Restoration of hepatic glycogen deposition reduces hyperglycaemia, hyperphagia and gluconeogenic enzymes in a streptozotocin-induced model of diabetes in rats.

AIMS/HYPOTHESIS: Glycogen deposition is impaired in diabetes, thus contributing to the development of hyperglycaemia. Several glucose-lowering strategies have attempted to increase liver glycogen deposition by modulating targets, which eventually trigger the activation of liver glycogen synthase (LGS). However, these targets also alter several other biological processes, and therefore their therapeutic use may be limited. Here we tested the approach of directly activating LGS and evaluated the potential of this strategy as a possible treatment for diabetes. METHODS: In this study, we examined the efficacy of directly overproducing a constitutively active form of LGS in the liver to ameliorate streptozotocin-induced diabetes in rats. RESULTS: Activated mutant LGS overproduction in the liver of streptozotocin-induced diabetic rats normalised liver glycogen content, despite low levels of glucokinase and circulating insulin. Moreover, this overproduction led to a decrease in food intake and in the production of the main gluconeogenic enzymes, glucose-6-phosphatase, fructose-1,6-bisphosphatase and phosphoenolpyruvate carboxykinase. The resulting combined effect was a reduction in hyperglycaemia. CONCLUSIONS/INTERPRETATION: The restoration of liver glycogen ameliorated diabetes and therefore is considered a potential strategy for the treatment of this disease.

Intracellular distribution of glycogen synthase and glycogen in primary cultured rat hepatocytes.

Changes in the intracellular distribution of liver glycogen synthase (GS) might constitute a new regulatory mechanism for the activity of this enzyme at cellular level. Our previous studies indicated that incubation of isolated hepatocytes with glucose activated GS and resulted in its translocation from a homogeneous cytosolic distribution to the cell periphery. These studies also suggested a relationship with insoluble elements of the cytoskeleton, in particular actin. Here we show the translocation of GS in a different experimental model that allows the analysis of this phenomenon in long-term studies. We describe the reversibility of translocation of GS and its effect on glycogen distribution. Incubation of cultured rat hepatocytes with glucose activated GS and triggered its translocation to the hepatocyte periphery. The relative amount of the enzyme concentrated near the plasma membrane increased with time up to 8 h of incubation with glucose, when the glycogen stores reached their maximal value. The lithium-induced covalent activation of GS was not sufficient to cause its translocation to the cell periphery. The intracellular distribution of GS closely resembled that of glycogen. Our results showed an interaction between GS and an insoluble element of the hepatocyte matrix. Although no co-localization between actin filaments and GS was observed in any condition, disruption of actin cytoskeleton resulted in a significantly lower percentage of cells in which the enzyme translocated to the cell periphery in response to glucose. This observation suggests that the microfilament network has a role in the translocation of GS.

Evidence for a functional glycogen metabolism in mature mammalian spermatozoa.

The glycogen content in fresh raw dog spermatozoa was 0.22+/-0.03 micromol/mg protein. This matched with the presence of a glycogen-like staining in the head and midpiece. Glycogen levels lowered to 0.05 micromol/mg protein after incubation for 60 min without sugars. Addition of either 10 mM fructose or 10 mM glucose increased glycogen content to 0.70 micromol/mg protein. On the other hand, glycogen synthase activity ratio of fresh dog sperm (0.35+/-0.07, measured in the absence and the presence of glucose 6-P) increased to 0.55 with 10 mM fructose for 20 min, whereas glucose had a smaller effect. Spermatozoa extracts had also a protein of about 100 Kd, which reacted against a rat liver glycogen synthase antibody. This was located in sperm head and midpiece. Furthermore, glycogen phosphorylase activity ratio measured in presence and absence of AMP (0.25+/-0.03 in fresh samples) decreased to 0.15 by 10 mM glucose for 20 min, whereas fructose was less potent in this regard. The maximal effect of glucose and fructose were observed from 10-20 mM onwards. This work is the first indication for a functional glycogen metabolism in mammal spermatozoa, which could play an important role in regulating sperm survival in vivo.

Adenovirus-mediated introduction of DNA into pig sperm and offspring.

The ability of adenoviral vectors to transfer DNA into boar spermatozoa and to offspring was tested. Exposure of spermatozoa to adenovirus bearing the E. coli lacZ gene resulted in the transfer of the gene to the head of the spermatozoa. Treatment did not affect either viability or acrosomal integrity of boar sperm. Of the 2-to 8-cell embryos obtained after in vitro fertilization with adenovirus-exposed sperm, 21.7% expressed the LacZ product. Four out of 56 piglets (about 7%) obtained after artificial insemination with adenovirus-exposed spermatozoa were positive in PCR analyses, even though none of the piglets showed the LacZ gene after southern blot analysis. RT-PCR analysis performed in tissues from two positive stillborn piglets showed the presence of the LacZ mRNA in all of the tissues tested. The offspring obtained after mating two positive animals did not show LacZ gene presence. Our results indicate that adenovirus could be a feasible mechanism for the delivery of DNA into spermatozoa, even though the transfer of the transgene may be limited to the first generation.

Protein kinase C-dependent in vivo phosphorylation of prourokinase leads to the formation of a receptor competitive antagonist.

We recently reported that in vivo phosphorylation of urokinase-type plasminogen activator on Ser138/303 prevents its catalytic-independent ability to promote myelomonocytic cell adherence and motility. We now show that Ca2+ activated, phospholipid-dependent protein kinase C from rat brain phosphorylates in vitro a peptide corresponding to prourokinase residues 133-143 (DGKKPSSPPEE) and the full-length molecule on Ser138/139. The in vivo involvement of the protein kinase C isoenzyme family is supported by the finding that inhibition of kinase C activity prevents prourokinase phosphorylation on Ser138/303 in A431 human carcinoma cells. Conversely, a short treatment of A431 cells with phorbol myristate acetate increases the extent of phosphorylated prourokinase and, concomitantly, affects its function; under these conditions, the capability of prourokinase to up-regulate U937 monocyte-like cell adherence is severely impaired, although receptor binding is unaltered. By the aid of a "phosphorylation-like" variant (Ser138 to Glu) we show that modification of Ser138 is sufficient to confer to prourokinase the antagonistic properties observed following in vivo stimulation of protein kinase C activity. These observations provide the first evidence that protein kinase C directs the formation of a receptor competitive antagonist by regulating the in vivo phosphorylation state of prourokinase.

The zeta isozyme of protein kinase C binds to tubulin through the pseudosubstrate domain.

It has been suggested that the protein kinase C zeta (zeta PKC) isoform is involved in mitogenic signaling in Xenopus oocytes and mammalian cells. Thus, the characterization of potential regulatory molecules that bind to zeta PKC is of great interest. We report here the identification by affinity chromatography of tubulin as a zeta PKC-binding protein. Further immunofluorescence and microtubule copolymerization studies are consistent with this interaction. It is suggested that tubulin binds to zeta PKC through its pseudosubstrate domain. Furthermore, results demonstrate that treatment of cells with nocodazole, which disrupts microtubule structures, severely impairs the activity of native zeta PKC, stressing the potential functional relevance of zeta PKC binding to tubulin.

Protein kinases involved in the phosphorylation of human tau protein in transfected COS-1 cells.

Human tau phosphorylation has been studied in transfected COS-1 cells. Treatment with okadaic acid alters the electrophoretic mobility of human tau protein transiently expressed in transfected cells, due to an increase in the level of phosphorylation. Treatment with okadaic acid also results in an increased phosphorylation of Alzheimer's disease-type phosphoepitopes. Tau phosphorylation within COS-1 cells is partially inhibited by in vivo treatment with DRB, a protein kinase inhibitor. Double treatment of transfected cells with okadaic acid and DRB reveals that phosphorylation of tau protein at the AT8 epitope is achieved by a DRB-resistant protein kinase which is different from that responsible for tau phosphorylation at the SMI-31 epitope, which appears to be sensitive to DRB.

Characterisation of antimitotic products from marine organisms that disorganise the microtubule network: ecteinascidin 743, isohomohalichondrin-B and LL-15.

The effect of selected marine compounds with anti-tumoral activity on the cell microtubule network was tested by immunofluorescence analyses, or by other in vitro analyses involving competition with colchicine or with GTP for tubulin binding and tubulin polymerisation, studies that were carried out in parallel with other microtubule poisons used as controls. Three compounds were found to disorganise the microtubule network: isohomohalichondrin B, LL-15 and ecsteinascidin 743. The first two compounds prevent microtubule assembly and GTP binding to tubulin. Ecteinascidin 743 disorganises the microtubule network but it does not seem to interact directly with tubulin.

The antitumoral compound Kahalalide F acts on cell lysosomes.

The target for the antitumoral peptidic drug, Kahalalide F, has been studied in cultured cells. In the presence of the compound, the cells became impressively swollen, showing the formation of large vacuoles. The formation of these vacuoles appears to be the consequence of changes in lysosomal membranes. Thus, lysosomes are a target for Kahalalide F action.

Characterization of microtubule-associated protein phosphoisoforms present in isolated growth cones.

The presence of microtubule-associated proteins (MAPs) in growth cones has been analyzed by isolation of these structures and characterization of their proteins by immunofluorescence studies. Two major MAPs, MAP1B and tau, were present in growth cones of cerebellum neurons isolated from 5-day-old rats. Both MAPs could be modified by proline-dependent protein kinases (PDPK) with opposite effects. PDPK-modified MAP1B isoforms are present at the growth cones whereas PDPK-modified tau isoforms are absent. This result suggests a different role for each phosphoMAP. To look for a possible PDPK involved in the modification of MAP1B at the growth cone, the localization of MAP and cdc2 kinases was studied. Our results indicate that the distribution in neuronal cells of MAP kinase is compatible with a possible role of this protein in modifying MAP1B.

Tissue-type plasminogen activator (tPA) is the main plasminogen activator associated with isolated rat nerve growth cones.

Different studies in tissue culture have shown the involvement of plasminogen activators (PAs) in nerve growth-cone migration. We have studied PA activity associated with isolated rat brain growth cones. Fibrin-agarose zymographies show that tissue-type PA (tPA) is the main PA associated with these structures. After fractionation of growth cones, a slightly higher tPA activity was found associated with the particulate fraction. The present findings support the requirement of this protease for neurite growth.