Freeze-clamped Liver Research Articles

Profiling Carbohydrate Metabolism in Liver and Hepatocellular Carcinoma with [13C]-Glycerate Probes.

The interplay between glycolysis and gluconeogenesis is central to carbohydrate metabolism. Here, we describe novel methods to assess carbohydrate metabolism using [13C]-probes derived from glycerate, a molecule whose metabolic fate in mammals remains underexplored. Isotope-based studies were conducted via NMR and mass spectrometry analyses of freeze-clamped liver tissue extracts after [2,3-13C2]glycerate infusion. The ex vivo investigations were correlated with in vivo measurements using hyperpolarized [1-13C]glycerate. Application of [13C]glycerate to N-nitrosodiethylamine (DEN)-treated rats provided further assessments of intermediary carbohydrate metabolism in hepatocellular carcinoma. This method afforded direct analyses of control versus DEN tissues, and altered ratios of 13C metabolic products as well as unique glycolysis intermediates were observed in the DEN liver/tumor. Isotopomer studies showed increased glycerate uptake and altered carbohydrate metabolism in the DEN rats.

A Look into Liver Mitochondrial Dysfunction as a Hallmark in Progression of Brain Energy Crisis and Development of Neurologic Symptoms in Hepatic Encephalopathy.

Background: The relationship between liver disease and neuropathology in hepatic encephalopathy is well known, but the genesis of encephalopathy in liver failure is yet to be elucidated. Conceptually, the main cause of hepatic encephalopathy is the accumulation of brain ammonia due to impaired liver detoxification function or occurrence of portosystemic shunt. Yet, as well as taking up toxic ammonia, the liver also produces vital metabolites that ensure normal cerebral function. Given this, for insight into how perturbations in the metabolic capacity of the liver may be related to brain pathology, it is crucial to understand the extent of ammonia-related changes in the hepatic metabolism that provides respiratory fuel for the brain, a deficiency of which can give rise to encephalopathy. Methods: Hepatic encephalopathy was induced in starved rats by injection of ammonium acetate. Ammonia-induced toxicity was evaluated by plasma and freeze-clamped liver and brain energy metabolites, and mitochondrial, cytoplasmic, and microsomal gluconeogenic enzymes, including mitochondrial ketogenic enzymes. Parameters of oxidative phosphorylation were recorded polarographically with a Clark-type electrode, while other measures were determined with standard fluorometric enzymatic methods. Results: Progressive impairment of liver mitochondrial respiration in the initial stage of ammonia-induced hepatotoxicity and the subsequent energy crisis due to decreased ATP synthesis lead to cessation of gluconeogenesis and ketogenesis. Reduction in glucose and ketone body supply to the brain is a terminal event in liver toxicity, preceding the development of coma. Conclusions: Our study provides a framework to further explore the relationship between hepatic dysfunction and progression of brain energy crisis in hepatic encephalopathy.

Abstract 244: The Flavonoids Naringenin and Nobiletin Stimulate the AMPKinase Pathway in Primary Mouse Hepatocytes

Dyslipidemia associated with insulin resistance and obesity are core features of the metabolic syndrome and type 2 diabetes, which contribute significantly to atherosclerosis. In mouse models of diet-induced metabolic dysregulation, the citrus flavonoids naringenin and nobiletin prevent obesity, hepatic steatosis, apoB100 overproduction, dyslipidemia, insulin resistance and atherosclerosis. To elucidate the mechanism of action in liver we assessed flavonoid-induced activation of AMP-activated protein kinase (AMPK), the major regulator of cellular energy homeostasis, in primary mouse hepatocytes. Stimulated AMPK activity promotes catabolic, ATP-generating processes such as fatty acid (FA) oxidation while inhibiting anabolic processes such as FA synthesis. In primary C57BL/6 (WT) hepatocytes, naringenin and nobiletin increased phosphorylation (P) of AMPK and its downstream target acetyl-CoA carboxylase (ACC) in a time- and dose-dependent manner. This was associated with decreased apoB100 secretion. Phosphorylation of ACC by AMPK inhibits the formation of malonyl-CoA reducing substrate for FA synthesis in the cytosol while relieving inhibition of mitochondrial FA oxidation by malonyl-CoA. Under insulin resistant conditions stimulated by high glucose media, reduced pAMPK and pACC were reversed by flavonoid treatment in WT hepatocytes, whereas these effects were lost in Ampkβ1-/- hepatocytes. Sterol receptor element binding protein-1c, which stimulates lipogenesis, was also phosphorylated (inhibited) by flavonoid-induced AMPK activation. BAPTA, a calcium chelator or STO609, an inhibitor of Ca2+/calmodulin-dependent protein kinase kinase-beta (CaMKKβ), did not block flavonoid-induced pACC, suggesting that CaMKKβ is not required for AMPK activation by flavonoids. In chow-fed Ldlr-/- mice, acute i.p. injection of nobiletin following a fasting-refeeding protocol, depressed the respiratory exchange ratio indicative of a switch to FA oxidation. Freeze-clamped liver samples from these mice sacrificed 90 min. post injection showed marked induction of pAMPK and pACC. These results suggest that naringenin and nobiletin attenuate hepatic steatosis and metabolic dysregulation, in part, through activation of hepatic AMPK.

Physiological Regulation of Phospholipid Methylation Alters Plasma Homocysteine in Mice

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-alpha (CTalpha) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20-40% in mice lacking hepatic CTalpha. CTalpha-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.

Prevention of hepatic steatosis and hepatic insulin resistance in mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 knockout mice

In order to investigate the role of mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase 1 (mtGPAT1) in the pathogenesis of hepatic steatosis and hepatic insulin resistance, we examined whole-body insulin action in awake mtGPAT1 knockout (mtGPAT1(-/-)) and wild-type (wt) mice after regular control diet or three weeks of high-fat feeding. In contrast to high-fat-fed wt mice, mtGPAT1(-/-) mice displayed markedly lower hepatic triacylglycerol and diacylglycerol concentrations and were protected from hepatic insulin resistance possibly due to a lower diacylglycerol-mediated PKC activation. Hepatic acyl-CoA has previously been implicated in the pathogenesis of insulin resistance. Surprisingly, compared to wt mice, mtGPAT1(-/-) mice exhibited increased hepatic insulin sensitivity despite an almost 2-fold elevation in hepatic acyl-CoA content. These data suggest that mtGPAT1 might serve as a novel target for treatment of hepatic steatosis and hepatic insulin resistance and that long chain acyl-CoA's do not mediate fat-induced hepatic insulin resistance in this model.

A Crucial Role of MafA as a Novel Therapeutic Target for Diabetes*♦

MafA, a recently isolated pancreatic beta-cell-specific transcription factor, is a potent activator of insulin gene transcription. In this study, we show that MafA overexpression, together with PDX-1 (pancreatic and duodenal homeobox factor-1) and NeuroD, markedly increases insulin gene expression in the liver. Consequently, substantial amounts of insulin protein were induced by such combination. Furthermore, in streptozotocin-induced diabetic mice, MafA overexpression in the liver, together with PDX-1 and NeuroD, dramatically ameliorated glucose tolerance, while combination of PDX-1 and NeuroD was much less effective. These results suggest a crucial role of MafA as a novel therapeutic target for diabetes.

Ca2+‐Dependent Elevation of Inositol 1,4,5‐Trisphosphate Level Induced by Freezing or Homogenization of Tissues and Cells

Various cells and tissues contain high basal levels of inositol 1,4,5-trisphosphate, raising questions about the functional significance of inositol 1,4,5-trisphosphate in some tissues such as the heart. We used intact tissue and isolated cells from heart and liver of adult rats to examine if different fixation procedures might artificially elevate the level of inositol 1,4,5-trisphosphate. The basal level of inositol 1,4,5-trisphosphate in intact, freeze-clamped cardiac tissue from adult rats was 10 times higher than in isolated, non-frozen cardiomyocytes, while freeze-clamped liver contained approximately 4 times higher inositol 1,4,5-trisphosphate levels than isolated, non-frozen hepatocytes. Stimulation with norepinephrine induced a significant increase in the inositol 1,4,5-trisphosphate level in isolated cardiomyocytes, whereas no significant increase was observed in freeze-clamped cardiac tissue. Freezing of isolated cardiomyocytes or hepatocytes before extraction increased basal inositol 1,4,5-trisphosphate levels 3 times. In cellular homogenates prepared in the presence of EGTA and stored at 4 degrees , readdition of calcium resulted in a time-dependent increase in inositol 1,4,5-trisphosphate mass and a decrease in the mass of phosphatidylinositol 4,5-bisphosphate (PIP(2)). The reaction was essentially complete within 30 sec. in homogenates from cardiomyocytes, while PIP(2) hydrolysis was slower in hepatocyte homogenates. Perfusion of intact rat hearts with EGTA present during the last 2 min. of perfusion, followed by freeze-clamping, resulted in basal inositol 1,4,5-trisphosphate levels comparable to those in isolated cardiomyocytes, and norepinephrine stimulation increased inositol 1,4,5-trisphosphate mass by approximately 80%. The presence of EGTA did not significantly affect PIP(2) levels in perfused hearts. The results suggest that freezing or homogenization of intact tissue and isolated cells may result in Ca(2+)-dependent activation of phospholipase C, leading to high basal inositol 1,4,5-trisphosphate levels that may mask agonist-induced changes.

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.

The atrial natriuretic peptide and cGMP: Novel activators of the heat shock response in rat livers

Preischemic treatment with atrial natriuretic peptide (ANP) attenuates ischemia-reperfusion injury of the rat liver via cyclic guanosine monophosphate (cGMP). The attenuated activation of nuclear factor κB (NF-κB) seems to contribute to this effect. The aim of this study was to determine whether heat shock proteins are involved in these molecular pathways. Livers of male Sprague-Dawley rats were continuously perfused with Krebs-Henseleit (KH) buffer with or without ANP or 8-Br-cGMP. In different experiments livers were perfused with or without ANP for 20 minutes, kept in cold storage solution for 24 hours, and reperfused. Activation of heat shock transcription factor (HSF) (by electrophoretic mobility shift assay), heat shock protein 70 (HSP70), and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (by reverse transcription polymerase chain reaction [RT-PCR]), as well as HSP70 (by Western blot) were investigated in freeze-clamped liver samples. During continuous perfusion ANP as well as 8-Br-cGMP activated HSF, HSP70 protein concentrations paralleled HSF-activation. ANP pretreated livers exhibited elevated HSF after 24 hours of ischemia and elevated HSP70 mRNA levels during reperfusion. ANP prevented the marked decrease of HSP70 protein during reperfusion. Coimmunoprecipitation studies showed increased binding of HSP70 to inhibitory factor κB (IκB) in ANP-treated livers. In conclusion, we showed the cGMP-mediated activation of HSF by ANP, which resulted in elevated HSP70 mRNA and protein concentrations and correlated with enhanced binding of HSP70 to IκB. This could be an important mechanism of ANP-mediated prevention of hepatic preservation damage. (H EPATOLOGY 2002;35:88-94.)

Moderate hypothermia ameliorates liver energy failure after intestinal ischaemia-reperfusion in anaesthetised rats

Background/Purpose: Intestinal ischaemia-reperfusion (IR) can cause liver failure. The aims of this work were to study the effects of intestinal IR on liver energy metabolism and to evaluate the effects of moderate hypothermia. Methods: Intestinal IR (90-minute intestinal ischaemia plus 60-minute or 240-minute reperfusion) was achieved by clamping and unclamping the superior mesenteric artery in rats. Normothermia or moderate hypothermia (30° to 33°C) was maintained by adjusting the environmental temperature. The ratio of hepatic inorganic phosphate to adenosine triphosphate (ATP) was monitored continuously during intestinal IR using in vivo phosphorus (31P) magnetic resonance spectroscopy. Phosphorus metabolites also were measured in extracts prepared from freeze-clamped liver and intestine. Results: Mortality occurred exclusively during normothermic intestinal IR. A progressive increase in the hepatic inorganic phosphate to ATP ratio after normothermic intestinal IR was observed. Moderate hypothermia delayed this effect. Analysis of liver extracts confirmed above findings. However, there was no difference in intestinal phosphocreatine or ATP between normothermic and hypothermic rats undergoing intestinal IR. Conclusions: Intestinal IR at normothermia was associated with liver energy failure and high mortality rate. Moderate hypothermia ameliorated liver energy failure but did not attenuate intestinal energy failure after intestinal IR. Hypothermia may prove to be useful in the management of patients with intestinal IR injuries in the future. J Pediatr Surg 36:269-275. Copyright © 2001 by W.B. Saunders Company.

Atrial natriuretic peptide reduces expression of TNF-α mRNA during reperfusion of the rat liver upon decreased activation of NF-κB and AP-1

Background/Aims: The cardiovascular hormone Atrial Natriuretic Peptide (ANP) attenuates activation of the pro-inflammatory transcription factor NF-κB in macrophages. ANP was also shown to protect from ischemia-reperfusion injury of the rat liver. This study aimed to investigate the effects of this immunomodulatory hormone and its second messenger cGMP on the activation of the two redox-sensitive transcription factors AP-1 and NF-κB and the expression of corresponding pro-inflammatory target genes during ischemia and reperfusion of the liver. The identification of the mechanisms underlying the protection by ANP should reveal new aspects concerning the pathomechanisms of ischemia/reperfusion injury.Methods: Rat livers were perfused with and without ANP or 8-Br-cGMP preceding 24 h of cold storage in University of Wisconsin solution. During reperfusion NF-κB and AP-1 DNA binding activities were determined in freeze-clamped liver samples by electrophoretic mobility shift assay. Protein levels of p50, p65, and of IκB were determined by Western blot. mRNA coding for inducible nitric oxide synthase, cyclooxygenase-2, and TNF-α was determined by RT-PCR and Northern blot.Results: After 45 min of reperfusion DNA binding activities of NF-κB were increased, whereas in ANP pre-treated livers this effect was markedly reduced. AP-1, another important redox-sensitive transcription factor, was activated and in the course of reperfusion the subunit composition of AP-1 changed as assessed by supershift assays. ANP markedly reduced binding activities of both forms of AP-1. 8-Br-cGMP mimicked the effects of ANP on NF-κB and AP-1. Neither inducible nitric oxide synthase nor cyclooxygenase-2 mRNA could be detected. In contrast, a profound expression of transcripts coding for TNF-α was detected in the course of reperfusion and ANP markedly reduced TNF-α mRNA expression.Conclusion: ANP seems to mediate its protective effect during ischemia and reperfusion by reducing the activation of NF-κB and AP-1 via cGMP. The reduced binding activity of these redox-sensitive transcription factors was accompanied by a diminished mRNA expression of TNF-α, a cytokine known to be involved in cellular damage in ischemia reperfusion injury.

Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain

Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin (50 μM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K0.5, 79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, can also be explained in this way.

Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain

Although metformin is widely used for the treatment of non-insulin-dependent diabetes, its mode of action remains unclear. Here we provide evidence that its primary site of action is through a direct inhibition of complex 1 of the respiratory chain. Metformin(50 microM) inhibited mitochondrial oxidation of glutamate+malate in hepatoma cells by 13 and 30% after 24 and 60 h exposure respectively, but succinate oxidation was unaffected. Metformin also caused time-dependent inhibition of complex 1 in isolated mitochondria, whereas in sub-mitochondrial particles inhibition was immediate but required very high metformin concentrations (K(0.5),79 mM). These data are compatible with the slow membrane-potential-driven accumulation of the positively charged drug within the mitochondrial matrix leading to inhibition of complex 1. Metformin inhibition of gluconeogenesis from L-lactate in isolated rat hepatocytes was also time- and concentration-dependent, and accompanied by changes in metabolite levels similar to those induced by other inhibitors of gluconeogenesis acting on complex 1. Freeze-clamped livers from metformin-treated rats exhibited similar changes in metabolite concentrations. We conclude that the drug's pharmacological effects are mediated, at least in part, through a time-dependent, self-limiting inhibition of the respiratory chain that restrains hepatic gluconeogenesis while increasing glucose utilization in peripheral tissues. Lactic acidosis, an occasional side effect, canal so be explained in this way.

Relationship of glycogen synthase and glycogen phosphorylase to protein phosphatase 2C and cAMP-dependent protein kinase in liver of obese rhesus monkeys.

The regulation of glycogen synthase (GS) and glycogen phosphorylase (GP) activity by phosphorylation/ dephosphorylation has been proposed to be via changes in activities of several different protein (serine/threonine) phosphatases and kinases, including protein phosphatase (PP) 1/2A, PP2C, and cAMP-dependent protein kinase (PKA). In order to determine whether PP1/2A, PP2C, and/or PKA activities are related to GS and/or GP activities, these enzymes were measured in freeze-clamped liver biopsies obtained under basal fasting conditions from 16 obese monkeys. Four monkeys were normoglycemic and normoinsulinemic, five were hyperinsulinemic, and seven had type 2 diabetes (NIDDM). Liver glycogen and glucose 6-phosphate (G6P) contents were also determine. Basal enzyme activities and basal substrate concentrations were not significantly different between the three group of obese monkeys; however, there were several significant linear relationships observed when the monkeys were treated as one group. Therefore, multiple regression was used to determine the correlation between key variables. GS fractional activity was correlated to GP fractional activity (p < 0.05) and to PP2C activity (p = 0.005) (adjusted R2, 53%). GP independent activity was correlated to GS independent activity (p < 0.07) and to PKA fractional activity (p = 0.005) (adjusted R2, 64%). PP2C activity was correlated to GS fractional activity (p < 0.0005) and to PP1/2A activity (p < 0.0001) (adjusted R2, 83%). PKA fractional activity was correlated to GP total activity (p < 0.0005) and to age (p = 0.001) (adjusted R2, 82%). G6P content was correlated to glycogen content (p < 0.05) and to PP2C activity (p = 0.0005) (adjusted R2, 73%). In conclusion, PP2C and PKA are involved in the regulation of GS and GP activity in the basal state in liver of obese monkeys with a wide range of glucose tolerance.

Validated High-Performance Liquid Chromatography-Electrochemical Method for Determination of Glutathione and Glutathione Disulfide in Small Tissue Samples

Glutathione (GSH) and glutathione disulfide (GSSG) are biologically important intracellular thiols; alterations in the GSH/GSSG ratio are often used to assess exposure of cells to oxidative stress. Although several methods are available for measuring GSH and GSSG, all have some disadvantages including the need to generate derivatives, the inability to conveniently measure both GSH and GSSG, and a lack of sufficient sensitivity to allow detection in very small samples/cells of extrahepatic tissue. These studies present a rapid, validated HPLC-electrochemical method for determining GSH and GSSG in small samples such as those from microdissected airways of the mouse containing 50–200 μg protein which is suitable for routine use. GSH and GSSG can be measured at levels of 1 and 2 pmol on column, respectively, with acceptable accuracy and precision and without the need to generate derivatives. In microdissected airways from the mouse, the intraday assay coefficient of variation for GSH varied from 4.7 to 5.9% and for GSSG was 4.4 to 5.7%. The interday assay coefficient of variation ranged from 6.0 to 7.6% for GSH and 5.5 to 23% for GSSG. The effects of repeated freezing and thawing on the concentrations of GSH and GSSG indicate that multiple cycles do not significantly alter the GSH or GSSG concentration as the number of cycles increases. Addition of GSH or GSSG to samples increased the peak areas appropriately, without altering the peak shape, retention time, or peak area of the corresponding reduced (oxidized) thiol. The ratio of GSH/GSSG in freeze-clamped liver ranged from 46 to 248, while liver tissue which was homogenized fresh had GSH/GSSG ratios of 62–150. The technique appears to be capable of reproducibly measuring GSH and GSSG in small quantities of nonhepatic tissue.

Energy Depletion in the Liver and in Isolated Hepatocytes of Tumor-Bearing Animals

Cancer cachexia has a great impact on morbidity and mortality in patients undergoing surgery. Failure to maintain lean tissue against tumor-induced hypermetabolism results in cumulative weight loss and ultimate failure of the host. Cellular free energy is among the factors that regulates metabolic pathways and may be altered in the tumor-bearing state. We studied in-vivo and in-vitro ATP levels and metabolic parameters in Fischer rats bearing a methylcholanthrene-induced sarcoma to elucidate the energy state. Tissue ATP was measured in freeze-clamped liver and muscle in 15 tumor-bearing rats (TBR) at different tumor burdens and their pair-fed controls (CTR) by bioluminescence assay. Plasma metabolites, hormones, and enzymes were determined in the same animals. Liver ATP level was lower in TBR with a 5% tumor burden: 3.07 ± 0.56 (SE) nmole/mg tissue vs CTR: 5.33 ± 0.60 ( P < 0.05), 10% TBR: 2.48 ± 0.54 vs CTR: 4.05 ± 0.63 (n.s.), and 20% TBR: 1.91 ± 0.21 vs CTR: 3.86 ± 0.40 ( P < 0.01). Muscle ATP was not different between TBR and CTR. This progressive loss of liver ATP was associated with decreased plasma insulin level ( P < 0.001) and with increased alkaline phosphatase level ( P < 0.01) by multiple regression. In vitro, hepatocytes were isolated from 8 TBR and 8 CTR by in-situ liver perfusion with collagenase and the cellular ATP was measured before and after 60 min incubation with glucogenic substrates. Hepatocytes from TBR decreased ATP by 42% ( P < 0.05) in 10 m M lactate with higher gluconeogenesis, while control hepatocytes maintained the ATP. These results demonstrate a progressive loss of energy generating capacity in the tumor-bearing liver with increased metabolic activity. Insulin resistance is postulated as a potential mechanism for these effects.

Importance of substrate changes in the decrease of hepatic glucose cycling during insulin infusion and declining glycemia in the depancreatized dog.

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G<-->G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6-9 h at a starting dose of 45-150 pmol.kg-1.min-1 to normalize plasma glucose from 23.9 +/- 1.4 to 5.0 +/- 0.4 mmol/l and gradually decreased to and maintained at a basal rate (1.7 +/- 1.0 pmol.kg-1.min-1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 +/- 2.7 and normalized at 1.3 +/- 0.6 mumol.kg-1.min-1 (P < 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 +/- 3.1 to 11.6 +/- 1.2) than did glucose production (from 25.9 +/- 1.9 to 10.3 +/- 1.0 mumol.kg-1.min-1) (both P < 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 +/- 24 vs. 71 +/- 13 nmol/g, P < 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 +/- 0.8 to 19.6 +/- 2.6 U/g) and glucokinase (from 1.1 +/- 0.2 to 1.0 +/- 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P < 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content.(ABSTRACT TRUNCATED AT 250 WORDS)

Importance of Substrate Changes in the Decrease of Hepatic Glucose Cycling During Insulin Infusion and Declining Glycemia in the Depancreatized Dog

We wished to determine whether the elevated glucose cycling (GC) between glucose and glucose-6-phosphate (G⇆G6P) in diabetes can be reversed with acute insulin treatment. In six insulin-deprived, anesthetized, depancreatized dogs, insulin was infused for 6–9 h at a starting dose of 45–150 pmol-kg−1-min−1 to normalize plasma glucose from 23.9 ± 1.4 to 5.0 ± 0.4 mmol/1 and gradually decreased to and maintained at a basal rate (1.7 ± 1.0 pmol · kg−1 · min−1) during the last 3 h. GC, measured with [2-3H]- and [6-3H]glucose, fell markedly from 15.3 ± 2.7 and normalized at 1.3 ± 0.6 μmol · kg−1 · min−1 (P &amp;lt; 0.001). This occurred because total hepatic glucose output fell much more (from 41.2 ± 3.1 to 11.6 ± 1.2) than did glucose production (from 25.9 ± 1.9 to 10.3 ± 1.0 μmol · kg−1 · min−1) (both P &amp;lt; 0.01). Freeze-clamped liver biopsies were taken at timed intervals for measurements of hepatic enzymes and substrates. The elevated hepatic hexose-6-phosphate levels decreased with insulin infusion (151 ± 24 vs. 71 ± 13 nmol/g, P &amp;lt; 0.01). Maximal activities of glucose-6-phosphatase (G6Pase) (from 17.6 ± 0.8 to 19.6 ± 2.6 U/g) and glucokinase (from 1.1 ± 0.2 to 1.0 ± 0.2 U/g) did not change. Insulin infusion resulted in a threefold increase (P &amp;lt; 0.05) in the activity of glycogen synthase (active form), but had no effect on hepatic glycogen content. We therefore conclude that in the depancreatized dog, 1) acute insulin infusion with a concurrent correction of hyperglycemia can markedly reduce and normalize GC, and 2) the acute reduction in GC is primarily mediated by reduced substrate fluxes through glucokinase and G6Pase and does not require stable changes in maximal activities of the two enzymes. However, in vivo allosteric changes in enzyme activities (particularly G6Pase), immeasurable in vitro, cannot be excluded. In addition to enzyme measurements, the importance of tracer methods that allow for in vivo measurements of fluxes through glucokinase and G6Pase is emphasized.

Mitochondrial metabolism in different thyroid states

The protonmotive force, as well as the mitochondrial and cytosolic concentrations of malate, 2-oxoglutarate, glutamate and aspartate, were determined in livers from hypo-, eu- and hyper-thyroid rats, by density-gradient centrifugation of freeze-clamped livers in non-aqueous solvents [Soboll, Akerboom, Schwenke, Haase & Sies (1980) Biochem. J. 192, 951-954]. The mitochondrial/cytosolic pH difference and the membrane potential were significantly enhanced in hyperthyroid livers compared with the hypothyroid state, resulting in an increased protonmotive force in the presence of thyroid hormones [Soboll & Sies (1989) Methods Enzymol. 174, 118-130]. The mitochondrial concentrations of 2-oxoglutarate, glutamate and aspartate were significantly higher in the euthyroid than in the hypothyroid state, but only slightly higher in the hyperthyroid state. Mitochondrial malate, on the other hand, increased significantly from the hypothyroid to the hyperthyroid state. The mitochondrial/cytosolic concentration gradients were significantly increased in the presence of thyroid hormones only for malate. The changes in steady-state metabolite concentrations reflect a higher substrate supply and a stimulation of mitochondrial metabolism. However, a clear relationship between the increased protonmotive force, as the driving force for mitochondrial metabolite transport, and the subcellular metabolite concentrations is not observable in different thyroid states.

Evidence that AMP triggers phosphorylation as well as direct allosteric activation of rat liver AMP‐activated protein kinase

1. In freshly isolated rat hepatocytes, the activity of the AMP-activated protein kinase is high, but decreases by 5-10-fold during incubation of the cells for 60 min. The expressed activity of acetyl-CoA carboxylase is initially very low, then rises in a reciprocal manner to the AMP-activated protein kinase activity. For both enzymes, treatment of partially purified preparations under dephosphorylating conditions abolishes the difference in activity between freshly isolated and preincubated cells. Thus, both the high activity of the AMP-activated protein kinase and the low activity of acetyl-CoA carboxylase in freshly isolated cells can be explained by phosphorylation. 2. Immediately after isolation, the hepatocytes have AMP/ATP ratios that are unphysiologically high (approximately 1:1.5). During incubation of the cells for 60 min, AMP levels fall and ATP levels rise so that the ratio becomes about 1:15, close to previous estimates of the ratio in freeze-clamped liver. The fall in AMP/ATP ratio precedes the decrease in AMP-activated protein kinase activity. 3. In cells which have been incubated for 60 min, treatment with 20 mM fructose, which causes a large but transient increase in the AMP/ATP ratio, also causes concomitant activation of the AMP-activated protein kinase and inactivation of acetyl-CoA carboxylase. 4. In all cases described above, the increases in activity of acetyl-CoA carboxylase were blocked by treatment with the cell-permeable protein phosphatase inhibitor, okadaic acid. However, the decreases in activity of the AMP-activated protein kinase were not blocked by this inhibitor. This is consistent with the finding that okadaic-acid-insensitive protein phosphatase 2C is the most effective at dephosphorylating the kinase in cell-free assays. 5. The results above suggested that AMP either promotes phosphorylation, or inhibits dephosphorylation, of the kinase. Studies in a partially purified cell-free system suggested that the former hypothesis was correct; reactivation of dephosphorylated AMP-activated protein kinase by kinase kinase was completely dependent on the presence of AMP. 6. Our results, obtained in both intact cells and a cell-free system, suggest that rises in the AMP/ATP ratio promote phosphorylation of the AMP-activated protein kinase by the kinase kinase, as well as causing direct allosteric activation. This represents a very sensitive system for switching off lipid biosynthetic pathways when ATP levels are limiting. The results with okadaic acid also suggest that protein phosphatase 2C is mainly responsible for dephosphorylation of the AMP-activated protein kinase in intact hepatocytes.