Effects of glucagon on hepatic microsomal glucose-6-phosphatase in vivo

The smooth endoplasmic reticulum (SER) has been implicated in glycogen deposition and depletion. It has been suggested that SER proliferation plays a role in elevated glucose release during rapid glycogenolysis (Striffler et al., Am. J. Anat. 160: 363, 1981). In these studies, the role of SER in glucagon-stimulated hepatic glucose release was examined by assessing changes in microsomal glucose-6-phosphatase (G-6-Pase) and membrane cholesterol to phospholipid ratios. In control fed rats given 6 i.p. injections of glucagon 350 micrograms/injection) at hourly intervals, percentage hepatic glycogen decreased nearly 30 fold, with liver homogenate G-6-Pase (U/mg protein) increasing 50% (p less than .02 relative to vehicle-injected controls) from .055 +/- .003 at 0h (n = 12) to .081 +/- .004 at 6h (n = 11). The increase in homogenate G-6-Pase was reflected in the isolated SER fraction by a 48% rise (p less than .01 relative to controls) in activity from a 0h value of .210 +/- .010 (n = 10) to a peak activity of .310 +/- .027 U/mg microsomal protein at 5 h (n = 12). G-6-Pase also increased in the rough endoplasmic reticulum (RER) reaching .242 +/- .023 U/mg protein at 4h (n = 14), but then declining to .209 +/- .019 U/mg protein at 6 h (n = 11). The changes in G-6-Pase were accompanied by alterations in the ratio of microsomal cholesterol to phospholipid, which decreased by 50% (p less than .002) in both RER and SER fractions signifying changes in membrane lipid environment. Ultrastructural analysis revealed a marked reduction in hepatic glycogen and conspicuous presence of elements of the SER in regions of the cytoplasm where glycogen was or presumably had been located.     

Effects of fasting and cortisol administration on carbohydrate metabolism in Xenopus laevis Daudin.

The concentrations of most intermediates of glycolysis and gluconeogenesis were measured in livers from juvenile and adult Xenopus laevis. Comparison of the mass action ratios of the enzymes of these pathways with their apparent equilibrium constants suggests that flow through the pathways is most likely to be regulated at the hexokinase/glucose-6-phosphatase (G-6-Pase) or the pyruvate kinase/pyruvate carboxylase + phosphoenolpyruvate carboxykinase (PEPCK) steps. Injection of 10 mg of cortisol/kg daily for 7 days or fasting for up to 130 days had no effect upon the mass action ratio of any enzyme. In juvenile X. laevis fasted for 50 days, liver weight, blood glucose, and liver glycogen all decreased. Fructose-1,6-diphosphatase (FDPase) activity increased slightly while G-6-Pase activity decreased. Injection of cortisol (25 mg/kg daily for 7 days) was followed by increases in liver glycogen and blood glucose and in the rate of urea excretion. G-6-Pase activity rose above that in fed toads. These results are consistent with cortisol causing an increase in glucose synthesis, regulated at G-6-Pase. In adult X. laevis fasted for 60 days, hepatic alanine aminotransferase (AAT) activity fell by half but there were no changes in the activity of other enzymes or in the levels of glycolytic intermediates. After 130 days fasting, plasma free fatty acid and urea levels were reduced but there were no changes in plasma glucose or amino acid concentration or in liver or muscle glycogen content. The activities of G-6-Pase, FDPase, AAT, and carbamyl phosphate synthetase were reduced but glutamate dehydrogenase and total PEPCK were unchanged. In fed adult toads, injection of cortisol (10 mg/kg daily for 7 days) had no effect on any of the parameters measured while in toads fasted for 60 days, the only changes following injection of cortisol were increases in the concentration of urea in the plasma and in its rate of excretion, together with an increase in the activity of hepatic PEPCK. The results suggest that the effects of fasting and cortisol injection vary with the age of the animals. Considerable variability in the results, probably attributable to the low metabolic rate of the toads and seasonal effects, may well be masking the effects of administration of cortisol.     

The role of insulin and glucocorticoids in the regulation of hepatic glycogen metabolism: effect of fasting, refeeding, and adrenalectomy

The roles of insulin, adrenal corticol hormones, and nutritional factors in the regulation of hepatic glycogen metabolism were investigated by means of fasting and refeeding normal and adrenalectomized (ADX) rats. More specifically, the hypothesis in question in this study is that certain hepatic phosphoprotein phosphatases are targets of insulin action in liver. In anesthetized rats, the hepatic glycogen concentration and the activities of hepatic glycogen synthase, glycogen synthase phosphatase, glycogen phosphorylase, and phosphorylase phosphatase were correlated with peripheral plasma glucose and immunoreactive insulin levels. Hepatic phosphatase activities were measured in (soluble) the high speed supernatant and smooth endoplasmic reticulum (SER). Fasting resulted in expected diminutions in circulating glucose and insulin levels and loss of hepatic glycogen. These changes were greater in ADX rats. The percentage of hepatic glycogen synthase in the active or I form increased with fasting in normal rats, but did not change in ADX rats. Hepatic synthase phosphatase activities were decreased in SER by fasting in both normal and ADX rats, but to a much greater extent in the latter; soluble synthase phosphatase was much less affected by fasting. The percentage of phosphorylase in the active or a form was significantly decreased in normal, but not ADX, rats. Phosphorylase phosphatase activities were not significantly changed by fasting in any of the subcellular fractions in normal liver, but were increased in the hepatic SER of ADX rats. Refeeding fasted rats for 2 and 6 h resulted in increased hepatic glycogen, activation of glycogen synthase, and increased circulating levels of both insulin and glucose. Refeeding also caused increases in SER-associated synthase phosphatase activity in ADX animals. SER phosphorylase phosphatase activities were significantly increased by refeeding in normal rats, but were decreased in ADX rats. Regression analysis of the data suggested statistically significant positive correlations between insulin levels and SER synthase phosphatase activity in ADX animals, on the one hand, and SER synthase phosphatase and the percentage of synthase in the I form, on the other. No statistically significant correlation between insulin levels and phosphorylase phosphatase activities could be demonstrated. These results are compatible with the hypothesis that glycogen synthase phosphatase activity in liver, especially that associated with SER, is subject to physiological regulation by circulating levels of insulin. In contrast, phosphorylase phosphatase activity seems to be much less influenced by changes in the circulating insulin level. The results are compatible with the proposition that SER-associated phosphoprotein phosphatases are physiologically relevant in the regulation of hepatic glycogen metabolism.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of sub-total gastro-intestinal pancreatectomy of the rat foetus.

Sub-total pancreatectomy in utero was performed in 18-day-old rat foetuses. Pancreatectomized, sham-operated and control foetuses were collected 3 days later and body weight, glucose and insulin levels in blood, and glycogen content and glucose-6-phosphatase (G-6-Pase) activity of the liver were determined. Pancreatectomized foetuses showed only very small pancreatic remnants (less than or equal to 1 mg) and accordingly their insulin levels were much lower (four to five times) than those of sham-operated or control foetuses; their blood glucose levels were slightly increased and liver glycogen content and G-6-Pase activity were slightly reduced; their body weights were also reduced. These results are discussed in relation to other relevant data in the literature. They afford direct experimental evidence of the endogenous origin of insulin in the foetal blood. It is suggested that during the last days of intra-uterine life insulin merely completes the action of the glucocorticoids on glycogen storage in rat foetal liver and probably contributes to foetal body growth. Its relative ineffectiveness on the foetal blood glucose level is not explained. As pancreatectomized foetuses develop sub-normal liver G-6-Pase activity, glucagon is probably not responsible for the increase in this activity occurring during normal development before birth.     

Mechanisms by which bis(maltolato)oxovanadium(IV) normalizes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression in streptozotocin-diabetic rats in vivo

Vanadium treatment normalizes plasma glucose levels in streptozotocin-diabetic rats in vivo, but the mechanism(s) involved are still unclear. Here, we tested the hypothesis that the in vivo effects of vanadium are mediated by changes in gluconeogenesis. Diabetic rats were treated with bis(maltolato)oxovanadium(IV) (BMOV) in the drinking water (0.75-1 mg/ml, 4 wk) or, for comparison, with insulin implants (4 U/d) for the final week of study. As with insulin, BMOV lowered plasma glucose and normalized phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase) mRNA in the liver and kidney of diabetic rats. To determine the importance of reducing hyperglycemia per se, diabetic rats were treated either with a single ED(50) dose of BMOV (0.1 mmol/kg, ip) or with phlorizin (900 mg/kg.d, 5 d). BMOV rapidly restored PEPCK and G-6-Pase mRNA and normalized plasma glucose in responsive (50%) diabetic rats but had no effect on the nonresponsive hyperglycemic rats. Phlorizin corrected plasma glucose but had no effect on PEPCK mRNA and only partially normalized G-6-Pase mRNA. In conclusion, 1) BMOV inhibits PEPCK mRNA expression and activity by rapid mechanisms that are not reproduced simply by correction of hyperglycemia; and 2) BMOV inhibits G-6-Pase expression by complex mechanisms that depend, in part, on correction of hyperglycemia.     

Morphological and biochemical observations on hepatic glycogen metabolism in mice on a controlled feeding schedule

Changes in hepatocyte morphology were correlated with chemically measured liver glycogen, blood glucose, and plasma insulin levels in control-fed mice (6 hr fed, 18 hr fasted) sacrificed at various time intervals after initiation of a 6-hr meal. At initiation of feeding hepatic glycogen was low (0.05%) but deposition proceeded rapidly, reaching a maximum of 6.99±0.13% by the sixth hour. Glycogen was depleted during the subsequent fasting period, reaching the prefeeding levels by 24 hr. A relative hyperglycemia (140–192 mg/100 ml) predominated during all stages of glycogen deposition and depletion until the 21 st hour. Plasma insulin levels were maximum during feeding (63±7 U/ml, 3 hr) with mild hyperinsulinemia (insulin>16 U/ml) occurring during glycogen depletion (9–21 hr). Histochemical determinations (PAS) showed lobular patterns of hepatic glycogen which correlated with chemically measured glycogen levels. Six hours after initiation of feeding, periportal cells showed intensely stained masses of glycogen while centrilobular cells showed relatively diffuse staining. At 24 hours after initiation of feeding (18 hr of fasting), no significant staining was observed in the hepatocytes. Ultrastructurally, during all stages of glycogen deposition and depletion, centrilobular cells were characterized by the presence of dispersed glycogen particles with elements of smooth endoplasmic reticulum (SER) between the particles, while periportal cells showed dense glycogen deposits with SER restricted to the periphery of the glycogen masses.Supported by grants AM-06013, AM-27097 and in part by The University of Virginia Diabetes Research Training Center (AM-22125) from USPHS.     

Gluconeogenesis in non-obese diabetic (NOD) mice: in vivo effects of vandadate treatment on hepatic glucose-6-phoshatase and phosphoenolpyruvate carboxykinase

The contribution of gluconeogenesis to hyperglycemia in non-obese diabetic (NOD) mice has been investigated using oral vanadate administration. Vanadate compounds have been shown to mimic many actions of insulin; however, the exact mechanism is poorly understood. The aims of the present study were (1) to elucidate vanadate's action in vivo, and to assess the possibility that its glucose-reducing effect is dependent on the presence of a minimal concentration of insulin; and (2) to evaluate the effects of vanadate administration on the key hepatic gluconeogenesis enzymes, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK), as well as glucose-6-phosphate dehydrogenase (G-6-PDH). Vanadate caused a significant reduction in blood glucose but failed to normalize it, despite effective serum vanadate concentrations (26.2 +/- 1.6 micromol/L). Two weeks after initiation of treatment, blood glucose levels were 26.0 +/- 1.8, 21.7 +/- 3.0, 16.0 +/- 1.6, and 14.3 +/- 2.3 mmol/L in the control (C), insulin (I), vanadate (V), and combined vanadate and insulin (V + I) groups, respectively (P < .001). G-6-Pase activity was significantly reduced by vanadate (622 +/- 134 v365 +/- 83 nmol/min/mg protein in C vV, P < .05). PEPCK activity was also significantly reduced (844 +/- 370, 623 +/- 36, 337 +/- 43, and 317 +/- 75 nmol/min/mg in the C, I, V, and V + I groups, respectively, P < .001). No significant differences in the hepatic glycogen stores and G-6-PDH activity were noted between treatment groups. Our study suggests that the inhibition of hepatic G-6-Pase and PEPCK activity by vanadate plays an important role in reducing blood glucose levels in NOD mice.     

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.     

肝脏中糖代谢关键酶作为糖尿病药物靶点的研究进展

葡萄糖激酶、葡萄糖-6-磷酸酶、磷酸烯醇式丙酮酸羧激酶、果糖-1,6-二磷酸酶和糖原磷酸化酶是肝脏中精代谢的关键酶.糖尿病的一个特征就是葡萄糖激酶活性下降和葡萄糖-6-磷酸酶、磷酸烯醇式丙酮酸羧激酶、果糖-1,6-二磷酸酶和糖原磷酸化酶活性的升高.最近的研究都致力于通过激活或抑制这儿种酶来降低血糖并使胰岛素正常分泌.因此这5种酶将有可能是治疗糖尿病的全新靶点.     

Effect of feeding, fasting, and diabetes on liver glycogen synthase activity, protein, and mRNA in rats

Summary Hepatic glycogen synthase activity is increased in diabetic animals. However, the relationship between enzymic activity, enzyme protein mass, and mRNA abundance has not been well characterized. In the present study, these relationships were determined in 3- and 8-day diabetic, fed and fasted rats. The results were compared to data obtained in normal fed and fasted animals. In normal rats, total synthase specific activity and protein mass were similar in the fed and fasted state. However, in fed animals, the synthase mRNA abundance was increased 1.7-fold. In 3-day diabetic rats, total synthase specific activity was increased approximately 29 % compared to normal controls. It was unaffected by feeding and fasting and was associated with an approximate 15 % increase in enzyme mass. Synthase mRNA was increased 1.8 and 2.6-fold in fasted and fed animals, respectively. In 8-day diabetic rats, total synthase specific activity was increased more than 2-fold compared to controls. However, the enzyme protein mass was decreased by approximately 20 %. The mRNA abundance in 8-day diabetic fasted rats was only 30 % of controls, while in fed rats it was increased by 40 %. These data indicate that feeding and fasting have a major effect on synthase mRNA abundance which is independent of synthase activity, or protein mass, or both, in normal and diabetic animals. Total synthase specific activity increased with duration of diabetes. This was associated with only a modest change in protein mass. Thus, diabetes induces an increase in synthase catalytic efficacy. The specific activity of phosphorylase is decreased in diabetic rats. [Diabetologia (1997) 40: 758–763] Received: 18 October 1996 and in revised form: 23 January 1997     

Stable overexpression of the glucose-6-phosphatase catalytic subunit attenuates glucose sensitivity of insulin secretion from a mouse pancreatic beta-cell line

Glucose-6-phosphatase (G-6-Pase) hydrolyzes glucose-6-phosphate to glucose, reciprocal with the so-called glucose sensor, glucokinase, in pancreatic beta cells. To study the role of G-6-Pase in glucose-stimulated insulin secretion from beta cells, we have introduced rat G-6-Pase catalytic subunit cDNA and have established permanent clones with 3-, 7- and 24-fold G-6-Pase activity of the mouse beta-cell line, MIN6. In these clones, glucose usage and ATP production in the presence of 5.5 or 25 mM glucose were reduced, and glucose-stimulated insulin secretion was decreased in proportion to the increased G-6-Pase activity. In addition, insulin secretory capacity in response to d-fructose and pyruvate was unchanged; however, 25 mM glucose-stimulated insulin secretion and intracellular calcium response were completely inhibited. In the clone with 24-fold G-6-Pase activity, changes in intracellular NAD(P)H autofluorescence in response to 25 mM glucose were reduced, but the changes with 20 mM fructose and 20 mM pyruvate were not altered. Stable overexpression of G-6-Pase in beta cells resulted in attenuation of the overall glucose-stimulated metabolic responses corresponding to the degree of overexpression. This particular experimental manipulation shows that the possibility exists of modulating glucose-stimulated insulin release by thoroughly altering glucose cycling at the glucokinase/G-6-Pase step.     

Autoregulation by glucose of hepatic glucose balance: Permissive effect of insulin

To study the pathways involved and the effect of insulin on the autoregulation of hepatic glucose balance, isolated hepatocytes from normal, diabetic and treated diabetic rats were incubated with varying concentrations of glucose (100–400 mg/dl) with and without pyruvate (10 mM). Net glucose production or utilization was calculated from the change in media glucose concentrations before and after incubation, net glycogenolysis by the change in tissue glycogen levels before and after incubation, net glycolysis by following the incorporation of glucose-C¹⁴ into lactate C¹⁴ and gluconeogenesis by the difference in glucose production in the presence and absence of pyruvate. Hepatocytes from control and insulin-treated animals manifested autoregulation of glucose balance. Glucose modulated the glycogen and glycolytic pathways but did not affect gluconeogenesis. In hepatocytes from diabetic rats, there was no autoregulation, tissue glycogen was unmeasurable both before and after incubation, glycolysis was markedly curtailed and gluconeogenesis was increased. It may be concluded that (1) glucose autoregulates its own production or utilization by modulating the glycogen and glycolytic pathways, (2) autoregulation is lost in severe diabetes leading to fasting hyperglycemia, and (3) insulin has a permissive effect on the autoregulation of glucose balance by maintaining the rate-limiting enzymes, glycogen synthase and glucokinase, so that glucose can exert its effect on these pathways.     

Studies on glucose-6-phosphatase, fructose-1,6-diphosphatase activity, glycogen distribution and endoplasmic reticulum changes during hexachlorocyclohexane induced hepatocarcinogenesis in pure inbred Swiss mice

Inbred Swiss mice were fed hexachlorocyclohexane (BHC) at 500 ppm dose level in diet for 2, 4, 6 and 8 months. Later BHC was discontinued for 4 months and subsequently the animals were refed BHC for 1 month. Glucose-6-phosphatase (G6Pase) and fructose-1,6-diphosphatase (FDPase) activity was studied at different time intervals accompanied with changes in glycogen distribution and endoplasmic reticulum (ER) proliferation in hepatocytes. G6Pase and FDPase showed a decline in activities on BHC feeding. The activities of these enzymes showed recovery on BHC discontinuation. The changes were progressive with duration of exposure. After 6 months exposure the biochemical changes became more resistant to recovery. Maximal changes occurred in 8 month-exposure and the changes were irreversible. Glycogen accumulation and depletion followed a definite pattern. After two months of BHC feeding, increase in parenchymal glycogen storage zones was observed. In the later stage of hepatocarcinogenesis and specially in tumors, glycogen was depleted considerably. Smooth endoplasmic reticulum (SER) proliferation was recorded around the 3rd and 4th month. The correlation between glycogen accumulation, SER proliferation, G6Pase and FDPase activity is discussed.     

White rice vinegar improves pancreatic beta-cell function and fatty liver in streptozotocin-induced diabetic rats

Vinegar is a traditional remedy for ailments including diabetes. This study was conducted to investigate the beneficial effects of vinegar in streptozotocin (STZ)-induced diabetic rats. STZ-induced diabetic rats were orally administered with white rice vinegar (WRV, 2?ml/kg body weight per day, n?=?6) or with an equal volume of drinking water (n?=?6) for 1?month. Fasting and random blood glucose was measured from tail vein samples. Body weight, 24-h food and water intake were monitored 1?week and 1?month after STZ injection. Fasting serum insulin concentrations were assayed using ELISA. Pancreatic beta- and alpha-cell proportions were measured using immunofluorescence microscopy. Periodic acid Schiff staining was performed to access glycogen contents and histological changes in liver tissues. Compared with control animals, the WRV-treated rats had less weight loss, lower fasting and random blood glucose, higher fasting serum insulin and higher beta-cell proportion. The WRV treatment also improved fatty changes and glycogen storages in the liver of STZ rats. Oral intake of WRV improved fasting hyperglycemia and body weight loss through attenuating insulin deficiency, pancreatic beta-cell deficit, and hepatic glycogen depletion and fatty changes in STZ-induced diabetic rats.     

Pyruvate kinase activity in isolated rat hepatocytes during a feeding cycle and during fasting

The short-term regulation of pyruvate kinase in rat hepatocytes was studied during a feeding cycle or progressive fasting. In fed and fasted rats, the activity ratio of pyruvate kinase (V0.5-mMPEP/Vmax) in crude extracts was directly correlated with the concentration of glucose 1,6-bisphosphate++ in hepatocytes. Precipitation of the enzyme from homogenates with ammonium sulphate, which removes fructose 1,6-bisphosphate, induced in both groups of animals a low activity ratio of pyruvate kinase which remained unchanged during the whole experiment. These results show that in absence of added glucagon in hepatocytes, the activity of pyruvate kinase is mainly controlled by the intracellular level of fructose 1,6-bisphosphate. Addition of glucagon to hepatocytes from fed or fasted rats inactivated pyruvate kinase and decreased the concentration of fructose 1,6-bisphosphate in cells. However, in crude extracts both the activity ratio of pyruvate kinase and fructose 1,6-biphosphate levels were higher in fed rats than in starved rats. These findings suggest that glucagon-induced inactivation of pyruvate kinase also depends upon the concentration of fructose 1,6-biphosphate in hepatocytes.     

Enzymehistochemical studies on the pancreatic islets in mice injected with anti-insulin serum

Summary Islets of Langerhans in white mice with a diabetic syndrome after single and repeated injections of anti-insulin serum were studied by enzymehistochemical methods and compared with controls. Changes in activity of some enzymes were noted in the islet area in accordance with the histopathological findings of severe degranulation and hypersecretory changes of the beta cells. — The -glycerophosphate oxidase (GPOX) showed the most sensitive and severe decrease of activity whereas the acid phosphatase (ACPase) was only slightly decreased. Compared with the enzyme activity in normal mice, no significant changes of glucose-6-phosphate dehydrogenase (G-6-PD) could be observed. The only enzyme showing an increased activity was the glucose-6-phosphatase (G-6-Pase). The studies of the cytochrome oxidase (CCOX), lactic dehydrogenase (LDH), succinic dehydrogenase (SDH), acid phosphatase (ACPase), alkaline phosphatase (APase) and adenosine triphosphatase (ATPase) revealed no alterations of the enzyme patterns under these experimental conditions. The findings are discussed with regard to the hypothetical relations between the pentose phosphate shunt (indicator enzyme: G-6-PD), the glycerophosphate cycle (indicator enzyme: GPOX) and the phosphorylation of glucose (indicator enzyme: G-6-Pase) to insulin synthesis and release.Supported by Deutsche Forschungsgemeinschaft, Bad Godesberg, Germany.     

Increased insulin action in cultured hepatocytes from rats with diabetes induced by neonatal streptozotocin

Previous studies have shown that Wistar rats injected at birth (n0) with STZ (n0-STZ) develop as adults a noninsulin-dependent diabetic state characterized by a lack of insulin response to glucose in vivo, a mild basal hyperglycemia, and an impaired glucose tolerance. Our former in vivo studies using the insulin-glucose clamp technique revealed an increased insulin action upon hepatic glucose production in these animals. We have now cultured hepatocytes from these mildly diabetic rats in parallel with hepatocytes from control rats, to examine more closely basal and insulin-regulated glucose production and glucose incorporation into glycogen. In addition, we extended our investigation to other hepatic functions such as lipid synthesis and amino acid transport, which could not be studied in vivo. Although glucose production from glycogenolysis or gluconeogenesis in absence or presence of glucagon was identical in the two cell populations, glucagon-stimulated glycogenolysis was more sensitive to insulin action in diabetic hepatocytes. Similarly, insulin action on glucose incorporation into glycogen, lipogenesis, and amino acid transport were enhanced in diabetic hepatocytes. The hormone effect was manifested by an increase in the sensitivity and/or in the responsiveness, reflecting the multiplicity of the pathways whereby the insulin signal is transduced through the insulin receptor to multiple postreceptor sites. To gain insight into the possible mechanism of these disturbances, we evaluated the initial insulin receptor interaction and the kinase activity of the receptor beta-subunit. In accordance with our previous study on intact livers, we found no alteration in either of these parameters in n0-STZ rat hepatocytes. Thus, the present study clearly demonstrates that these diabetic rats exhibit a postreceptor hyperresponsiveness to insulin at the cellular level. It strengthens the notion that a beta-cell deficiency with glucose intolerance does not necessarily lead to a hepatic insulin resistance.     

Expression of the low Km GLUT-1 glucose transporter is turned on in perivenous hepatocytes of insulin-deficient diabetic rats

In normal fed rats the low Km glucose transporter GLUT-1 is expressed only in one row of hepatocytes immediately surrounding a terminal hepatic venule, while the high Km GLUT-2 is expressed in every hepatocyte. Previously, we showed that additional perivenous hepatocytes express GLUT-1 in fasting animals. In diabetes, as in starvation, the liver functions to release glucose into the circulation, but unlike starvation, circulating extracellular glucose is high in diabetes. By immunofluorescence and Western blotting we studied whether glucose or insulin is the primary extracellular signal for inducing GLUT-1 expression in hepatocytes. We observed that streptozocin-induced diabetes causes induction of GLUT-1 expression in the plasma membrane of hepatocytes within four cell rows of a terminal hepatic venule; GLUT-2 expression is unaltered. Chronic insulin treatment of diabetic rats reduces the number of rows of hepatocytes expressing GLUT-1 from approximately four to approximately two. In contrast, chronic insulin infusion into nondiabetic rats does not affect the number of hepatocytes expressing GLUT-1. Thus, both fasting and diabetes induce GLUT-1 expression in specific hepatocytes that normally do not express this gene. This induction correlates with low insulin levels in the blood, and not with circulating glucose levels.     

Ultrastructural heterogeneity of glycogen in human liver

The ultrastructure of the lobular patterns of hepatic glycogen was studied in four human liver surgical biopsies, obtained 14 h after the last meal. In centrilobular hepatocytes, glycogen particles were regularly dispersed with elements of SER between the particles. Periportal hepatocytes showed dense glycogen deposits with SER restricted to the periphery of the glycogen masses. The ultrastructural heterogeneity of the lobular distribution of glycogen in our human cases is similar to that found in control-fed rats and mice. The metabolic signification of these findings is as yet unclear.