Palbinone and triterpenes from Moutan Cortex (Paeonia suffruticosa,Paeoniaceae) stimulate glucose uptake and glycogen synthesis via activation of AMPK in insulin-resistant human HepG2 Cells

Moutan Cortex is a well-known herb in traditional Korean, Chinese, and Japanese anti-diabetic formulae. In the current study, we investigated the metabolic effects of isolated triterpenes (1–7) in HepG2 cells under high glucose conditions. These compounds remakably stimulated AMP-activated protein kinase (AMPK), GSK-3β, and ACC phosphorylation. The compounds also increased glucose uptake and enhanced glycogen synthesis. Among these, compound 1 displayed the greatest potential anti-diabetic activity though the AMPK activation pathway. Compound 1 significantly increased the levels of phospho-AMPK, phospho-ACC, and phospho-GSK-3β and stimulated glucose uptake and glycogen synthesis in a dose-dependent manner. In conclusion, our results suggest that these compounds, especially compound 1, may have beneficial roles in glucose metabolism via the AMPK pathway.     

Manganese sulfate-dependent glycosylation of endogenous glycoproteins in human skeletal muscle is catalyzed by a nonglucose 6-P-dependent glycogen synthase and not glycogenin

Glycogenin, a Mn2+-dependent, self-glucosylating protein, is considered to catalyze the initial glucosyl transfer steps in glycogen biogenesis. To study the physiologic significance of this enzyme, measurements of glycogenin mediated glucose transfer to endogenous trichloroacetic acid precipitable material (protein-bound glycogen, i.e., glycoproteins) in human skeletal muscle were attempted. Although glycogenin protein was detected in muscle extracts, activity was not, even after exercise that resulted in marked glycogen depletion. Instead, a MnSO4-dependent glucose transfer to glycoproteins, inhibited by glycogen and UDP-pyridoxal (which do not affect glycogenin), and unaffected by CDP (a potent inhibitor of glycogenin), was consistently detected. MnSO4-dependent activity increased in concert with glycogen synthase fractional activity after prolonged exercise, and the MnSO4-dependent enzyme stimulated glucosylation of glycoproteins with molecular masses lower than those glucosylated by glucose 6-P-dependent glycogen synthase. Addition of purified glucose 6-P-dependent glycogen synthase to the muscle extract did not affect MnSO4-dependent glucose transfer, whereas glycogen synthase antibody completely abolished MnSO4-dependent activity. It is concluded that: (1) MnSO4-dependent glucose transfer to glycoproteins is catalyzed by a nonglucose 6-P-dependent form of glycogen synthase; (2) MnSO4-dependent glycogen synthase has a greater affinity for low molecular mass glycoproteins and may thus play a more important role than glucose 6-P-dependent glycogen synthase in the initial stages of glycogen biogenesis; and (3) glycogenin is generally inactive in human muscle in vivo.     

Influence of glycogen storage on vascular smooth muscle metabolism

The role of glycogen as an oxidative substrate for vascular smooth muscle (VSM) remains controversial. To elucidate the importance of glycogen as an oxidative substrate and the influence of glycogen flux on VSM substrate selection, we systematically altered glycogen levels and measured metabolism of glucose, acetate, and glycogen. Hog carotid arteries with glycogen contents ranging from 1 to 11 micromol/g were isometrically contracted in physiological salt solution containing 5 mM [1-(13)C]glucose and 1 mM [1, 2-(13)C]acetate at 37 degrees C for 6 h. [1-(13)C]glucose, [1, 2-(13)C]acetate, and glycogen oxidation were simultaneously measured with the use of a (13)C-labeled isotopomer analysis of glutamate. Although oxidation of glycogen increased with the glycogen content of the tissue, glycogen oxidation contributed only approximately 10% of the substrate oxidized by VSM. Whereas [1-(13)C]glucose flux, [3-(13)C]lactate production from [1-(13)C]glucose, and [1, 2-(13)C]acetate oxidation were not regulated by glycogen content, [1-(13)C]glucose oxidation was significantly affected by the glycogen content of VSM. However, [1-(13)C]glucose remained the primary ( approximately 40-50%) contributor to substrate oxidation. Therefore, we conclude that glucose is the predominate substrate oxidized by VSM, and glycogen oxidation contributes minimally to substrate oxidation.     

EFFECTS OF GAMMA-HYDROXYBUTYRIC ACID AND OTHER HYPNOTICS ON GLUCOSE UPTAKE IN VIVO AND IN VITRO

Abstract— (1) The effects of gamma-hydroxybutyrate, imidazole-4-acetic acid and pento-barbitone on mouse brain glucose, glycogen and lactate levels have been studied. All the drugs significantly increased the brain glucose content, but did not significantly alter brain glycogen levels. The increase in brain glucose following imidazole-4-acetic acid or hypnotic doses of pentobarbitone was matched by corresponding decreases in the lactate level; this was not the case with gamma-hydroxybutyrate where the total glucose equivalents in the brain, expressed as the tissue level of (glucose) + (lactate/2), were significantly increased.
(2) All drugs except imidazole-4-acetic acid significantly decreased the rate of appearance of [¹⁴C]glucose into the bloodstream in vivo but had no effect on the uptake of glucose into rat diaphragm in vitro when present at 2·5 mM concentration.
(3) Only imidazole-4-acetic acid significantly inhibited glucose uptake into the brain in vivo but at 2·5 mM had no significant effect on glucose uptake into rat cerebral cortical slices in vitro.
(4) It is concluded that the very large increase in brain glucose level observed following the injection of hypnotic doses of gamma-hydroxybutyrate cannot be explained in terms of an increased net uptake of glucose into the brain.     

Palmitate action to inhibit glycogen synthase and stimulate protein phosphatase 2A increases with risk factors for type 2 diabetes

Recent studies have suggested that abnormal regulation of protein phosphatase 2A (PP2A) is associated with Type 2 diabetes in rodent and human tissues. Results with cultured mouse myotubes support a mechanism for palmitate activation of PP2A, leading to activation of glycogen synthase kinase 3. Phosphorylation and inactivation of glycogen synthase by glycogen synthase kinase 3 could be the mechanism for long-chain fatty acid inhibition of insulin-mediated carbohydrate storage in insulin-resistant subjects. Here, we test the effects of palmitic acid on cultured muscle glycogen synthase and PP2A activities. Palmitate inhibition of glycogen synthase fractional activity is increased in subjects with high body mass index compared with subjects with lower body mass index (r = -0.43, P = 0.03). Palmitate action on PP2A varies from inhibition in subjects with decreased 2-h plasma glucose concentration to activation in subjects with increased 2-h plasma glucose concentration (r = 0.45, P < 0.03) during oral glucose tolerance tests. The results do not show an association between palmitate effects on PP2A and glycogen synthase fractional activity. We conclude that subjects at risk for Type 2 diabetes have intrinsic differences in palmitate regulation of at least two enzymes (PP2A and glycogen synthase), contributing to abnormal insulin regulation of glucose metabolism.     

Direct enzymatic procedure for the determination of liver glycogen

A method is proposed to measure glycogen content in liver homogenates without extraction and acid hydrolysis of tissue glycogen. Homogenates were treated with amyloglucosidase, which degrades glycogen to glucose, and the glucose was the determined enzymatically by the use of glucose oxidase and peroxidase. The method was shown to yield nearly complete (99%) recoveries of standard glycogen, while 5 hr of acid hydrolysis of standard glycogen were required to obtain comparable recoveries. When compared to an acid hydrolysis method for liver, amyloglucosidase degradation of rat liver glycogen and subsequent determination of glucose resulted in higher values for glycogen content. The amyloglucosidase, glucose oxidase: peroxidase method has the advantage of rapidity, whereas the traditional method consisting of extraction, precipitation, and acid hydrolysis is not only time consuming, but may also be subject to losses of glycogen in each step.     

Glucose and fatty acid metabolism in McA-RH7777 hepatoma cells vs. rat primary hepatocytes: responsiveness to nutrient availability

The overabundance of dietary fats and simple carbohydrates contributes significantly to obesity and metabolic disorders associated with obesity. The liver balances glucose and lipid distribution, and disruption of this balance plays a key role in these metabolic syndromes. We investigated (1) how hepatocytes balance glucose and fatty acid metabolism when one or both nutrients are supplied in abundance and (2) whether rat hepatoma cells (McA-RH7777) reflect nutrient partitioning in a similar manner as compared with primary hepatocytes. Increasing media palmitate concentration increased fatty acid uptake, triglyceride synthesis and beta-oxidation. However, hepatoma cells had a 2-fold higher fatty acid uptake and a 2-fold lower fatty acid oxidation as compared with primary hepatocytes. McA-RH7777 cells did not synthesize significant amounts of glycogen and preferentially metabolized the glucose into lipids or into oxidation. In primary hepatocytes, the glucose was mostly spared from oxidation and instead partitioned into both de novo glycogen and lipid synthesis. Overall, lipid production was rapidly induced in response to either glucose or fatty acid excess and this may be one of the earliest indicators of metabolic syndrome development associated with nutrient excess.     

Effects of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, on glucose transport and metabolism in skeletal muscle.

The effect of okadaic acid, an inhibitor of protein phosphatases-1 and -2A, was studied on glucose transport and metabolism in soleus muscles isolated from lean and insulin-resistant obese mice. In muscles from lean mice, the uptake of 2-deoxyglucose, an index of glucose transport and phosphorylation, was increased by okadaic acid in a concentration-dependent manner. At 5 microM, okadaic acid was as efficient as a maximally effective insulin concentration. Glucose metabolism (glycolysis and glycogen synthesis) was also measured. Whereas glycolysis was stimulated by okadaic acid, glycogen synthesis was unchanged. When okadaic acid and insulin were added together in the incubation medium, the rates of glucose transport, glycolysis, and glycogen synthesis were similar to those obtained with insulin alone, whether maximal or submaximal insulin concentrations were used. Furthermore, okadaic acid did not activate the kinase activity of the insulin receptor studied in an acellular system or in intact muscles. These results indicate that a step in the insulin-induced stimulation of muscle glucose transport involves a serine/threonine phosphorylation event that is regulated by protein phosphatases-1 and/or -2A. In muscles of insulin-resistant obese mice, the absolute values of deoxyglucose uptake stimulated by okadaic acid were lower than in muscles from lean mice. However, the okadaic acid effect, expressed as a fold stimulation, was normal. These observations suggest that in the insulin-resistant state linked to obesity, the serine/threonine phosphorylation event is likely occurring normally, but a defect at the level of the glucose transporter itself would prevent a normal response to insulin or okadaic acid.     

Increased gluconeogenesis in rats exposed to hyper-G stress

The role of gluconeogenesis on the increase in plasma glucose and liver glycogen of rats exposed to hyper-G (radial acceleration) stress was determined. Overnight-fasted, male Sprague-Dawley rats (250-300 g) were injected i.p. with uniformly labeled 1 4C lactate, alanine, or glycerol (5 microCi/rat) and immediately exposed to 3.1G for 0.25, 0.50, and 1.0 hr. 1 4C incorporation of the labeled substrates into plasma glucose and liver glycogen was measured and compared to uncentrifuged control rats injected in a similar manner. Significant increases in 1 4C incorporation of all three labeled substrates into plasma glucose were observed in centrifuged rats at all exposure periods; 1 4C incorporation into liver glycogen was significantly increased only at 0.50 and 1.0 hr. The i.p. administration (5 mg/100-g body wt) of 5-methoxyindole-2-carboxylic acid, a potent gluconeogenesis inhibitor, prior to centrifugation blocked the increase in plasma glucose and liver glycogen during the first hour of centrifugation. The increase in plasma glucose and liver glycogen was also abolished in adreno-demedullated rats exposed to centrifugation for 1.0 hr. Propranolol, a beta-adrenergic blocker, suppressed the increase in plasma glucose of rats exposed to centrifugation for 0.25 hr. From the results of this study, it is concluded that the initial, rapid rise in plasma glucose as well as the increase in liver glycogen of rats exposed to hyper-G stress can be attributed to an increased rate of gluconeogenesis, and that epinephrine plays a dominant role during the early stages of exposure to centrifugation.     

Interactions of okadaic acid with insulin action in hepatocytes: role of protein phosphatases in insulin action.

The regulation of carbohydrate metabolism involves changes in the phosphorylation state of enzymes. We used okadaic acid, a potent inhibitor of protein phosphatases type 2A (IC50 0.05-2 nM) and type 1 (IC50 10-20 nM) to determine the role of these phosphatases in the control of carbohydrate metabolism by insulin in rat hepatocytes. In the absence of insulin, okadaic acid caused total inhibition of glycogen synthesis at 100 nM and half-maximal inhibition at 8-9 nM. In the presence of insulin, lower concentrations of okadaic acid (to which type 2A phosphatases are sensitive) were effective at inhibiting glycogen synthesis. 2.5 nM okadaic acid caused total inhibition of the 2-fold stimulation of glycogen synthesis by insulin but had no effect on the basal unstimulated rate of glycogen synthesis. This suggests the involvement of type 2A protein phosphatases in the stimulation of glycogen synthesis by insulin. Okadaic acid (5 nM), partially suppressed but did not abolish the increase in glucokinase mRNA levels caused by insulin, indicating that dephosphorylation mechanisms may be involved in the control of glucokinase mRNA levels by insulin. It is concluded that activation of protein phosphatases type 1 and/or type 2A by insulin may have a widespread role in the control of glucose metabolism at various sites.     

鳜对葡萄糖和糊精利用差异比较研究

研究通过比较鳜(Siniperca chuatsi)对不同碳水化合物的利用差异, 探究肉食性鱼类对碳水化合物利用的分子机制。按照1670 mg/kg剂量对鳜灌喂葡萄糖和糊精后, 分别在0、1h、2h、3h、4h、8h、12h和24h收集水样、血浆、肝脏和肌肉, 检测尿糖、血糖、血甘油三酯、血胰岛素、肝糖原、肌糖原含量及糖代谢相关基因表达水平等指标。结果显示: (1) 灌喂后1—12h内, 两组鳜相比, 葡萄糖组尿糖显著高于糊精组, 血糖及胰岛素含量在两组间无显著差异; (2) 两组鳜甘油三酯含量在2h时达到最大值, 糊精组甘油三酯含量在4h时显著高于葡萄糖组, 糊精组肝糖原含量在1h时显著高于葡萄糖组, 且糊精组肌糖原含量在24h内均显著高于葡萄糖组; (3) 灌喂后1h, 灌喂糊精组葡萄糖激酶(Glucokinase, GK)、脂肪酸合成酶(Fatty Acid Synthetase, FAS)、乙酰辅酶A羧化酶Ⅰ型(Acetyl-CoA Carboxylase Type Ⅰ, ACC1)、柠檬酸合成酶(Citroyl Synthetase, CS)基因表达水平显著高于葡萄糖组, 而在灌喂后8h, 糊精组糖原合酶(Glycogen Synthase, GS)和CS基因表达水平却显著低于葡萄糖组。结果表明, 肉食性鱼类鳜摄入糖后可以促进糖原和脂肪的合成, 转化为糖原和甘油三酯, 从而减少未利用糖的排出, 且鳜对葡萄糖的利用效率低于糊精。     

Specific reduction of hepatic glucose 6-phosphate transporter-1 ameliorates diabetes while avoiding complications of glycogen storage disease

D-Glucose-6-phosphatase is a key regulator of endogenous glucose production, and its inhibition may improve glucose control in type 2 diabetes. Herein, 2'-O-(2-methoxy)ethyl-modified phosphorothioate antisense oligonucleotides (ASOs) specific to the glucose 6-phosphate transporter-1 (G6PT1) enabled reduction of hepatic D-Glu-6-phosphatase activity in diabetic ob/ob mice. Treatment with G6PT1 ASOs decreased G6PT1 expression, reduced G6PT1 activity, blunted glucagon-stimulated glucose production, and lowered plasma glucose concentration in a dose-dependent manner. In contrast to G6PT1 knock-out mice and patients with glycogen storage disease, excess hepatic and renal glycogen accumulation, hyperlipidemia, neutropenia, and elevations in plasma lactate and uric acid did not occur. In addition, hypoglycemia was not observed in animals during extended periods of fasting, and the ability of G6PT1 ASO-treated mice to recover from an exogenous insulin challenge was not impaired. Together, these results demonstrate that effective glucose lowering by G6PT1 inhibitors can be achieved without adversely affecting carbohydrate and lipid metabolism.     

Metabolic responses to acute handling by fingerling inland and anadromous striped bass

Fed and 3-day fasted inland (average mass: 6.97 g) and anadromous (average mass: 6.54 g) striped bass Morone saxatilis fingerlings were held in dipnets above water for 5 min in groups of six. Severity of the response to this handling was measured by whole-body glucose, glycogen, and lactic acid in non-handled bass (considered control level), and then at 30 min, 1, 6, 12, 24 and 48 h recovery. At resting levels, both fed and fasted inland bass showed significantly higher concentrations of the whole body variables than anadromous bass. All four groups of bass showed an increase in lactic acid and glucose immediately after handling, with a concomitant decrease in glycogen. Peak levels of glucose and lactic acid were similar in the four groups. Fasting did not have an effect on the glucose and lactic acid responses, but did affect the glycogen response. The two fasted groups did not return to control glycogen concentrations during the 48-h recovery period. By 48 h, both glucose and lactic acid had returned to control levels. It is concluded that inland and anadromous strains of fingerling striped bass do not differ in their sensitivity to an acute handling stress. Recovery of glycogen energy stores following handling is much better if fish are not fasted before handling.     

The role of glucose 6-phosphate in mediating the effects of glucokinase overexpression on hepatic glucose metabolism

Pharmacological activation or overexpression of glucokinase in hepatocytes stimulates glucose phosphorylation, glycolysis and glycogen synthesis. We used an inhibitor of glucose 6-phosphate (Glc6P) hydrolysis, namely the chlorogenic derivative, 1-[2-(4-chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4,5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid (also known as S4048), to determine the contribution of Glc6P concentration, as distinct from glucokinase protein or activity, to the control of glycolysis and glycogen synthesis by glucokinase overexpression. The validity of S4048 for testing the role of Glc6P was supported by its lack of effect on glucokinase binding and its nuclear/cytoplasmic distribution. The stimulation of glycolysis by glucokinase overexpression correlated strongly with glucose phosphorylation, whereas glycogen synthesis correlated strongly with Glc6P concentration. Metabolic control analysis was used to determine the sensitivity of glycogenic flux to glucokinase or Glc6P at varying glucose concentrations (5-20 mm). The concentration control coefficient of glucokinase on Glc6P (1.4-1.7) was relatively independent of glucose concentration, whereas the flux control coefficients of Glc6P (2.4-1.0) and glucokinase (3.7-1.8) on glycogen synthesis decreased with glucose concentration. The high sensitivity of glycogenic flux to Glc6P at low glucose concentration is consistent with covalent modification by Glc6P of both phosphorylase and glycogen synthase. The high control strength of glucokinase on glycogenic flux is explained by its concentration control coefficient on Glc6P and the high control strength of Glc6P on glycogen synthesis. It is suggested that the regulatory strength of pharmacological glucokinase activators on glycogen metabolism can be predicted from their effect on the Glc6P content.     

Carbohydrate energy reserves and effects of food deprivation in male and female rainbow trout

We investigated the effects of nutritional state on carbohydrate, lipid, and protein stores in the heart, liver, and white skeletal muscle of male and female rainbow trout. For fed animals we also partitioned glycogen into fractions based on acid solubility. Fish (10-14 months-old, ~400-500 g) were held at 14 °C and either fed (1% of body weight, every other day) or deprived of food for 14 days. Under fed conditions, glycogen was increased 54% in ventricles from males compared with females, and elevated in the liver (87%) and white muscle (70%) in sexually-maturing versus immature males. Acid soluble glycogen predominated over the acid insoluble fraction in all tissues and was similar between sexes. Food deprivation 1) selectively reduced glycogen and free glucose in male ventricles by ~30%, and 2) did not change glycogen in the liver or white muscle, or triglyceride, protein or water levels in any tissues for both sexes. These data highlight sex differences in teleost cardiac stores and the metabolism of carbohydrates, and contrast with mammals where cardiac glycogen increases during fasting and acid insoluble glycogen is a significant fraction. Increased glycogen in the hearts of male rainbow trout appears to pre-empt sex-specific cardiac growth while storage of acid soluble glycogen may reflect a novel strategy for efficient synthesis and mobilization of glycogen in fishes.     

Mechanism of muscle glycogen autoregulation in humans

To examine the mechanism by which muscle glycogen limits its own synthesis, muscle glycogen and glucose 6-phosphate (G-6-P) concentrations were measured in seven healthy volunteers during a euglycemic ( approximately 5.5 mM)-hyperinsulinemic ( approximately 450 pM) clamp using (13)C/(31)P nuclear magnetic resonance spectroscopy before and after a muscle glycogen loading protocol. Rates of glycogen synthase (V(syn)) and phosphorylase (V(phos)) flux were estimated during a [1-(13)C]glucose (pulse)-unlabeled glucose (chase) infusion. The muscle glycogen loading protocol resulted in a 65% increase in muscle glycogen content that was associated with a twofold increase in fasting plasma lactate concentrations (P < 0.05 vs. basal) and an approximately 30% decrease in plasma free fatty acid concentrations (P < 0.001 vs. basal). Muscle glycogen loading resulted in an approximately 30% decrease in the insulin-stimulated rate of net muscle glycogen synthesis (P < 0.05 vs. basal), which was associated with a twofold increase in intramuscular G-6-P concentration (P < 0.05 vs. basal). Muscle glycogen loading also resulted in an approximately 30% increase in whole body glucose oxidation rates (P < 0.05 vs. basal), whereas there was no effect on insulin-stimulated rates of whole body glucose uptake ( approximately 10.5 mg. kg body wt(-1). min(-1) for both clamps) or glycogen turnover (V(syn)/V(phos) was approximately 23% for both clamps). In conclusion, these data are consistent with the hypothesis that glycogen limits its own synthesis through feedback inhibition of glycogen synthase activity, as reflected by an accumulation of intramuscular G-6-P, which is then shunted into aerobic and anaerobic glycolysis.     

Overexpression of protein targeting to glycogen in cultured human muscle cells stimulates glycogen synthesis independent of glycogen and glucose 6-phosphate levels

There is growing evidence that glycogen targeting subunits of protein phosphatase-1 play a critical role in regulation of glycogen metabolism. In the current study, we have investigated the effects of adenovirus-mediated overexpression of a specific glycogen targeting subunit known as protein targeting to glycogen (PTG) in cultured human muscle cells. PTG was overexpressed both in muscle cells cultured at high glucose (glycogen replete) or in cells incubated for 18 h in the absence of glucose and then incubated in high glucose (glycogen re-synthesizing). In both glycogen replete and glycogen resynthesizing cells, PTG overexpression caused glycogen to be synthesized at a linear rate 1-5 days after viral treatment, while in cells treated with a virus lacking a cDNA insert (control virus), glycogen content reached a plateau at day 1 with no further increase. In the glycogen replete PTG overexpressing cells, glycogen content was 20 times that in controls at day 5. Furthermore, in cells undergoing glycogen resynthesis, PTG overexpression caused a doubling of the initial rate of glycogen synthesis over the first 24 h relative to cells treated with control virus. In both sets of experiments, the effects of PTG on glycogen synthesis were correlated with a 2-3-fold increase in glycogen synthase activity state, with no changes in glycogen phosphorylase activity. The alterations in glycogen synthase activity were not accompanied by changes in the intracellular concentration of glucose 6-phosphate. We conclude that PTG overexpression activates glycogen synthesis in a glucose 6-phosphate-independent manner in human muscle cells while overriding glycogen-mediated inhibition. Our findings suggest that modulation of PTG expression in muscle may be a mechanism for enhancing muscle glucose disposal and improving glucose tolerance in diabetes.     

Glycogen-targeting subunits and glucokinase differentially affect pathways of glycogen metabolism and their regulation in hepatocytes

Overexpression of the glucose-phosphorylating enzyme glucokinase (GK) or members of the family of glycogen-targeting subunits of protein phosphatase-1 increases hepatic glucose disposal and glycogen synthesis. This study was undertaken to evaluate the functional properties of a novel, truncated glycogen-targeting subunit derived from the skeletal muscle isoform G(M)/R(Gl) and to compare pathways of glycogen metabolism and their regulation in cells with overexpressed targeting subunits and GK. When overexpressed in hepatocytes, truncated G(M)/R(Gl) (G(M)DeltaC) was approximately twice as potent as full-length G(M)/R(Gl) in stimulation of glycogen synthesis, but clearly less potent than GK or two other native glycogen-targeting subunits, G(L) and PTG. We also found that cells with overexpressed G(M)DeltaC are unique in that glycogen was efficiently degraded in response to lowering of media glucose concentrations, stimulation with forskolin, or a combination of both maneuvers, whereas cells with overexpressed G(L), PTG, or GK exhibited impairment in one or both of these glycogenolytic signaling pathways. (2)H NMR analysis of purified glycogen revealed that hepatocytes with overexpressed GK synthesized a larger portion of their glycogen from triose phosphates and a smaller portion from tricarboxylic acid cycle intermediates than cells with overexpressed glycogen-targeting subunits. Additional evidence for activation of distinct pathways of glycogen synthesis by GK and targeting subunits is provided by the additive effect of co-overexpression of the two types of proteins upon glycogen synthesis and a much larger stimulation of glucose utilization, glucose transport, and lactate production elicited by GK. We conclude that overexpression of the novel targeting subunit G(M)DeltaC confers unique regulation of glycogen metabolism. Furthermore, targeting subunits and GK stimulate glycogen synthesis by distinct pathways.     

Inhibition of adipose tissue lipolysis increases intramuscular lipid and glycogen use in vivo in humans

This study investigates the consequences of inhibition of adipose tissue lipolysis on skeletal muscle substrate use. Ten subjects were studied at rest and during exercise and subsequent recovery under normal, fasting conditions (control trial, CON) and following administration of a nicotinic acid analog (low plasma free fatty acid trial, LFA). Continuous [U-13C]palmitate and [6,6-2H2]glucose infusions were applied to quantify plasma free fatty acid (FFA) and glucose oxidation rates and to estimate intramuscular triacylglycerol (IMTG) and glycogen use. Muscle biopsies were collected to measure 1) fiber type-specific IMTG content; 2) allosteric regulators of hormone-sensitive lipase (HSL), glycogen phosphorylase, and pyruvate dehydrogenase; and 3) the phosphorylation status of HSL at Ser563 and Ser565. Administration of a nicotinic acid analog (acipimox) substantially reduced plasma FFA rate of appearance and subsequent plasma FFA concentrations (P < 0.0001). At rest, this substantially reduced plasma FFA oxidation rates, which was compensated by an increase in the estimated IMTG use (P < 0.05). During exercise, the progressive increase in FFA rate of appearance, uptake, and oxidation was prevented in the LFA trial and matched by greater IMTG and glycogen use. Differential phosphorylation of HSL or relief of its allosteric inhibition by long-chain fatty acyl-CoA could not explain the increase in muscle TG use, but there was evidence to support the contention that regulation may reside at the level of the glucose-fatty acid cycle. This study confirms the hypothesis that plasma FFA availability regulates both intramuscular lipid and glycogen use in vivo in humans.