scholarly journals The contribution of pyruvate cycling to loss of [6-3H]glucose during conversion of glucose to glycogen in hepatocytes: effects of insulin, glucose and acinar origin of hepatocytes

1993 ◽  
Vol 289 (1) ◽  
pp. 255-262 ◽  
Author(s):  
L Agius ◽  
D Tosh ◽  
M Peak
Keyword(s):  
Guinea Pig ◽  
High Glucose ◽  
Glucose Concentration ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Human Hepatocytes ◽  
Pyruvate Cycling ◽  
Stimulation Of ◽  
In Cells

1. During conversion of [6-3H,U-14C]glucose to glycogen in liver, loss of 6-3H can occur either by cycling via pyruvate (between glycolysis and gluconeogenesis) or by other mechanisms. We used mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, to determine the extent to which pyruvate cycling contributes to loss of 6-3H during glucose conversion to glycogen in hepatocytes. 2. Mercaptopicolinate increased the 3H/14C ratio in glycogen during incubation of rat, guinea pig, pig and human hepatocytes with [6-3H,U-14C]glucose. The increase in the 3H/14C ratio in glycogen caused by mercaptopicolinate was greater in periportal than in perivenous rat hepatocytes, indicating that cycling of glucose via pyruvate is more prominent in cells with a higher gluconeogenic relative to glycolytic capacity. 3. The effect of mercaptopicolinate on the 3H/14C ratio in glycogen was observed both in the absence and in the presence of insulin, indicating that stimulation of glycogen synthesis by insulin is not associated with inhibition of pyruvate cycling. In rat and guinea pig but not in pig hepatocytes, the effects of mercaptopicolinate on the 3H/14C ratio in glycogen were greater at 10-15 mM glucose than at 30 mM glucose, suggesting diminished cycling via pyruvate at high glucose concentrations. 4. Insulin increased the loss of 6-3H during stimulation of conversion of glucose to glycogen in hepatocytes from all species. This was due in part to an increase in pyruvate cycling and in part to other mechanisms that are not inhibited by mercaptopicolinate. 5. These results suggest that pyruvate cycling is a significant, but not exclusive, component of the loss of 6-3H in the hepatocyte during glucose conversion to glycogen. The extent of pyruvate cycling is dependent on the acinar origin of the hepatocytes and on the glucose concentration and presence of insulin.

scholarly journals Positive and Negative Regulation of Phosphoinositide 3-Kinase-Dependent Signaling Pathways by Three Different Gene Products of the p85α Regulatory Subunit

2000 ◽  
Vol 20 (21) ◽  
pp. 8035-8046 ◽  
Author(s):  
Kohjiro Ueki ◽  
Petra Algenstaedt ◽  
Franck Mauvais-Jarvis ◽  
C. Ronald Kahn
Keyword(s):  
Glucose Transport ◽  
Kinase Activity ◽  
Glycogen Synthesis ◽  
Catalytic Subunit ◽  
Regulatory Subunit ◽  
Regulatory Subunits ◽  
Irs Proteins ◽  
Terminal Structure ◽  
Stimulation Of ◽  
In Cells

ABSTRACT Phosphoinositide (PI) 3-kinase is a key mediator of insulin-dependent metabolic actions, including stimulation of glucose transport and glycogen synthesis. The gene for the p85α regulatory subunit yields three splicing variants, p85α, AS53/p55α, and p50α. All three have (i) a C-terminal structure consisting of two Src homology 2 domains flanking the p110 catalytic subunit-binding domain and (ii) a unique N-terminal region of 304, 34, and 6 amino acids, respectively. To determine if these regulatory subunits differ in their effects on enzyme activity and signal transduction from insulin receptor substrate (IRS) proteins under physiological conditions, we expressed each regulatory subunit in fully differentiated L6 myotubes using adenovirus-mediated gene transfer with or without coexpression of the p110α catalytic subunit. PI 3-kinase activity associated with p50α was greater than that associated with p85α or AS53. Increasing the level of p85α or AS53, but not p50α, inhibited both phosphotyrosine-associated and p110-associated PI 3-kinase activities. Expression of a p85α mutant lacking the p110-binding site (Δp85) also inhibited phosphotyrosine-associated PI 3-kinase activity but not p110-associated activity. Insulin stimulation of two kinases downstream from PI-3 kinase, Akt and p70 S6 kinase (p70S6K), was decreased in cells expressing p85α or AS53 but not in cells expressing p50α. Similar inhibition of PI 3-kinase, Akt, and p70S6K was observed, even when p110α was coexpressed with p85α or AS53. Expression of p110α alone dramatically increased glucose transport but decreased glycogen synthase activity. This effect was reduced when p110α was coexpressed with any of the three regulatory subunits. Thus, the three different isoforms of regulatory subunit can relay the signal from IRS proteins to the p110 catalytic subunit with different efficiencies. They also negatively modulate the PI 3-kinase catalytic activity but to different extents, dependent on the unique N-terminal structure of each isoform. These data also suggest the existence of a mechanism by which regulatory subunits modulate the PI 3-kinase-mediated signals, independent of the kinase activity, possibly through subcellular localization of the catalytic subunit or interaction with additional signaling molecules.

scholarly journals Regulation of angiotensin II receptors and PKC isoforms by glucose in rat mesangial cells

1999 ◽  
Vol 276 (5) ◽  
pp. F691-F699 ◽  
Author(s):  
Farhad Amiri ◽  
Raul Garcia
Keyword(s):  
Angiotensin Ii ◽  
High Glucose ◽  
Glucose Concentration ◽  
Mesangial Cells ◽  
Competitive Binding ◽  
Ang Ii ◽  
Pkc Isoforms ◽  
Normal Glucose ◽  
Elevated Glucose ◽  
In Cells

It has been shown that glomerular angiotensin II (ANG II) receptors are downregulated and protein kinase C (PKC) is activated under diabetic conditions. We, therefore, investigated ANG II receptor and PKC isoform regulation in glomerular mesangial cells (MCs) under normal and elevated glucose concentrations. MCs were isolated from collagenase-treated rat glomeruli and cultured in medium containing normal or high glucose concentrations (5.5 and 25.0 mM, respectively). Competitive binding experiments were performed using the ANG II antagonists losartan and PD-123319, and PKC analysis was conducted by Western blotting. Competitive binding studies showed that the AT1 receptor was the only ANG II receptor detected on MCs grown to either subconfluence or confluence under either glucose concentration. AT1 receptor density was significantly downregulated in cells grown to confluence in high-glucose medium. Furthermore, elevated glucose concentration enhanced the presence of all MC PKC isoforms. In addition, PKCβ, PKCγ and PKCε were translocated only in cells cultured in elevated glucose concentrations following 1-min stimulation by ANG II, whereas PKCα, PKCθ, and PKCλ were translocated by ANG II only in cells grown in normal glucose. Moreover, no changes in the translocation of PKCδ, PKCι, PKCζ, and PKCμ were detected in response to ANG II stimulation under euglycemic conditions. We conclude that MCs grown in high glucose concentration show altered ANG II receptor regulation as well as PKC isoform translocation compared with cells grown in normal glucose concentration.

scholarly journals Signalling pathways involved in the stimulation of glycogen synthesis by insulin in rat hepatocytes

Diabetologia ◽  
1998 ◽  
Vol 41 (1) ◽  
pp. 16-25 ◽  
Author(s):  
M. Peak ◽  
J. J. Rochford ◽  
A. C. Borthwick ◽  
S. J. Yeaman ◽  
L. Agius
Keyword(s):  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Signalling Pathways ◽  
Stimulation Of

scholarly journals Induction of mRNA for phosphoenolpyruvate carboxykinase (GTP) by dexamethasone in cultured rat hepatocytes requires on-going protein synthesis

1987 ◽  
Vol 246 (1) ◽  
pp. 237-240 ◽  
Author(s):  
V L Nebes ◽  
S M Morris
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Rat Hepatocytes ◽  
Serum Free Medium ◽  
Free Medium ◽  
Serum Free ◽  
Maximum Induction ◽  
Cultured Rat Hepatocytes ◽  
Time Required ◽  
Necessary And Sufficient ◽  
Stimulation Of

Dexamethasone is necessary and sufficient to induce mRNA for phosphoenolpyruvate carboxykinase (GTP) (PEPCK) by 19-fold in rat hepatocytes cultured in serum-free medium. However, the time required for maximum induction is 16 h. The slow induction suggested that glucocorticoids regulate the expression of an intermediate gene product(s) which is required for glucocorticoid stimulation of PEPCK-gene expression. Consistent with this notion was the finding that cycloheximide completely blocked the response to dexamethasone. In contrast, cycloheximide did not block the response to a cyclic AMP analogue.

scholarly journals Physiological concentrations of 2-oxoglutarate regulate the activity of phosphoenolpyruvate carboxykinase in liver

1992 ◽  
Vol 285 (3) ◽  
pp. 767-771 ◽  
Author(s):  
M A Titheradge ◽  
R A Picking ◽  
R C Haynes
Keyword(s):  
Pyruvate Kinase ◽  
Rat Liver ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Rat Hepatocytes ◽  
Fasted State ◽  
Stimulation Of

2-Oxoglutarate was found to inhibit purified rat liver phosphoenolpyruvate carboxykinase when the assay was performed in the direction of either phosphoenolpyruvate or oxaloacetate synthesis. The inhibition was competitive with respect to oxaloacetate or phosphoenolpyruvate, the Ki values being 0.32 +/- 0.04 mM 0.63 +/- 0.19 mM respectively. 2-Oxoglutarate inhibited non-competitively when tested against GTP or Mn2+. The reported cytosolic concentrations of 2-oxoglutarate in rat hepatocytes are such that the enzyme is likely to be significantly inhibited under basal conditions. The cytosolic concentration of 2-oxoglutarate is known to fall precipitously under the influence of glucagon and other hormones that stimulate gluconeogenesis, and it is suggested that the hormone-induced decrease in 2-oxoglutarate content would alleviate the inhibition of phosphoenolpyruvate carboxykinase and stimulate flux from oxaloacetate to phosphoenolpyruvate. The implications of this finding to the rationalization of the role of pyruvate kinase in the stimulation of gluconeogenesis in the fasted state are discussed.

scholarly journals Comparison of the effects of various amino acids on glycogen synthesis, lipogenesis and ketogenesis in isolated rat hepatocytes

1991 ◽  
Vol 273 (1) ◽  
pp. 57-62 ◽  
Author(s):  
A Baquet ◽  
A Lavoinne ◽  
L Hue
Keyword(s):  
Amino Acids ◽  
Time Course ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Isolated Rat Hepatocytes ◽  
Aminoisobutyric Acid ◽  
Malonyl Coa ◽  
Lag Period ◽  
Isolated Rat ◽  
Stimulation Of

Several amino acids were found to stimulate glycogen synthesis and lipogenesis, and to inhibit ketogenesis in isolated rat hepatocytes. When hepatocytes were incubated in the presence of 20 mM-glucose, the amino acids could be classified in decreasing order of efficiency as follows: glutamine and proline, alanine, aminoisobutyric acid, asparagine and histidine for stimulation of glycogen synthesis; glutamine, proline and alanine for stimulation of lipogenesis; proline and glutamine for inhibition of ketogenesis. The study of the time course revealed that the rates were not linear and were preceded by a lag period. In all conditions studied, glutamine and proline were found to have similar quantitative effects on glycogen synthesis and lipid metabolism. However, their effects differ qualitatively. Indeed, the effects of proline on glycogen synthesis, lipogenesis and glutamate and aspartate content were faster. Moreover, proline increased the hydroxybutyrate/acetoacetate ratio, whereas glutamine did not change it. Incubation of hepatocytes with aminoisobutyric acid or under hypo-osmotic conditions, which increased cell volume and mimicked the amino acid-induced stimulation of glycogen synthesis, had little effect on lipogenesis. In hepatocytes incubated without glucose, ketogenesis was inhibited, in decreasing order of efficiency, by alanine, asparagine, glutamine and proline. Under these conditions, glutamine increased, alanine decreased and asparagine did not affect the concentration of malonyl-CoA. This indicates that the latter cannot be responsible for the inhibition of ketogenesis by alanine and asparagine.

scholarly journals Nitric oxide inhibits glycogen synthesis in isolated rat hepatocytes

1998 ◽  
Vol 330 (2) ◽  
pp. 1045-1049 ◽  
Author(s):  
Fleur SPRANGERS ◽  
P. Hans SAUERWEIN ◽  
A. Johannes ROMIJN ◽  
M. George van WOERKOM ◽  
J. Alfred MEIJER
Keyword(s):  
Nitric Oxide ◽  
Glucose Metabolism ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Rat Hepatocytes ◽  
Inhibitory Effect ◽  
No Donor ◽  
Isolated Rat Hepatocytes ◽  
Intracellular Glucose

There is increasing evidence for the existence of intrahepatic regulation of glucose metabolism by Kupffer cell products. Nitric oxide (NO) is known to inhibit gluconeogenic flux through pyruvate carboxylase and phosphoenolpyruvate carboxykinase. However, NO may also influence glucose metabolism at other levels. Using hepatocytes from fasted rats incubated with the NO-donor S-nitroso-N-acetylpenicillamine, we have now found that the synthesis of glycogen from glucose is even more sensitive to inhibition by NO than gluconeogenesis. Inhibition of glycogen production by NO was accompanied by a rise in intracellular glucose 6-phosphate and UDPglucose. Activity of glycogen synthase, as measured in extracts of hepatocytes after the cells had been exposed to NO, was decreased. Experiments with gel-filtered liver extracts revealed that inhibition of glycogen synthase was caused by an inhibitory effect of NO on the conversion of glycogen synthase b into glycogen synthase a.

scholarly journals Stimulation of [1-14C]oleate oxidation to 14CO2 in isolated rat hepatocytes by the catecholamines, vasopressin and angiotensin. A possible mechanism of action

1983 ◽  
Vol 212 (1) ◽  
pp. 85-91 ◽  
Author(s):  
M C Sugden ◽  
D I Watts
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Tricarboxylic Acid Cycle ◽  
Rat Hepatocytes ◽  
Tricarboxylic Acid ◽  
Isolated Rat Hepatocytes ◽  
Acid Cycle ◽  
14Co2 Production ◽  
Oxoglutarate Dehydrogenase ◽  
Stimulation Of

Adrenaline, noradrenaline, vasopressin and angiotensin increased 14CO2 production from [1-14C]oleate by hepatocytes from fed rats but not by hepatocytes from starved rats. The hormones did not increase 14CO2 production when hepatocytes from fed rats were depleted of glycogen in vitro. Increased 14CO2 production from]1-14C]oleate in response to the hormones was observed when hepatocytes from starved rats were incubated with 3-mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase. 3-Mercaptopicolinate inhibited uptake and esterification of [1-14C]oleate, slightly increased 14CO2 production from [1-14C]oleate and greatly increased the [3-hydroxybutyrate]/[acetoacetate] ratio. In the presence of 3-mercaptopicolinate 14CO2 production in response to the catecholamines was blocked by the α-antagonist phentolamine and required extracellular Ca2+. The effects of vasopressin and angiotensin were also Ca2+-dependent. The actions of the hormones of 14CO2 production from [I-14C]oleate by hepatocytes from starved rats in the presence of 3-mercaptopicolinate thus have the characteristics of the response to the hormones found with hepatocytes from fed rats incubated without 3-mercaptopicolinate. The stimulatory effects of the hormones on 14CO2 production from [1-14C]oleate were not the result of decreased esterification (as the hormones increased esterification) or increased β-oxidation. It is suggested that the effect of the hormones to increase 14CO2 production from [1-14C]oleate are mediated by CA2+-activation of NAD+-linked isocitrate dehydrogenase, the 2-oxoglutarate dehydrogenase complex, and/or electron transport. The results also demonstrate that when the supply of oxaloacetate is limited it is utilized for gluconeogenesis rather than to maintain tricarboxylic acid-cycle flux.

scholarly journals Amylin impairment of insulin effects on glycogen synthesis and phosphoenolpyruvate carboxykinase gene expression in rat primary cultured hepatocytes

1994 ◽  
Vol 304 (2) ◽  
pp. 449-453 ◽  
Author(s):  
S Baqué ◽  
J J Guinovart ◽  
A M Gómez-Foix
Keyword(s):  
Gene Expression ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Mrna Level ◽  
Rat Hepatocytes ◽  
Synthesis Rate ◽  
Cultured Hepatocytes ◽  
Glycogen Accumulation ◽  
Primary Cultured Hepatocytes

The ability of amylin to impair hepatic insulin action is controversial. We have found that the effect of amylin in primary cultured hepatocytes is strongly dependent on the culture conditions. Only in hepatocytes preincubated in the presence of fetal serum did amylin, at concentrations ranging from 1 to 100 nM, reduce insulin-stimulated glycogen synthesis rate and glycogen accumulation without showing direct effects. Neither basal glycogen synthase nor glycogen phosphorylase activity was modified by amylin treatment. Nevertheless, amylin (100 nM) blocked the activation of glycogen synthase by insulin. Amylin also proved capable of opposing the reduction in the expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene induced by insulin, whereas the basal mRNA level of PEPCK was unaffected by amylin treatment. Thus, these results show that, in cultured rat hepatocytes, amylin is indeed able to interfere with insulin regulation of glycogenesis and PEPCK gene expression, favouring the hypothesis that amylin may modulate liver sensitivity to insulin.

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