A User and Their Family's Perspective of The Use of a Low-Tech Vs A High-Tech AAC System.

This qualitative case study describes a 9-year-old child, diagnosed with homonymous hemianopia, left side weakness and seizures that has been followed by Access to Communication and Technology Unit in Malta for 5 years. The child previously used a communication book and now uses an iPad as a speech generating device. A semi-structured interview was utilised with the parent to explore preference for each AAC system and the reasons for it. The impact of each AAC system on the family and on the child's communication skills, and perceived barriers in the implementation of the AACs were also explored. The child's own experience using the AAC systems was also investigated using a structured interview format. Talking Mats was used to support the child's understanding of the questions and to explore her perspectives on the two AAC systems using Yes-No responses. The parent interview was analysed thematically and represented visually using a thematic network. This was compared with child responses. Four organising themes emerged including barriers, benefits, facilitators, and expectations. Specific barriers included self-funding in order to provide the child with the best fit high-tech AAC. Perceived benefits for both AAC systems were that it increased her communicative intent. The child's mother perceived access to increased vocabulary and capacity for sentence building, operational autonomy as well as voice output as a benefit of the SGD. The child's results indicated a preference for the high-tech AAC because she found it easier to navigate than the low-tech AAC.

Stimulation of glycogen synthesis and inactivation of phosphorylase in hepatocytes by serotonergic mechanisms, and counter-regulation by atypical antipsychotic drugs.

AIMS/HYPOTHESIS: Intraportal infusion of serotonin (5-hydroxytryptamine, 5-HT) or inhibitors of its cellular uptake stimulate hepatic glucose uptake in vivo by either direct or indirect mechanisms. The aims of this study were to determine the direct effects of 5-HT in hepatocytes and to test the hypothesis that atypical antipsychotic drugs that predispose to type 2 diabetes counter-regulate the effects of 5-HT. MATERIALS AND METHODS: Rat hepatocytes were studied in short-term primary culture. RESULTS: Serotonin (5-HT) stimulated glycogen synthesis at nanomolar concentrations but inhibited it at micromolar concentrations. The stimulatory effect was mimicked by alpha-methyl-5-HT, a mixed 5-HT1/5-HT2 receptor agonist, whereas the inhibition was counteracted by a 5-HT2B/2C receptor antagonist. alpha-Methyl-5-HT stimulated glycogen synthesis additively with insulin, but unlike insulin, did not stimulate glucose phosphorylation and glycolysis, nor did it cause Akt (protein kinase B) phosphorylation. Stimulation of glycogen synthesis by alpha-methyl-5-HT correlated with depletion of phosphorylase a. This effect could not be explained by elevated levels of glucose 6-phosphate, which causes inactivation of phosphorylase, but was explained, at least in part, by decreased phosphorylase kinase activity in situ. The antipsychotic drugs clozapine and olanzapine, which bind to 5-HT receptors, counteracted the effect of alpha-methyl-5-HT on phosphorylase inactivation. CONCLUSIONS/INTERPRETATION: This study provides evidence for both stimulation and inhibition of glycogen synthesis in hepatocytes by serotonergic mechanisms. The former effects are associated with the inactivation of phosphorylase and are counteracted by atypical antipsychotic drugs that cause hepatic insulin resistance. Antagonism of hepatic serotonergic mechanisms may be a component of the hepatic dysregulation caused by antipsychotic drugs that predispose to type 2 diabetes.

The role of protein kinase B/Akt in insulin-induced inactivation of phosphorylase in rat hepatocytes.

AIMS/HYPOTHESIS: An insulin signalling pathway leading from activation of protein kinase B (PKB, also known as Akt) to phosphorylation (inactivation) of glycogen synthase kinase-3 (GSK-3) and activation of glycogen synthase is well characterised. However, in hepatocytes, inactivation of GSK-3 is not the main mechanism by which insulin stimulates glycogen synthesis. We therefore tested whether activation of PKB causes inactivation of glycogen phosphorylase. MATERIALS AND METHODS: We used a conditionally active form of PKB, produced using recombinant adenovirus, to test the role of acute PKB activation in the control of glycogen phosphorylase and glycogen synthesis in hepatocytes. RESULTS: Conditional activation of PKB mimicked the inactivation of phosphorylase, the activation of glycogen synthase, and the stimulation of glycogen synthesis caused by insulin. In contrast, inhibition of GSK-3 caused activation of glycogen synthase but did not mimic the stimulation of glycogen synthesis by insulin. PKB activation and GSK-3 inhibition had additive effects on the activation of glycogen synthase, indicating convergent mechanisms downstream of PKB involving inactivation of either phosphorylase or GSK-3. Glycogen synthesis correlated inversely with the activity of phosphorylase-a, irrespective of whether this was modulated by insulin, by PKB activation or by a selective phosphorylase ligand, supporting an essential role for phosphorylase inactivation in the glycogenic action of insulin in hepatocytes. CONCLUSIONS/INTERPRETATION: In hepatocytes, the acute activation of PKB, but not the inhibition of GSK-3, mimics the stimulation of glycogen synthesis by insulin. This is explained by a pathway downstream of PKB leading to inactivation of phosphorylase, activation of glycogen synthase, and stimulation of glycogen synthesis, independent of the GSK-3 pathway.

Justification of glioma biology beyond a cellular basis of interpretation.

Gliomas as neoplasms primarily arising from and constituted by glial cells would appear to implicate cell types that inherently reflect variation of aspects of a putative reparative process. The prominence of an astrocytic type cell of origin would further perhaps constitute a system of malignant transformation based on aberrant progression in cell proliferation and of cell pathology related to aspects on one hand of a gliosis and on the other of an autonomous process of progressiveness. In such terms, perhaps, one might consider the molecular aspects of gliomatous pathogenesis as simply a process of integral aberration of various aspects of astrocytic or glial cell responsiveness outside the normal confines of the normal reparative process and inherently beyond a strict cellular basis of interpretation in pathobiologic terms of such processes as anti-apoptosis and amplification of growth factor receptivity.

Towards a pathogenetic definition of multiple sclerosis in terms of integrative transformation of generic pathologic processes.

The multiple sclerosis disease process is presented in the light of a generic pathobiologic phenomenon on equal terms with such other phenomena as ischemia, neoplasia, infection and congenital malformation, with the added improviso that it would also constitute a true form of integrative resolution of these other generic phenomena towards a real transformation of events in terms of both cellular forms of injury and also in terms of pathogenesis and evolution. Perhaps, in the final analysis, a putative infectious agent is implicated in multiple sclerosis whose peculiar attributes are reflected in a distinctive immunologic response beyond even the conventional concepts of either hypersensitivity or of immunologic surveillance. In a sense, perhaps, a particular patient immunologic constitution in an environment conducive to exposure to a putative agent results in a form of integration of this agent leading to persistent demyelinative relapse of the affected oligodendrocyte on an essential background of a distinct plaque centered on a specific vessel of supply. The recognition of the oligodendrocyte as a specialized astrocyte might in itself strictly characterize such a generic demyelinative process of multiple sclerosis in terms of its essential remitting/relapsing course.

Is beta-amyloid fibrillogenesis a strict process of deposition inherently interactive in molecular terms?

Amyloid fibrillogenesis as a process of interactive molecular processes of deposition in Alzheimer's disease might function as a phenomenon that transforms intracellular amyloid segregation to a state of equilibration with extracellular deposition. beta-Amyloidosis might dynamically implicate loss of viability of vascular tunica media myofibers as a strict reflection of loss of viability of neurons in such an overall system of equilibration between intracellular and extracellular amyloid fibrillogenesis. In terms beyond simple concepts of strict biophysical equilibration, deposition of beta-amyloid in Alzheimer's disease might constitute a phenomenon of congophilic angiopathy as a strict pathobiologic index of activity of the Alzheimer process; such a correlate would perhaps involve a quantitative index that would qualitatively characterize the Alzheimer process as an interactive series of reactions between the intracellular and extracellular microenvironment.

A concept of essentially secondary factors central to definitive subtype characterization of Hodgkin's disease.

Conceptually, Hodgkin's disease would appear to constitute a neoplasm that integrally incorporates responsive cellular elements in terms strictly of morphologic patterns of interaction resulting especially in a predictable scale of therapeutic and prognostic implications as related particularly to pathobiologic course and response to treatment. In this sense, Hodgkin's disease might in a real sense constitute a valid point of reference in terms of processes not only in the generation of the neoplastic process but particularly in terms of how such a neoplastic process interacts with host systems in specifically characterizing the very nature of the intrinsic neoplastic process itself. In this manner, therefore, it might be valid to consider the totality of manifestations of Hodgkin's disease as a fundamental series of processes that integrally determines the genesis and nature of the neoplastic process as a function especially of various host systems in response to that neoplasm. In terms strictly related to a viral or Epstein-Barr virus-related development of Hodgkin's disease, it might perhaps be true that transcription factor NF-kappaB might induce the signaling of a CD30 receptor pathway that is intrinsically linked with anti-apoptosis of germinal center lymphocytes. In overall terms, perhaps, the Epstein-Barr viral nuclear antigens such as Latent membrane protein 1 would activate NF-kappaB as a mechanism that induces anti-apoptotic effect by multiple pathways in the added context particularly of a concerted series of cytokines that secondarily regulate T lymphocyte response. Indeed, in simple terms, Hodgkin's disease would appear to involve a basic mechanism of induced transcription as an effective anti-apoptotic mechanism as exerted on Reed-Sternberg cells. The Epstein-Barr viral nuclear antigens might be pivotal in orchestrating a full series of cytokine and T lymphocyte responses that would perhaps contribute significantly to the effective perpetuation of such anti-apoptotic effect or effects as exerted on the Reed-Sternberg cells.

Hepatic glycogen synthesis is highly sensitive to phosphorylase activity: evidence from metabolic control analysis.

We used metabolic control analysis to determine the flux control coefficient of phosphorylase on glycogen synthesis in hepatocytes by titration with a specific phosphorylase inhibitor (CP-91149) or by expression of muscle phosphorylase using recombinant adenovirus. The muscle isoform was used because it is catalytically active in the b-state. CP-91149 inactivated phosphorylase with sequential activation of glycogen synthase. It increased glycogen synthesis by 7-fold at 5 mm glucose and by 2-fold at 20 mm glucose with a decrease in the concentration of glucose causing half-maximal rate (S(0.5)) from 26 to 19 mm. Muscle phosphorylase was expressed in hepatocytes mainly in the b-state. Low levels of phosphorylase expression inhibited glycogen synthesis by 50%, with little further inhibition at higher enzyme expression, and caused inactivation of glycogen synthase that was reversed by CP-91149. At endogenous activity, phosphorylase has a very high (greater than unity) negative control coefficient on glycogen synthesis, regardless of whether it is determined by enzyme inactivation or overexpression. This high control is attenuated by glucokinase overexpression, indicating dependence on other enzymes with high control. The high control coefficient of phosphorylase on glycogen synthesis affirms that phosphorylase is a strong candidate target for controlling hyperglycemia in type 2 diabetes in both the absorptive and postabsorptive states.

Occurrence of paradoxical or sustained control by an enzyme when overexpressed: necessary conditions and experimental evidence with regard to hepatic glucokinase.

It is widely assumed that the control coefficient of an enzyme on pathway flux decreases as the concentration of enzyme increases. However, it has been shown [Kholodenko and Brown (1996) Biochem. J. 314, 753-760] that enzymes with sigmoidal kinetics can maintain or even gain control with an increase in enzyme activity or concentration. This has been described as 'paradoxical control'. Here we formulate the general requirements for allosteric enzyme kinetics to display this behaviour. We show that a necessary condition is that the Hill coefficient of the enzyme should increase with an increase in substrate concentration or decrease with an increase in product concentration. We also describe the necessary and sufficient requirements for the occurrence of paradoxical control in terms of the flux control coefficients and the derivatives of the elasticities. The derived expression shows that the higher the control coefficient of an allosteric enzyme, the more likely it is that the pathway will display this behaviour. Control of pathway flux is generally shared between a large number of enzymes and therefore the likelihood of observing sustained or increased control is low, even if the kinetic parameters are in the most favourable range to generate the phenomenon. We show that hepatic glucokinase, which has a very high flux control coefficient and displays sigmoidal behaviour within the hepatocyte in situ as a result of interaction with a regulatory protein, displays sustained or increased control over an extended range of enzyme concentrations when the regulatory protein is overexpressed.

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

We investigated the subcellular localization, mobility, and activity of glucokinase in MIN6 cells, a glucose-responsive insulin-secreting beta-cell line. Glucokinase is present in the cytoplasm and a vesicular/granule compartment that is partially colocalized with insulin granules. The granular staining of glucokinase is preserved after permeabilization of the cells with digitonin. There was no evidence for changes in distribution of glucokinase between the cytoplasm and the granule compartment during incubation of the cells with glucose. The rate of release of glucokinase and of phosphoglucoisomerase from digitonin-permeabilized cells was slower when cells were incubated at an elevated glucose concentration (S0.5 approximately 15 mmol/l). This effect of glucose was counteracted by competitive inhibitors of glucokinase (5-thioglucose and mannoheptulose) but was unaffected by fructose analogs and may be due to changes in cell shape or conformation of the cytoskeleton that are secondary to glucose metabolism. Based on the similar release of glucokinase and phosphoglucoisomerase, we found no evidence for specific binding of cytoplasmic digitonin-extractable glucokinase. The affinity of beta-cells for glucose is slightly lower than that in cell extracts and, unlike that in hepatocytes, is unaffected by fructose, tagatose, or a high-K+ medium, which is consistent with the lack of change in glucokinase distribution or release. We conclude that glucokinase is present in two locations, cytoplasm and the granular compartment, and that it does not translocate between them. This conclusion is consistent with the lack of adaptive changes in the glucose phosphorylation affinity. The glucokinase activity associated with the insulin granules may have a role in either direct or indirect coupling between glucose phosphorylation and insulin secretion.

Use of alpha-toxin from Staphylococcus aureus to test for channelling of intermediates of glycolysis between glucokinase and aldolase in hepatocytes.

We investigated whether hepatocytes permeabilized with alpha-toxin from Staphylococcus aureus are a valid model for studying the channelling of intermediates of glycolysis between glucokinase and triosephosphate isomerase. These cells are permeable to 2-aminoisobutyrate, ATP, glucose 6-phosphate (Glc6P) and fructose 2, 6-bisphosphate [Fru(2,6)P(2)], but maintain cell integrity in the presence of ATP as judged by the retention of cytoplasmic enzymes. During incubation with 25 mM glucose, an ATP-generating system and saturating concentrations of Fru(2,6)P(2), rates of detritiation of [2-(3)H]glucose and [3-(3)H]glucose were similar. Exogenous Glc6P (1 mM) and to a lesser extent fructose 6-phosphate, but not Fru(1, 6)P(2), decreased the rate of detritiation of [3-(3)H]glucose. During incubation with 25 mM glucose and Glc6P (0.2-1 mM), with either [3-(3)H]glucose or [3-(3)H]Glc6P as labelled substrate, there was dilution of metabolism of [3-(3)H]glucose with increasing Glc6P but no overall increase in glycolytic flux from glucose and Glc6P, indicating that glycolysis is apparently saturated with Glc6P despite the permeability of the cells to this metabolite. These findings could be explained by partial channelling of Glc6P between glucokinase and glycolysis in the presence of saturating concentrations of Fru(2,6)P(2). They provide an alternative explanation for the concept that there is more than one Glc6P pool.

Impaired glycogen synthesis in hepatocytes from Zucker fatty fa/fa rats: the role of increased phosphorylase activity.

AIMS/HYPOTHESIS: The Zucker fatty fa/fa rat develops hyperinsulinaemia, insulin-resistance and severe obesity as a result of a homozygous mutation in the leptin receptor gene. The aim was to characterise the metabolic defect(s) in hepatocytes from fa/fa rats. METHODS: Glucose metabolism and key regulatory enzymes were investigated in hepatocytes from fa/fa and Fa/? rats after short-term culture in the absence of insulin. RESULTS: Hepatocytes from fa/fa rats have higher glucokinase activity and expression of the glucokinase regulatory protein and higher rates of glycolysis and lipogenesis, but lower rates of glycogen synthesis than hepatocytes from Fa/? controls. Insulin caused a similar stimulation of glycogen synthesis in hepatocytes from fa/fa rats as in controls ( > twofold) but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Adenovirus-mediated glucokinase overexpression stimulated glycogen synthesis and glycolysis but aggravated rather than abolished the relative impairment of glycogen synthesis in cells from fa/fa rats. Inhibition of glycolysis with 2,5-anhydromannitol, an inhibitor of glycolysis and gluconeogenesis, increased glucose 6-phosphate concentrations and glycogen synthesis in hepatocytes from Fa/? and fa/fa rats but did not restore the impaired glycogen synthesis in cells from fa/fa rats. Hepatocytes from fa/fa rats had a higher activity of phosphorylase a in the basal state and after incubation with insulin or glucagon and higher total phosphorylase. CONCLUSION/INTERPRETATION: The increased activity of phosphorylase is a major contributing factor to the impaired glycogen synthesis in hepatocytes from fa/fa rats and could contribute to the lipogenic state by a glycogenolytic-glycolytic-lipogenic pathway.

The role of the regulatory protein of glucokinase in the glucose sensory mechanism of the hepatocyte.

Glucokinase has a very high flux control coefficient (greater than unity) on glycogen synthesis from glucose in hepatocytes (Agius et al., J. Biol. Chem. 271, 30479-30486, 1996). Hepatic glucokinase is inhibited by a 68-kDa glucokinase regulatory protein (GKRP) that is expressed in molar excess. To establish the relative control exerted by glucokinase and GKRP, we applied metabolic control analysis to determine the flux control coefficient of GKRP on glucose metabolism in hepatocytes. Adenovirus-mediated overexpression of GKRP (by up to 2-fold above endogenous levels) increased glucokinase binding and inhibited glucose phosphorylation, glycolysis, and glycogen synthesis over a wide range of concentrations of glucose and sorbitol. It decreased the affinity of glucokinase translocation for glucose and increased the control coefficient of glucokinase on glycogen synthesis. GKRP had a negative control coefficient of glycogen synthesis that is slightly greater than unity (-1.2) and a control coefficient on glycolysis of -0.5. The control coefficient of GKRP on glycogen synthesis decreased with increasing glucokinase overexpression (4-fold) at elevated glucose concentration (35 mM), which favors dissociation of glucokinase from GKRP, but not at 7.5 mM glucose. Under the latter conditions, glucokinase and GKRP have large and inverse control coefficients on glycogen synthesis, suggesting that a large component of the positive control coefficient of glucokinase is counterbalanced by the negative coefficient of GKRP. It is concluded that glucokinase and GKRP exert reciprocal control; therefore, mutations in GKRP affecting the expression or function of the protein may impact the phenotype even in the heterozygote state, similar to glucokinase mutations in maturity onset diabetes of the young type 2. Our results show that the mechanism comprising glucokinase and GKRP confers a markedly extended responsiveness and sensitivity to changes in glucose concentration on the hepatocyte.

Investigation of the mechanism by which glucose analogues cause translocation of glucokinase in hepatocytes: evidence for two glucose binding sites.

Glucokinase translocates between the cytoplasm and nucleus of hepatocytes where it is bound to a 68 kDa protein. The mechanism by which glucose induces translocation of glucokinase from the nucleus was investigated using glucose analogues that are not phosphorylated by glucokinase. There was strong synergism on glucokinase translocation between effects of glucose analogues (glucosamine, 5-thioglucose, mannoheptulose) and sorbitol, a precursor of fructose 1-phosphate. In the absence of glucose or glucose analogues, sorbitol had a smaller effect than glucose on translocation. However, sorbitol potentiated the effects of glucose analogues. In the absence of sorbitol the effect of glucose on glucokinase translocation is sigmoidal with a Hill coefficient of 1.9 suggesting involvement of two glucose-binding sites. The effects of glucosamine and 5-thioglucose were also sigmoidal but with lower Hill Coefficients. In the presence of sorbitol, the effects of glucose, glucosamine and 5-thioglucose were hyperbolic. Mannoheptulose, unlike the other glucose analogues, had a hyperbolic effect on glucokinase translocation in the absence of sorbitol suggesting interaction with one site and was synergistic rather than competitive with glucose. The results favour a two-site model for glucokinase translocation involving either two glucose-binding sites or one binding-site for glucose and one for fructose 1-phosphate. The glucose analogues differed in their effects on the kinetics of purified glucokinase. Mannoheptulose caused the greatest decrease in co-operativity of glucokinase for glucose whereas N-acetylglucosamine had the smallest effect. The anomalous effects of mannoheptulose on glucokinase translocation and on the kinetics of purified glucokinase could be explained by a second glucose-binding site on glucokinase.

Glucose-6-phosphatase overexpression lowers glucose 6-phosphate and inhibits glycogen synthesis and glycolysis in hepatocytes without affecting glucokinase translocation. Evidence against feedback inhibition of glucokinase.

In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)(1) have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4-fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.

Inhibition of rat hepatocyte proliferation by transforming growth factor beta and glucagon is associated with inhibition of ERK2 and p70 S6 kinase.

Stimulation of hepatocyte proliferation by epidermal growth factor (EGF) and insulin is inhibited by transforming growth factor beta (TGF-beta) and by glucagon. It is also suppressed by inhibitors of various protein kinases, including rapamycin, which blocks activation of p70 S6 kinase (p70(S6k)), PD98059, which inhibits the activation of extracellular-regulated kinase (ERK), and SB 203580, an inhibitor of the p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated whether the inhibition of proliferation by TGF-beta involves these protein kinase cascades. Culture of hepatocytes with TGF-beta for 16 hours decreased the stimulation by EGF of ERK2 and p70(S6k) (by 50% and 35%, respectively), but did not affect the stimulation of either p38 MAPK, c-jun NH2-terminal kinase (JNK), or protein kinase B (PKB). Culture of hepatocytes with glucagon for 16 hours also inhibited the stimulation by EGF of activation of ERK2 and p70(S6k) (by approximately 50%). The inhibitory effects of glucagon were observed when the hormone was added either 10 minutes or 60 minutes before EGF addition, whereas no effects of TGF-beta were observed after 10-minute or 60-minute incubation. These results suggest that the inhibition of hepatocyte proliferation by TGF-beta may be in part mediated by inhibition of ERK2 and p70(S6k), but does not involve PKB, JNK, or p38 MAPK. Unlike glucagon, the effects of TGF-beta are not elicited in response to short-term treatment.

Leptin enhances glycogen storage in hepatocytes by inhibition of phosphorylase and exerts an additive effect with insulin.

The effects of the adipocyte-derived hormone leptin on glucose metabolism in hepatocytes were investigated. Incubation of hepatocytes from Wistar rats with leptin for 16 h caused a dose-dependent increase in incorporation of [14C]glucose into glycogen, with a maximal effect at 30 nmol/l leptin. This effect of leptin was observed over a range of glucose concentrations (10-25 mmol/l) and was associated with stimulation of net glycogen deposition. It was not counteracted by mercaptopicolinate, an inhibitor of phosphoenolpyruvate carboxykinase, indicating that it is not due to increased gluconeogenic flux. Leptin also enhanced the short-term stimulation of glycogen synthesis by insulin. These effects of leptin were associated with inhibition of phosphorylase a, which occurred after 4 h of exposure to leptin. Culture with leptin for 16 h did not affect the activities of glucose-6-phosphatase or glucokinase or the activation state of glycogen synthase. Leptin did not affect glycolysis determined from the detritiation of [3-(3)H]glucose. The inhibitory effects of leptin on phosphorylase a were counteracted by short-term incubation with glucagon but were additive with the inhibitory effects of insulin and also with the inhibition caused by resorcinol (25 pmol/l), which inhibits phosphorylase kinase by a mechanism analogous to the antidiabetic drug proglycosyn. These results show that leptin has additive effects with insulin in inhibiting phosphorylase and stimulating glycogen storage in hepatocytes, indicating that these hormones act by separate but convergent mechanisms. It is concluded that the primary action of leptin in hepatocytes is to enhance glycogen storage. This may be an important compensatory mechanism for the inhibition of insulin secretion.

Evidence for glucose and sorbitol-induced nuclear export of glucokinase regulatory protein in hepatocytes.

Glucokinase is rapidly exported from the nucleus of hepatocytes in response to a rise in glucose or fructose 1-P. We demonstrate using confocal microscopy and quantitative imaging that in contrast to previous findings, the regulatory protein of glucokinase (GKRP) also translocates from the nucleus during substrate-induced translocation of glucokinase. However, the fractional decrease in nuclear GKRP is smaller than for glucokinase and is determined by the metabolic state and not by the distribution of glucokinase. Translocation of glucokinase and GKRP is not inhibited by leptomycin B, an inhibitor of exportin-1 function. These findings highlight the importance of quantitative imaging for determining nuclear export of proteins and suggest that GKRP may have a role in nuclear export or import of glucokinase.

The physiological role of glucokinase binding and translocation in hepatocytes.

The compartmentation of glucokinase in the hepatocyte is regulated by the extracellular glucose concentration and by substrates that alter the concentration of fructose 1-phosphate in the hepatocyte. At low glucose concentrations, that mimic the fasted state, glucokinase is sequestered in an inactive state bound to the 68 kDa regulatory protein in the nucleus. In these conditions the rate of glucose phosphorylation is less than 15% of the total glucokinase activity. An increase in extracellular glucose concentration, within the range occurring in the portal vein in the absorptive state, or low concentrations of fructose or sorbitol (precursors of fructose 1-phosphate), cause the translocation of glucokinase from the nucleus to the cytoplasm and this is associated with a corresponding increase in glucose phosphorylation. The effect of glucose on translocation is mimicked by mannose which is also phosphorylated by glucokinase as well as by competitive inhibitors of glucokinase (mannoheptulose and 5-thioglucose) which are not phosphorylated. Various lines of evidence suggest that the action of these analogues is most likely due to binding to an allosteric or non-catalytic site. The saturation curve of glucose phosphorylation in intact hepatocytes is sigmoidal with an S0.5 of approximately 20 mM and a Hill coefficient approximately 2. This saturation curve can be explained by the activity of glucokinase in the cytoplasmic compartment. Translocation of glucokinase from the nucleus to the cytoplasm in response to precursors of fructose 1-phosphate (which cause dissociation of glucokinase from the regulatory protein) is associated with stimulation of glucose phosphorylation, glycolysis and glycogen synthesis. Using Metabolic Control Analysis to determine the Control Coefficient (Control Strength) of cytoplasmic (free) glucokinase on glucose metabolism it can be shown that the free glucokinase activity has a very high control strength on glycogen synthesis (CFGKJ > 1), indicating a major role of translocation of glucokinase in the control of hepatic glycogen synthesis. Overexpression of glucokinase in hepatocytes by adenovirus-mediated glucokinase overexpression is associated with a marked increase in glycogen synthesis. The relation between glycogen synthesis and enzyme overexpression is sigmoidal with an enzyme concentration causing half-saturation (S0.5) in the physiological range. The high Control Coefficient of glucokinase on hepatic glycogen synthesis explains the abnormalities of hepatic glycogen synthesis in patients with a single mutant allele of the glucokinase gene (Maturity Onset Diabetes of the Young, type 2).