scholarly journals Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport

2003 ◽  
Vol 370 (1) ◽  
pp. 47-56 ◽  
Author(s):  
Richard R. WHITESELL ◽  
Hossein ARDEHALI ◽  
Richard L. PRINTZ ◽  
Joseph M. BEECHEM ◽  
Susan M. KNOBEL ◽  
...  
Keyword(s):  
Glucose Transport ◽  
Insulin Action ◽  
Cytochalasin B ◽  
L6 Myotubes ◽  
Test Models ◽  
Different Types ◽  
Extracellular Glucose ◽  
Low Glucose ◽  
Glucose Phosphorylation ◽  
Intracellular Glucose

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426—1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and d-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15±4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

scholarly journals Hexose metabolism in pancreatic islets. Inhibition of hexokinase

1984 ◽  
Vol 223 (2) ◽  
pp. 447-453 ◽  
Author(s):  
M H Giroix ◽  
A Sener ◽  
D G Pipeleers ◽  
W J Malaisse
Keyword(s):  
Major Fraction ◽  
Intracellular Space ◽  
Extracellular Glucose ◽  
Low Glucose ◽  
Glucose Phosphorylation ◽  
Metabolic Data ◽  
A Minor ◽  
Glycolytic Rate ◽  
State Content ◽  
Hexose Metabolism

In islet homogenates, hexokinase-like activity (Km 0.05 mM; Vmax. 1.5 pmol/min per islet) accounts for the major fraction of glucose phosphorylation. Yet the rate of glycolysis in intact islets incubated at low glucose concentrations (e.g. 1.7 mM) sufficient to saturate hexokinase only represents a minor fraction of the glycolytic rate observed at higher glucose concentrations. This apparent discrepancy between enzymic and metabolic data may be attributable, in part at least, to inhibition of hexokinase in intact islets. Hexokinase, which is present in both islet and purified B-cell homogenates, is indeed inhibited by glucose 6-phosphate (Ki 0.13 mM) and glucose 1,6-bisphosphate (Ki approx. 0.2 mM), but not by fructose 2,6-bisphosphate. In intact islets, the steady-state content of glucose 6-phosphate (0.26-0.79 pmol/islet) and glucose 1,6-bisphosphate (5-48 fmol/islet) increases, in a biphasic manner, at increasing concentrations of extracellular glucose (up to 27.8 mM). From these measurements and the intracellular space of the islets, it was estimated that the rate of glucose phosphorylation as catalysed by hexokinase represents, in intact islets, no more than 12-24% of its value in islet homogenates.

scholarly journals A role for AMPK in increased insulin action after serum starvation

2010 ◽  
Vol 299 (5) ◽  
pp. C1171-C1179 ◽  
Author(s):  
James Kain Ching ◽  
Pooja Rajguru ◽  
Nandhini Marupudi ◽  
Sankha Banerjee ◽  
Jonathan S. Fisher
Keyword(s):  
Glucose Transport ◽  
Insulin Action ◽  
Cellular Response ◽  
Dominant Negative ◽  
Serum Starvation ◽  
Protein Levels ◽  
L6 Myotubes ◽  
Compound C ◽  
Atm Protein ◽  
Starvation Effect

Serum starvation is a common cell culture procedure for increasing cellular response to insulin, though the mechanism for the serum starvation effect is not understood. We hypothesized that factors known to potentiate insulin action [e.g., AMP-activated protein kinase (AMPK) and p38] or to be involved in insulin signaling leading to glucose transport [e.g., Akt, PKCζ, AS160, and ataxia telangiectasia mutated (ATM)] would be phosphorylated during serum starvation and would be responsible for increased insulin action after serum starvation. L6 myotubes were incubated in serum-containing or serum-free medium for 3 h. Levels of phosphorylated AMPK, Akt, and ATM were greater in serum-starved cells than in control cells. Serum starvation did not affect p38, PKCζ, or AS160 phosphorylation or insulin-stimulated Akt or AS160 phosphorylation. Insulin had no effect on glucose transport in control cells but caused an increase in glucose uptake for serum-starved cells that was preventable by compound C (an AMPK inhibitor), by expression of dominant negative AMPK (AMPK-DN), and by KU55933 (an ATM inhibitor). ATM protein levels increased during serum starvation, and this increase in ATM was prevented by compound C and AMPK-DN. Thus, it appears that AMPK is required for the serum starvation-related increase in insulin-stimulated glucose transport, with ATM as a possible downstream effector.

Glucose transport and phosphorylation in single cardiac myocytes: rate-limiting steps in glucose metabolism

1994 ◽  
Vol 266 (3) ◽  
pp. E326-E333 ◽  
Author(s):  
J. Manchester ◽  
X. Kong ◽  
J. Nerbonne ◽  
O. H. Lowry ◽  
J. C. Lawrence
Keyword(s):  
Glucose Metabolism ◽  
Glucose Transport ◽  
Glucose Concentration ◽  
Adult Rat ◽  
Freeze Dried ◽  
Extracellular Glucose ◽  
Order Of Magnitude ◽  
Rate Limiting ◽  
Intracellular Glucose ◽  
In Cells

Microanalytic methods were used to investigate the regulation of glucose metabolism by insulin in single myocytes isolated from adult rat ventricles. Cultured myocytes were incubated with or without insulin and, with either glucose or 2-deoxyglucose (2-DG), rinsed, and freeze-dried. Individual cells were weighed and levels of 2-DG-6-phosphate (2-DG-6-P) or glucose and glucose 6-phosphate (G-6-P) were determined after enzymatic amplification. In cells incubated with 2-DG, insulin increased the level of 2-DG-6-P by as much as 30-fold, indicative of dramatic activation of glucose transport. In cells incubated with glucose, insulin increased the levels of G-6-P by approximately threefold. Increasing extracellular glucose without insulin also increased G-6-P; however, intracellular glucose concentrations were not increased, indicating that glucose transport is rate limiting in nonstimulated myocytes. In contrast, intracellular glucose concentrations were increased by over an order of magnitude by insulin, reaching 60% of the extracellular glucose concentration. Measurements of glucose and G-6-P in the same insulin-treated cells revealed that accumulation of G-6-P reached a plateau when extracellular glucose was increased > 2 mM. At this point the estimated intracellular glucose concentration was 300 microM, or approximately 10 times the Michaelis constant of hexokinase for glucose. These results indicate that in the presence of insulin and physiological concentrations of glucose, hexokinase is saturated with glucose. Consequently, the rate-limiting step for insulin-stimulated glucose utilization is glucose phosphorylation rather than glucose transport.

A model to measure insulin effects on glucose transport and phosphorylation in muscle: a three-tracer study

1996 ◽  
Vol 270 (1) ◽  
pp. E170-E185 ◽  
Author(s):  
M. P. Saccomani ◽  
R. C. Bonadonna ◽  
D. M. Bier ◽  
R. A. DeFronzo ◽  
C. Cobelli
Keyword(s):  
Glucose Transport ◽  
Nonlinear Least Squares ◽  
Distribution Volume ◽  
Human Muscle ◽  
Transmembrane Transport ◽  
Model Parameters ◽  
Identifiability Analysis ◽  
Glucose Phosphorylation ◽  
Intracellular Glucose

We studied five healthy subjects with perfused forearm and euglycemic clamp techniques in combination with a three-tracer (D-[12C]mannitol, not transportable; 3-O-[14C]methyl-D-glucose, transportable but not metabolizable; D-[3-3H]glucose, transportable and metabolizable) intra-arterial pulse injection to assess transmembrane transport and intracellular phosphorylation of glucose in vivo in human muscle. The washout curves of the three tracers were analyzed with a multicompartmental model. A priori identifiability analysis of the tracer model shows that the rate constants of glucose transport into and out of the cells and of glucose phosphorylation are uniquely identifiable. Tracer model parameters were estimated by a nonlinear least-squares parameter estimation technique. We then solved for the tracee model and estimated bidirectional transmembrane transport glucose fluxes, glucose intracellular phosphorylation, extracellular and intracellular volumes of glucose distribution, and extracellular and intracellular glucose concentrations. Physiological hyperinsulinemia (473 +/- 22 pM) caused 2.7-fold (63.1 +/- 7.2 vs. 23.4 +/- 6.1 mumol.min-1.kg-1, P < 0.01) and 5.1-fold (42.5 +/- 5.8 vs. 8.4 +/- 2.2 mumol.min-1.kg-1, P < 0.01) increases in transmembrane influx and intracellular phosphorylation of glucose, respectively. Extracellular distribution volume and concentration of glucose were unchanged, whereas intracellular distribution volume of glucose was increased (approximately 2-fold) and intracellular glucose concentration was almost halved by hyperinsulinemia. In summary, 1) a multicompartment model of three-tracer kinetic data can quantify transmembrane glucose fluxes and intracellular glucose phosphorylation in human muscle; and 2) physiological hyperinsulinemia stimulates both transport and phosphorylation of glucose and, in doing so, amplifies the role of glucose transport as a rate-determining step of muscle glucose uptake.

scholarly journals Effects of insulin on glucose transport and glucose transporters in rat heart

1988 ◽  
Vol 250 (1) ◽  
pp. 277-283 ◽  
Author(s):  
D Zaninetti ◽  
R Greco-Perotto ◽  
F Assimacopoulos-Jeannet ◽  
B Jeanrenaud
Keyword(s):  
Glucose Transport ◽  
Rat Heart ◽  
Plasma Membranes ◽  
Cytochalasin B ◽  
Glucose Transporters ◽  
Hill Coefficient ◽  
Maximal Velocity ◽  
Microsomal Membranes ◽  
The Hill ◽  
Intracellular Glucose

The effect of insulin on glucose transport and glucose transporters was studied in perfused rat heart. Glucose transport was measured by the efflux of labelled 3-O-methylglucose from hearts preloaded with this hexose. Insulin stimulated 3-O-methylglucose transport by: (a) doubling the maximal velocity (Vmax); (b) decreasing the Kd from 6.9 to 2.7 mM; (c) increasing the Hill coefficient toward 3-O-methylglucose from 1.9 to 3.1; (d) increasing the efficiency of the transport process (k constant). Glucose transporters in enriched plasma and microsomal membranes from heart were quantified by the [3H]cytochalasin-B-binding assay. When added to normal hearts, insulin produced the following changes in the glucose transporters: (a) it increased the translocation of transporters from an intracellular pool to the plasma membranes; (b) it increased (from 1.6 to 2.7) the Hill coefficient of the transporters translocated into the plasma membranes toward cytochalasin B, suggesting the existence of a positive co-operativity among the transporters appearing in these membranes; (c) it increased the affinity of the transporters (and hence, possibly, of glucose) for cytochalasin B. The data provide evidence that the stimulatory effect of insulin on glucose transport may be due not to the sole translocation of intracellular glucose transporters to the plasma membrane, but to changes in the functional properties thereof.

scholarly journals Compartmentalization of transport and phosphorylation of glucose in a hepatoma cell line

2005 ◽  
Vol 386 (2) ◽  
pp. 245-253 ◽  
Author(s):  
Richard R. WHITESELL ◽  
Hossein ARDEHALI ◽  
Joseph M. BEECHEM ◽  
Alvin C. POWERS ◽  
Wieb VAN DER MEER ◽  
...  
Keyword(s):  
Cell Line ◽  
Glucose Transport ◽  
Hepatoma Cell ◽  
Compartment Model ◽  
Hepatoma Cell Line ◽  
L6 Myotubes ◽  
H4iie Cells ◽  
Two Compartment Model ◽  
Measured Activity ◽  
Glucose Phosphorylation

The first steps of glucose metabolism are carried out by members of the families of GLUTs (glucose transporters) and HKs (hexokinases). Previous experiments using the inhibitor of glucose transport, CB (cytochalasin B), revealed that compartmentalization of GLUTs and HKs is a major factor in the control of glucose uptake in L6 myotubes [Whitesell, Ardehali, Printz, Beechem, Knobel, Piston, Granner, Van Der Meer, Perriott and May (2003) Biochem. J. 370, 47–56]. In the present paper, we evaluate compartmentalization of GLUTs and HKs in a hepatoma cell line, H4IIE, which is characterized by excess GLUT activity, HKI in a particulate and a cytosolic fraction, and insignificant G6Pase (glucose-6-phosphatase) activity. The measured activity of glucose transport exceeded the rate of phosphorylation approx. 30-fold. Treatment with 25 μM CB (Ki∼3 μM in H4IIE cells) paradoxically increased the excess of GLUTs over phosphorylation (GLUTs are inhibited 80%, while phosphorylation is inhibited 98%). The global relationships of the data could be reconciled most simply by a two-compartment model. In this model, phosphorylation of glucose is carried out by a subset of HK molecules supplied by a subset of GLUTs that are more sensitive to CB than the other GLUTs. The agent, DCC (dicyclohexylcarbodi-imide) caused HKI to translocate from the particulate compartment to the cytosolic compartment and potently inhibited glucose phosphorylation. The particulate compartment may represent the mitochondria, to which the more CB-sensitive GLUTs may control the transport of glucose.

Roles of glucose transport and glucose phosphorylation in muscle insulin resistance of NIDDM

Diabetes ◽  
1996 ◽  
Vol 45 (7) ◽  
pp. 915-925 ◽  
Author(s):  
R. C. Bonadonna ◽  
S. Del Prato ◽  
E. Bonora ◽  
M. P. Saccomani ◽  
G. Gulli ◽  
...  
Keyword(s):  
Insulin Resistance ◽  
Glucose Transport ◽  
Muscle Insulin Resistance ◽  
Glucose Phosphorylation

scholarly journals Effect of Insulin on Proximal Tubules Handling of Glucose: A Systematic Review

2020 ◽  
Vol 2020 ◽  
pp. 1-17 ◽  
Author(s):  
Ricardo Pereira-Moreira ◽  
Elza Muscelli
Keyword(s):  
Glucose Transport ◽  
Insulin Action ◽  
Current Knowledge ◽  
Sglt2 Inhibitor ◽  
Proximal Tubules ◽  
Human Studies ◽  
Glucose Disposal ◽  
Diabetic Patients ◽  
Insulin Effect ◽  
Renal Gluconeogenesis

Renal proximal tubules reabsorb glucose from the glomerular filtrate and release it back into the circulation. Modulation of glomerular filtration and renal glucose disposal are some of the insulin actions, but little is known about a possible insulin effect on tubular glucose reabsorption. This review is aimed at synthesizing the current knowledge about insulin action on glucose handling by proximal tubules. Method. A systematic article selection from Medline (PubMed) and Embase between 2008 and 2019. 180 selected articles were clustered into topics (renal insulin handling, proximal tubule glucose transport, renal gluconeogenesis, and renal insulin resistance). Summary of Results. Insulin upregulates its renal uptake and degradation, and there is probably a renal site-specific insulin action and resistance; studies in diabetic animal models suggest that insulin increases renal SGLT2 protein content; in vivo human studies on glucose transport are few, and results of glucose transporter protein and mRNA contents are conflicting in human kidney biopsies; maximum renal glucose reabsorptive capacity is higher in diabetic patients than in healthy subjects; glucose stimulates SGLT1, SGLT2, and GLUT2 in renal cell cultures while insulin raises SGLT2 protein availability and activity and seems to directly inhibit the SGLT1 activity despite it activating this transporter indirectly. Besides, insulin regulates SGLT2 inhibitor bioavailability, inhibits renal gluconeogenesis, and interferes with Na+K+ATPase activity impacting on glucose transport. Conclusion. Available data points to an important insulin participation in renal glucose handling, including tubular glucose transport, but human studies with reproducible and comparable method are still needed.

Insulin Action on Glucose Transport in Isolated Skeletal Muscle from Patients with Liver Cirrhosis

1994 ◽  
Vol 29 (1) ◽  
pp. 71-76 ◽  
Author(s):  
U. Johansson ◽  
L. S. Eriksson ◽  
D. Galuska ◽  
J. R. Zierath ◽  
H. Wallberg-henriksson
Keyword(s):  
Skeletal Muscle ◽  
Liver Cirrhosis ◽  
Glucose Transport ◽  
Insulin Action
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