[A national meeting for medical students--new pedagogic approach. High tempo visits to different scientific fields].
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Education, Medical↗
Biomedical subjects
P Strålfors
Publications and source records attributed to P Strålfors.
Insulin second messengers.
The molecular pathways for insulin's signal transduction from its cell surface receptor to the cell's interior metabolic machinery remain in many ways uncharted. Lately two molecules have been proposed as second messengers transducing the insulin signal into the target cell. One is a phospho-oligosaccharide/inositolphosphoglycan and the other is diacylglycerol, both deriving from the same plasma membrane glycolipid, which is hydrolysed in response to insulin treatment. The phospho-oligosaccharide appears to mediate many metabolic effects of insulin through control of the phosphorylation state of key regulatory metabolic enzymes. Diacylglycerol may mediate insulin's stimulation of glucose transport over the plasma membrane. The glycolipid precursor of these putative second messengers, as well as the receptor for insulin, appear to be localized in caveolae microdomains of the plasma membrane, and glucose transporters accumulate in caveolae in response to insulin treatment, suggesting a focal role for caveolae in insulin signalling.
Animals↗
Cytoplasmic CREB alpha-like antigens in specific regions of the rat brain.
We have investigated the expression of the alpha-region of CREB (amino acids 88-101) in the rat brain. This region is encoded by a separate exon and has been reported to enhance the transcriptional efficiency of CREB. Light microscopic immunohistochemistry showed labeled neurons in several brain regions, such as the spinal cord, pontine reticular formation, cerebellum, hypothalamus, hippocampus and olfactory cortex. The labeling was exclusively cytoplasmic, and electron microscopy revealed that the CREB alpha-immunoreactivity was localized to the Golgi apparatus and axon terminals. Immunoblotting after SDS-PAGE showed specifically detected proteins of 21 and 160 kD. We suggest that neuronal CREB alpha-proteins represent alternatively spliced isoforms of CREB that lack the nuclear translocation signal. These isoforms may be involved in the regulation of CREB-mediated transcription.
Animals↗
Insulin-stimulated glucose uptake involves the transition of glucose transporters to a caveolae-rich fraction within the plasma membrane: implications for type II diabetes.
BACKGROUND: Adipose and muscle tissues express an insulin-sensitive glucose transporter (GLUT4). This transporter has been shown to translocate from intracellular stores to the plasma membrane following insulin stimulation. The molecular mechanisms signalling this event and the details of the translocation pathway remain unknown. In type II diabetes, the cellular transport of glucose in response to insulin is impaired, partly explaining why blood-glucose levels in patients are not lowered by insulin as in normal individuals. MATERIALS AND METHODS: Isolated rat epididymal adipocytes were stimulated with insulin and subjected to subcellular fractionation and to measurement of glucose uptake. A caveolae-rich fraction was isolated from the plasma membranes after detergent solubilization and ultracentrifugal floatation in a sucrose gradient. Presence of GLUT4 and caveolin was determined by immunoblotting after SDS-PAGE. RESULTS: In freshly isolated adipocytes, insulin induced a rapid translocation of GLUT4 to the plasma membrane fraction, which was followed by a slower transition of the transporter into a detergent resistant caveolae-rich region of the plasma membrane. The insulin-stimulated appearance of transporters in the caveolae-rich fraction occurred in parallel with enhanced glucose uptake by cells. Treatment with isoproterenol plus adenosine deaminase rapidly inhibited insulin-stimulated glucose transport by 40%, and at the same time GLUT4 disappeared from the caveolae-rich fraction and from plasma membranes as a whole. CONCLUSIONS: Insulin stimulates glucose uptake in adipocytes by rapidly translocating GLUT4 from intracellular stores to the plasma membrane. This is followed by a slower transition of GLUT4 to the caveolae-rich regions of the plasma membrane, where glucose transport appears to take place. These results have implications for an understanding of the defect in glucose transport involved in type II diabetes.
Adenosine Deaminase↗
Uptake and metabolism of long-chain 1,2-diacylglycerols by rat adipocytes and H4IIE hepatoma cells.
The physiological long-chain diacylglycerols 1,2-dioleoylglycerol and 1,2-dimyristoylglycerol were taken up and metabolized, both by adipocytes and by hepatoma cells, in amounts sufficient to elicit biological responses, without use of emulsifier or any special cell treatment. In adipocytes the only metabolic lipid product of either diacylglycerol was triacylglycerol. In hepatoma cells both diacylglycerols were converted to a number of identified products, mainly phosphatidylcholine and triacylglycerol. This has implications for the interpretation of reports in which long-chain diacylglycerols have been found ineffective in stimulation of cells.
Adipocytes↗
Isolation of phosphooligosaccharide/phosphoinositol glycan from caveolae and cytosol of insulin-stimulated cells.
A phosphooligosaccharide has been proposed as a second messenger of insulin. It is believed to be structurally related to the carbohydrate moiety of phosphatidylinositol glycan anchors of many cell surface proteins. Herein we demonstrate that [32]phosphate in freshly isolated adipocytes and [3H]galactose in cultured hepatoma cells (H4IIE) labeled the same set of three different glycolipids. With all three, the radiolabel was made water soluble by phosphatidylinositol(glycan)-specific phospholipase C or D catalyzed hydrolysis. We isolated the three phospholipase C-released substances. One of them was susceptible to nitrous acid deamination, indicative of a hexosamine with a free amino group. This phosphooligosaccharide structure had an apparent molecular mass between tetra- and pentaglucose by gel filtration. By anion-exchange chromatography it was separated into two differently charged and interconvertible species. Adipocytes stimulated with insulin accumulated the nitrous acid sensitive phosphooligosaccharide: after stimulation the intracellular level of free phosphooligosaccharide increased threefold within 5 min, fell off during the next few minutes and then remained at a slightly elevated level. After insulin stimulation the intracellular concentration of free phosphooligosaccharide was > 1,000-fold higher than in the incubation medium. When prepared from rat livers on a preparative scale, the oligosaccharide was also found to exhibit insulinomimetic effects on protein phosphorylation of insulin target proteins in intact adipocytes. After subcellular fractionation of adipocytes the lipid-bound [32P]phosphooligosaccharide of the plasma membrane was found to be localized in plasma membrane domains apparently corresponding to caveolae. Lipid-bound [32P]phosphooligosaccharide was found also in the microsomal fraction.
Adipocytes↗
Translocation of insulin-regulated glucose transporter is stimulated by long-chain 1,2-diacylglycerol in rat adipocytes.
The long-chain diacylglycerol 1,2-dimyristoylglycerol emulsified with taurodeoxycholate has been shown to potently stimulate glucose transport in isolated rat adipocytes (Stråfors, Nature 335, 554-556 (1988)). We now report that this 1,2-diacylglycerol in the presence of taurodeoxycholate, similarly to insulin, induced translocation of the insulin-regulated glucose transporter (GLUT-4) from a microsomal membrane compartment to the plasma membrane. H4IIE hepatoma cells expressed mRNA for GLUT-1, but not for GLUT-4. In these, otherwise insulin-responsive, cells diacylglycerol or insulin had only a marginal effect on glucose transport.
Adipocytes↗
Inhibition of Raf-1 kinase expression abolishes insulin stimulation of DNA synthesis in H4IIE hepatoma cells.
The involvement of Raf-1 kinase in the insulin signal transduction chain leading to control of cell proliferation was studied in the H4IIE rat hepatoma cell line by inhibiting expression of the kinase with antisense oligodeoxyribonucleotide directed against Raf-1 mRNA. Antisense oligonucleotide was found to reduce (at 2 microM) or completely block (at 15 microM) the stimulation by insulin of DNA synthesis, measured as thymidine incorporation. The residual DNA synthesis seen in the absence of insulin stimulation was also inhibited by the Raf-1 kinase antisense oligonucleotide.
Animals↗
Phosphorylation control by insulin in adipocytes is interfered with at a post-receptor step by phosphoinositol and glucosamine.
Inositol-phosphates, glucosamine and glucose-6-phosphate blocked the effects of insulin on target protein phosphorylation in adipocytes, but the unsubstituted or sulphated derivatives of inositol or of glucose, or N-acetyl-glucosamine were without effect. The insulin stimulated tyrosine phosphorylation of the insulin receptor was not affected. The sugar-phosphates inositol-phosphate and glucose-6-phosphate did not enter into the cells. They also blocked the insulin-like effects of a potential second messenger of insulin, a phospho-oligosaccharide (POS), which has previously been shown to mimic the effects of insulin on protein phosphorylation in intact cells.
Adipose Tissue↗
Autolysis of isolated adipocytes by endogenously produced fatty acids.
Isolated rat adipocytes, in which lipolysis was maximally stimulated by isoproterenol, continued to produce fatty acids in excess of the high-affinity binding capacity of the fatty acid acceptor bovine serum albumin in the incubation medium. At an average of 17 mol fatty acids/mol serum albumin, there was a burst of lactate dehydrogenase activity recovered in the incubation medium, indicating cell lysis. It is concluded that endogenously produced fatty acids will cause autolysis of adipocytes in vitro. Actively lipolysing adipocytes were more fragile than resting cells, since increasing amounts of lactate dehydrogenase activity was recovered in the medium during active lipolysis.
Adipose Tissue↗
Insulin-induced dephosphorylation of hormone-sensitive lipase. Correlation with lipolysis and cAMP-dependent protein kinase activity.
The effect of insulin on the state of phosphorylation of hormone-sensitive lipase, cellular cAMP-dependent protein kinase activity and lipolysis was investigated in isolated adipocytes. Increased phosphorylation of hormone-sensitive lipase in response to isoproterenol stimulation was closely paralleled by increased lipolysis. Maximal phosphorylation and lipolysis was obtained when the cAMP-dependent protein kinase activity ratio was greater than or equal to 0.1, and this corresponded to a 50% increase in the state of phosphorylation of hormone-sensitive lipase. Insulin (1 nM) reduced cAMP-dependent protein kinase activity and also reduced lipolysis with both cAMP-dependent and cAMP-independent antilipolytic effects up to an activity ratio of approximately 0.4, above which the antilipolytic effect was lost. Insulin caused a decrease in the state of phosphorylation of hormone-sensitive lipase at all levels of cAMP-dependent protein kinase activity. Under basal conditions, with cAMP-dependent protein kinase activity at a minimum, this reflected a dephosphorylation of the basal phosphorylation site of hormone-sensitive lipase in a manner not mediated by cAMP. When the cAMP-dependent protein kinase was stimulated to phosphorylate the regulatory phosphorylation site of hormone-sensitive lipase, the insulin-induced dephosphorylation occurred both at the basal and regulatory sites. At low levels of cAMP-dependent protein kinase activity ratios (0.05-0.1), dephosphorylation of the regulatory site correlated with reduced cAMP-dependent protein kinase activity, but not at higher activity ratios (greater than 0.1). Stimulation of cells with isoproterenol produced a transient (1-5 min) peak of cAMP-dependent protein kinase activity and of phosphorylation of hormone-sensitive lipase. The state of phosphorylation also showed a transient peak when the protein kinase was maximally and constantly activated. In the presence of raised levels of cellular cAMP, insulin (1 nM) caused a rapid (t1/2 approximately 1 min) dephosphorylation of hormone-sensitive lipase. In unstimulated cells the reduction in phosphorylation caused by insulin was distinctly slower (t1/2 approximately 5 min). These findings are interpreted to suggest that insulin affects the state of phosphorylation of hormone-sensitive lipase and lipolysis through a cAMP-dependent pathway, involving reduction of cAMP, and through a cAMP-independent pathway, involving activation of a protein phosphatase activity that dephosphorylates both the regulatory and basal phosphorylation sites of hormone-sensitive lipase.
Adipose Tissue↗
Inhibitors of protein phosphatase-1. Inhibitor-1 of bovine adipose tissue and a dopamine- and cAMP-regulated phosphoprotein of bovine brain are identical.
Protein phosphatase inhibitor-1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP-dependent protein kinase on a threonyl residue; this phosphorylation was necessary for inhibition of protein phosphatase-1. Bovine adipose tissue inhibitor-1 was compared directly with rabbit skeletal muscle inhibitor-1 and with a 32000-Mr, dopamine- and cAMP-regulated phosphoprotein from bovine brain (DARPP-32), also an inhibitor of protein phosphatase-1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor-1 was found to be very similar and possibly identical to DARPP-32 and was clearly distinct from skeletal muscle inhibitor-1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus-aureus-V8-protease-generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two-dimensional thin-layer electrophoresis/chromatography; elution on reverse-phase HPLC; chymotryptic peptide maps as analysed by reverse-phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase-1 (DARPP-32), which is distinct from that of skeletal muscle (inhibitor-1).
Adipose Tissue↗
Insulin stimulation of glucose uptake can be mediated by diacylglycerol in adipocytes.
An early effect of insulin in adipocytes is to stimulate glucose uptake. The increased uptake appears to be due to mobilization of glucose transporters from an intracellular location to the plasma membrane and to enhanced intrinsic activity of the transporters. Little is known about the insulin-generated signals causing these changes. Phorbol esters have been shown to mimic the insulin effect, but phosphorylation of the transporter does not seem to be involved. A phospho-oligosaccharide was recently shown to mimic the effects of insulin on protein phosphorylation, suggesting that it could be a mediator for some intracellular metabolic effects of the hormone, but it did not affect glucose uptake. A diacyglycerol is produced in the plasma membrane in conjunction with the generation of the phospho-oligosaccharide. Here I show that added 1,2-diacylglycerols potently increase glucose transporter-mediated uptake of glucose in rat adipocytes, but without activation of protein kinase C.
Adipose Tissue↗
Adipose tissue protein phosphatase inhibitor-2.
Rat fat cells contain three species of spontaneously active inhibitor proteins of protein phosphatase 1, as resolved by SDS-PAGE, with apparent molecular masses of 40 kDa, and 28 kDa respectively. The 33-kDa, thermostable inhibitor was highly purified from bovine adipose tissue and shown to be very similar to inhibitor-2 of skeletal muscle. It was phosphorylated, on threonine only, by glycogen synthase kinase 3. It formed an inactivated complex with protein phosphatase 1, that was reactivated by incubation with ATP-Mg and glycogen synthase kinase 3. By gel filtration it had a Stokes radius of 3.4 nm. Peptide and phosphopeptide maps, generated by Staphylococcus aureus V8 proteinase, trypsin or thermolysin, of the inhibitor and of the skeletal muscle inhibitor-2 were similar. The 40-kDa inhibitor, which was denatured by boiling, represents a novel protein phosphatase inhibitor protein or an undegraded precursor of inhibitor-2. The total activity of inhibitor-2-like material (thermostable and macromolecular) in an adipocyte cytosol extract corresponded to an intracellular concentration of 0.3 microM inhibitor-2.
Adipose Tissue↗
Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle.
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Amino Acid Sequence↗
Isoproterenol and insulin control the cellular localization of ATP citrate-lyase through its phosphorylation in adipocytes.
The enzyme ATP citrate-lyase of the fatty acid synthesis pathway is phosphorylated in vitro and in isolated cells. However, no effect of phosphorylation on the enzyme activity has been detected. It is demonstrated that the beta-adrenergic agonist isoproterenol or insulin both promote an immobilization of ATP citrate-lyase, detected in digitonin-permeabilized adipocytes. This effect was reproduced by the cyclic AMP analog cyclic 8-bromo-AMP. The beta-adrenergic antagonist propranolol blocked, but failed to reverse, the isoproterenol-directed effect. Propranolol also failed to reverse the isoproterenol-induced increased phosphorylation of ATP citrate-lyase specifically. In response to increasing concentrations of isoproterenol, an increased extent of phosphorylation of ATP citrate-lyase was paralleled by an increased immobilization of the enzyme. It is suggested that the state of phosphorylation of ATP citrate-lyase in adipocytes controls the localization in the cell.
8-Bromo Cyclic Adenosine Monophosphate↗
Phosphorylation of the basal site of hormone-sensitive lipase by glycogen synthase kinase-4.
In rat adipocytes hormone-sensitive lipase is phosphorylated at two sites termed 'regulatory' and 'basal', in the former case by cyclic AMP-dependent protein kinase causing an activation of the lipase [(1984) Proc. Natl. Acad. Sci. USA 81, 3317-3321]. Here, the basal phosphorylation site was found to be phosphorylated by glycogen synthase kinase-4 without any effects on lipase activity, or on the extent of its activation subsequent to phosphorylation of the regulatory site. Glycogen synthase kinase-3, casein kinase-I, and casein kinase-II did not phosphorylate the lipase. Phosphorylase kinase phosphorylated it to a very low extent at a third phosphorylation site not phosphorylated in the fat cell.
Adipose Tissue↗
[The mechanism of action of insulin is an unsolved problem--new discoveries can explain the effects].
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Animals↗