Gérald van de Werve

Influence of long-term diabetes on liver glycogen metabolism in the rat.

Diabetes acutely impairs the ability of the liver to synthesize glycogen. However, the effect of chronic diabetes on the glycogenic function of the liver is not known. We measured hepatic glycogen contents in streptozotocin (STZ)-diabetic rats 3 weeks or 9 months after the induction of diabetes, in the fed state and following a 24-hour fast. In the fed state, liver glycogen levels were markedly decreased in short-term diabetic animals (5.8 +/- 2.0 v 33.9 +/- 2.3 mg/g, P less than .001), but not in long-term diabetic rats (18.3 +/- 4.4 v 20.7 +/- 1.3 mg/g, P = NS) as compared with age-matched nondiabetic animals, despite comparable hyperglycemia (portal plasma glucose levels of 424 +/- 21 and 449 +/- 24 mg/100 mL, short- and long-term diabetics, respectively). In the fasted state, on the other hand, liver glycogen was depleted in acute diabetes (4.5 +/- 2.2 mg/g v 1.9 +/- 0.5 of control rats), but significantly increased in chronic diabetes (10.1 +/- 3.1 v 0.2 +/- 0.03 mg/g, P less than .001). The latter finding was confirmed by electron-microscopical examination of liver cells. Furthermore, the percentage of hepatic glycogen synthase in the active form (synthase a) was lower than normal in short-term diabetic rats and in old nondiabetic rats. In long-term diabetic animals, on the other hand, synthase a was significantly higher than in old controls (P less than .01).(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetes Affects Similarly the Catalytic Subunit and Putative Glucose-6-phosphate Translocase of Glucose-6-phosphatase

The effect of streptozocin diabetes on the expression of the catalytic subunit (p36) and the putative glucose-6-phosphate translocase (p46) of the glucose-6-phosphatase system (G6Pase) was investigated in rats. In addition to the documented effect of diabetes to increase p36 mRNA and protein in the liver and kidney, a ∼2-fold increase in the mRNA abundance of p46 was found in liver, kidney, and intestine, and a similar increase was found in the p46 protein level in liver. In HepG2 cells, glucose caused a dose-dependent (1–25 mm) increase (up to 5-fold) in p36 and p46 mRNA and a lesser increase in p46 protein, whereas insulin (1 μm) suppressed p36 mRNA, reduced p46 mRNA level by half, and decreased p46 protein by about 33%. Cyclic AMP (100 μm) increased p36 and p46 mRNA by >2- and 1.5-fold, respectively, but not p46 protein. These data suggest that insulin deficiency and hyperglycemia might each be responsible for up-regulation of G6Pase in diabetes. It is concluded that enhanced hepatic glucose output in insulin-dependent diabetes probably involves dysregulation of both the catalytic subunit and the putative glucose-6-phosphate translocase of the liver G6Pase system.

Glucose Transport and Glucose 6-Phosphate Hydrolysis in Intact Rat Liver Microsomes

Glucose transport was investigated in rat liver microsomes in relation to glucose 6-phosphatase (Glu-6-Pase) activity using a fast sampling, rapid filtration apparatus. 1) The rapid phase in tracer uptake and the burst phase in glucose 6-phosphate (Glu-6-P) hydrolysis appear synchronous, while the slow phase of glucose accumulation occurs during the steady-state phase of glucose production. 2) [14C]Glucose efflux from preloaded microsomes can be observed upon addition of either cold Glu-6-P or Glu-6-Pase inhibitors, but not cold glucose. 3) Similar steady-state levels of intramicrosomal glucose are observed under symmetrical conditions of Glu-6-P or vanadate concentrations during influx and efflux experiments, and those levels are directly proportional to Glu-6-Pase activity. 4) The rates of both glucose influx and efflux are characterized by t values that are independent of Glu-6-P concentrations. 5) Glucose efflux in the presence of saturating concentrations of vanadate was not blocked by 1 mM phloretin, and the initial rates of efflux appear directly proportional to intravesicular glucose concentrations. 6) It is concluded that glucose influx into microsomes is tightly linked to Glu-6-Pase activity, while glucose efflux may occur independent of hydrolysis, so that microsomal glucose transport appears unidirectional even though it can be accounted for by diffusion only over the accessible range of sugar concentrations.

Activation of mitogen-activated protein kinase in freshly isolated rat hepatocytes by both a calcium- and a protein kinase C—dependent pathway

In the present study, we investigated the role of calcium and protein kinase C (PKC) in the activation of mitogen-activated protein kinase (MAPK) in isolated rat hepatocytes. We found that the glycogenolytic hormone norepinephrine (NE), acting through the alpha1-adrenergic receptor and the G protein Gq, was able to induce a dose- and time-dependent activation of MAPK in hepatocytes. Vasopressin, which acts through a different receptor but also through stimulation of the Gq-dependent pathway, also caused a twofold activation of MAPK. Activation of MAPK by both agonists required the presence of free extracellular calcium and was blocked by the specific PKC inhibitor, Ro 31-8220. MAPK activation was also induced by phorbol myristate acetate (PMA), confirming that a PKC-dependent pathway exists for MAPK activation in liver. Furthermore, calcium-mobilizing agents such as thapsigargin and ionomycin were able to induce an activation of MAPK by a PKC-independent pathway that was totally abolished by preincubation of cells with EGTA. A second pathway for MAPK activation that relies solely on calcium may therefore exist. Ro 31-8220 did not affect phosphorylase activation by NE, vasopressin, thapsigargin, and ionomycin, indicating that PKC inhibition did not interfere with the signaling pathway leading to inositol-1,4,5-triphosphate (IP3)-induced calcium mobilization or with changes in calcium fluxes. The role of MAPK activation by NE and vasopressin in the regulation of hepatic carbohydrate metabolism is discussed.

Evidence for a Membrane Exchangeable Glucose Pool in the Functioning of Rat Liver Glucose-6-phosphatase

We have investigated the kinetics of tracer uptake into rat liver microsomes in relation to [14C]glucose 6-phosphate (Glu-6-P) hydrolysis by glucose 6-phosphatase (Glu-6-Pase). 1) The steady-state levels of intravesicular tracer accumulated during the rapid (AMP1) and slow (AMP2) phases of uptake both demonstrate Michaelis-Menten kinetics relative to outside Glu-6-P concentrations with K values similar to those observed for the initial burst (V) and steady-state (V) rates of Glu-6-P hydrolysis. 2) The AMP1/AMP2 ratio is constant (mean value = 0.105 ± 0.018) over the whole range of outside Glu-6-P concentrations and is equal to the AMP/AMP ratio (0.109 ± 0.032). 3) Linear relationships are observed between the initial rates of glucose transport during the slow uptake phase (V2) and [AMP1], and between [V] and [AMP2]. 4) The value of V exceeds by more than 10-fold that of V. 5) It is concluded that the substrate transport model is incompatible with those results and that AMP1 represents a membrane exchangeable glucose pool. 6) We propose a new version of the conformational model in which the catalytic site lies deep within a hydrophilic pocket of an intrinsic membrane protein and communicates with the extra- and intravesicular spaces through channels with different glucose permeabilities.

Influence of Long-Term Diabetes on Renal Glycogen Metabolism in the Rat

Background/Aims: The effects of acute insulin deficiency on the kidney have been investigated in animal models of experimental diabetes; however, the impact of long-term diabetes has not been determined. Methods: We measured renal glycogen contents in streptozotocin (STZ)-diabetic rats 3 weeks (n = 12) or 9 months (n = 12) after the induction of diabetes, and in 2 groups of control rats of similar age (n = 16 and n = 12, respectively), in the fed state and after a 24-hour fast. Results: Diabetic rats had high glucose levels, low insulin but normal glucagon concentrations in portal blood. In the fasting state, kidney glycogen content was very low in both young control and young diabetic rats (54 ± 15 and 189 ± 26 µg/g, respectively, mean ± SD); in contrast, glycogen levels were markedly elevated in rats with long-standing diabetes as compared to old nondiabetic animals (2,628 ± 1,023 ± and 1,968 ± 989 µg/g of diabetic rat, fasting and fed, respectively, p < 0.001 vs. 0 ± 0 and 4 ± 6 µg/g of control rats). On electron microscopy, large glycogen clusters were localized to the renal tubules. Kidney phosphorylase activity was higher, and synthase activity lower in diabetic than control rats (p < 0.05 for both), whereas kidney glycogen was strongly related to plasma glucose levels, suggesting that the enzyme changes were secondary to glycogen accumulation itself. Renal hexosephosphates and fructose-2,6-bisphosphate contents were both increased in long-term diabetic rats (p < 0.05), implying enhanced fluxes through both glycolysis and gluconeogenesis. Conclusion: In chronic, untreated diabetes glycogen accumulates in the renal tubules; prolonged hyperglycemia is the sole driving force for this phenomenon.

Insulin-induced Ca2+ entry in hepatocytes is important for PI 3-kinase activation, but not for insulin receptor and IRS-1 tyrosine phosphorylation

Insulin produces an influx of Ca(2+) into isolated rat hepatocyte couplets that is important to couple its tyrosine kinase receptor to MAPK activity (Benzeroual et al., Am. J. Physiol. 272, (1997) G1425-G1432. In the present study, we have examined the implication of Ca(2+) in the phosphorylation state of the insulin receptor (IR) beta-subunit and of insulin receptor substrate-1 (IRS-1), as well as in the stimulation of PI 3-kinase activity in cultured hepatocytes. External Ca(2+) chelation (EGTA 4 mM) or administration of Ca(2+) channel inhibitors gadolinium 50 microM or nickel 500 microM inhibited insulin-induced PI 3-kinase activation by 85, 50 and 50%, respectively, whereas 200 microM verapamil was without effect. In contrast, the insulin-induced tyrosine phosphorylation of IR beta-subunit and of IRS-1 was not affected by any of the experimental conditions. Our data demonstrate that the stimulation of PI 3-kinase activity by the activated insulin receptor, but not the phosphorylation of IR beta-subunit and IRS-1, requires an influx of Ca(2+). Ca(2+) thus appears to play an important role as a second messenger in insulin signaling in liver cells.

Dietary P i deprivation in rats affects liver cAMP, glycogen, key steps of gluconeogenesis and glucose production

We previously reported [Xie, Li, Méchin and van de Werve (1999) Biochem. J. 343, 393-396] that dietary phosphate deprivation for 2 days up-regulated both the catalytic subunit and the putative glucose-6-phosphate translocase of the rat liver microsomal glucose-6-phosphatase system, suggesting that increased hepatic glucose production might be responsible for the frequent clinical association of hypophosphataemia and glucose intolerance. We now show that liver cAMP was increased in rats fed with a diet deficient in P(i) compared with rats fed with a control diet. Accordingly, in the P(i)-deficient group pyruvate kinase was inactivated, the concentration of phosphoenolpyruvate was increased and fructose 2, 6-bisphosphate concentration was decreased. Phosphoenolpyruvate carboxykinase activity was marginally increased and glucokinase activity was unchanged by P(i) deprivation. The liver glycogen concentration decreased in the P(i)-deficient group. In the fed state, plasma glucose concentration was increased and plasma P(i) and insulin concentrations were substantially decreased in the P(i)-deficient group. All of these changes, except decreased plasma P(i), were cancelled in the overnight fasted P(i)-deficient group. In the fasted P(i)-deficient group, immediately after a glucose bolus, the plasma glucose level was elevated and the inhibition of endogenous glucose production was decreased. However, this mild glucose intolerance was not sufficient to affect the rate of fall of the glucose level after the glucose bolus. Taken together, these changes are compatible with a stimulation of liver gluconeogenesis and glycogenolysis by the P(i)-deficient diet and further indicate that the liver might contribute to impaired glucose homeostasis in P(i)-deficient states.

Opposite Effects of Hyperglycemia and Insulin Deficiency on Liver Glycogen Synthase Phosphatase Activity in the Diabetic Rat

The specific effect of hyperglycemia on the reported decrease in liver glycogen synthase phosphatase activity was studied in STZ-induced diabetic rats with normal fasting insulinemia. Four groups of animals were investigated: control (nondiabetic), diabetic hyperglycemic (STZ), diabetic normoglycemic (STZ followed by 3-day phloridzin treatment), and a diabetic normoglycemic group injected with glucose to reinstate hyperglycemia. None of the treatments significantly altered fasting plasma insulin and glucagon concentrations. We found that hepatic synthase phosphatase activity decreased in STZ-induced diabetic rats and was further markedly reduced when glycemia was normalized in the diabetic animals. This additional decrease in phosphatase activity was almost fully reversed when hyperglycemia was restored by acute glucose infusion of the normoglycemic diabetic rats. In parallel, the levels of liver G6P and F6P were markedly reduced in the diabetic normoglycemic rats and restored with reinstatement of hyperglycemia. In contrast, liver microsomal glucose-6-phosphatase activity was enhanced and glucokinase activity was lowered in all diabetic groups, regardless of glycemia. Our results indicate that hyperglycemia per se counteracts part of the loss of hepatic synthase phosphatase in diabetic animals and provokes the stable conversion of synthase phosphatase from a less active to a more active form.

Hormone-stimulated glucose production from glycogen in hepatocytes from streptozotocin diabetic rats

The contribution of hormone-stimulated glycogenolysis to hepatic glucose production was studied in hepatocytes from streptozotocin diabetic rats. To this end, the activation of glycogen phosphorylase by glucagon, vasopressin, and the alpha 1-adrenergic agonist phenylephrine was compared in hepatocytes from normal and diabetic rats and related to glycogen content, glucose production, and microsomal glucose-6-phosphatase activity. Streptozotocin-induced diabetes reduced the glycogen content and the amount of total (a + b) phosphorylase in hepatocytes proportionally to the severity of the disease. In cells from severely diabetic rats (group 1), the responsiveness of activation of phosphorylase to the hormones was reduced by about half, consistent with a 45% reduction in total phosphorylase. In addition, the sensitivity of phosphorylase activation to all hormones investigated was decreased by about 1 order of magnitude or more in cells of this group. In hepatocytes from rats with milder diabetes (group 2), maximal phosphorylase activation reached an intermediate value between that of the control group and of group 1. In response to all hormones investigated, group 2 diabetic rat hepatocytes produced less glucose than control rat liver cells, while in group 1 there was no increase in glucose production at all, presumably because glycogen concentration was too low. However, in group 2 diabetic rat hepatocytes, glucagon-stimulated glucose production, unlike phosphorylase activation, did not show decrease sensitivity, presumably because glucose-6-phosphatase activity is increased by diabetes. Our results thus indicate that hormone-stimulated liver glycogenolysis is unlikely to contribute to enhanced glucose production in insulin-deficient diabetes, despite increased glucose-6-phosphatase activity.