maximal effect of Na+ on Ca2+ accumulation was achieved at 14 mM. The Ca2+ efflux rate constant in the absence of Na+ was 0.16 +/- 0.01 min-1, whereas the efflux rate constant in the presence of 50 mM Na+ was 0.25 +/- 0.02 min-1. Liver homogenate sedimentation fractions from 1,500 to 105,000 X g were assayed for azide-insensitive Mg2+-ATP-dependent Ca2+ accumulation. Na+-sensitive Ca2+ uptake activity was found to specifically co-sediment with the plasma membrane-associated enzymes, 5'-nucleotidase and Na+/K+-ATPase, whereas Na+-insensitive Ca2+ uptake was found to co-sediment with the endoplasmic reticulum-associated enzyme, glucose-6-phosphatase. The plasma membrane Ca2+ pump was also distinguished from the endoplasmic reticulum Ca2+ pump by its sensitivity to inhibition by vanadate. Half-maximal inhibition of plasma membrane Ca2+ uptake occurred at 0.8 microM VO4(3-), whereas half-maximal inhibition of microsomal Ca2+ uptake occurred at 40 microM.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Studies of the Ca2+ requirements for glucose- and carbachol-induced augmentation of inositol trisphosphate and inositol tetrakisphosphate accumulation in digitonin-permeabilized islets. Evidence for a glucose recognition site in insulin secretion

The metabolism of D-glucose is believed to initiate and regulate insulin secretion by islet beta-cells, although the identity of the metabolite which couples glucose metabolism to the cellular events involved in insulin secretion is unknown. An alternative hypothesis involves the presence of a glucoreceptor for which there has been no biochemical evidence. We have investigated whether glucose recognition by the beta-cell is coupled to phospholipase C. We have used digitonin-permeabilized, [3H]inositol-prelabeled islets to study glucose and carbachol activation of phospholipase C. In this model, carbachol recognition by its muscarinic receptor was coupled to phospholipase C activation. D-Glucose (but not L-glucose) also stimulated phospholipase C activity in these permeabilized islets. This effect was not due to glucose metabolism since glucose 6-phosphate did not affect phospholipase C activity and since phosphorylation of [3H]glucose was not detectable in digitonin-permeabilized islets. Glucose had no effect on the myo-inositol-1,4,5-trisphosphate-5-phosphatase or 3-kinase activities. In the absence of agonist, free Ca2+ concentrations between 0.1 and 1 microM (as determined with a Ca2+-specific electrode) did not influence phospholipase C activity. Stimulation of phospholipase C activity by either carbachol or glucose required Ca2+ in the submicromolar range and was optimal at 0.5 microM free Ca2+.myo-Inositol-1,3,4,5-tetrakisphosphate production from permeabilized islets was synergistically augmented by Ca2+ (0.5-10 microM) and glucose. Phospholipase C activity in islets is therefore not directly activated by free Ca2+ concentrations in the submicromolar range. Furthermore, glucose per se activates phospholipase C activity independently of glucose metabolism. A working hypothesis based on these findings is that glucose is recognized by a site which is coupled to phospholipase C in islets.     

Effect of thyroid hormone on intracellular Ca2+ mobilization by noradrenaline and vasopressin in relation to glycogenolysis in rat liver

The relation between Ca2+ efflux, Ca2+ mobilization from mitochondria and glycogenolysis was studied in perfused euthyroid and hypothyroid rat livers stimulated by Ca2+-mobilizing hormones. Ca2+ efflux, induced by noradrenaline (1 microM) in the absence or presence of DL-propranolol (10 microM) from livers perfused with medium containing a low concentration of Ca2+ (approx. 24 microM), was decreased by more than 50% in hypothyroidism. This correlated with an equal decrease of the fractional mobilization of mitochondrial Ca2+, which could account for 65% of the difference between the net amounts of Ca2+ expelled from the euthyroid and hypothyroid livers. With vasopressin (10 nM) similar results were found, suggesting that hypothyroidism has a general effect on mobilization of internal Ca2+. In normal Ca2+ medium (1300 microM), however, the effect of vasopressin on net Ca2+ fluxes and phosphorylase activation was not impaired in hypothyroidism, indicating that Ca2+ mobilization from the mitochondria in this case plays a minor role in phosphorylase activation. The alpha 1-adrenergic responses of Ca2+ efflux, phosphorylase activation and glucose output, glucose-6-phosphatase activity and oxygen consumption in hypothyroid rat liver were completely restored by in vivo T3 injections (0.5 micrograms per 100 g body weight, daily during 3 days). Perfusion with T3 (100 pM) during 19 min did not influence hypothyroid rat liver oxygen consumption and alpha 1-receptor-mediated Ca2+ efflux. However, this in vitro T3 treatment showed a completely recovered alpha 1-adrenergic response of phosphorylase and a partly restored glucose-6-phosphatase activity and glucose output. The results indicate that thyroid hormones may control alpha 1-adrenergic stimulation of glycogenolysis by at least two mechanisms, i.e., a long-term action on Ca2+ mobilization, and a short-term action on separate stages of the glycogenolytic process.     

Antagonistic regulation of the glucose/glucose 6-phosphate cycle by insulin and glucagon in cultured hepatocytes.

Flux through the glucose/glucose 6-phosphate cycle in cultured hepatocytes was measured with radiochemical techniques. Utilization of [2-3H]glucose was taken as a measure of glucokinase flux. Liberation of [14C]glucose from [U-14C]glycogen and from [U-14C]lactate, as well as the difference between the utilization of [2-3H]glucose and of [U-14C]glucose, were taken as measures of glucose-6-phosphatase flux. At constant 5 mM-glucose and 2 mM-lactate concentrations insulin increased glucokinase flux by 35%; it decreased glucose-6-phosphatase flux from glycogen by 50%, from lactate by 15% and reverse flux from external glucose by 65%, i.e. overall by 40%. Glucagon had essentially no effect on glucokinase flux; it enhanced glucose-6-phosphatase flux from glycogen by 700%, from lactate by 45% and reverse flux from external glucose by 20%, i.e. overall by 110%. At constant glucose concentrations cellular glucose 6-phosphate concentrations were essentially not altered by insulin, but were increased by glucagon by 230%. In conclusion, under basic conditions without added hormones the glucose/glucose 6-phosphate cycle showed only a minor net glucose uptake, of 0.03 mumol/min per g of hepatocytes; this flux was increased by insulin to a net glucose uptake of 0.21 mumol/min per g and reversed by glucagon to a net glucose release of 0.22 mumol/min per g. Since the glucose 6-phosphate concentrations after hormone treatment did not correlate with the glucose-6-phosphatase flux, it is suggested that the hormones influenced the enzyme activity directly.     

Effect of extracellular Ca2+ on plasma membrane Ca2+ inflow and cytoplasmic free Ca2+ in isolated hepatocytes

An initial rapid phase and a subsequent slow phase of 45Ca2+ uptake were observed following the addition of 45Ca2+ to Ca2+-deprived hepatocytes. The magnitude of the rapid phase increased 15-fold over the range 0.1-11 mM extracellular Ca2+ (Ca2+o) and was a linear function of [Ca2+]o. The increases in the rate of 45Ca2+ uptake were accompanied by only small increases in the intracellular free Ca2+ concentration. In cells made permeable to Ca2+ by treatment with saponin, the rate of 45Ca2+ uptake (measured at free Ca2+ concentrations equal to those in the cytoplasm of intact cells) increased as the concentration of saponin increased from 1.4 to 2.5 micrograms per mg wet weight cells. Rates of 45Ca2+ uptake by cells permeabilized with an optimal concentration of saponin were comparable with those of intact cells incubated at physiological [Ca2+o], but were substantially lower than those for intact cells incubated at high [Ca2+o]. It is concluded that Ca2+ which enters the hepatocyte across the plasma membrane is rapidly removed by binding and transport to intracellular sites and by the plasma membrane (Ca2+ + Mg2+)-ATPase and the plasma membrane Ca2+ inflow transporter is not readily saturated with Ca2+o.     

The flavonoid silibinin decreases glucose-6-phosphate hydrolysis in perfused rat hepatocytes by an inhibitory effect on glucose-6-phosphatase.

BACKGROUND/AIMS: The flavonoid silibinin has been reported to be beneficial in several hepatic disorders. Recent evidence also suggests that silibinin could be beneficial in the treatment of type 2 diabetes, owing to its anti-hyperglycemic properties. However, the mechanism(s) underlying these metabolic effects remains unknown. METHODS: The effects of silibinin on liver gluconeogenesis were studied by titrating hepatocytes from starved rats with sub-saturating concentrations of various exogenous substrates in a perifusion system. Hepatocytes from fed rats were also used to investigate glycogenolysis from endogenous glycogen. The effect of silibinin on glucose-6-phosphatase kinetics was determined in intact and permeabilized rat liver microsomes. RESULTS: Silibinin induced a dose-dependent inhibition of gluconeogenesis associated with a potent decrease in glucose-6-phosphate hydrolysis. This effect was demonstrated whatever the gluconeogenic substrates used, i.e. dihydroxyacetone, lactate/pyruvate, glycerol and fructose. In addition, silibinin decreased the glucagon-induced stimulation of both gluconeogenesis and glycogenolysis, this being associated with a reduction of glucose-6-phosphate hydrolysis. Silibinin inhibits glucose-6-phosphatase in rat liver microsomes in a concentration-dependent manner that could explain the decrease in glucose-6-phosphate hydrolysis seen in intact cells. CONCLUSION: The inhibitory effect of silibinin on both hepatic glucose-6-phosphatase and gluconeogenesis suggests that its use may be interesting in treatment of type 2 diabetes.     

Linked oscillations of free Ca2+ and the ATP/ADP ratio in permeabilized RINm5F insulinoma cells supplemented with a glycolyzing cell-free muscle extract

We show in the accompanying paper that the steady-state level of free Ca2+ maintained by the organelles of permeabilized RINm5F insulinoma cells varies inversely with the ATP/ADP ratio when this ratio is set by addition of creatine phosphokinase and fixed ratios of creatine to creatine phosphate. We, therefore, asked whether acute cyclic alterations in the cytosolic ATP/ADP ratio in the range known to modulate O2 consumption might be involved in regulating the physiological activity of Ca2+ -ATPases and the cytosolic free Ca2+ level. To explore this hypothesis we combined two experimental systems: 1) permeabilized RINm5F insulinoma cells that can maintain a low medium Ca2+ concentration and 2) a cell-free extract of rat skeletal muscle that spontaneously exhibits oscillatory behavior of glycolysis and linked oscillations in the ATP/ADP ratio, when provided with glucose. The free Ca2+ level maintained by the permeabilized cells oscillated in phase with the glycolytic oscillations and correlated closely with the ATP/ADP ratio but not with glucose 6-phosphate, fructose 6-phosphate, orthophosphate, or pH. When glucokinase replaced hexokinase as the glucose phosphorylating enzyme, Ca2+ oscillations were induced by increasing the glucose concentration from 2 to 8 mM. The results demonstrate a link between metabolite changes and free Ca2+ levels in a reconstituted physiological system. They support a model in which oscillations in glycolysis and the ATP/ADP ratio may cause oscillations in cytosolic free Ca2+, beta-cell electrical activity, and insulin release.     

Molecular pathology of glucose-6-phosphatase

It was known in the 1950s that hepatic microsomal glucose-6-phosphatase plays an important role in the regulation of blood glucose levels. All attempts since then to purify a single polypeptide with glucose-6-phosphatase activity have failed. Until recently, virtually nothing was known about the molecular basis of glucose-6-phosphatase or its regulation. Recent studies of the type 1 glycogen storage diseases, which are human genetic deficiencies that result in impaired glucose-6-phosphatase activity, have greatly increased our understanding of glucose-6-phosphatase. Glucose-6-phosphatase has been shown to comprise at least five different polypeptides, the catalytic subunit of glucose-6-phosphatase with its active site situated in the lumen of the endoplasmic reticulum; a regulatory Ca2+ binding protein; and three transport proteins, T1, T2, and T3, which respectively allow glucose-6-phosphate, phosphate, and glucose to cross the endoplasmic reticulum membrane. Purified glucose-6-phosphatase proteins, immunospecific antibodies, and improved assay techniques have led to the diagnosis of a variety of new type 1 glycogen storage diseases. Recent studies of the type 1 glycogen storage diseases have led to a much greater understanding of the role and regulation of each of the glucose-6-phosphatase proteins.