Knockout of hyaluronidase Spam1 reduces age-related bone and cartilage changes in mouse knee.

OBJECTIVE: To investigate the role of Spam1 hyaluronidase in age-related bone and cartilage changes in the mouse knee. DESIGN: Spam1-/- and WT mice were euthanised at different ages from 10 to 52 weeks. The right hindlimbs were dissected, scanned with peripheral Quantitative Computed Tomography (pQCT) and then decalcified for histological analysis (modified Mankin score). In other mice, cartilages of both tibiae were sampled at 10, 30 and 52 weeks of age for RNA extraction and qPCR analysis. We assessed the expression of hyaluronidases Hyal1 and Hyal2, hyaluronan synthase HAS2, extracellular matrix proteases Mmp13 and Adamts-5, and type 2 collagen. RESULTS: Spam1-/- mice did not exhibit specific morphological characters up to 52 weeks of age. From 20 weeks, the proximal tibia of Spam1-/- mice had a significantly lower bone mineral density than WT mice. At 52 weeks, the modified Mankin score was significantly lower in Spam1-/- than WT mice. Spam1-/- chondrocytes expressed significantly less Hyal2 than WT ones at all ages and less Mmp13 at 52 weeks. Through all the experiment, the Hyal1 expression of Spam1-/- chondrocytes remained similar as that of WT chondrocytes. CONCLUSION: Spam1 knockout reduced significantly cartilage degradation in mouse knee whereas the chondrocyte expression of Hyal 1, Hyal 2 and Mmp13 was modified, suggesting a role of this hyaluronidase in cartilage metabolism.

Long chain fatty acyl-CoA synthetase 5 expression is induced by insulin and glucose: involvement of sterol regulatory element-binding protein-1c.

In a screen for sterol regulatory element-binding protein (SREBP)-1c target genes in the liver, we identified long chain fatty acyl-CoA synthetase 5 (ACS-5). Hepatic ACS-5 mRNA is poorly expressed during fasting and diabetes and strongly induced by carbohydrate refeeding and insulin treatment. In cultured hepatocytes, insulin and a high glucose concentration induce ACS-5 mRNA. Adenoviral overexpression of a nuclear form of SREBP-1c in liver of diabetic mice or in cultured hepatocytes mimics the effect of insulin to induce ACS-5. By contrast, a dominant negative form of SREBP-1c abolishes the effect of insulin on ACS-5 expression. The dietary and SREBP-1c-mediated insulin regulation of ACS-5 expression indicate that ACS-5 is involved in the anabolic fate of fatty acids.

Role of cysteine in the dietary control of the expression of 3-phosphoglycerate dehydrogenase in rat liver.

Shifting rats to a protein-free, carbohydrate-rich diet, although not starvation, resulted in the appearance of mRNA for, and activity of, 3-phosphoglycerate dehydrogenase (3-PGDH) in liver as well as in a marked decrease in plasma cystine concentration. Refeeding with protein caused a 50% decrease in the mRNA in 8 h and its complete disappearance within 24 h, followed by a slower disappearance of the enzymic activity. Intraperitoneal administration of cysteine or methionine to protein-starved rats decreased the mRNA by 50-60% after 8 h. However, the repeated administration of cysteine failed to cause the complete disappearance of this mRNA in 24 h. In hepatocytes in primary culture, cysteine plus methionine and glucagon had, independently, an approx. 4-fold inhibitory effect on the abundance of the 3-PGDH mRNA and caused its almost complete disappearance when tested together. Insulin had an approx. 2-fold stimulatory effect, which was antagonized by cysteine plus methionine but was still apparent in the presence of glucagon. Nuclear run-on experiments and analysis of the stability of the mRNA with 5,6-dichlorobenzimidazole riboside, an inhibitor of RNA polymerase II, suggested that the effect of cysteine plus methionine was due to destabilization of the mRNA, whereas the effect of glucagon was exerted on transcription. Cysteine, but not methionine, inhibited the accumulation of 3-PGDH mRNA in FTO2B hepatoma cells. In conclusion, the dietary control of the expression of the 3-PGDH gene in liver seems to involve the negative effects of cysteine and glucagon and the positive effect of insulin.

Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes.

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet. 19, 88-92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

Cloning, sequencing and expression of rat liver 3-phosphoglycerate dehydrogenase.

Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human D- or L-hydroxyglutaric aciduria.

Sequence of a putative glucose 6-phosphate translocase, mutated in glycogen storage disease type Ib.

We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6-phosphate translocase that is functionally associated with glucose-6-phosphatase.