The effects of insulin replacement and withdrawal on hepatic ultrastructure and biochemistry

This study examines the early hepatic biochemical and ultrastructural responses to insulin replacement in streptozotocin-diabetic rats and insulin withdrawal from insulin-maintained diabetic rats. Insulin administration rapidly lowered plasma glucose and the elevated glucose-6-phosphatase (G-6-Pase) specific activity of the diabetic rats. However, hepatic glycogen did not increase until after 3 hr of insulin treatment. Hepatic ultrastructure responded to insulin replacement after the decline in glucose and G-6-Pase. This was seen in periportal hepatocytes as a reduction in the close association between smooth endoplasmic reticulum (SER) and glycogen particles in the diabetic animals. The treated rats showed hepatic SER restricted to the periphery of glycogen masses, as is characteristic of these cells from normal rats, in many cells by 6 hr and all cells by 18 hr. Insulin withdrawal from insulin-treated diabetic rats elicited nearly a total reversal of the above events. Plasma insulin declined to a value half that of the normal rats by 6 hr after withdrawal; concurrently, plasma glucose rose sharply to hyperglycemic values as hepatic glycogen content dropped. Following the rise in plasma glucose and fall in glycogen content, G-6-Pase specific activity increased and by 16 hr reached the high values characteristic of the diabetic animal. Hepatic ultrastructure was also changed as evidenced by an intrusion of elements of the SER into the dense glycogen masses; the result was dispersed glycogen closely associated with SER as seen in the diabetic animal. It is concluded that the hepatic response to insulin replacement in diabetic animals and diabetic onset in insulin-withdrawn animals is rapid and occurs through defined stages.     

Activation of liver G-6-Pase in response to insulin-induced hypoglycemia or epinephrine infusion in the rat

This study was conducted to test the hypothesis of the activation of glucose-6-phosphatase (G-6-Pase) in situations where the liver is supposed to sustain high glucose supply, such as during the counterregulatory response to hypoglycemia. Hypoglycemia was induced by insulin infusion in anesthetized rats. Despite hyperinsulinemia, endogenous glucose production (EGP), assessed by [3-(3)H]glucose tracer dilution, was paradoxically not suppressed in hypoglycemic rats. G-6-Pase activity, assayed in a freeze-clamped liver lobe, was increased by 30% in hypoglycemia (P < 0.01 vs. saline-infused controls). Infusion of epinephrine (1 microg x kg(-1) x min(-1)) in normal rats induced a dramatic 80% increase in EGP and a 60% increase in G-6-Pase activity. In contrast, infusion of dexamethasone had no effect on these parameters. Similar insulin-induced hypoglycemia experiments performed in adrenalectomized rats did not induce any stimulation of G-6-Pase. Infusion of epinephrine in adrenalectomized rats restored a stimulation of G-6-Pase similar to that triggered by hypoglycemia in normal rats. These results strongly suggest that specific activatory mechanisms of G-6-Pase take place and contribute to EGP in situations where the latter is supposed to be sustained.     

Enzymatic characterization of the pancreatic islet-specific glucose-6-phosphatase-related protein (IGRP)

Glucose is the main physiological stimulus for insulin biosynthesis and secretion by pancreatic beta-cells. Glucose-6-phosphatase (G-6-Pase) catalyzes the dephosphorylation of glucose-6-phosphate to glucose, an opposite process to glucose utilization. G-6-Pase activity in pancreatic islets could therefore be an important factor in the control of glucose metabolism and, consequently, of glucose-dependent insulin secretion. While G-6-Pase activity has been shown to be present in pancreatic islets, the gene responsible for this activity has not been conclusively identified. A homolog of liver glucose-6-phosphatase (LG-6-Pase) specifically expressed in islets was described earlier; however, the authors could not demonstrate enzymatic activity for this protein. Here we present evidence that the previously identified islet-specific glucose-6-phosphatase-related protein (IGRP) is indeed the major islet glucose-6-phosphatase. IGRP overexpressed in insect cells possesses enzymatic activity comparable to the previously described G-6-Pase activity in islets. The K(m) and V(max) values determined using glucose-6-phosphate as the substrate were 0.45 mm and 32 nmol/mg/min by malachite green assay, and 0.29 mm and 77 nmol/mg/min by glucose oxidase/peroxidase coupling assay, respectively. High-throughput screening of a small molecule library led to the identification of an active compound that specifically inhibits IGRP enzymatic activity. Interestingly, this inhibitor did not affect LG-6-Pase activity, while conversely LG-6-Pase inhibitors did not affect IGRP activity. These data demonstrate that IGRP is likely the authentic islet-specific glucose-6-phosphatase catalytic subunit, and selective inhibitors to this molecule can be obtained. IGRP inhibitors may be an attractive new approach for the treatment of insulin secretion defects in type 2 diabetes.     

The Protective Effects of Eugenol on Carbon Tetrachloride induced Hepatotoxicity in Rats

Our earlier studies in vitro have shown that eugenol inhibits liver microsomal monooxygenase activities and carbon tetrachloride (CCl₄)-induced lipid peroxidation (Free Rad. Res. 20,253-266,1994). The objective of the present investigation was to study the in vivo protective effect of eugenol against CCI₄ toxicity. Eugenol (5 or 25 mg/kg body wt) given orally for 3 consecutive days did not alter the levels of serum glutamic oxalacetic transaminase (SGOTJ, microsomal enzymes such as cytochrome P450 reductase, glucose-6-phosphatase (G-6-Pase) xenobiotic-metabolizing enzymes (aminopyrine-N-demethylase, N-nitrosodimethylamine-demethylase and ethoxyresorufin-O-deethylase) and liver histology. Doses of eugenol (5 or 25 mg/kg) administered intragastrically to each rat on three consecutive days i.e. 48 hr, 24 hr and 30 min before a single oral dose of CCU (2.5 ml/kg body wt) prevented the rise in SGOT level without appreciable improvement in morphological changes in liver. Eugenol pretreatment also did not influence the decrease in microsomal cytochrome P₄₅₀ content, G-6-Pase and xenobiotic-metabolizing enzymes brought about by CCI₄. Since eugenol is metabolized and cleared rapidly from the body, the dose schedule was modified in another experiment. Eugenol (0.2,1.0,5.0 or 25 mg/kg) when given thrice orally i.e. prior to (-1 hr) along with (0 hr) and after (+ 3 hr) the i.p. administration of CCI₄ (0.4 ml/kg) prevented significantly the rise in SGOT activity as well as liver necrosis. The protective effect was more evident at 1 mg and 5 mg eugenol doses. However, the decrease in microsomal G-6-Pase activity by CCI₄ treatment was not prevented by eugenol suggesting that the damage to endoplasmic reticulum is not protected. The protective effect of eugenol against CC1₄ induced hepatotoxicity is more evident when it is given concurrently or soon after rather than much before CCU treatment.     

Kinetic analysis of glucose-6-phosphatase: an investigative approach to carbohydrate metabolism and kinetics

The enzyme glucose-6-phosphatase (G-6-Pase) catalyzes the hydrolysis of glucose-6-phosphate (G-6-P) to glucose. This is one of the key steps in gluconeogenesis and is critically important in maintaining stable blood glucose levels in most mammals. G-6-Pase is primarily found in the endoplasmic reticulum (ER) of hepatocytes and can easily be studied using isolated microsomes prepared from liver ER. A three-part undergraduate laboratory exercise uses rat liver microsomes to focus on the enzymatic analysis of G-6-Pase. The assessment of G-6-Pase activity is conducted using a stopped assay protocol combined with a colorimetric determination of inorganic phosphate (P_i) levels. The laboratory exercise was designed to carry out an independent inhibition investigation using orthovanadate, a competitive inhibitor of G-6-Pase with potential clinical importance. The format of the three-part investigation provides a useful mechanism for demonstrating enzyme kinetics and competitive inhibition using an enzyme that is important for carbohydrate metabolism and glycogen storage disease.     

Elevated insulin/glucagon ratios and decreased cyclic AMP levels accompany the glycogen and triglyceride storage syndrome in the hypothyroid chick

The role of endogenous glucagon and insulin on the hepatic glycogen and triglyceride storage syndrome in propylthiouracil (PTU)-induced hypothyroidism was investigated in the chick. PTU feeding in the diet resulted in a progressive increase in liver glycogen concentration associated with a concomitant decrease in hepatic glucose-6-phosphatase (G-6-Pase) activity. Plasma glucagon level was significantly decreased and insulin significantly increased after two days of PTU administration. These enzyme and hormone changes were associated with a significant increase in hepatic glucose-6-phosphate (G-6-P) and a decrease in cyclic AMP levels. Although our results do not directly prove, the data does suggest that the hepatic glycogen storage syndrome observed in the PTU-induced hypothyroidism in the chick is mediated through changes in pancreatic glucagon and insulin secretion. The extent of glycogen accumulation was inversely related to G-6-Pase which is a rate limiting glycogenolytic enzyme. A significant increase in the plasma insulin/glucagon ratio, along with a significant decrease in the hepatic cyclic AMP concentration, could most likely also account for the excessive hepatic triglyceride accumulation in the PTU-treated chicks.     

Diets enriched in sucrose or fat increase gluconeogenesis and G-6-Pase but not basal glucose production in rats

High-fat (HFD) and high-sucrose diets (HSD) reduce insulin suppression of glucose production in vivo, increase the capacity for gluconeogenesis in vitro, and increase glucose-6-phosphatase (G-6-Pase) activity in whole cell homogenates. The present study examined the effects of HSD and HFD on in vivo gluconeogenesis, the catalytic and glucose-6-phosphate translocase subunits of G-6-Pase, glucokinase (GK) translocation, and glucose cycling. Rats were fed a high-starch control diet (STD; 68% cornstarch), HSD (68% sucrose), or HFD (45% fat) for 7-13 days. The ratio of 3H in C6:C2 of glucose after 3H2O injection into 6- to 8-h-fasted rats was significantly increased in HSD (0.68 +/- 0.07) and HFD (0.71 +/- 0.08) vs. STD (0.40 +/- 0.10). G-6-Pase activity was significantly higher in HSD and HFD vs. STD in both intact and disrupted liver microsomes. HSD and HFD significantly increased the amount of the p36 catalytic subunit protein, whereas the p46 glucose-6-phosphate translocase protein was increased in HSD only. Despite increased nonglycerol gluconeogenesis and increased G-6-Pase, basal glucose and insulin levels as well as glucose production were not significantly different among groups. Hepatocyte cell suspensions were used to ascertain whether diet-induced adaptations in glucose phosphorylation and GK might serve to compensate for upregulation of G-6-Pase. Tracer-estimated glucose phosphorylation and glucose cycling (glucose <--> glucose 6-phosphate) were significantly higher in cells isolated from HSD only. After incubation with either 5 or 20 mM glucose and no insulin, GK activity (nmol. mg protein(-1). min(-1)) in digitonin-treated eluates (translocated GK) was significantly higher in HSD (32 +/- 4 and 146 +/- 6) vs. HFD (4 +/- 1 and 83 +/- 10) and STD (9 +/- 2 and 87 +/- 9). Thus short-term, chronic exposure to HSD and HFD increase in vivo gluconeogenesis and the G-6-Pase catalytic subunit. Exposure to HSD diet also leads to adaptations in glucose phosphorylation and GK translocation.     

Cytochemical changes in hepatocytes of rats with endotoxemia or sepsis: localization of fibronectin, calcium, and enzymes

Bacterial lipopolysaccharide (LPS) is known to be implicated in the pathogenesis of endotoxemia and septic shock. The liver is the first vital organ to exhibit pathological alterations in shock. The present studies include immunoelectron microscopic localization of tissue fibronectin and cytochemical localization of calcium and enzymes in hepatocytes of animals with LPS-induced endotoxemia or cecal ligation-induced septic shock. The results showed increased staining of fibronectin in the basal (perisinusoidal) surfaces and in the cisternae of rough endoplasmic reticulum and the Golgi complex of hepatocytes in rats with endotoxemia or septic shock. Intracellular calcium content was significantly increased in the LPS-treated or septic rats. Calcium pyroantimonate precipitate was deposited predominantly on the outer surfaces of the RER of hepatocytes. In addition, diminution or depletion of glycogen, reduction of catalase-containing peroxisomes, increase of G-6-Pase activity, and depletion of cytochrome c oxidase in many mitochondria were also observed in hepatocytes of experimental animals. The overall results suggest that LPS stimulates: (a) hepatic synthesis and secretion of fibronectin; (b) uptake of calcium by hepatocytes; and (c) G-6-Pase activity. LPS treatment also leads to reduced numbers of peroxisomes and depletion of cytochrome c oxidase.     

Morphometric analysis of hepatocytes from rats subjected to compound 48/80-induced anaphylactic shock

Morphometric and biochemical techniques were used to analyze hepatic glycogen, endoplasmic reticulum, and mitochondrial matrix granules in rats treated with compound 48/80 to induce an anaphylactic-like state of shock. Thirty minutes after insult there was a significant decrease in glycogen and mitochondrial matrix granules, an increase in rough endoplasmic reticulum (RER), and no change in smooth endoplasmic reticulum (SER). Less glycogen in experimental rats substantiated a previously described glycogenolytic response to compound 48/80. The decrease in matrix granules implies a loss and/or shift in intramitochondrial calcium as occurs in epinephrine-induced glycogenolysis in the rat. Since other glycogenolytic agents, e.g. glucagon, and starvation stimulate an increase in SER presumably from RER, the present morphological data suggest the increase in RER may precede proliferation of SER from RER.     

Endoplasmic reticulum associated glucose-6-phosphatase activity is developmentally regulated and enriched in microsomes of endo/mesoderm in sea urchins

Summary Glucose-6-phosphatase (G-6-Pase) activity was analyzed during early embryogenesis of the sea urchinS. purpuratus. This activity is detected in very low levels in unfertilized eggs and early embryos but is present at high levels in preparations of endoplasmic reticulum (microsomes) from gastrula stage embryos. The approximately eight-fold increase in the relative activity of G-6-Pase associated with the ER occurs abruptly during a 12 h interval at gastrulation, and thereafter remains at a level comparable to that found in mammalian liver microsomes. The enzyme activity associated with the ER of gastrula stage embryos was completely eliminated from the microsomal pellet when cell lysates were first treated with non-ionic detergent. Analysis of germlayer tissues from late stage pluteus embryos revealed that G-6-Pase activity was more highly enriched in microsomes of endo/mesoderm tissues as compared to microsomes from ectoderm. The increase in ER associated G-6-Pase activity during embryonic development and its enriched activity in the ER of endo/mesoderm, as well as the observation that the signal recognition particle becomes associated with the ER at gastrulation (Le Blanc and Infante 1989), opens the question that this cellular organelle may be differentiating during embryogenesis in sea urchins.     

Nuclear membranes from mammalian liver. VI. Glucose-6-phosphatase in rat liver, a cytochemical and biochemical study

Activities of glucose-6-phosphatase (G-6-Pase) and other phosphatases were determined in nuclei, nuclear membrane and microsomal fractions and subfractions, and condensed chromatin isolated from the liver of adult, newly born and prenatal rats. The purity of the fractions was controlled by electron microscopic morphometry and by measurement of various marker enzymes. The specific G-6-Pase activity of the nuclear membranes was found to be about 60% that of the microsomes. However, when calculated on the basis of the phospholipid content, all fractions had similar activities. Determinations of G-6-Pase enrichments and recoveries were also made. The correspondence of the hydrolysing activities of glucose-6-phosphate, mannose-6-phosphate, and inorganic pyrophosphate, together with various phosphotransferases, showed the same association of the G-6-Pase with these enzymes in the nuclear envelope as in the microsomal membranes. G-6-Pase was also demonstrated in the fractions by cytochemistry, and the activity was localized alongside the cisternal surfaces of both, inner and outer, nuclear membrane. ‘Free’ inner nuclear membrane fragments contained also G-6-Pase. No activity was observed at the nuclear pore complexes. Both, nuclear and microsomal membranes revealed a parallel rapid perinatal increase of G-6-Pase activity climaxing at 23 to 28 h after birth. Triton-X-100 treatment of isolated nuclei, which was found not to selectively release outer nuclear membranes, resulted in a great decrease of G-6-Pase activity as well as in losses of membrane phospholipids. The results clarify the divergence of earlier reports concerning the presence of G-6-Pase in the perinuclear cisterna and add biochemical evidence to the morphologically derived view of the nuclear envelope as being a special form of the ER system.     

Induction of control genes in intestinal gluconeogenesis is sequential during fasting and maximal in diabetes

We studied in rats the expression of genes involved in gluconeogenesis from glutamine and glycerol in the small intestine (SI) during fasting and diabetes. From Northern blot and enzymatic studies, we report that only phosphoenolpyruvate carboxykinase (PEPCK) activity is induced at 24 h of fasting, whereas glucose-6-phosphatase (G-6-Pase) activity is induced only from 48 h. Both genes then plateau, whereas glutaminase and glycerokinase strikingly rebound between 48 and 72 h. The two latter genes are fully expressed in streptozotocin-diabetic rats. From arteriovenous balance and isotopic techniques, we show that the SI does not release glucose at 24 h of fasting and that SI gluconeogenesis contributes to 35% of total glucose production in 72-h-fasted rats. The new findings are that 1) the SI can quantitatively account for up to one-third of glucose production in prolonged fasting; 2) the induction of PEPCK is not sufficient by itself to trigger SI gluconeogenesis; 3) G-6-Pase likely plays a crucial role in this process; and 4) glutaminase and glycerokinase may play a key potentiating role in the latest times of fasting and in diabetes.     

Chlorogenic acid reduces the plasma glucose peak in the oral glucose tolerance test: effects on hepatic glucose release and glycaemia

The effects of chlorogenic acid (CA) on hepatic glucose output, blood glucose levels and on glucose tolerance were analysed. Hepatic uptake of CA and its effects on hepatic catabolism of L-alanine and glucose-6-phosphatase (G-6-Pase) activity were also evaluated. CA (1 mM) inhibited about 40% of G-6-Pase activity (p < 0.05) in the microsomal fraction of hepatocytes, but no effect was observed on production of glucose from gluconeogenesis or on L-alanine catabolism, at various concentrations of CA (0.33, 0.5 and 1 mM), in liver perfusion experiments. Since there were indications of a lack of uptake of CA by the liver, it is possible that this compound did not reach sufficiently high intracellular levels to inhibit the target enzyme. Accordingly, intravenous administration of CA also failed to provoke a reduction in blood glucose levels. However, CA did promote a significant reduction (p < 0.05) in the plasma glucose peak at 10 and 15 min during the oral glucose tolerance test, probably by attenuating intestinal glucose absorption, suggesting a possible role for it as a glycaemic index lowering agent and highlighting it as a compound of interest for reducing the risk of developing type 2 diabetes.     

Effect of triiodo-L-thyronine treatment on Ca2+ sequestration in rat liver microsomes

T3 administration to rats exerts quite different effects on enzyme activities associated to liver microsomal membranes such as G-6-Pase, Mg ATPase and Ca2(+)-dependent ATPase: in fact G-6-Pase activity is significantly enhanced, Mg ATPase is not affected whereas Ca2(+)-dependent ATPase is drastically inhibited. The T3 induced decrease in Ca2(+)-dependent ATPase activity is associated with a net reduction (to about 50% with respect to controls) of the Ca2+ sequestration in liver microsomal vesicles. The enhanced level of inorganic phosphate in the endoplasmic reticulum due to the stimulation of G-6-Pase activity does not significantly affect the uptake of calcium in microsomal vesicles. The decreased Ca2(+)-dependent ATPase activity is associated to an enhanced level of the enzyme in the phosphorylated form (E-P). This suggests that in liver preparations from T3 treated rats the turnover of ATP and cleavage of E-P is reduced, thus resulting in the accumulation of the phosphorylated intermediate. The accumulation of E-P is in agreement with the inhibition of the calcium sequestration since the active transport of this cation in microsomal membranes requires the hydrolysis of the E-P complex.     

Glucose-6-phosphatase proteins of the endoplasmic reticulum (Review)

Summary

Hepatic glucose-6-phosphatase (G-6-Pase) catalyses the terminal step of hepatic glucose production and it plays a key role in the maintenance of blood glucose homeostasis. Hepatic G-6-Pase is an integral resident endoplasmic reticulum (ER) protein and it is part of a multicomponent system. Its active site is situated inside the lumen of the ER and transport proteins are needed to allow its substrates, glucose-6-phosphate (G-6-P) (and pyrophosphate), and its products, phosphate and glucose, to cross the ER membrane. In addition, a calcium-binding protein is also associated with the G-6-Pase enzyme. Recent immunological studies have shown that G-6-Pase (which has conventionally been thought to be present only in the gluconeogenic organs) is present in minor cell types in a variety of human tissues and that its distribution changes dramatically during human development. In all the tissues, enzymatic analysis, direct transport assays and/or immunological detection of the ER glucose and phosphate transport proteins have been used to demonstrate the presence and activity of the whole G-6-Pase system. The G-6-Pase protein is very hydrophobic and has proved difficult to purify to homogeneity. Four proteins of the system have now been isolated and polyclonal antibodies have been raised against them; two have also been cloned. The available sequences, together with topologicai studies, have given some information about both the topology of the proteins in the ER and the probable mechanisms by which the proteins are retained in the ER.     

Interference of antioxidants E/O of some free radical "scavengers" with the activity of glucose-6-phosphatase after administration of carbon tetrachloride]

G-6-Pase activity was investigated in the microsomal fraction from rat liver in the presence of carbon tetrachloride and/or propyl gallate (PG), reduced glutathione (GSH) and superoxide dismutase. Results obtained "in vitro" demonstrated that CCl4 induced a 60% inhibition of the microsomal enzyme activity. Moreover, a marked inhibition of G-6-Pase activity was found also when propyl gallate and reduced glutathione were added, at different concentrations, to incubation mixture. In addition, these drugs were unable to interfere with the dangerous effect exerted on the enzymatic activity by the haloalkane. Additional experiments carried out "in vivo" with propyl gallate produced evidence that intraperitoneal administration of the antioxidant was followed by a significant inhibition of G-6-Pase activity, while the damaging action of CCl4 was unaffected. Some possible explanations of these results are reported.     

Ultrastructural localization of intestinal glucose-6-phosphatase activity during the postnatal development of the mouse

The development of the endoplasmic reticulum (ER) and the ultrastructural localization of glucose-6-phosphatase activity have been studied in the proximal jejunum and distal ileum during the postnatal period. One day after birth, the amount and the repartition of ER in the jejunal enterocytes are similar to that observed in postweaning period. In the following days an extensive proliferation of SER is noted in the supranuclear zone of the absorbing cells. From day 7 till postweaning period a gradual decrease of the amount of SER is observed and after weaning, the ultrastructure of the enterocytes is similar to that in the adult mouse enterocytes. At all time, a positive reaction for G-6-Pase activity is observed in the cisternae of the endoplasmic reticulum and in the nuclear envelope. In the distal ileum, the SER is poorly developed one day after birth. During the first two weeks, the ER increases but no extensive proliferation of SER can be noted as in the jejunum. The G-6-Pase activity can be visualized in the rough and smooth endoplasmic reticulum as well as in the nuclear envelope. It appears that the proliferation of SER could be interpreted as the morphologic expression of an increased G-6-Pase activity.