Net Glycogen Breakdown Research Articles

Insulin-induced hypoglycemia increases hepatic sensitivity to glucagon in dogs

In individuals with type 1 diabetes, hypoglycemia is a common consequence of overinsulinization. Under conditions of insulin-induced hypoglycemia, glucagon is the most important stimulus for hepatic glucose production. In contrast, during euglycemia, insulin potently inhibits glucagon's effect on the liver. The first aim of the present study was to determine whether low blood sugar augments glucagon's ability to increase glucose production. Using a conscious catheterized dog model, we found that hypoglycemia increased glucagon's ability to overcome the inhibitory effect of insulin on hepatic glucose production by almost 3-fold, an effect exclusively attributable to marked enhancement of the effect of glucagon on net glycogen breakdown. To investigate the molecular mechanism by which this effect comes about, we analyzed hepatic biopsies from the same animals, and found that hypoglycemia resulted in a decrease in insulin signaling. Furthermore, hypoglycemia and glucagon had an additive effect on the activation of AMPK, which was associated with altered activity of the enzymes of glycogen metabolism.

Molecular Characterization of Insulin-Mediated Suppression of Hepatic Glucose Production In Vivo

OBJECTIVEInsulin-mediated suppression of hepatic glucose production (HGP) is associated with sensitive intracellular signaling and molecular inhibition of gluconeogenic (GNG) enzyme mRNA expression. We determined, for the first time, the time course and relevance (to metabolic flux) of these molecular events during physiological hyperinsulinemia in vivo in a large animal model.RESEARCH DESIGN AND METHODS24 h fasted dogs were infused with somatostatin, while insulin (basal or 8× basal) and glucagon (basal) were replaced intraportally. Euglycemia was maintained and glucose metabolism was assessed using tracer, 2H2O, and arterio-venous difference techniques. Studies were terminated at different time points to evaluate insulin signaling and enzyme regulation in the liver.RESULTSHyperinsulinemia reduced HGP due to a rapid transition from net glycogen breakdown to synthesis, which was associated with an increase in glycogen synthase and a decrease in glycogen phosphorylase activity. Thirty minutes of hyperinsulinemia resulted in an increase in phospho-FOXO1, a decrease in GNG enzyme mRNA expression, an increase in F2,6P2, a decrease in fat oxidation, and a transient decrease in net GNG flux. Net GNG flux was restored to basal by 4 h, despite a substantial reduction in PEPCK protein, as gluconeogenically-derived carbon was redirected from lactate efflux to glycogen deposition.CONCLUSIONSIn response to acute physiologic hyperinsulinemia, 1) HGP is suppressed primarily through modulation of glycogen metabolism; 2) a transient reduction in net GNG flux occurs and is explained by increased glycolysis resulting from increased F2,6P2 and decreased fat oxidation; and 3) net GNG flux is not ultimately inhibited by the rise in insulin, despite eventual reduction in PEPCK protein, supporting the concept that PEPCK has poor control strength over the gluconeogenic pathway in vivo.

Fiber-specific responses of muscle glycogen repletion in fasted rats physically active during recovery from high-intensity physical exertion

Mild physical activity performed immediately after a bout of intense exercise in fasting humans results in net glycogen breakdown in their slow oxidative (SO) muscle fibers and glycogen repletion in their fast twitch (FT) fibers. Because several animal species carry a low proportion of SO fibers, it is unclear whether they can also replenish glycogen in their FT fibers under these conditions. Given that most skeletal muscles in rats are poor in SO fibers (<5%), this issue was examined using groups of 24-h fasted Wistar rats (n=10) that swam for 3 min at high intensity with a 10% weight followed by either a 60-min rest (passive recovery, PR) or a 30-min swim with a 0.5% weight (active recovery, AR) preceding a 30-min rest. The 3-min sprint caused 61-79% glycogen fall across the muscles examined, but not in the soleus (SOL). Glycogen repletion during AR without food was similar to PR in the white gastrocnemius (WG), where glycogen increased by 71%, and less than PR in both the red and mixed gastrocnemius (RG, MG). Glycogen fell by 26% during AR in the SOL. Following AR, glycogen increased by 36%, 87%, and 37% in the SOL, RG, and MG, respectively, and this was accompanied by the sustained activation of glycogen synthase and inhibition of glycogen phosphorylase in the RG and MG. These results suggest that mammals with a low proportion of SO fibers can also replenish the glycogen stores of their FT fibers under extreme conditions combining physical activity and fasting.

Exercise in the fasted state facilitates fibre type‐specific intramyocellular lipid breakdown and stimulates glycogen resynthesis in humans

The effects were compared of exercise in the fasted state and exercise with a high rate of carbohydrate intake on intramyocellular triglyceride (IMTG) and glycogen content of human muscle. Using a randomized crossover study design, nine young healthy volunteers participated in two experimental sessions with an interval of 3 weeks. In each session subjects performed 2 h of constant-load bicycle exercise ( approximately 75% ), followed by 4 h of controlled recovery. On one occasion they exercised after an overnight fast (F), and on the other (CHO) they received carbohydrates before ( approximately 150 g) and during (1 g (kg bw)(-1) h(-1)) exercise. In both conditions, subjects ingested 5 g carbohydrates per kg body weight during recovery. Fibre type-specific relative IMTG content was determined by Oil red O staining in needle biopsies from m. vastus lateralis before, immediately after and 4 h after exercise. During F but not during CHO, the exercise bout decreased IMTG content in type I fibres from 18 +/- 2% to 6 +/- 2% (P = 0.007) area lipid staining. Conversely, during recovery, IMTG in type I fibres decreased from 15 +/- 2% to 10 +/- 2% in CHO, but did not change in F. Neither exercise nor recovery changed IMTG in type IIa fibres in any experimental condition. Exercise-induced net glycogen breakdown was similar in F and CHO. However, compared with CHO (11.0 +/- 7.8 mmol kg(-1) h(-1)), mean rate of postexercise muscle glycogen resynthesis was 3-fold greater in F (32.9 +/- 2.7 mmol kg(-1) h(-1), P = 0.01). Furthermore, oral glucose loading during recovery increased plasma insulin markedly more in F (+46.80 microU ml(-1)) than in CHO (+14.63 microU ml(-1), P = 0.02). We conclude that IMTG breakdown during prolonged submaximal exercise in the fasted state takes place predominantly in type I fibres and that this breakdown is prevented in the CHO-fed state. Furthermore, facilitated glucose-induced insulin secretion may contribute to enhanced muscle glycogen resynthesis following exercise in the fasted state.

Effects of short-term improvement of insulin treatment and glycemia on hepatic glycogen metabolism in type 1 diabetes.

Insufficiently treated type 1 diabetic patients exhibit inappropriate postprandial hyperglycemia and reduction in liver glycogen stores. To examine the effect of acute improvement of metabolic control on hepatic glycogen metabolism, lean young type 1 diabetic (HbA1c 8.8 +/- 0.3%) and matched nondiabetic subjects (HbA1c 5.4 +/- 0.1%) were studied during the course of a day with three isocaloric mixed meals. Hepatic glycogen concentrations were determined noninvasively using in vivo 13C nuclear magnetic resonance spectroscopy. Rates of net glycogen synthesis and breakdown were calculated from linear regression of the glycogen concentration time curves from 7:30-10:30 P.M. and from 10:30 P.M. to 8:00 A.M., respectively. The mean plasma glucose concentration was approximately 2.4-fold higher in diabetic than in nondiabetic subjects (13.6 +/- 0.4 vs. 5.8 +/- 0.1 mmol/l, P < 0.001). Rates of net glycogen synthesis and net glycogen breakdown were reduced by approximately 74% (0.11 +/- 0.02 vs. 0.43 +/- 0.04 mmol/l liver/min, P < 0.001) and by approximately 47% (0.10 +/- 0.01 vs. 0.19 +/- 0.01 mmol/l liver/min, P < 0.001) in diabetic patients, respectively. During short-term (24-h) intensified insulin treatment, the mean plasma glucose level was not different between diabetic and nondiabetic subjects (6.4 +/- 0.1 mmol/l). Net glycogen synthesis and breakdown increased by approximately 92% (0.23 +/- 0.04 mmol/l liver/min, P = 0.017) and by approximately 40% (0.14 approximately 0.01 mmol/l liver/min, P = 0.011), respectively. In conclusion, poorly controlled type 1 diabetic patients present with marked reduction in both hepatic glycogen synthesis and breakdown. Both defects in glycogen metabolism are improved but not normalized by short-term restoration of insulinemia and glycemia.

Lipid-dependent control of hepatic glycogen stores in healthy humans.

Non-esterified fatty acids and glycerol could stimulate gluconeogenesis and also contribute to regulating hepatic glycogen stores. We examined their effect on liver glycogen breakdown in humans. After an overnight fast healthy subjects participated in three protocols with lipid/heparin (plasma non-esterified fatty acids: 2.2 +/- 0.1 mol/l; plasma glycerol: 0.5 +/- 0.03 mol/l; n = 7), glycerol (0.4 +/- 0.1 mol/l; 1.5 +/- 0.2 mol/l; n = 5) and saline infusion (control; 0.5 +/- 0.1 mol/l; 0.2 +/- 0.02 mol/l; n = 7). Net rates of glycogen breakdown were calculated from the decrease of liver glycogen within 9 h using 13C nuclear magnetic resonance spectroscopy. Endogenous glucose production was measured with infusion of D-[6,6-2H2]glucose. Endogenous glucose production decreased by about 25 % during lipid and saline infusion (p < 0.005) but not during glycerol infusion (p < 0.001 vs lipid, saline). An increase of plasma non-esterified fatty acids or glycerol reduced the net glycogen breakdown by about 84 % to 0.6 +/- 0.3 micromol x kg(-1) x min(-1) (p < 0.001 vs saline: 3.7 +/- 0.5 micromol x kg(-1) x min(-1)) and by about 46 % to 2.0 +/- 0.4 micromol x kg(-1) x min(-1) (p < 0.01 vs saline and lipid), respectively. Rates of gluconeogenesis increased to 11.5 +/- 0.8 micromol x kg(-1) x min(-1) (p < 0.01) and 12.8 +/- 1.0 micromol x kg(-1) min(-1) (p < 0.01 vs saline: 8.2 +/- 0.7 micromol x l(-1) x min(-1)), respectively. An increase of non-esterifled fatty acid leads to a pronounced inhibition of net hepatic glycogen breakdown and increases gluconeogenesis whereas glucose production does not differ from the control condition. We suggest that this effect is not due to increased availability of glycerol alone but rather to lipid-dependent control of hepatic glycogen stores.

Inhibition of myocardial glucose uptake by cGMP.

Guanosine 3',5'-cyclic monophosphate (cGMP), a second messenger of nitric oxide (NO), regulates myocardial contractility. It is not known whether this effect is accompanied by a change in heart metabolism. We report here the effects of 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP), a cGMP analog, on regulatory steps of glucose metabolism in isolated working rat hearts perfused with glucose as the substrate. When glucose uptake was stimulated by increasing the workload, addition of the cGMP analog totally suppressed this stimulation and accelerated net glycogen breakdown. 8-BrcGMP did not affect pyruvate dehydrogenase activity but activated acetyl-CoA carboxylase, the enzyme that produces malonyl-CoA, an inhibitor of long-chain fatty acid oxidation. To test whether glucose metabolism could also be affected by altering the intracellular concentration of cGMP, we perfused hearts with NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NO synthase, or with S-nitroso-N-acetylpenicillamine (SNAP), a NO donor. Perfusion with L-NAME decreased cGMP and increased glucose uptake by 30%, whereas perfusion with SNAP resulted in opposite effects. None of these conditions affected adenosine 3',5'-cyclic monophosphate concentration. Limitation of glucose uptake by SNAP or 8-BrcGMP decreased heart work, and this was reversed by adding alternative oxidizable substrates (pyruvate, beta-hydroxybutyrate) together with glucose. Therefore, increased NO production decreases myocardial glucose utilization and limits heart work. This effect is mediated by cGMP, which is thus endowed with both physiological and metabolic properties.

Control of glucose utilization in working perfused rat heart.

Metabolic control analyses of glucose utilization were performed for four groups of working rat hearts perfused with Krebs-Henseleit buffer containing 10 mM glucose only, or with the addition of 4 mM D-beta-hydroxybutyrate/1 mM acetoacetate, 100 nM insulin (0.05 unit/ml), or both. Net glycogen breakdown occurred in the glucose group only and was converted to net glycogen synthesis in the presence of all additions. The flux of [2-3H]glucose through P-glucoisomerase (EC 5.3.1.9) was reduced with ketones, elevated with insulin, and unchanged with the combination. Net glycolytic flux was reduced in the presence of ketones and the combination. The flux control coefficients were determined for the portion of the pathway involving glucose transport to the branches of glycogen synthesis and glycolysis. Major control was divided between the glucose transporter and hexokinase (EC 2.7.1.1) in the glucose group. The distribution of the control was slightly shifted to hexokinase with ketones, and control at the glucose transport step was abolished in the presence of insulin. Analysis of the pathway from 3-P-glycerate to pyruvate determined that the major control was shared by enolase (EC 4.2.1.1) and pyruvate kinase (EC 2.7.1.40) in the glucose group. Addition of ketones, insulin, or the combination shifted the control to P-glycerate mutase (EC 5.4.2.1) and pyruvate kinase. These results illustrate that the control of the metabolic flux in glucose metabolism of rat heart is not exerted by a single enzyme but variably distributed among enzymes depending upon substrate availability, hormonal stimulation, or other changes of conditions.

Glycogen metabolism in dog inner medullary collecting ducts.

The regulation of glycogen degradation and synthesis in canine inner medullary collecting ducts (IMCD) was investigated using IMCD tubules suspensions prepared from dog papilla. A small but significant amount of glycogen was found in dog IMCD. Under aerobic condition and especially when no exogenous substrate is available, glycogen breakdown can support IMCD glycolysis for a short period of time. Increasing concentration of exogenous glucose but not lactate was able to reduce and even to suppress (20 mM glucose) the glycogen breakdown. A net synthesis of glycogen was observed only when the endogenous glycogen pool was previously partially or totally depleted. Under anaerobic condition, glycogenolysis was stimulated. The addition of up to 20 mM glucose now reduced but never suppressed this process. Glycogen metabolism responded to variation in the cells energy needs, since the net glycogen breakdown was diminished and glycogen synthesis increased when the cellular ATP turnover was reduced. The reverse effects were observed when the ATP turnover was increased. At all times, glycogen metabolism correlated well with changes in tissue glucose 6-phosphate concentration. The energy requirement of IMCD cells and the availability of alternative energy sources (active mitochondria, exogenous glucose) are therefore capable of eliciting an integrated and appropriate response of glycogen phosphorylase and synthase in IMCD tubules in suspension studied in vitro.

Insulin and glucose suppress hepatic glycogenolysis by distinct enzymatic mechanisms

Both insulin and hyperglycemia can effectively suppress hepatic glucose output (HGO). We examined whether insulin and hyperglycemia specifically suppress liver net glycogen breakdown in a rat model in which glycogen is the major source of HGO. We further examined whether insulin and hyperglycemia act by similar or distinct enzymatic mechanisms. HGO, the rate of net glycogen loss, and glycogen phosphorylase and synthase activities were measured in fed, anesthetized rats infused with saline or insulin (7 mU/min/kg) while either maintaining plasma glucose at basal (7.8 ± 0.2 mmol/L, euglycemic clamp [EC]) or at 10 mmol/L above basal (18 ± 0.4 mmol/L, hyperglycemic clamp [HC]). During the basal period, the rate of HGO in each group was comparable to the rate of net glycogen breakdown, averaging 76 ± 9 and 75 ± 5 μmol/min/kg, respectively. Thus glycogen breakdown appeared to be a major source of ongoing HGO. Over the last 60 minutes of the experimental period, the rate of glycogenolysis averaged 69 ± 8 μmol/min/kg in saline-treated rats; this could account for about 80% of the total HGO. During both EC and HC studies, HGO was suppressed (5.5 ± 3 and −3.6 ± 10 μmol/min/kg, respectively; P < .001 for each). Net glycogen breakdown decreased by 50% in EC rats ( P < .05) and ceased in HC rats ( P < .001). Glycogen synthase was predominantly in the active form in all three experimental groups (87% ± 2%, 89% ± 2%, and 95% ± 3% in saline, EC, and HC rats, respectively). The fraction of glycogen phosphorylase in the active form was similar in saline (41% ± 2%) and EC (40% ± 2%) rats, but lower (23% ± 5%, P < .03) in HC rats. In summary, insulin fully suppresses HGO and partially inhibits net glycogenolysis in vivo without affecting the phosphorylation state of glycogen phosphorylase. Addition of hyperglycemia enhances phosphorylase a to b conversion and fully suppresses net glycogenolysis. Glucose produced by residual glycogenolysis during EC does not reach the systemic circulation and must be disposed of glycolytically in liver.

Acute myocardial ischemia causes a transmural gradient in glucose extraction but not glucose uptake

We assessed the relationship between myocardial glucose metabolism and blood flow during ischemia in eight open-chest swine. Coronary flow was controlled by an extracorporeal perfusion circuit. Left anterior descending coronary arterial (LAD) flow was reduced by 60%, while left circumflex flow was normally perfused. The rate of glucose uptake (Rg) was measured with a coronary infusion of 2-deoxy-D-[14C]glucose and myocardial blood flow with radiolabeled microspheres. Myocardial biopsies were taken after 50 min of ischemia. Regional arterial-venous glucose difference was calculated as Rg per myocardial blood flow. Subendocardial blood flow decreased from 1.27 +/- 0.19 to 0.25 +/- 0.11 ml.g-1.min-1 (P less than 0.0001). The subendocardial arterial-venous glucose difference was greater in the LAD bed (1.38 +/- 0.35 mumol/ml) than the left circumflex coronary arterial perfusion bed (0.10 +/- 03; P less than 0.01); however, there was no statistically significant difference in the rate of glucose uptake between the two beds. Subendocardial glycogen concentration in the LAD perfusion bed was reduced to 26% of circumflex bed values. In conclusion, acute ischemia stimulated a dramatic increase in glucose extraction; however, this did not compensate for the decrease in blood flow, and thus the rate of glucose uptake did not increase significantly. The high rate of glycolysis is primarily supported by accelerated net glycogen breakdown rather than increased glucose uptake.

Substrate cycling between glucose 6-phosphate and glycogen occurs in Schistosoma mansoni

The regulation of glycogen metabolism in Schistosoma mansoni was studied in vitro with special emphasis on the possible occurrence of substrate (‘futile’) cycling. The partition of label between carbon atoms 1 and 6 of the glucose units in glycogen was analysed after the incubation of intact worm pairs in the presence of [6- 14C]glucose. Under all conditions tested, more than 99% of the label in glycogen was still in the 6 position, demonstrating that glycogen was synthesised not via an indirect pathway involving 3-carbon units, but directly, from glucose. Increasing the glucose concentration stimulated glycogen synthase and decreased the activity of glycogen phosphorylase. An inverse relationship was shown between the actual glycogen content and the rate of glycogenesis. Substrate cycling occurred between glucose 6-phosphate and glycogen. Glucose was incorporated into glycogen during periods of net glycogen breakdown, and vice versa: glycogen degradation occurred during periods of net glycogen synthesis. Under our experimental conditions of net glycogen degradation, the rate of glycogen synthesis as a percentage of that of glycogen breakdown was dependent on the external glucose concentration and ranged from 5 to 68% for 2 to 100 mM glucose, respectively. The synthesis of glycogen during periods of net glycogen breakdown was shown to occur in each individual worm pair.

Regulation of Glycogen Metabolism in Polymorphonuclear Leukocytes

Abstract When polymorphonuclear leukocytes were incubated in medium without glucose for 1 hour, glycogen content declined at an essentially constant rate, whereas with 10 mm glucose there was no net change in glycogen during this period. Intracellular glucose 6-phosphate rose during the first 10 min of incubation in both media, but the rate was more than doubled in the presence of glucose. At the same time, ATP levels tended to be higher and those of AMP and orthophosphate lower when glucose was present in the medium than in its absence. The concentration of cyclic AMP (adenosine 3',5'-monophosphate) decreased by about 45% in 5 min whether or not glucose was present. The percentage of glycogen synthetase in the glucose-6-P-independent form was slightly but significantly higher in cells incubated for 10 min with glucose, whereas the total activity was not influenced by the presence of glucose in the medium. Glucose caused a rapid (5 min) decrease in phosphorylase activity which remained depressed for about 30 min and then rose gradually reaching the control level in 60 min. 2-Deoxyglucose procuced a greater depression which was maintained for 60 min. When cells were incubated with latex particles (in the absence of glucose), the rate of glycogen breakdown was accelerated during the first 15 min, but afterward (when phagocytosis was completed), approximated that of the resting cells. During phagocytosis, there was a delay in the accumulation of glucose-6-P, but levels of ATP, ADP, AMP, Pi, cyclic AMP, phosphorylase, and glycogen synthetase (glucose-6-P independent and total) were not significantly different from those in resting cells during the first 15 min. Theophylline, 1 mm, decreased leukocyte phosphorylase activity by 36% in 5 min without altering levels of cyclic AMP or the activation state of glycogen synthetase. Prostaglandin E1 in the presence of theophylline increased phosphorylase activity and cyclic AMP levels and decreased the percentage of glucose-6-P-independent glycogen synthetase. The effect of prostaglandin E1 on phosphorylase activity was observed whether or not either glucose or theophylline, or both, was present in the medium. In cells incubated with 20 mm NaF plus theophylline, levels of phosphorylase and cyclic AMP were increased. In the absence of theophylline, NaF decreased the percentage of glucose-6-P-independent glycogen synthetase and had no measurable effect on cyclic AMP levels. Neither NaF nor prostaglandin E1 significantly increased net glycogen breakdown in 1 hour of incubation. Although the leukocyte phosphorylase and glycogen synthetase activities can be influenced by agents acting through the cyclic AMP system, exogenous glucose modifies the activity of these enzymes without changing cyclic AMP levels. The marked acceleration in glycogen breakdown associated with phagocytosis is probably secondary to increased utilization of glucose-6-P and is not accompanied by changes in these enzymes or in cyclic AMP concentrations. Some data on the effects of glucose-6-P, Mg++, ATP, and orthophosphate on leukocyte glycogen synthetase activity are reported.