Inhibition of cyclic AMP-dependent protein kinase by the polar head group of an insulin-sensitive glycophospholipid

A glycophospholipid has been purified from rat liver membranes and shown to copurify with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this glycophospholipid is a phospho-oligosaccharide generated by treatment with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus. It has been proposed that this phospho-oligosaccharide, which is also generated in response to insulin, may play a role in insulin action. Incubation of the catalytic subunit of cyclic AMP-dependent protein kinase with this phospho-oligosaccharide inhibited the activity of the kinase to phosphorylate histone IIA, a purified preparation of phospholipid methyltransferase and kemptide, a phosphate-accepting peptide. Inhibition of kinase activity was dose-dependent and 50% inhibition of histone phosphorylation was demonstrated with a concentration of phospho-oligosaccharide of around 2 microM. This effect was demonstrated in the presence of ATP at concentrations up to 1 mM, indicating that the phospho-oligosaccharide acts at physiological concentrations of ATP and that it does not compete with this nucleotide for the same binding site in the kinase. Inhibition by the phospho-oligosaccharide of kinase activity could be reversed by dilution or dialysis and was not reproduced by up to 50 microM myo-inositol, glucosamine, galactose, myo-inositol 1-phosphate, glucosamine 1-phosphate, galactose 1-phosphate or phosphorylcholine. The inhibitory activity was resistant to mild acid treatment but was labile to treatment with alkali, exposure to nitrous acid or incubation with sodium periodate. The phospho-oligosaccharide had no effect on the phosphorylation of lysine-rich histone by rat brain protein kinase C and on the binding of cyclic AMP to a cyclic AMP-dependent protein kinase. In conclusion, the data in this study suggested that a phospho-oligosaccharide generated from an insulin-sensitive glycophospholipid may play a role in insulin action by modulating cyclic AMP-dependent protein kinase activity.     

A phospho-oligosaccharide mimics insulin action on glycogen phosphorylase and pyruvate kinase activities in isolated rat hepatocytes

A phospho-oligosaccharide which is the polar head group of a novel insulin-sensitive glycophospholipid has recently been involved in insulin action. We have investigated the insulin-like effects of this phospho-oligosaccharide on both glycogen phosphorylase a and pyruvate kinase activities of hepatocytes incubated in the presence of glucagon (0.1 nM). Similarly to insulin, the phospho-oligosaccharide antagonized glucagon-dependent activation of glycogen phosphorylase, as well as the inactivation of pyruvate kinase caused by this hormone. The antagonistic action of the phospho-oligosaccharide on glucagon effects was dose-dependent. Furthermore, it partially antagonized glucagon-stimulated cyclic AMP levels. These results support the hypothesis that this phospho-oligosaccharide mediates at least some insulin actions in hepatocytes.     

Initial characterization of a polyclonal antibody to an insulin-sensitive glycophospholipid

This paper describes the production of a rabbit polyclonal antibody against an insulin-sensitive glycophospholipid from rat liver membranes. The immunogen was a highly purified glycophospholipid-tetanus toxoid conjugate. Immunoglobulin purified from immune serum reacted with a glycophospholipid-ovalbumin conjugate, indicating specificity for the glycophospholipid hapten and not the protein carrier. By radioimmunoassay the antibody recognized the purified glycophospholipid antigen but not other phospholipids including phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol. The antigenic site appears to be the carbohydrate portion of the glycophospholipid. The antibody also reacted with glycophospholipid purified from two rat hepatoma cell lines. Analysis of partially purified liver glycophospholipid by thin-layer chromatography revealed over 20 orcinol- or fluorescamine-positive bands, but immunostaining identified only 1 band. The latter had an Rf identical to those of the original glycophospholipid isolated from rat liver and metabolically labeled material isolated from hepatoma cells. The antibody should prove useful in determining the role of the glycophospholipid and its metabolites in insulin action.     

The insulinomimetic effects of the polar head group of an insulin-sensitive glycophospholipid on pyruvate dehydrogenase in both subcellular and whole cell assays

The polar head group that was released by treating an insulin-sensitive glycophospholipid with a phosphatidylinositol-specific phospholipase C (PI-PLC) stimulated pyruvate dehydrogenase (PDH) in both subcellular and whole cell assays. Stimulation of PDH activity in the subcellular assay was detected after gel filtration chromatography of the polar head group. This stimulation was not due to the presence of contaminating calcium and magnesium. The PDH-stimulating activity was proportional to the amount of polar head group added to the assay. The effect of the polar head group on PDH in the subcellular assay was blocked by sodium fluoride, suggesting that the polar head group activated the PDH phosphatase. In the whole cell assay, the polar head group stimulated PDH activity to an equal or greater extent as a physiological concentration of insulin. The effect of the polar head group was detected at 5 min, peaked at 10 min, and declined thereafter. In contrast, insulin stimulated PDH activity more slowly, but consistently. The PDH-stimulating activity eluted after bacitracin but ahead of ATP during gel filtration chromatography, and it was destroyed by exposure to NH4OH or alkaline phosphatase and by boiling in water. These data support the proposal that an early step in insulin action is the release of insulinomimetic polar head group from the insulin-sensitive glycophospholipid.     

Asymmetric distribution of the phosphatidylinositol-linked phospho-oligosaccharide that mimics insulin action in the plasma membrane

We have investigated the topography of a glycosyl-phosphatidylinositol implicated in insulin action by a combination of two complementary methods: (a) chemical labelling with a non-permeable (isethionyl acetimidate) and a permeable (ethyl acetimidate) probe; and (b) enzymatic modifications with beta-galactosidase (EC 3.2.1.23) or phosphatidylinositol-specific phospholipase C (EC 3.1.4.3). Using the first approach the majority of the glycosyl-phosphatidylinositol is found in the outer surface of intact hepatocytes, adipocytes, fibroblasts and lymphocytes, but not in erythrocytes which presented only a 20% of the total labelled glycosyl-phosphatidylinositol to the exterior. Upon insulin addition (10 nM), about 60% of the total glycosyl-phosphatidylinositol was hydrolysed in both hepatocytes and adipocytes but not in erythrocytes. In agreement with the extracellular localization in hepatocytes and with the proposed role of this glycolipid in insulin action, treatment of rat hepatocytes with beta-galactosidase from Escherichia coli, an enzyme that hydrolyses the oligosaccharide moiety of the glycosyl-phosphatidylinositol, cleaved 65% of the total glycophospholipid and blocked the effect of insulin (but not of glucagon) on pyruvate kinase (EC 2.7.1.40). Similar treatment with phosphatidylinositol-specific phospholipase C from Bacillus cereus hydrolysed 62% of the total glycosyl-phosphatidylinositol. From the various approaches used it is concluded that the majority of this glycophospholipid is at the outer surface in a variety of insulin-sensitive cells.     

Identification of a novel insulin-sensitive glycophospholipid from H35 hepatoma cells

This study identifies and partially characterizes an insulin-sensitive glycophospholipid in H35 hepatoma cells. The incorporation of [3H]glucosamine into cell lipids was investigated. A major labeled lipid was purified by sequential thin layer chromatography using first an acid followed by a basic solvent system. After hydrochloric acid hydrolysis and sugar analysis by thin layer chromatography, 80% of the radioactivity in the purified lipid was found to comigrate with glucosamine. H35 cells were prelabeled with [3H]glucosamine for either 4 or 24 h and treated with insulin causing a dose-dependent stimulation of turnover of the glycophospholipid which was detected within 1 min. The purified glycolipid was cleaved by nitrous acid deamination indicating that the glucosamine C-1 was linked to the lipid moiety through a glycosidic bond. [14C]Ethanolamine, [3H]inositol, and [3H]sorbitol were not incorporated into the purified glycolipid. The incorporation of various fatty acids into this glycolipid was also studied. [3H]Palmitate was found to be preferentially incorporated while myristic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, and arachidonic acid were either not incorporated or incorporated less than 10% of palmitate. The purified glycolipid labeled with [3H]palmitate was cleaved by treatment with phospholipase A2 but was resistant to mild alkali hydrolysis suggesting the presence of a 1-hexadecyl,2-palmitoyl-glyceryl moiety in the purified lipid. Treatment of labeled glycophospholipid with phosphatidylinositol-specific phospholipase C from Staphylococcus aureus generated a compound migrating as 1-alkyl,2-acyl-glycerol and a polar head group with a size in the range from 800 to 3500. These findings coupled with the nitrous acid deamination demonstrate that glucosamine was covalently linked through a phosphodiester bond to the glyceryl moiety of the purified glycolipid. These findings suggest that insulin acts on this glycophospholipid by stimulating an insulin-sensitive phospholipase C. This unique glycophospholipid may play an important role in insulin action by serving as precursor of insulin-generated mediators.     

The polar head group of a novel insulin-sensitive glycophospholipid mimics insulin action on phospholipid methyltransferase

A phospholipid has been purified from rat liver membranes which copurified with an insulin-sensitive glycophospholipid isolated from H35 hepatoma cells. The polar head group of this phospholipid was generated by treatment with a phosphatidylinositol-specific phospholipase C from Staphylococcus aureus and purified through a C18 extraction column. Like insulin, the addition of this polar head group to isolated rat adipocytes inhibited the stimulatory effect of isoproterenol on phospholipid methyltransferase. The polar head group was also active on a subcellular fraction. The addition of the polar head group to microsomes isolated from isoproterenol-treated adipocytes produced a time-dependent inactivation of phospholipid methyltransferase, approaching basal activity. It is proposed that the effects of insulin on phospholipid methyltransferase may be mediated by this polar head group.     

Skeletal muscle cells from insulin-resistant (non-diabetic) individuals are susceptible to insulin desensitization by palmitate.

We recently demonstrated that in vivo insulin resistance is not retained in cultured skeletal muscle cells. In the present study, we tested the hypothesis that treating cultured skeletal muscle cells with fatty acids has an effect on insulin action which differs between insulin-sensitive and insulin-resistant subjects. Insulin effects were examined in myotubes from 8 normoglycemic non-obese insulin-resistant and 8 carefully matched insulin-sensitive subjects after preincubation with or without palmitate, linoleate, and 2-bromo-palmitate. Insulin-stimulated glycogen synthesis decreased by 27 +/- 5 % after palmitate treatment in myotubes from insulin-resistant, but not from insulin-sensitive subjects (1.50 +/- 0.08-fold over basal vs. 1.81 +/- 0.09-fold, p = 0.042). Despite this observation, we did not find any impairment in the PI 3-kinase/PKB/GSK-3 pathway. Furthermore, insulin action was not affected by linoleate and 2-bromo-palmitate. In conclusion, our data provide preliminary evidence that insulin resistance of skeletal muscle does not necessarily involve primary defects in insulin action, but could represent susceptibility to the desensitizing effect of fatty acids and possibly other environmental or adipose tissue-derived factors.     

Partial structure of an insulin-sensitive glycophospholipid

The structure of a glycophospholipid, which has been involved in insulin action, has been investigated using H35 cells and rat liver membranes. The present evidence indicates that this molecule contains a phosphatidyl-chiro-inositol moiety, glycosidically linked to a non-N-acetylated glucosamine. In addition, the polar head group of the lipid contains galactose, probably four residues, and a total number of three phosphates.     

Protein kinase C modulates insulin action in human skeletal muscle

There is good evidence from cell lines and rodents that elevated protein kinase C (PKC) overexpression/activity causes insulin resistance. Therefore, the present study determined the effects of PKC activation/inhibition on insulin-mediated glucose transport in incubated human skeletal muscle and primary adipocytes to discern a potential role for PKC in insulin action. Rectus abdominus muscle strips or adipocytes from obese, insulin-resistant, and insulin-sensitive patients were incubated in vitro under basal and insulin (100 nM)-stimulated conditions in the presence of GF 109203X (GF), a PKC inhibitor, or 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA), a PKC activator. PKC inhibition had no effect on basal glucose transport. GF increased (P < 0.05) insulin-stimulated 2-deoxyglucose (2-DOG) transport approximately twofold above basal. GF plus insulin also increased (P < 0.05) insulin receptor tyrosine phosphorylation 48% and phosphatidylinositol 3-kinase (PI 3-kinase) activity approximately 50% (P < 0.05) vs. insulin treatment alone. Similar results for GF on glucose uptake were observed in human primary adipocytes. Further support for the hypothesis that elevated PKC activity is related to insulin resistance comes from the finding that PKC activation by dPPA was associated with a 40% decrease (P < 0.05) in insulin-stimulated 2-DOG transport. Incubation of insulin-sensitive muscles with GF also resulted in enhanced insulin action ( approximately 3-fold above basal). These data demonstrate that certain PKC inhibitors augment insulin-mediated glucose uptake and suggest that PKC may modulate insulin action in human skeletal muscle.     

Phospholipase C mimics insulin action on pyruvate dehydrogenase and insulin mediator generation but not glucose transport or utilization

This study investigated the extent to which a purified phosphatidylinositol-specific and a commercial non-specific phospholipase C mimicked acute insulin action in rat adipocytes. The enzymes mimicked insulin stimulation of pyruvate dehydrogenase (PDH) and breakdown of a glycophospholipid proposed as a precursor for an intracellular mediator of insulin action, but were much less effective in stimulating glucose transport and utilization. These observations corroborate recent suggestions that insulin may activate a phospholipase C to generate a mediator that can account for insulin activation of PDH from a mediator precursor with a phosphatidylinositol anchor. This mediator precursor is probably an outer membrane component since effects were obtained with intact cells. It is unlikely that this mechanism accounts fully for insulin action since phosphatidylinositol-specific and commercial phospholipase C stimulation of glucose transport was significantly less than that elicited by insulin.     

Regulation of insulin secretion and GLUT4 trafficking by the calcium sensor synaptotagmin VII

Insulin regulates blood glucose by promoting uptake by fat and muscle, and inhibiting production by liver. Insulin-stimulated glucose uptake is mediated by GLUT4, which translocates from an intracellular compartment to the plasma membrane. GLUT4 traffic and insulin secretion both rely on calcium-dependent, regulated exocytosis. Deletion of the voltage-gated potassium channel Kv1.3 results in constitutive expression of GLUT4 at the plasma membrane. Inhibition of channel activity stimulated GLUT4 translocation through a calcium dependent mechanism. The synaptotagmins (Syt) are calcium sensors for vesicular traffic, and Syt VII mediates lysosomal and secretory granule exocytosis. We asked if Syt VII regulates insulin secretion by pancreatic beta cells, and GLUT4 translocation in insulin-sensitive tissues mouse model. Syt VII deletion (Syt VII -/-) results in glucose intolerance and a marked decrease in glucose-stimulated insulin secretion in vivo. Pancreatic islet cells isolated from Syt VII -/- cells secreted significantly less insulin than islets of littermate controls. Syt VII deletion disrupted GLUT4 traffic as evidenced by constitutive expression of GLUT4 present at the plasma membrane of fat and skeletal muscle cells and unresponsiveness to insulin. These data document a key role for Syt VII in peripheral glucose homeostasis through its action on both insulin secretion and GLUT4 traffic.     

Approaches to the molecular cloning of protein-tyrosine phosphatases in insulin-sensitive tissues

The intrinsic tyrosyl kinase activity of the insulin receptor is regulated by a balance between insulin-induced receptor autophosphorylation, which stimulates the receptor kinase, and enzymatic dephosphorylation of the receptor, which deactivates its kinase activity. The cellular protein-tyrosine phosphatase (PTPase) enzymes responsible for reversing the activated state of the insulin receptor have not been characterized. Our laboratory is interested in identifying and cloning the specific PTPase(s) that regulate the phosphorylation state of the insulin receptor. This chapter will summarize the design and results of our initial molecular cloning studies to identify specific PTPases in insulin-sensitive tissues that may have a potential physiological role in insulin action and clinical insulin resistance.     

Phylogenesis and Biological Characterization of a New Glucose Transporter in the Chicken (Gallus gallus), GLUT12

In mammals, insulin-sensitive GLUTs, including GLUT4, are recruited to the plasma membrane of adipose and muscle tissues in response to insulin. The GLUT4 gene is absent from the chicken genome, and no functional insulin-sensitive GLUTs have been characterized in chicken tissues to date. A nucleotide sequence is predicted to encode a chicken GLUT12 ortholog and, interestingly, GLUT12 has been described to act as an insulin-sensitive GLUT in mammals. It encodes a 596 amino acid protein exhibiting 71% identity with human GLUT12. First, we present the results of a phylogenetic study showing the stability of this gene during evolution of vertebrates. Second, tissue distribution of chicken SLC2A12 mRNA was characterized by RT-PCR. It was predominantly expressed in skeletal muscle and heart. Protein distribution was analysed by Western blotting using an anti-human GLUT12 antibody directed against a highly conserved region (87% of identity). An immuno-reactive band of the expected size (75kDa) was detected in the same tissues. Third a physiological characterization was performed: SLC2A12 mRNA levels were significantly lowered in fed chickens subjected to insulin immuno-neutralization. Finally, recruitment of immuno-reactive GLUT12 to the muscle plasma membrane was increased following 1h of intraperitoneal insulin administration (compared to a control fasted state). Thus insulin administration elicited membrane GLUT12 recruitment. In conclusion, these results suggest that the facilitative glucose transporter protein GLUT12 could act in chicken muscle as an insulin-sensitive transporter that is qualitatively similar to GLUT4 in mammals.     

Insulin-like effect of epidermal growth factor in isolated rat hepatocytes. Modulation of the alpha-1-adrenergic stimulation of ureagenesis

Insulin and epidermal growth factor (EGF) inhibit the stimulation of ureagenesis induced by adrenaline (alpha 1-adrenergic effect) in hepatocytes from control rats incubated in medium without calcium and in cells from hypothyroid rats. In hepatocytes from euthyroid rats incubated in normal buffer neither insulin or EGF diminished the alpha 1-adrenergic stimulation of ureagenesis. No effect of EGF or insulin on the alpha 1-adrenergic stimulation of phosphatidylinositol labeling was observed under any conditions. It is suggested that EGF mimics the action of insulin on one of the pathways of the alpha 1-adrenergic action: the calcium-independent, insulin-sensitive pathway which predominates in hepatocytes from hypothyroid rats.     

The insulin receptor: structure and function

Promising progress in understanding the molecular basis of insulin action has been achieved by demonstrating that the insulin receptor is an insulin-sensitive tyrosine kinase. Here we discuss the structure of this receptor kinase and compare it with receptors for related growth factors. We review the known modes to regulate the receptor kinase activity, either through its autophosphorylation (on tyrosine residues) or through its phosphorylation by other kinases (on serine and threonine residues). We discuss the role of the receptor kinase activity in hormone signal transduction in light of results indicating a reduced kinase activity in insulin-resistant states. Finally, studies to identify natural substrates for the insulin receptor kinase are presented. The possible physiological role of these phosphorylated substrates in mediating insulin action is evaluated.     

Insulin increases the intracellular concentration of total oxalyl thiolesters in BC3H-1 myocytes: potential anti-insulin mediators.

Oxalyl thiolesters (RS-CO-COOH) may represent negative intracellular messengers for insulin action. Using a reverse-phase, ion-pair high pressure liquid chromatographic technique, total intracellular oxalyl thiolesters were measured in insulin-sensitive BC3H-1 myocytes after the addition of insulin. The total oxalyl thiolester concentration increased to a maximum of 2.9 times the basal concentration by 30 min after the addition of 100 microU/ml insulin and decreased to 1.8 times by 180 min. Insulin's stimulation of pyruvate dehydrogenase as measured by lactate oxidation ([1-14C]-lactate --> 14CO2) in intact BC3H-1 myocytes reached a maximum at 15-30 min and returned to basal activity during the 60-90 min measurement interval. These results suggest that oxalyl thiolesters are increased in concentration following insulin-induced signal transduction to reverse insulin-stimulated metabolic events.     

St. John's Wort inhibits insulin signaling in murine and human adipocytes

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.     

St. John's Wort inhibits insulin signaling in murine and human adipocytes

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.     

Modulation of casein kinase II activity by the polar head group of an insulin-sensitive glycosyl-phosphatidylinositol

A phospho-oligosaccharide, whose production is stimulated by insulin, modulated the activity of partially purified casein kinase II. Whereas at 2 microM the phospho-oligosaccharide stimulated casein kinase II 1.3-fold, higher concentrations of this molecule were inhibitory. 50% inhibition of the enzyme was obtained at 15 microM phospho-oligosaccharide. This biphasic effect of the phospho-oligosaccharide on casein kinase II activity was observed using as substrate both casein or the specific peptide for casein kinase II, Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu. The effect of the phospho-oligosaccharide on casein kinase II was still observed after gel filtration. Deamination of the phospho-oligosaccharide with nitrous acid abolished both the activation and the inhibition of casein kinase II. The glycophospholipid precursor of the phospho-oligosaccharide did not affect casein kinase II activity. Moreover, modulation of casein kinase II activity was not observed with other compounds structurally related to the phospho-oligosaccharide, when used in the micro-molar range. In conclusion, the present results indicate that the phospho-oligosaccharide that mimics and might mediate some of the actions of insulin modulates casein kinase II activity in vitro.