The role of the cytoplasmic redox potential in the control of fatty acid synthesis from glucose, pyruvate and lactate in white adipose tissue

The metabolism of lactate, pyruvate and glucose was studied in epididymal adipose tissue of starved, normally fed and starved-re-fed rats. Lactate conversion into fatty acid occurred at an appreciable rate only in the adipocyte of starved-re-fed animals. NNN'N'-Tetramethyl-p-phenylenediamine, an agent that transports reducing power from the cytoplasm to the mitochondria, caused large increments of fatty acid synthesis from lactate and a smaller one from glucose but a decrease in that from pyruvate. Glucose (1.0mm) increased fatty acid synthesis from lactate 4.3-fold but only 1.67-fold from pyruvate in adipocytes from normally fed animals. 2-Deoxyglucose decreased fatty acid synthesis from lactate to a greater degree (threefold) compared to that from pyruvate in adipocytes from starved-re-fed animals. l-Glycerol 3-phosphate contents were approximately equal in epididymal fat-pads, incubated in the presence of lactate or pyruvate, from normally fed animals, whereas the addition of 1mm-glucose resulted in a tenfold increase in l-glycerol 3-phosphate content only in the presence of lactate. The l-glycerol 3-phosphate content was tenfold higher in adipose tissue from starved-re-fed animals incubated in the presence of lactate than in the presence of pyruvate. 2-Deoxyglucose caused these values to be slightly lowered in the presence of lactate. We suggest that lactate metabolism is limited by the rate of NADH removal from the cytoplasm. In the starved-re-fed state, this occurs by reduction of dihydroxyacetone phosphate formed from glycogen to produce l-glycerol 3-phosphate, thus permitting lactate conversion into fatty acid. When glucose is the substrate, and rates of transport are not limiting, the rate of removal of cytoplasmic NADH limits glucose conversion into fatty acid.     

Control of glycogen metabolism by gluconeogenic and ketogenic substrates in isolated hepatocytes from fed rats.

1. This study was conducted to examine the effects of gluconeogenic and ketogenic substrates on the activities of the glycogen-metabolizing enzymes and on glycogenolysis in isolated hepatocytes from fed rats. 2. Gluconeogenic substrates like fructose, dihydroxyacetone or lactate turned out to stimulate the glucose-induced activation of glycogen synthase and this effect may be linked, to some extent, to the increase of the cellular glucose 6-phosphate concentration. 3. The effect of fructose was accompanied by the onset of glycogen synthesis. 4. Energetic substrates like fatty acids were also potent activators of glycogen synthase, especially in the presence of glucose. 5. When fatty acids were added alone or together with a physiological concentration of glucose, they induced or potentiated the inhibition of glycogen phosphorylase-a. 6. This inhibitory effect was mediated by a decrease of lactate release. 7. The stimulatory effect of amino acids on glycogen synthase seemed to be direct, non mediated by an inhibition of the phosphorylase-a activity although hepatic glycogenolysis markedly decreased. 8. Moreover, the amino acid action could be linked to their capacities to induce cell swelling and/or to limit proteolysis.     

Stimulation of glycogen synthesis by proglycosyn (LY177507) by isolated hepatocytes of normal and streptozotocin diabetic rats

The phenacylimidazolium compound LY177507 was shown by Harris et al. (Harris, R. A., Yamanuchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J., and Stephens, T. W. (1989) J. Biol. Chem. 264, 14674-14680) to stimulate glycogen synthesis greatly in isolated rat hepatocytes. We extended studies with this compound, designated proglycosyn (Yamaguchi, K., Stephens, T. W., Chikadar, K., Depaoli-Roach, A., And Harris, R. A. (1991) Diabetes 40, (Suppl. 1) 102 (abstr.] employing hepatocytes from normal and streptozotocin diabetic rats. Proglycosyn is more effective than amino acids in stimulating glycogen synthesis. In cells incubated with glucose, lactate, or dihydroxyacetone the effect of glutamine and proglycosyn was synergistic. In cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, the stimulation by the two agonists was additive. Proglycosyn diverted the gluconeogenic flux from glucose to glycogen. The maximal rates of glycogen deposition attained in the presence of glutamine and proglycosyn from cells incubated with glucose plus lactate, or glucose plus dihydroxyacetone, where about 80 and 110 mumols/h/g of liver, respectively. Proglycosyn depressed glycogenolysis in hepatocytes of fed rats and stimulated glycogen synthesis from lactate and dihydroxyacetone. The incorporation of [U-14C]glucose and [U-14C]lactate in these cells occurred in the presence of glycogen breakdown or exceeded net production, indicating the occurrence of recycling of glycogen in hepatocytes of fed rats. Hepatocytes from fasted streptozotocin diabetic rats contained high levels of glycogen. Glycogenolysis was markedly depressed by proglycosyn. Glycogen synthesis from lactate and dihydroxyacetone in these cells was stimulated by glutamine and proglycosyn in a fashion similar to that in cells from fasted control rats, and the rates of glycogen synthesis were similar in cells of control and diabetic rats. With glucose as sole substrate, glutamine did not stimulate glycogen synthesis. When both agonists were present, there was a marked synergism and substantial glycogen formation. Streptozotocin diabetic rats prior to the onset of cachexia have a normal capacity for glycogen synthesis.     

Effect of glycerol and dihydroxyacetone on hepatic lipogenesis

Glycerol is a dietary component which is metabolized primarily by the liver and kidney where it is used mainly for glucose synthesis. The metabolism of glycerol is very similar to that of dihydroxyacetone which can be considered its more oxidized counterpart. The effects of these substrates on hepatic lipogenesis and gluconeogenesis were examined. In isolated hepatocytes, 10 mM dihydroxyacetone caused a large increase in glucose output and stimulated lipogenesis without affecting the lactate/pyruvate ratio or the total ATP content of the cells. (As compared to dihydroxyacetone, 10 mM glycerol was less effective as a gluconeogenic substrate, increased the lactate/pyruvate ratio, caused a slight decrease in the total ATP content, and inhibited lipogenesis by at least 40% depending on the type of diet fed to the rats.) The fall in ATP levels was very small and did not correlate with the changes in fatty acid synthesis. The immediate cause of the inhibition of lipogenesis, brought about by glycerol in hepatocytes from sucrose fed rats, seemed to be a large decrease in pyruvate levels. This did not result from impairment of glycolysis but from a rise in the cytosolic NADH/NAD ratio.     

Glucokinase overexpression restores glucose utilization and storage in cultured hepatocytes from male Zucker diabetic fatty rats.

Zucker diabetic fatty rats develop type 2 diabetes concomitantly with peripheral insulin resistance. Hepatocytes from these rats and their control lean counterparts have been cultured, and a number of key parameters of glucose metabolism have been determined. Glucokinase activity was 4.5-fold lower in hepatocytes from diabetic rats than in hepatocytes from healthy ones. In contrast, hexokinase activity was about 2-fold higher in hepatocytes from diabetic animals than in healthy ones. Glucose-6-phosphatase activity was not significantly different. Despite the altered ratios of glucokinase to hexokinase activity, intracellular glucose 6-phosphate concentrations were similar in the two types of cells when they where incubated with 1-25 mM glucose. However, glycogen levels and glycogen synthase activity ratio were lower in hepatocytes from diabetic animals. Total pyruvate kinase activity and its activity ratio as well as fructose 2,6-bisphosphate concentration and lactate production were also lower in cells from diabetic animals. All of these data indicate that glucose metabolism is clearly impaired in hepatocytes from Zucker diabetic fatty rats. Glucokinase overexpression using adenovirus restored glucose metabolism in diabetic hepatocytes. In glucokinase-overexpressing cells, glucose 6-phosphate levels increased. Moreover, glycogen deposition was greatly enhanced due to the activation of glycogen synthase. Pyruvate kinase was also activated, and fructose-2,6-bisphosphate concentration and lactate production were increased in glucokinase-overexpressing diabetic hepatocytes. Overexpression of hexokinase I did not increase glycogen deposition. In conclusion, hepatocytes from Zucker diabetic fatty rats showed depressed glycogen and glycolytic metabolism, but glucokinase overexpression improved their glucose utilization and storage.     

Ammonia overloading in hepatocytes isolated from liver of fetal and adult rats

Ammonia overloading was investigated during glucose and fructose metabolism in isolated hepatocytes under a variety of metabolic conditions. In all assay conditions, the glycolytic flux and oxygen uptake was not modified by 10 mM ammonia. In hepatocytes isolated from rats fed as libitum, the presence of ammonia caused a decrease in the production of lactate (pyruvate); this effect was not observed in anaerobic incubations, in hepatocytes isolated from starved animals, or in fetal hepatocytes. In spite of an overproduction of urea, ammonia detoxification also takes place by the synthesis of alanine, glutamate and aspartate. Addition of 1 mM aminooxyacetate, an inhibitor of aminotransferases, to the incubation medium prevents the formation of these amino acids, and also prevents the decrease of lactate in hepatocytes isolated from fed animals.     

Desensitization of cyclic GMP-mediated regulation of fatty acid metabolism in hepatocytes from ethanol-fed rats

The mechanisms by which ethanol causes accumulation of hepatic triacylglycerols are complex. It has been proposed that nitric oxide/cyclic GMP signaling pathway may be involved in regulation of fatty acid metabolism in the liver. Here, we investigated if this mechanism may have a role in adaptation to ethanol consumption. Hepatocytes were isolated from rats fed with an ethanol-containing liquid diet and pair-fed control rats, and incubated with a range of concentrations of 8-bromo-cyclic GMP. In both types of cells, this cyclic GMP analog inhibited in parallel fatty acid synthesis de novo and acetyl-CoA carboxylase activity. Addition of 8-bromo-cyclic GMP also decreased the rate of palmitate esterification to triacylglycerols and phospholipids, whereas palmitate oxidation was increased. However, in all these metabolic effects, hepatocytes from ethanol-fed rats were significantly less sensitive to the addition of 8-bromo-cyclic GMP. In order to know if this may be a more general mechanism of adaptation to ethanol, we also studied the effects on glucose metabolism. Similarly, hepatocytes from ethanol-fed rats showed a decreased sensitivity in the inhibition by 8-bromo-cyclic GMP of glycogen synthesis, fatty acid synthesis and the synthesis of glycerol backbone of hepatic triacylglycerols. These data suggest that ethanol consumption induces a desensitization of the regulatory effects mediated by cyclic GMP in fatty acid metabolism, contributing to triacylglycerol accumulation in the liver.     

Pathway of glycogen synthesis from glucose in hepatocytes maintained in primary culture

Glycogen synthesis was examined in primary cultures of adult rat hepatocytes that had been isolated from rats following a 24-h fast. Glycogen synthesis was dependent on the concentration of glucose in the culture medium and also required the presence of insulin. The addition of dexamethasone to the culture medium also increased the amount of glycogen synthesis. When the culture medium was supplemented with [U-14C,3-3H]glucose, it was found that approximately 60% of the glucose incorporated into glycogen was not derived from the pool of labeled glucose. In addition, the relative ratio of 3H/14C in the newly synthesized glycogen was approximately 50% of the ratio of the two isotopes in glucose in the culture medium, indicating that the glucose had undergone metabolism prior to its incorporation into glycogen. However, when hepatocytes were isolated from rats that had been fed ad libitum and the synthesis of glycogen from [U-14C,3-3H]glucose was followed, the relative ratio of the two isotopes in glycogen was similar to that measured for glucose in the culture medium, indicating that the glucose was directly incorporated into glycogen without any apparent metabolism. These results indicate that the synthesis of glycogen from glucose may, at least in part, follow an indirect pathway whereby glucose is metabolized prior to incorporation of the carbon into glycogen, but that the pathway followed for the synthesis of glycogen is dependent on the prior metabolic state of the animal.     

Metabolic effects of proglycosyn.

Proglycosyn, a phenylacyl imidazolium compound that lowers blood glucose levels, was demonstrated previously to promote hepatic glycogen synthesis, stabilize hepatic glycogen stores, activate glycogen synthase, inactivate glycogen phosphorylase, and inhibit glycolysis. In the present study proglycosyn was found to inhibit fatty acid synthesis, stimulate fatty acid oxidation, and lower fructose 2,6-bisphosphate levels, but to have no significant effects on cell swelling and the levels of cAMP in hepatocytes prepared from fed rats. Verapamil and atropine blocked the effects of proglycosyn on glycogen metabolism, but these compounds inhibit proglycosyn accumulation by hepatocytes. Proglycosyn stimulated phosphoprotein phosphatase activity in postmitochondrial extracts, as measured by dephosphorylation of phosphorylase a and glycogen synthase D, but this action required a very high concentration of the compound, making it unlikely to be the actual mechanism involved. It is proposed that a metabolite of proglycosyn is responsible for its metabolic effects.     

A comparison of lipid metabolism in hepatocytes isolated from fed and starved neonatal and adult rats.

1. The utilization of [1-14C]palmitate by hepatocytes prepared from fed and starved neonatal and adult rats has been examined by measuring isotopic incorporation into various products. 2. In cells from fed adult rats the principal products were esters (triglycerides and phospholipids) but ketone bodies were the main metabolic end products in cells from starved adult and fed and starved neonatal rats. Production of triglycerides exceeded that of phospholipids in fed adult cells whereas phospholipid formation always predominated in neonatal cells. 3. The high rate of fatty acid oxidation and hence NADH formation by neonatal cells is reflected by a lower acetoacetate--3-hydroxybutyrate ratio at the earlier stages of incubation of neonatal cells. 4. The addition of glycerol modified quantitatively the products of palmitate metabolism by adult hepatocytes but no such effects were observed with neonatal cells. 5. Compared with adult cells, neonatal hepatocytes showed very low rates of lipogenesis that were only enhanced a little by addition of lactate/pyruvate and did not show any effects of glucose concentration upon incorporation of tritium from 3H2O into lipids.     

Likely individuality of the enzymes catalyzing the phosphorylation of choline and ethanolamine.

Dichloroacetate has effects upon hepatic metabolism which are profoundly different from its effects on heart, skeletal muscle, and adipose tissue metabolism. With hepatocytes prepared from meal-fed rats, dichloroacetate was found to activate pyruvate dehydrogenase, to increase the utilization of lactate and pyruvate without effecting an increase in the net utilization of glucose, to increase the rate of fatty acid synthesis, and to decrease slightly [1-¹⁴C]oleate oxidation to ¹⁴CO₂ without decreasing ketone body formation. With hepatocytes isolated from 48-h-starved rats, dichloroacetate was found to activate pyruvate dehydrogenase, to have no influence on net glucose utilization, to inhibit gluconeogenesis slightly with lactate as substrate, and to stimulate gluconeogenesis significantly with alanine as substrate. The stimulation of fatty acid synthesis by dichloroacetate suggests that the activity of pyruvate dehydrogenase can be rate determining for fatty acid synthesis in isolated liver cells. The minor effects of dichloroacetate on gluconeogenesis suggest that the regulation of pyruvate dehydrogenase is only of marginal importance in the control of gluconeogenesis.     

Fructose-induced hepatic gluconeogenesis: effect of L-carnitine

High fructose feeding (60 g/100 g diet) in rodents induces alterations in both glucose and lipid metabolism. The present study was aimed to evaluate whether intraperitoneal carnitine (CA), a transporter of fatty acyl-CoA into the mitochondria, could attenuate derangements in carbohydrate metabolizing enzymes and glucose overproduction in high fructose-diet fed rats. Male Wistar rats of body weight 150-160 g were divided into 4 groups of 6 rats each. Groups 1 and 4 animals received control diet while the groups 2 and 3 rats received high fructose-diet. Groups 3 and 4 animals were treated with CA (300 mg/Kg body weight/day, i.p.) for 30 days. At the end of the experimental period, levels of carnitine, glucose, insulin, lactate, pyruvate, glycerol, triglycerides and free fatty acids in plasma were determined. The activities of carbohydrate metabolizing enzymes and glycogen content in liver and muscle were assayed. Hepatocytes isolated from liver were studied for the gluconeogenic activity in the presence of substrates such as pyruvate, lactate, glycerol, fructose and alanine. Fructose-diet fed animals showed alterations in glucose metabolizing enzymes, increased circulating levels of gluconeogenic substrates and depletion of glycogen in liver and muscle. There was increased glucose output from hepatocytes of animals fed fructose-diet alone with all the gluconeogenic substrates. The abnormalities associated with fructose feeding such as increased gluconeogenesis, reduced glycogen content and other parameters were brought back to near normal levels by CA. Hepatocytes from these animals showed significant inhibition of glucose production from pyruvate (74.3%), lactate (65.4%), glycerol (69.6%), fructose (56.2%) and alanine (63.6%) as compared to CA untreated fructose-fed animals. The benefits observed could be attributed to the effect of CA on fatty acyl-CoA transport.     

Stimulation of hepatic lipogenesis and acetyl-coenzyme A carboxylase by vasopressin.

The effect of vasopressin on the short-term regulation of fatty acid synthesis was studied in isolated hepatocytes from rats fed ad libitum. Vasopressin stimulates fatty acid synthesis by 30-110%. This increase is comparable with that obtained with insulin. Angiotensin also stimulates fatty acid synthesis, whereas phenylephrine does not. The dose-response curve for vasopressin-stimulated lipogenesis is similar to the dose-response curve for glycogenolysis and release of lactate plus pyruvate. Vasopression also stimulates acetyl-CoA carboxylase activity in a dose-dependent manner. Vasopressin does not relieve glucagon-inhibited lipogenesis, whereas insulin does. The action of vasopressin on hepatic lipogenesis is decreased, but not suppressed, in Ca2+-depleted hepatocytes. The results suggest that vasopressin acts on lipogenesis by increasing availability of lipogenic substrate (lactate + pyruvate) and by activating acetyl-CoA carboxylase.     

Glucose metabolism in perfused skeletal muscle. Effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition.

1. The regulation of glucose uptake and disposition in skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin and exercise, induced by sciatic-nerve stimulation, enhanced glucose uptake about tenfold in fed and starved rats, but were without effect in rats with diabetic ketoacidosis. 3. At rest, the oxidation of lactate (0.44 mumol/min per 30 g muscle in fed rats) was decreased by 75% in both starved and diabetic rats, whereas the release of alanine and lactate (0.41 and 1.35 mumol/min per 30 g respectively in the fed state) was increased. Glycolysis, defined as the sum of lactate+alanine release and lactate oxidation, was not decreased in either starvation or diabetes. 4. In all groups, exercise tripled O2 consumption (from approximately 8 to approximately 25 mumol/min per 30 g of muscle) and increased the release and oxidation of lactate five- to ten-fold. The differences in lactate release between fed, starved and diabetic rats observed at rest were no longer apparent; however, lactate oxidation was still several times greater in the fed group. 5. Perfusion of the hindquarter of a fed rat with palmitate, octanoate or acetoacetate did not alter glucose uptake or lactate release in either resting or exercising muslce; however, lactate oxidation was significantly inhibited by acetoacetate, which also increased the intracellular concentration of acetyl-CoA. 6. The data suggest that neither that neither glycolysis nor the capacity for glucose transport are inhbitied in the perfused hindquarter during starvation or perfusion with fatty acids or ketone bodies. On the other hand, lactate oxidation is inhibited, suggesting diminished activity of pyruvate dehydrogenase. 7. Differences in the regulation of glucose metabolism in heart and skeletal muscle and the role of the glucose/fatty acid cycle in each tissue are discussed.     

Glycogen synthesis by hepatocytes from diabetic rats.

Hepatocytes prepared from streptozotocin- and alloxan-diabetic rats starved for 24 h contain 0.5--2% wet wt. of glycogen. Glycogen synthesis in the hepatocytes from such rats, after prior depletion of the glycogen by glucagon injection, was studied. As distinct from cells from normal animals, there was no glycogen synthesis from glucose as sole substrate, even at concentrations of 60 mM. When supplied with glucose, a gluconeogenic precursor (lactate, dihydroxyacetone or fructose), and with glutamine there was concurrent synthesis of glucose and of glycogen. Without glutamine there was little or no glycogen synthesis. The rate of glycogen formation was in the same range as for cells from control rats. Glutamine addition markedly activated glycogen synthase in cells of starved diabetic rats, but there was no effect on phosphorylase. We obtained very little synthesis of glycogen with hepatocytes from fed diabetic rats, whereas with normal animals, synthesis by such cells equals or exceeds that obtained from starved rats. The conversion of synthase b (inactive) into the active form was studied in rat liver homogenates. The activation of the synthase in cells from starved diabetic rats is somewhat less than that from normal animals, but that from fed diabetic rats is markedly decreased compared with that in livers of fed control animals or that of starved diabetic animals.     

Importance of endogenous substrates for cultured adult rat cardiac myocytes

In Ca-tolerant adult cardiomyocytes the contribution of endogenous substrates (glycogen, tri- and diacylglycerol) to oxidative substrate metabolism was investigated. After 4 h in culture medium (M 199 plus 4% fetal calf serum) the cellular triacylglycerol content is 3.6-fold higher than in fresh myocardium and reflects the free fatty acid composition of the medium. When triacylglycerol is degraded, all long-chain fatty acids are hydrolysed at equal rates. In these quiescent cells, the activity of pyruvate dehydrogenase is low (10% of full activity, in Tyrode solution with 5 mM glucose). Up to 30% of full pyruvate dehydrogenase activity, the contribution of non-lipid substrates (glycogen, glucose, lactate and pyruvate) to oxidative energy production is correlated to pyruvate dehydrogenase activity. At 5 mM medium concentration, glucose, lactate and pyruvate share in energy production the proportions of 15, 36 and 50%, whereas endogenous lipolysis accounts for 78, 61 and 46%. It is concluded that these quiescent cardiomyocytes represent cardiac metabolism in a basal state in which the preference for fatty acids, especially from endogenous lipids, is very pronounced. The utilization of endogenous substrates therefore has to be considered in all studies investigating the oxidative metabolism of these isolated cells.     

Proline and hepatic lipogenesis

The effects of proline on lipogenesis in isolated rat hepatocytes were determined and compared with those of lactate, an established lipogenic precursor. Proline or lactate plus pyruvate increased lipogenesis (measured with 3H2O) in hepatocytes from fed rats depleted of glycogen in vitro and in hepatocytes from starved rats. Lactate plus pyruvate but not proline increased lipogenesis in hepatocytes from starved rats. ( - )-Hydroxycitrate, an inhibitor of ATP-citrate lyase, partially inhibited incorporation into saponifiable fatty acid of 3H from 3H2O and 14C from [U-14C]lactate with hepatocytes from fed rats. Incorporation of 14C from [U-14C]proline was completely inhibited. Similar complete inhibition of incorporation of 14C from [U-14C]proline by ( - )-hydroxycitrate was observed with glycogen-depleted hepatocytes or hepatocytes from starved rats. Inhibition of phosphoenolpyruvate carboxykinase by 3-mercaptopicolinate did not inhibit the incorporation into saponifiable fatty acid of 3H from 3H2O or 14C from [U-14C]proline or [U-14C]lactate. Both 3-mercaptopicolinate and ( - )-hydroxycitrate increased lipogenesis (measured with 3H2O) in the absence or presence of lactate or proline with hepatocytes from starved rats. The results are discussed with reference to the roles of phosphoenolpyruvate carboxykinase, mitochondrial citrate efflux, ATP-citrate lyase and acetyl-CoA carboxylase in proline- or lactate-stimulated lipogenesis.     

Studies on the alpha-adrenergic activation of hepatic glucose output. I. Studies on the alpha-adrenergic activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase in isolated rat liver parenchymal cells.

Epinephrine and the alpha-adrenergic agonist phenylephrine activated phosphorylase, glycogenolysis, and gluconeogenesis from lactate in a dose-dependent manner in isolated rat liver parenchymal cells. The half-maximally active dose of epinephrine was 10-7 M and of phenylephrine was 10(-6) M. These effects were blocked by alpha-adrenergic antagonists including phenoxybenzamine, but were largely unaffected by beta-adrenergic antagonists including propranolol. Epinephrine caused a transient 2-fold elevation of adenosine 3':5'-monophosphate (cAMP) which was abolished by propranolol and other beta blockers, but was unaffected by phenoxybenzamine and other alpha blockers. Phenoxybenzamine and propranolol were shown to be specific for their respective adrenergic receptors and to not affect the actions of glucagon or exogenous cAMP. Neither epinephrine (10-7 M), phenylephrine (10-5 M), nor glucagon (10-7 M) inactivated glycogen synthase in liver cells from fed rats. When the glycogen synthase activity ratio (-glucose 6-phosphate/+ glucose 6-phosphate) was increased from 0.09 to 0.66 by preincubation of such cells with 40 mM glucose, these agents substantially inactivated the enzyme. Incubation of hepatocytes from fed rats resulted in glycogen depletion which was correlated with an increase in the glycogen synthase activity ratio and a decrease in phosphorylase alpha activity. In hepatocytes from fasted animals, the glycogen synthase activity ratio was 0.32 +/- 0.03, and epinephrine, glucagon, and phenylephrine were able to lower this significantly. The effects of epinephrine and phenylephrine on the enzyme were blocked by phenoxybenzamine, but were largely unaffected by propranolol. Maximal phosphorylase activation in hepatocytes from fasted rats incubated with 10(-5) M phenylephrine preceded the maximal inactivation of glycogen synthase. Addition of glucose rapidly reduced, in a dose-dependent manner, both basal and phenylephrine-elevated phosphorylase alpha activity in hepatocytes prepared from fasted rats. Glucose also increased the glycogen synthase activity ratio, but this effect lagged behind the change in phosphorylase. Phenylephrine (10-5 M) and glucagon (5 x 10(-10) M) decreased by one-half the fall in phosphoryalse alpha activity seen with 10 mM glucose and markedly suppressed the elevation of glycogen synthase activity. The following conclusions are drawn from these findings. (a) The effects of epinephrine and phenylephrine on carbohydrate metabolism in rat liver parenchymal cells are mediated predominantly by alpha-adrenergic receptors. (b) Stimulation of these receptors by epinephrine or phenylephrine results in activation of phosphorylase and gluconeogenesis and inactivation of glycogen synthase by mechanisms not involving an increase in cellular cAMP. (c) Activation of beta-adrenergic receptors by epinephrine leads to the accumulation of cAMP, but this is associated with minimal activation of phosphorylase or inactivation of glycogen synthase...     

Regulation of glycogen metabolism in primary cultures of rat hepatocytes. Restoration of acute effects of insulin and glucose in cells from diabetic rats

Defects in the deposition of glycogen and the regulation of glycogen synthesis in the livers of severely insulin-deficient rats can be reversed, in vivo, within hours of insulin administration. Using primary cultures of hepatocytes isolated from normal and diabetic rats in a serum-free chemically defined medium, the present study addresses the chronic action of insulin to facilitate the direct effects of insulin and glucose on the short term regulation of the enzymes controlling glycogen metabolism. Primary cultures were maintained in the presence of insulin, triiodothyronine, and cortisol for 1-3 days. On day 1 in alloxan diabetic cultures, 10(-7) M insulin did not acutely activate glycogen synthase over a period of 15 min or 1 h, whereas insulin acutely activated synthase in cultures of normal hepatocytes. By day 3 in hepatocytes isolated from alloxan diabetic rats, insulin effected an approximate 30% increase in per cent synthase I within 15 min as was also the case for normal cells. The acute effect of insulin on synthase activation was independent of changes in phosphorylase alpha. Whereas glycogen synthase phosphatase activity could not be shown to be acutely affected by insulin, the total activity in diabetic cells was restored to normal control values over the 3-day culture period. The acute effect of 30 mM glucose to activate glycogen synthase in cultured hepatocytes from normal rats after 1 day of culture was missing in hepatocytes isolated from either alloxan or spontaneously diabetic (BB/W) rats. After 3 days in culture, glucose produced a 50% increase in glycogen synthase activity during a 10-min period under the same conditions. These studies clearly demonstrate that insulin acts in a chronic manner in concert with thyroid hormones and steroids to facilitate acute regulation of hepatic glycogen synthesis by both insulin and glucose.     

Effect of alloxan-diabetes on gluconeogenesis and ureogenesis in isolated rabbit liver cells.

1. Neither alloxan-diabetes nor starvation affected the rate of glucose production in hepatocytes incubated with lactate, pyruvate, propionate or fructose as substrates. In contrast, glucose synthesis with either alanine or glutamine was increased nearly 3- and 12-fold respectively, in comparison with that in fed rabbits. 2. The addition of amino-oxyacetate resulted in about a 50% decrease in glucose formation from lactate in hepatocytes isolated from fed, alloxan-diabetic and starved rats, suggesting that both mitochondrial and cytosolic forms of rabbit phosphoenolpyruvate carboxykinase function actively during gluconeogenesis. 3. Alloxan-diabetes resulted in about 2-3-fold stimulation of urea production from either amino acid studied or NH4Cl as NH3 donor, whereas starvation caused a significant increase in the rate of ureogenesis only in the presence of alanine as the source of NH3. 4. As concluded from changes in the [3-hydroxybutyrate]/[acetoacetate] ratio, in hepatocytes from diabetic animals the mitochondrial redox state was shifted toward oxidation in comparison with that observed in liver cells isolated from fed rabbits.