Effects of metformin on glucose and glucagon regulated gluconeogenesis in cultured normal and diabetic hepatocytes

The effects of glucose and glucagon on the anti-gluconeogenic action of metformin were investigated in normal and diabetic hepatocytes. Glucose production from lactate was elevated by 88% in hepatocytes from fasted normal rats compared with hepatocytes from fed animals. Diabetes caused 3.5- and 2.1-fold increases in hepatic gluconeogenesis under fasting and fed conditions, respectively. Metformin (250 microM) suppressed glucose production by 37% in normal and by 30% in diabetic hepatocytes from fed rats. This drug was more effective (up to 67%) with increasing concentrations of glucose in the medium. Potentiation by metformin of insulin action on gluconeogenesis was elevated significantly (P < 0.01 to 0.001) by glucose in vitro. Metformin (75-250 microM) also counteracted the effects of glucagon at optimal concentrations in normal (32-68%) as well as diabetic (8-46%) hepatocytes. The findings of this study indicate that (i) the anti-gluconeogenic effect of metformin is enhanced by glucose in vivo and in vitro; and (ii) the suppression of glucagon-induced gluconeogenesis by metformin could play a role in its glucose-lowering effects in diabetic conditions.     

Effects of lactate infusion on hepatic gluconeogenesis and glycogenolysis

Endogenous glucose production rate (EGPR) remains constant when lactate is infused in healthy humans. A decrease of glycogenolysis or of gluconeogenesis from endogenous precursors or a stimulation of glycogen synthesis, may all be involved; This autoregulation does not depend on changes in glucoregulatory hormones. It may be speculated that alterations in basal sympathetic tone may be involved. To gain insights into the mechanisms responsible for autoregulation of EGPR, glycogenolysis and gluconeogenesis were measured, with a novel method (based on the prelabelling of endogenous glycogen with 13C glucose, and determination of hepatic 13C glycogen enrichment from breath 13CO2 and respiratory gas exchanges) in healthy humans infused with lactate or saline. These measurements were performed with or without beta-adrenergic receptor blockade (propranolol). Infusion of lactate increased energy expenditure, but did not increase EGPR; the relative contributions of gluconeogenesis and glycogenolysis to EGPR were also unaltered. This indicates that autoregulation is attained, at least in part, by inhibition of gluconeogenesis from endogenous precursors. beta-adrenergic receptor blockade alone (with propranolol) did not alter EGPR, glycogenolysis or gluconeogenesis. During infusion of lactate, propranolol decreased the thermic effect of lactate but EGPR remained constant. This indicates that alterations of beta-adrenergic activity is not required for autoregulation of EGPR.     

Effects of fatty acids and hormones on fatty acid metabolism and gluconeogenesis in bovine hepatocytes

Primary cultures of hepatocytes were used to study the effects of extracellular oleate concentration and hormones on fatty acid metabolism and gluconeogenesis. Rates of oleate uptake and oxidation to acid-soluble products varied linearly as oleate concentrations increased (0.1 to 2 mM), but rates of triglyceride accumulation varied quadratically. Insulin increased the proportion of oleate that was esterified by 22% without affecting the formation of acid-soluble products. Cells incubated with 2 mM [1-(14)C]oleate for 24 h eliminated 9.6% of the labeled intracellular lipid as acid-soluble products in the following 24 h when no oleate was present during depletion and eliminated 7.7% when 2 mM oleate was present. Insulin reduced labeled triglyceride depletion by 49%. Gluconeogenesis from [2-(14)C] propionate was depressed by 24%, and formation of acid-soluble products was increased by 46% in cells infiltrated with lipid because of previous exposure to 2 mM oleate for 45 h. Rates of gluconeogenesis from propionate were reduced 23% when 2 mM oleate was present during the 3-h period that gluconeogenesis was measured, and the effect was not modified by lipid infiltration. Lipid infiltration influenced hepatic function, and insulin regulated hepatic triglyceride concentration.     

Quantification of gluconeogenesis in cirrhosis: response to glucagon

BACKGROUND & AIMS: Accelerated starvation and early recruitment of alternate fuels in cirrhosis have been attributed to reduced availability of hepatic glycogen. The aim of this study was to measure gluconeogenesis (as a marker of protein oxidation) in relation to total glucose production and glucagon-stimulated glycogenolysis. METHODS: Glucose and urea production, gluconeogenesis, and glycogenolysis were calculated using stable isotope methods before and during glucagon infusion (3 ng. kg-1. min-1) in 5 cirrhotic patients and 5 matched controls before and after glycogen repletion. RESULTS: In the basal state, cirrhotic patients had a normal rate of glucose production, but the contribution of gluconeogenesis was increased (74.3% +/- 4.1% vs. 55. 6% +/- 12.1%; P < 0.005). Glycogen repletion normalized the rate of gluconeogenesis. The glycemic response to glucagon (3 ng. kg-1. min-1) was blunted in cirrhotic patients because of a lower rate of glycogenolysis (0.63 +/- 0.23 vs. 1.22 +/- 0.23 mg. kg-1. min-1; P < 0.01) and was not affected by glycogen repletion. Despite increased gluconeogenesis, the simultaneously measured rate of urea synthesis was lower in cirrhotic patients (3.11 +/- 1.02 vs. 5.0 +/- 1.0 mg/kg; P < 0.05). CONCLUSIONS: These data show that in cirrhosis, glucose production is sustained by an increased rate of gluconeogenesis. The hepatic resistance to glucagon action is not caused by reduced glycogen stores.     

Ultrastructure of periportal and centrilobular hepatocytes in human fatty liver of various aetiology

The ultrastructure of periportal and centrilobular hepatocytes of fatty liver from 2 alcoholic patients, 2 diabetic patients and 2 obese patients is described and compared with that of hepatocytes in 2 patients with normal liver. A striking difference between periportal and centrilobular hepatocytes in all cases of fatty liver is deomonstrated. The periportal hepatocytes contain abnormal mitochondria, while centrilobular hepatocytes have normal mitochondria, but fatty vacuoles, peroxisomes and lysosomes are more abundant. The nature and degree of ultrastructural changes showed no correlation with aetiology of the fatty liver. The normal livers showed no such changes or distinct differences between periportal and centrilobular hepatocytes.     

Regulation of intracellular pH in periportal and perivenular hepatocytes isolated from ethanol-treated rats

The aim of this study was to gain information on intracellular pH (pHi) regulation in periportal (PP) and perivenular (PV) hepatocytes isolated from rats pair-fed liquid diets with either ethanol (T rats) or isocaloric carbohydrates (C rats). pHi was analyzed by the pH-sensitive dye BCECF in perfused subconfluent hepatocyte monolayers. Cells were acid-loaded by pulse exposure to NH4Cl and were alkali-loaded by suddenly reducing external CO2 and HCO3- (from 10% and 50 mM, respectively, to 5% and 25 mM) at constant pHout. In cells from C rats: (a) steady-state pHi was higher in PP than in PV hepatocytes in the presence, but not in the absence, of bicarbonate; (b) pHi recovery from an acid load was 35% higher in PP than in PV cells in the presence of HCO3-, whereas it was similar in HCO3(-)-free experiments; and, on the contrary, (c) pHi recovery from an alkaline load was 30% higher in PV than in PP cells. In cells from T rats: (a) steady-state pHi was always lower than in cells isolated from pair-fed animals; (b) steady-state pHi was similar in PP and PV hepatocytes either in the presence or absence of bicarbonate in the perfusate; (c) pHi recovery from an acid load was not significantly different in PP and PV cells either in the presence of HCO3- or in HCO3(-)-free experiments; and (d) pHi recovery from an alkaline load was similar in PP and PV cells. Our data suggest that chronic ethanol treatment selectively modifies pHi by affecting the activity of ion transport mechanisms regulating pHi in PP and PV hepatocytes isolated from rat liver.     

Effects of glucagon on pancreatic secretion in the dog

About 85-90% of cytoplasmic protein synthesized by young reticulocytes is globin, and about 10% is a polypeptide (I) of molecular weight 64,000 daltons. Maturation of reticulocytes is accompanied by selective reduction in the synthesis of polypeptide I, relative to globin; mature reticulocytes synthesize globin at a high level but make no detectable polypeptide I. Studies in which RNA from young and old reticulocytes was translated in a wheat germ cell-free extract showed that reduction in synthesis of polypeptide I was correlated with a reduction in the amount of translatable mRNA for this protein. Differential destruction of mRNA thus is an important factor in determining the types of proteins made during the final stages of erythropoiesis.     

Electron microscopic identification of putative liver stem cells and intermediate hepatocytes following periportal necrosis induced in rats by allyl alcohol

Plasminogen (Pg) in the synovial fluid of patients with acute inflammatory disease, including rheumatoid arthritis, osteoarthritis, and gout, was purified by affinity chromatography techniques. The Pg isolated from patients with osteoarthritis and gout has affinities for L-lysine Sepharose and structural properties similar to those of normal plasma Pg. However, Pg from rheumatoid synovial fluids is abnormal in that it does not bind to L-lysine Sepharose. Analyses of rheumatoid synovial fluid Pg purified by immunoaffinity chromatography indicate that the molecule has size and folding properties similar to those of normal plasma Pg. However, we detected abnormalities in the molecule using two different criteria. First, isolated kringles 1-3 fragments are unable to bind L-lysine Sepharose. Second, reactivity toward a monoclonal antibody against a region in kringles 1-3 is decreased. In addition, rheumatoid synovial fluid Pg competes with normal plasma Pg for binding to the receptor on rheumatoid synovial fibroblasts but not on normal synovial fibroblasts. We also found that binding and activation of rheumatoid synovial fluid derived Pg on the surface of rheumatoid synovial fibroblasts elicits a rise in the concentration of cytosolic free Ca2+ similar to that of normal plasma Pg. Our data suggest that the inability of rheumatoid synovial fluid Pg to bind to L-lysine Sepharose is due to minor structural changes in kringle 1-3. These changes do not affect the physiological properties or the binding ability of synovial fluid Pg to cellular receptors on cells obtained from rheumatoid synovial tissue.     

Effects of clofibrate withdrawal on peroxisomes in mouse hepatocytes

Adult male mice (NMRI strain) were initially treated with 0.5% clofibrate in the diet during four days to obtain a high level of peroxisomes in the hepatocytes. Groups of animals were then given control diet for one, two, three, or four days. Liver samples were cytochemically stained for catalase prior to examination by electron microscopy and subsequent morphometric analysis. Various enzyme activities were assayed in liver homogenates. A sixfold increase in peroxisomal area was found after four days of clofibrate feeding compared with untreated animals. The peroxisomal fractional area was unchanged after one day of refeeding control diet but was reduced with over 50% after two days on control diet and was similar to that in the untreated animals after four days. The size distribution of peroxisomes shifted to smaller values during the breakdown of peroxisomes and was similar to the untreated animals after four days of clofibrate withdrawal. During this process a more heterogeneous staining of peroxisomes was observed. The mitochondrial fractional area was not affected by the treatments, but a shift in size distribution to smaller values was observed in the most induced animals. Protein content was not affected by the treatments. The activity of catalase (twofold induction) decreased progressively to control values during recovery but enoyl-CoA hydratase activity (fourty-fold induction) decreased rapidly. NADPH-cytochrome c reductase activity (twofold induction) decreased rapidly to control levels. Citrate synthase activity was not affected by clofibrate treatment but decreased during recovery. Acid phosphatase activity (repressed) increased to control levels. Cathepsin D activity increased somewhat during recovery while proteolytic activity (towards casein) decreased transiently on control diet.     

Effects of glucagon on superior mesenteric artery and femoral artery haemodynamics in humans

OBJECTIVE: It remains unclear whether glucagon is a localized splanchnic arterial vasodilator in humans. This study examined this issue by assessing the haemodynamic effect of exogenous glucagon on splanchnic and extrasplanchnic arteries. METHODS: After an overnight fast, flow velocity of superior mesenteric artery and femoral artery was recorded by means of echo-Doppler in 10 controls and 10 patients with cirrhosis. Mean arterial pressure, heart rate and plasma glucagon level were also determined. These measurements were repeated after intramuscular injection of glucagon (1 mg) at 15 min and 30 min. RESULTS: Patients with cirrhosis had much higher glucagon levels than controls (P < 0.01). Plasma glucagon level rose following glucagon administration in controls (P < 0.01) and patients with cirrhosis (P < 0.01). Glucagon administration had no effect on mean arterial pressure, heart rate and femoral artery velocity in controls and patients with cirrhosis. In contrast, superior mesenteric artery velocity significantly increased after glucagon administration in both groups (P < 0.01, P < 0.01), although the effect was less pronounced in patients with cirrhosis than in controls (P < 0.05). CONCLUSION: These data suggest that glucagon might be a localized splanchnic arterial vasodilator. Thus, glucagon may be one of the factors contributing to the pathogenesis of the splanchnic hyperdynamic circulation seen in patients with cirrhosis.     

Gluconeogenesis and glycogenolysis in fluoride-treated rats

Intraperitonial administration of 10 mg fluoride (NaF)/kg body weight resulted in hyperglycemia in rats. Role of gluconeogenesis and glycogenolysis in this hyperglycemic response was evaluated. Results of the study indicate that the fluoride induced hyperglycemia is mainly due to increased hepatic glycogenolysis.     

Desensitization of alpha 1-, beta- and glucagon receptors in rat hepatocytes: influence of ageing

It is now clear that it is not necessary to use an HLA genotypically identical donor to have a successful marrow transplant. However, it is equally clear that the likelihood of complications increases with each increment in histoincompatibility. The implication is that histocompatibility testing must be of the highest possible precision to choose the optimal donor, and to predict the risk of adverse alloreactivity. Most clinicians would seriously consider transplantation from a one locus-mismatched relative or an HLA-matched unrelated donor in virtually any situation in which transplantation from a matched sibling would be felt to be the standard of care. More thought would need to go into transplantation from a two or three locus-mismatched relative or a mismatched unrelated donor.     

Effects of dietary manipulations and glucose infusion on glucagon response during exercise in rats

OBJECTIVE: To describe our initial experience with a computerized telecommunication system, termed the interactive voice-response system, to record resident performance of laparoscopic surgery. METHODS: After completing a laparoscopic procedure, the surgeon and resident telephone a toll-free number independently and respond to three prerecorded statements using a Likert scale of 1 to 5. The caller then is asked to describe the resident's response to critical incidents or elements of surprise that arose during the surgery. The ratings and verbal comments are compiled, transcribed, and forwarded to the respective resident. The resident (and program director) can hear the verbal comments by entering a four-digit code. RESULTS: Between May 1, 1995, and May 31, 1996, 430 cases were reported by 11 surgeons and 16 residents using the interactive voice-response system. One hundred ninety-five (45%) procedures were entered by both the resident and surgeon. A survey undertaken during the introductory phase of the project revealed that five of the seven residents exposed to the system found that it provided useful feedback and preferred the system to traditional in-service reporting methods. In addition, five residents thought that the system complemented the personal feedback they received in the operating room. CONCLUSION: The system has been accepted by both residents and surgeons and has addressed the important components of resident in-training evaluation, namely, evaluation on a case-by-case basis, timely feedback, and self-assessment of resident performance.     

Effects of curcumin on P-glycoprotein in primary cultures of rat hepatocytes

Curcumin is a natural phenolic compound found in the rhizomes of Curcuma longa and endowed with beneficial biological activities including antioxidant, anticarcinogenic and hepatoprotective effects. In this study curcumin was tested for its potential ability to interact in vitro with hepatic P-glycoprotein (Pgp), in a model system represented by primary cultures of rat hepatocytes, in which spontaneous overexpression of multidrug resistance (mdr) genes occurs. In both freshly-plated hepatocytes, containing low levels of Pgp, and 72 hour-cultured hepatocytes, containing high levels of Pgp, the Rhodamine-123 (R-123) efflux, which represents a specific functional test for Pgp-mediated transport, was inhibited by curcumin in a dose-dependent manner. Western blot analysis showed that 25microM curcumin, when included in the culture medium throughout the experimental observation (72 hours), was able to significantly lower the increase of mAb C219-immunoreactive protein spontaneously occurring in the cells during culture. Curcumin, at doses ranging from 50 to 150microM was cytotoxic for freshly-plated hepatocytes, as shown by the strong decrease in the cell ability to exclude trypan blue 24 hours later, but it was significantly less cytotoxic when added to 24 or 48 hour-cultured cells. The resistance to curcumin, progressively acquired by cells during culture, was significantly reduced by high concentrations of dexamethasone (DEX) or dimethyl-sulfoxide (DMSO), culture conditions known to inhibit the spontaneous overexpression of Pgp. In addition, in a concentration-dependent manner, verapamil reverted curcumin resistance in Pgp overexpressing hepatocytes. In photoaffinity labeling studies, curcumin competed with azidopine for binding to Pgp, suggesting a direct interaction with glycoprotein. These results suggest that curcumin is able to modulate in vitro both expression and function of hepatic Pgp and support the hypothesis that curcumin, a chemopreventive phytochemical, could reveal itself also as a compound endowed with chemosensitizing properties on mdr phenotype.     

Effects of oxidative stress on glycerolipid acyl turnover in rat hepatocytes

Lipid peroxidation was induced in freshly isolated rat hepatocytes by incubation in the presence of Fe3+, resulting in accumulation of thiobarbituric acid reactive substances. Analysis of lipid classes revealed that the levels and fatty acid compositions of the two major phospholipids, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), remained unchanged but the levels of triacylglycerols (TAG) were significantly reduced, and some of their polyunsaturated fatty acids were selectively lost as the result of oxidant treatment. Acyl turnover in PC and PE as determined by 18O incorporation from H2 (18)O-containing media remained largely unchanged during oxidant treatment, while some increased turnover of the saturated fatty acids in TAG was observed. We hypothesize that constitutive recycling of membrane phospholipids rather than selective in situ repair eliminates peroxidized species of PC and PE. TAG could serve as an expendable fatty acid reserve, providing a limited but very dynamic pool of polyunsaturated fatty acids for the resynthesis of phospholipids.     

Modulation of epinephrine-stimulated gluconeogenesis by insulin in hepatocytes isolated from genetically obese (fa/fa) Zucker rats

Genetically obese (fa/fa) Zucker rats present an impaired response of hepatic glucose production to the inhibition by insulin. In this work, we have investigated the modulation by this hormone of epinephrine-stimulated gluconeogenesis, in hepatocytes isolated from obese (fa/fa) rats and their lean (Fa/-) littermates. Epinephrine (1 microM) caused a maximal stimulation of [14C]lactate conversion to [14C]glucose in hepatocytes isolated from either obese or lean animals. The stimulation of gluconeogenesis by epinephrine was accompanied by a significant reduction of fructose 2,6-bisphosphate levels, an inactivation of both pyruvate kinase and 6-phosphofructo 2-kinase, and by a 2-fold increase in the cellular concentrations of cAMP. The presence of insulin in the incubation medium antagonized, in a concentration-dependent manner, the effects of epinephrine. In hepatocytes isolated from lean rats, the reversion caused by insulin was complete, the concentration required for half-maximal insulin action ranging from 0.22 to 0.56 nM. In contrast, in obese rat hepatocytes, insulin only partially blocked epinephrine-mediated effects, and the sensitivity to insulin was 2- to 4-fold lower, as indicated by the corresponding half-maximal insulin action values. Furthermore, insulin (10 nM) almost completely blocked the increase in cAMP levels induced by epinephrine in lean rat hepatocytes, whereas it only provoked a small and nonsignificant reduction of epinephrine-stimulated levels of the cyclic nucleotide in hepatocytes obtained from obese rats.     

Effects of dicarboxylic acids on fatty acid-metabolizing enzymes in cultured rat hepatocytes

A clear chain-length dependent effect was observed for peroxisomal fatty acid beta-oxidation and carnitine acetyltransferase and also for mitochondrial carnitine palmitoyltransferase in primary cultures of rat hepatocytes. The extent of modulation of peroxisomal beta-oxidation was higher with even-carbon numbered dicarboxylic acids than with odd-carbon numbered ones, although such a tendency was not detected in the mitochondrial reactions. These results indicate difference in the effect of fatty acid-derived dicarboxylates on peroxisomal and mitochondrial functions.     

A novel assay for evaluating glycogenolysis in rat adipocytes and the inability of insulin to antagonize glycogenolysis in this cell type

We report here on a novel procedure for measuring glycogenolysis in rat adipocytes. In this procedure, cells are incubated for 30 min at 37 degrees C with insulin or vanadate, and with [U-14C]glucose to label the glycogen pool with radioactive glucose. The cells are washed and preincubated for an additional 1 h, before being assayed. The extent of glycogenolysis is determined by the decrease in radioactivity in precipitated glycogen, which was quite substantial under experimental conditions facilitating glycogenolysis. From the assay, we determined the following. (a) Glycogenolysis is activated in rat adipocytes in response to lipolytic hormones (i.e. catecholamines and adrenocorticotropic hormone). (b) Other agents and conditions elevating intracellular adenosine 3',5'-monophosphate levels (i.e. cholera toxin, dibutyryladenosine 3',5'-monophosphate, and isobutylmethylxanthine) also activate glycogenolysis. (c) Glycogenolysis (as opposed to lipolysis) is activated at concentrations of adrenocorticotropic hormone or isoproterenol 7-11-fold lower and at adenosine 3',5'-monophosphate concentrations 7-fold lower. (d) Calyculin A, a specific inhibitor of protein phosphatase 1, activates glycogenolysis as well. Calyculin A also activates lipolysis at an equimolar potency. (e) Insulin does not antagonize glycogenolysis in rat adipocytes. In conclusion, the assay allowed us to compare glycogenolysis to lipolysis within the same cell, and to find that the sensitivity to hormones and adenosine 3',5'-monophosphate was about 1 order of magnitude higher for glycogenolysis than for lipolysis. A more striking finding was the inability of insulin to antagonize glycogenolysis in the rat adipose cell, an effect which occurs readily in liver and muscle cells via protein phosphatase 1-activating machinery. This rules out a role for adipose protein phosphatase 1 activation in the mechanism by which insulin antagonizes lipolysis and supports the contention that the insulin effect in lowering adenosine 3',5'-monophosphate levels is the central mechanism by which insulin antagonizes lipolysis.