花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

Glycosaminoglycans (GAGs) and their changes in early corneal development of Bufo raddei Strauch (from stage 16, neural tube, to stage 25, operculum completely closed) were studied with electron microscopic cytochemical method. Results show that synthesis of GAGs changes from non-sulfated to sulfated, and its content increased gradually with the development of cornea. Hyaluronic acid (HA) in each part of cornea begins to increase gradually from stage 16 to 21 (mouth open stage), with its peak at stage 20 (gill circulation stage) to 21, then decreases. In the mean time, contents of dermatam sulfate (DS), chondroitin sulfate (CS), heparan sulfate (HS) and heparin (Hep) increase gradually. It is considered that HA, HS and collagen may be related to the migration of mesenchymal cells, and HA promotes the expansion and hydration of corneal stroma; sulfated GAGs are correlated with dehydration of cornea, cell density and corneal transparency; DS, CS, HS and Hep deposited among collagen fibrils could adjust their arrangement. All these changes would enhance transparency of cornea.     

Effect of sulfated GAGs on the expression and activation of MMP-2 and MMP-9 in corneal and dermal explant cultures

It has been shown previously that hyaluronan (HA) added to fibroblast and keratocyte cell cultures or corneal explant cultures produces an up-regulation of MMP-2 and MMP-9 expression and activation. Here, we examine the effect of sulfated GAG-s, chondroitin 4 and 6 sulfate (CS4, CS6), dermatan sulfate (DS), keratan sulfate (KS) and heparan sulfate (HS) on MMP-2 and 9 expression and activation under the same culture conditions. It appears that CS4 has only minor effects, KS inhibits MMP-2 activation and CS6, DS and HS increase MMP-2 activation in corneal explant cultures. For skin explant cultures, DS, KS and HS strongly increase MMP-9 activation, whereas KS inhibits and DS increases MMP-2 activation. All these effects can be strongly inhibited by the addition of an antibody to CD44, except CS6 and DS. Activation by these two GAGs was only slightly affected, supporting the contention that the effects of HA, CS4, KS and HS are mediated by one of the isoforms of this CD44 receptor. The physio-pathological significance of these results is discussed for cornea and skin ageing, because of the divergent evolution with in vitro ageing of the relative proportions of GAGs synthesised by these two cell types.     

Interaction of epithelial cells and fibroblasts in culture: Effect on glycosaminoglycan levels

Twelve- to fifteen-day chick embryo liver cells (epithelial) were cultured on top of confluent chick embryo fibroblasts to produce an in vitro model of an epithelial-mesenchymal interacting system. This cocultivation resulted in a marked increase in hyaluronic acid (HA) levels and a decrease in chondroitin sulfate (CS) levels, either in total or in proportion to HA, compared with the two cell types cultured separately. The liver cells cultured alone produced little or no detectable glycosaminoglycans (GAGs). Cocultivation of increasing numbers of liver cells with fibroblasts resulted in a progressive increase and decrease of HA and CS levels, respectively, and the combined effect of these changes was a progressive increase in the HA/CS ratio. Fibroblasts cultured in liver-cell-conditioned growth medium also showed increased levels of HA, but in contrast to cocultivation, an increase in CS and no change in the HA/CS ratio. Liver cells cultured in fibroblast-conditioned growth medium showed no changes in GAG level. This suggests that under conditions of cocultivation the fibroblasts alone could be responsible for the increased HA levels and that the decreased CS levels are a result of conditions produced by the close proximity of the two cell types. In vivo most connective tissues immediately adjacent to epithelial tissues are also characterized by a matrix rich in HA and these results support the concept that some epithelial tissues are able to modulate the GAG composition of adjacent connective tissues and thereby affect their immediate extracellular environment.     

Application of fluorophore-assisted carbohydrate electrophoresis to analysis of disaccharides and oligosaccharides derived from glycosaminoglycans

Various combinations of fluorescent dyes, polyacrylamide gels, and electrophoresis buffers were tested by fluorophore-assisted carbohydrate electrophoresis (FACE) for the purpose of analyzing sulfated and nonsulfated glycosaminoglycan (GAG) oligosaccharides in which disaccharides and low-molecular weight oligosaccharides were included. A nonionic fluorescent dye was found to be suitable for analyzing sulfated disaccharides derived from sulfated GAGs (e.g., chondroitin sulfate, dermatan sulfate) because sulfated disaccharides themselves had enough anionic potential for electrophoresis. The migration rates of chondroitin sulfate (CS) disaccharides in polyacrylamide gels were affected by the number of sulfate residues and the conformation of each disaccharide. When an anionic fluorescent dye, 8-aminonaphthalene-1,3,6-trisulfonic acid disodium salt (ANTS), was coupled with sulfated GAG oligosaccharides, nearly all of the conjugates migrated at the electrophoretic front due to the added anionic potential. Nonsulfated hyaluronan (HA) oligosaccharides (2-16 saccharides) were subjected to electrophoresis by coupling with a nonionic fluorescent dye, 2-aminoacridone (AMAC), but did not migrate in the order of their molecular size. Especially di-, tetra-, hexa-, and octasaccharides of HA migrated in the reverse order of their molecular size. HA/CS oligosaccharides were able to migrate in the order of their chain lengths by coupling with an anionic fluorescent dye in a nonborate condition.     

Is a natural ligand of the T lymphocyte CD2 molecule a sulfated carbohydrate?

Recent studies have implicated sulfated polysaccharide (SP) recognition in a range of cell adhesion systems. Inasmuch as the CD2 (E rosette receptor, T11, LFA-2) molecule of human T lymphocytes is a cell surface glycoprotein involved in the adhesion of T cells to various target cells the possibility that CD2 binds SP was investigated. It was found that E rosetting of human T lymphocytes, a phenomenon involving CD2, was readily inhibited by the SP dextran sulfate (DxS) and, to a lesser extent, by the sulfated polymer polyvinyl sulfate whereas 11 other SP had no effect on E rosetting, this effect occurring at the T cell level. mAb binding studies revealed that DxS and polyvinyl sulfate, but none of the other SP tested, inhibited the binding to T cells of the anti-CD2 mAb OKT11 and anti-T112 but augmented expression of the T113 epitope of the CD2 molecule. In contrast, DxS had little or no effect on the binding of anti-CD3, -CD4, -CD8, -Pgp-1 and WT31 (TCR alpha/beta) mAb. Direct evidence that CD2 binds DxS was demonstrated by the ability of DxS-coupled fibers to totally deplete the CD2 Ag from lysates of radiolabeled human T lymphocytes and by the quantitative recovery of the CD2 Ag in fiber eluates. Control fibers coupled with other SP bound little or no CD2. Collectively, the data indicate that the CD2 molecule specifically binds DxS and suggest that a potential target cell ligand for CD2 is a sulfated carbohydrate structure.     

Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off of the sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glycosides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous according to electrophoresis and has M 45 +/- 1 kDa. The semiinactivation time of the enzyme at 60 degrees C is 20 min, and its Km value for the hydrolysis of p-nitrophenyl sulfate is 8.7 +/- 1 mM. It was shown that natural sulfated polyhydroxysteroids inhibit activity of the sulfatase; their I50 values depend on their structures and are within the range from 10(-3) to 10(-5) M.     

Effect of bitter gourd and spent turmeric on constituents of glycosaminoglycans in different tissues in streptozotocin induced diabetic rats

Diet is now one of the well established means in the management of diabetes. Bitter gourd and spent turmeric at 10% level were tested for their efficacy on glycosaminoglycan metabolism in various tissues viz., liver, spleen, lungs, heart and testis in control, diabetic and treated rats. The glycosaminoglycans (GAGs) were isolated from defatted and dried tissues. The contents of sulfated GAGs decreased in all the tissues and the decrease was more prominent in heart and testis. In the isolated GAGs, contents of total sugar, amino sugar, uronic acid and sulfate were studied. Decrease in total sugar content was maximum in testis. Amino sugar content decreased considerably in testis (38%) and lungs (15%). The content of uronic acid also decreased in testis (33%) besides heart (29%) and liver (25%). Sulfate groups in GAGs perform pivotal functions in many biological events and decrease in sulfate content was significant in heart (40%), testis (37%) and liver (37%). GAGs profile on the cellulose acetate electrophoresis revealed that heparan sulfate (HS), hyaluronic acid (HA) and chondroitin sulfate/dermatan sulfate (CS/DS) were present in liver, spleen and lungs. HS, CS were present in heart, DS/CS was observed in testis. The observed beneficial effects in GAGs metabolism during diabetes may be due to the presence of high amounts of dietary fibres present in bitter gourd and spent turmeric, besides, possible presence of bioactive compounds in one or both of them.     

Further characterization of axonally transported proteoglycans

We report further analysis of axonally transported proteoglycans in soluble and membranous subfractions of goldfish optic tectum. Distribution of transported³⁵SO₄ radioactivity was 35.2% soluble, 63.4% Triton-NaCl extractable and 1.4% unextracted. Proteoglycans isolated on DEAE cellulose were treated with chondroitinase AC or nitrous acid and remaining heparan sulfate proteoglycans (HSPGs) and chondroitin sulfate proteoglycans (CSPGs) were sized on Sepharose CL-6B. Kav values and estimated molecular weights were: Soluble CSPG-0.36 (160 kDa), Triton-NaCl extracted CSPG-.031 (200 kDa), Soluble HSPG-0.37 (150 kDa), Triton-NaCl extracted HSPG-0.37 (150 kDa). For constituent CS and HS chains the Kav values and estimated molecular weights on CL-6B were: Soluble CS-0.55 (15 kDa), Triton-NaCl extracted CS-0.55 (15 kDa), Soluble HS-0.59 (13 kDa) and Triton-NaCl extracted HS-0.65 (9 kDa). CS was shown to be sulfated exclusively at carbon 4 for both soluble and Triton NaCl extracted fractions.     

In vitro binding of human serum low-density lipoproteins by sulfated pectin derivatives

It was shown that sulfated pectin derivatives bind human serum low-density lipoproteins (LDLs) in vitro to a greater extent than native pectins. At the same time, the number of sulfate groups and the molecular weight of sulfated derivatives were crucial factors. The sulfated pectin derivatives with molecular weights more than 200 kDa containing 45 wt % sulfate groups had the greatest ability to bind LDLs, while the sulfated derivatives with molecular weights lower than 50 kDa containing 5% sulfate groups exhibited the lowest activity.     

High-performance liquid chromatography and on-line mass spectrometry detection for the analysis of chondroitin sulfates/hyaluronan disaccharides derivatized with 2-aminoacridone

In this study, we developed an on-line reverse-phase high-performance liquid chromatography-electrospray ionization-mass spectrometry (RP-HPLC-ESI-MS) separation and structural characterization of hyaluronan (HA)/chondroitin sulfate (CS)/dermatan sulfate (DS) disaccharides released by enzymatic treatment and derivatized with 2-aminoacridone (AMAC), providing a high-resolution system also applicable by using a further fluorimetric detector (Fp) before ESI-MS spectral acquisition. Isomeric nonsulfated HA and CS/DS disaccharides, isomeric monosulfated and isomeric disulfated CS/DS disaccharides, and the trisulfated species were distinctly separated and unambiguously identified by their retention times and mass spectra in negative ionization mode. In general, no multiply charged ions were detected even for highly charged disaccharides, but the presence of desulfonated products for highly sulfated species due to the relative instability of sulfo groups was observed. RP-HPLC-ESI-MS of each AMAC disaccharide was found to be linear from 3 to 500 ng with very high coefficient of correlation values due to the high efficiency of separation and the sharp outline of the peaks. Various CS/DS samples were characterized for disaccharide composition, and minor oligomer species identified as GalNAcSO₄ at the nonreducing end of chains was observed as a common component of these macromolecules. Furthermore, purified endogenous normal human plasma CS disaccharides were also evaluated by means of RP-HPLC-(Fp)-ESI-MS.     

花背蟾蜍角膜早期发育中氨基多糖的电镜细胞化学研究

本文用钌红显示氨基多糖的电镜细胞化学方法,较细致地研究了花背蟾蜍(Bufo raddeiStrauch)胚胎发育过程中(16—25期,即神经管至鳃盖完全封闭期)氨基多糖(GAG)在角膜早期发育中的变化。结果表明:GAG的合成由非硫酸化向硫酸化转变,且随着角膜的发育其含量逐渐增加。角膜各部位的HA在16—21期(胚胎开口期),其含量逐渐增加,且20(鳃血循环期)-21期达最高水平,此后含量下降,同时DS、CS、HS和Hep的含量上升。据分析,HA、HS和胶原与间充质细胞迁移有关,HA还可促使基质的膨胀和水化。硫酸化GAG在角膜的脱水、致密、细胞密度及角膜透明中起作用。胶原纤维间的硫酸化GAG能调节胶原纤维的规则化,故也有助于角膜的透明。     

Malaria sporozoites and circumsporozoite proteins bind specifically to sulfated glycoconjugates

Circumsporozoite (CS) proteins, which densely coat malaria (Plasmodia) sporozoites, contain an amino acid sequence that is homologous to segments in other proteins which bind specifically to sulfated glycoconjugates. The presence of this homology suggests that sporozoites and CS proteins may also bind sulfated glycoconjugates. To test this hypothesis, recombinant P. yoelii CS protein was examined for binding to sulfated glycoconjugate-Sepharoses. CS protein bound avidly to heparin-, fucoidan-, and dextran sulfate-Sepharose, but bound comparatively poorly to chondroitin sulfate A- or C-Sepharose. CS protein also bound with significantly lower affinity to a heparan sulfate biosynthesis-deficient mutant cell line compared with the wild-type line, consistent with the possibility that the protein also binds to sulfated glycoconjugates on the surfaces of cells. This possibility is consistent with the observation that CS protein binding to hepatocytes, cells invaded by sporozoites during the primary stage of malaria infection, was inhibited by fucoidan, pentosan polysulfate, and heparin. The effects of sulfated glycoconjugates on sporozoite infectivity were also determined. P. berghei sporozoites bound specifically to sulfatide (galactosyl[3-sulfate]beta 1-1ceramide), but not to comparable levels of cholesterol-3-sulfate, or several examples of neutral glycosphingolipids, gangliosides, or phospholipids. Sporozoite invasion into hepatocytes was inhibited by fucoidan, heparin, and dextran sulfate, paralleling the observed binding of CS protein to the corresponding Sepharose derivatives. These sulfated glycoconjugates blocked invasion by inhibiting an event occurring within 3 h of combining sporozoites and hepatocytes. Sporozoite infectivity in mice was significantly inhibited by dextran sulfate 500,000 and fucoidan. Taken together, these data indicate that CS proteins bind selectively to certain sulfated glycoconjugates, that sporozoite infectivity can be inhibited by such compounds, and that invasion of host hepatocytes by sporozoites may involve interactions with these types of compounds.     

Characterization of the recombinant rat 175-kDa hyaluronan receptor for endocytosis (HARE)

Hyaluronan (HA) and chondroitin sulfate (CS) clearance from lymph and blood in mammals is mediated by the HA receptor for endocytosis (HARE), which is present as two isoforms in rat and human (175/300 kDa and 190/315 kDa, respectively) in the sinusoidal endothelial cells of liver, spleen, and lymph nodes (Zhou, B., McGary, C. T., Weigel, J. A., Saxena, A., and Weigel, P. H. (2003) Glycobiology 13, 339-349). The small rat and human HARE proteins are not encoded directly by mRNA but are derived from larger precursors. Here we characterize the specificity and function of the 175-kDa HARE, expressed in the absence of the 300-kDa species, in stably transfected SK-Hep-1 cells. The HARE cDNA was fused with a leader sequence to allow correct orientation of the membrane protein. The recombinant rHARE contained approximately 25 kDa of N-linked oligosaccharides and, like the native protein, was able to bind HA in a ligand blot assay, even after de-N-glycosylation. SK-HARE cell lines demonstrated specific 125I-HA endocytosis, receptor recycling, and delivery of HA to lysosomes for degradation. The Kd for the binding of HA (number-average molecular mass approximately 133 kDa) to the 175-kDa HARE at 4 degrees C was 4.1 nm with 160,000 to 220,000 HA-binding sites per cell. The 175-kDa rHARE binds HA, dermatan sulfate, and chondroitin sulfates A, C, D, and E, but not chondroitin, heparin, heparan sulfate, or keratan sulfate. Surprisingly, recognition of glycosaminoglycans (GAGs) other than HA by native or recombinant HARE was temperature-dependent. Although competition was observed at 37 degrees C, none of the other GAGs competed for 125I-HA binding to SK-HARE cells at 4 degrees C. Anti-HARE monoclonal antibody-174 showed a similar temperature-dependence in its ability to block HA endocytosis. These data suggest that temperature-induced conformational changes may alter the GAG specificity of HARE. The results confirm that the 175-kDa rHARE does not require the larger HARE isoform to mediate endocytosis of multiple GAGs.     

Direct demonstration of heterogeneous,sulfatedO-linked carbohydrate chains on an endothelial ligand for L-selectin

We have previously identified endothelial ligands for L-selectin as sialylated, fucosylated and sulfated glycoproteins of approximately 50 kDa and 90 kDa (Sgp50 and Sgp90). In this report, we use the beta elimination reaction to demonstrate directly the presence of sulfatedO-linked sugar chains on one of these ligands, after metabolic labeling with radiolabeled sulfate or fucose. All of the sulfated and the majority of the fucosylatedO-linked sugar chains were shown to be sialylated by affinity chromatography on a Limax agglutinin column. Analyses by anion exchange and gel permeation chromatography revealed a complexity of sugar chains, which were heterogeneous both in charge and size. Charged groups other than sialic acid appeared to exert a predominant influence on the total charge of the sugar chains. The probable existence of a varying number of sulfate modifications per sugar chain is discussed.     

Cryoprotectants for the vitrification of corneal endothelial cells

The objective of this work was to select and test systematically possible cryoprotective agents (CPAs) and to obtain a suitable formula for vitrification of corneal endothelial cells (CECs). Fresh bovine CECs were isolated and tested with an optimized vitrification protocol with multi-step CPA loading and removal. Three types of CPAs components, i.e. the penetrating CPAs, sugars and macromolecular compounds, were experimentally evaluated using the viability assayed by trypan blue. Dimethyl sulfoxide, ethylene glycol (EG), 1,2-propanediol, 2,3-butanediol, acetamide and ethylene glycol monomethyl ether were chosen as the penetrating CPA components. Sugars including xylose, fructose, mannose, glucose, maltose, sucrose and trehalose were tested. Ficoll (MW 7 kDa), dextran (MW 7 kDa), chondroitin sulfate (CS, MW 18-30 kDa), bovine serum albumin (MW 68 kDa) and polyethylene glycol (MW 6 kDa, 10 kDa and 20 kDa) were chosen as the macromolecular compounds. CECs were also preserved by slow freezing as a control. The results showed that EG was the most suitable penetrating CPA component and glucose the most suitable sugar, and CS the most suitable macromolecule. The optimized concentrations for each component in the vitrification solution were 52% (w/w) EG, 8% (w/w) glucose and 3% (w/w) CS. The CEC survival rate of 89.4 ± 2.1% (mean ± SD) was obtained using this formula and established vitrification protocol which was comparable to that by slow freezing.     

Identification and tissue-specific distribution of sulfated glycosaminoglycans in the blood-sucking bug Rhodnius prolixus (Linnaeus)

We have previously characterized heparan sulfate (HS) as the major ovarian sulfated glycosaminoglycan (GAG) in females of Rhodnius prolixus, while chondroitin sulfate (CS) was the minor component. Using histochemical procedures we found that GAGs were concentrated in the ovarian tissue but not found inside the oocytes. Here, we extend our initial observations of GAG expression in R. prolixus by characterizing these molecules in other organs: the fat body, intestinal tract, and the reproductive tracts. Only HS and CS were found in the three organs analyzed, however CS was the major GAG species in these tissues. We also determined the compartmental distribution of GAGs in these organs by histochemical analysis using 1,9-dimethylmethylene blue, and evaluated the specific distribution of CS within both male and female reproductive tracts by immunohistochemistry using an anti-CS antibody. We also determined the GAG composition in eggs at days 0 and 6 of embryonic development. Only HS and CS were found in eggs at day 6, while no sulfated GAGs were detected at day 0. Our results demonstrate that HS and CS are the only sulfated GAG species expressed in the fat body and in the intestinal and reproductive tracts of Rhodnius male and female adults. Both sulfated GAGs were also identified in Rhodnius embryos. Altogether, these results show no qualitative differences in the sulfated GAG composition regarding tissue-specific or development-specific distribution.     

Aryl sulfatase of unusual specificity from the liver of marine mollusk Littorina kurila

An aryl sulfatase of unusual specificity has been isolated from the liver of marine mollusk Littorina kurila. It hydrolyzes p-nitrophenyl sulfate, does not affect the natural fucoidan, and catalyzes splitting off the sulfate group in position C4 of xylose residues within the carbohydrate chains of holostane triterpene glycosides from sea cucumbers. The properties of the enzyme were studied at pH 5.4. The protein is homogeneous according to electrophoresis and has M 45 ± 1 kDa. The semiinactivation time of the enzyme at 60°C is 20 min, and its K _m value for the hydrolysis of p-nitrophenyl sulfate is 8.7 ± 1 mM. It was shown that natural sulfated polyhydroxysteroids inhibit activity of the sulfatase; their I ₅₀ values depend on their structures and are within the range from 10^?3 to 10^?5 M.     

Pancreatic carcinoma is characterized by elevated content of hyaluronan and chondroitin sulfate with altered disaccharide composition

The amount and the types of glycosaminoglycans (GAGs) present in human pancreatic carcinoma were examined and compared with those in normal pancreas. Human pancreatic carcinoma contained increased levels (4-fold) of total GAGs. Particularly, this carcinoma is characterized by a 12-fold increase of hyaluronan (HA) and a 22-fold increase in chondroitin sulfate (CS) content. CS in pancreatic carcinoma exhibited an altered disaccharide composition which is associated with marked increase of non-sulfated and 6-sulfated disaccharides. Dermatan sulfate (DS) was also increased (1.5-fold) in carcinoma, whereas heparan sulfate (HS), the major GAG of normal pancreas, becomes the minor GAG in pancreatic carcinoma without significant changes in the content and in molecular size. In all cases, the galactosaminoglycans (GalGAGs, i.e. CS and DS) derived from pancreatic carcinomas were of lower molecular size compared to those from normal pancreas. The results in this study indicate, for the first time, that human pancreatic carcinoma is characterized by highly increased amounts of HA and of a structurally altered CS.     

Isolation and characterization of a novel chondroitin sulfate from squid liver integument rich in N-acetylgalactosamine(4,6-disulfate) and glucuronate(3-sulfate) residues

Novel chondroitin sulfate (CS) chains with an average molecular mass of 79.6 kDa were purified from squid liver integument. A compositional analysis of the CS chains using chondroitinases (CSases) ABC and AC-I revealed a range of variably sulfated disaccharides with GlcAβ1→3GalNAc(6-sulfate), GlcAβ1→3GalNAc(4-sulfate), and GlcAβ1→3GalNAc(4,6-disulfate) as the major ones, significant amounts of rare 3-sulfated GlcA-containing disaccharides, and a small amount of nonsulfated GlcAβ1→3GalNAc. The CS chains exhibited neurite outgrowth-promoting activity toward embryonic mouse hippocampal neurons, which was abolished completely by digestion with CSase ABC or AC-I. Consequently, whether these CS chains interact with heparin-binding growth factors was tested in a BIAcore system. All of the growth factors exhibited concentration-dependent and specific binding. CS chains from squid liver integument, with their unique composition and strong biological activities, may be a good candidate for therapeutic application.     

The antihyperglycemic effect of estrone sulfate in genetically obese-diabetic (ob/ob) mice is associated with reduced hepatic glucose-6-phosphatase.

Excessive glucose production by the liver contributes significantly to diabetic hyperglycemia. The enzyme system glucose-6-phosphatase plays a key role in regulating hepatic glucose production and therefore its inhibition is a potential therapeutic target for the correction of hyperglycemia. It has previously been shown that sulfated steroids, such as estrone sulfate and dehydroepiandrosterone sulfate, inhibit the glucose-6-phosphatase system in vitro, principally through inhibition of endoplasmic reticulum glucose-6-phosphate transport. We report here that in the obese/diabetic ob/ob mouse model, orally administered estrone sulfate reduces the abnormally elevated hepatic glucose-6-phosphatase enzyme activity and enzyme protein levels that are characteristic in the ob/ob mouse, and that this reduction is associated with normalization of blood glucose levels. Other sulfated and non-sulfated steroids also reduced, to a lesser extent, glucose-6-phosphatase enzyme activity - with the exception of dehydroepiandrosterone sulfate, which had no apparent effect on this system in ob/ob mice. Estrone sulfate is therefore an effective antihyperglycemic agent in ob/ob mice, and the glucose-6-phosphatase system can be successfully targeted for the therapeutic management of hyperglycemia in this animal model of non-insulin-dependent diabetes mellitus.