Enhydrin Regulates Postprandial Hyperglycemia in Diabetic Rats by Inhibition of α-Glucosidase Activity.

During the last few years, numerous attempts were made to identify effective α-glucosidase inhibitors from natural sources in order to develop new alternatives for diabetes management. Smallanthus sonchifolius (yacon) leaves were found to be effective in controlling postprandial hyperglycemia. Enhydrin, a constituent of yacon leaves, was noted for its significant hypoglycemic properties in diabetic rats. These properties were also demonstrated for yacon leaves decoction, which is rich in phenolic compounds such as chlorogenic acid and its derivatives. The purpose of the present study was to evaluate the potential of yacon leaves decoction and the isolated compound enhydrin to inhibit α-glucosidase enzyme, a possible mechanism of the above antihyperglycemic effect. In vitro assays showed that both 10% decoction and enhydrin significantly inhibited the activity of the yeast α-glucosidase enzyme in a dose-dependent manner, IC50 values being 50.40 and 134.17 µg/ml, respectively. In vivo experiments showed a rapid decrease in the hyperglycemic peak after sucrose load (2 g/kg body weight) in normal rats treated with the 10% decoction (140 mg/kg) and enhydrin (0.8 mg/kg). Both treatments caused a significant decrease in blood glucose levels in diabetic rats after sucrose load compared to diabetic control. These results suggest that both products assayed could be effective in the management of postprandial hyperglycemia through inhibition of α-glucosidase in the small intestine.

Association of lifestyle and demographic factors with estrogenic and glucocorticogenic activity in Mexican American women.

Breast cancer risk is higher in US-born than in foreign-born Hispanics/Latinas and also increases with greater length of US residency. It is only partially known what factors contribute to these patterns of risk. To gain new insights, we tested the association between lifestyle and demographic variables and breast cancer status, with measures of estrogenic (E) and glucocorticogenic (G) activity in Mexican American women. We used Chemical-Activated LUciferase gene eXpression assays to measure E and G activity in total plasma from 90 Mexican American women, without a history of breast cancer at the time of recruitment, from the San Francisco Bay Area Breast Cancer Study. We tested associations of nativity, lifestyle and sociodemographic factors with E and G activity using linear regression models. We did not find a statistically significant difference in E or G activity by nativity. However, in multivariable models, E activity was associated with Indigenous American ancestry (19% decrease in E activity per 10% increase in ancestry, P = 0.014) and with length of US residency (28% increase in E activity for every 10 years, P = 0.035). G activity was associated with breast cancer status (women who have developed breast cancer since recruitment into the study had 21% lower G activity than those who have not, P = 0.054) and alcohol intake (drinkers had 25% higher G activity than non-drinkers, P = 0.015). These associations suggest that previously reported breast cancer risk factors such as genetic ancestry and alcohol intake might in part be associated with breast cancer risk through mechanisms linked to the endocrine system.

Cell adhesion glycoprotein vitronectin during Xenopus laevis embryogenesis.

Vitronectin (vn) is a cell-adhesive glycoprotein present in blood and extracellular matrix of all vertebrates. In the present study we reported the cDNA cloning of Xenopus laevisvitronectin and its spatial and temporal expression pattern during the embryonic development of this important model organism. The deduced amino acid sequence of Xenopus laevis vn showed 49%, 47% and 43% identity with human, chicken and zebrafish orthologs, respectively, whereas the comparison with Xenopus tropicalis vn presented 85% identity. The structural organization consisting of a somatomedin B domain and two hemopexin-like domains was similar to higher vertebrate vitronectins. The vn transcripts were detected from stage 28 onward. At tadpole stages, vn is expressed in heart, gut derivatives and in the notochord. The protein was detected in heart, liver, foregut, pronephros and notochord at stages 43 and 47 of Xenopus embryos. Our results suggest that vitronectin is developmentally regulated and could participate in embryo organogenesis.

Ultrastructural changes in the follicular epithelium of Ceratophrys cranwelli previtellogenic oocytes.

In this work we carried out ultrastructural, autoradiographic and biochemical analyses of the follicular epithelium during C. cranwelli previtellogenesis. This study revealed that the follicular epithelium in early previtellogenesis is constituted of a single layer of squamous homogeneous cells. During mid-previtellogenesis two types of cells develop: dark cells and clear cells. The follicular dark cells are actively involved in the synthesis of RNA, which is transferred to the oocyte through the interface. In late previtellogenesis the dark cells show apoptotic characteristics such as chromatin condensation, DNA fragmentation and cytoplasm shrinkage. This process forms apoptotic bodies that seem to be engulfed by the oocyte. Our results show evidence that, during mid- and late C. cranwelli previtellogenesis, the follicular epithelium undergoes remodelling processes interacting with the oocyte.

Implication of gap junction coupling in amphibian vitellogenin uptake.

The aim of the present study was to investigate the physiological role and the expression pattern of heterologous gap junctions during Xenopus laevis vitellogenesis. Dye transfer experiments showed that there are functional gap junctions at the oocyte/follicle cell interface during the vitellogenic process and that octanol uncouples this intercellular communication. The incubation of vitellogenic oocytes in the presence of biotinylated bovine serum albumin (b-BSA) or fluorescein dextran (FDX), showed that oocytes develop stratum of newly formed yolk platelets. In octanol-treated follicles no sign of nascent yolk sphere formation was observed. Thus, experiments in which gap junctions were downregulated with octanol showed that coupled gap junctions are required for endocytic activity. RT-PCR analysis showed that the expression of connexin 43 (Cx43) was first evident at stage II of oogenesis and increased during the subsequent vitellogenic stages (III, IV and V), which would indicate that this Cx is related to the process that regulates yolk uptake. No expression changes were detected for Cx31 and Cx38 during vitellogenesis. Based on our results, we propose that direct gap junctional communication is a requirement for endocytic activity, as without the appropriate signal from surrounding epithelial cells X. laevis oocytes were unable to endocytose VTG.

Hypoglycemic effect of the water extract of Smallantus sonchifolius (yacon) leaves in normal and diabetic rats.

The hypoglycemic effect of the water extract of the leaves of Smallantus sonchifolius (yacon) was examined in normal, transiently hyperglycemic and streptozotocin (STZ)-induced diabetic rats. Ten-percent yacon decoction produced a significant decrease in plasma glucose levels in normal rats when administered by intraperitoneal injection or gastric tube. In a glucose tolerance test, a single administration of 10% yacon decoction lowered the plasma glucose levels in normal rats. In contrast, a single oral or intraperitoneal administration of yacon decoction produced no effect on the plasma glucose levels of STZ-induced diabetic rats. However, the administration of 2% yacon tea ad libitum instead of water for 30 days produced a significant hypoglycemic effect on STZ-induced diabetic rats. After 30 days of tea administration, diabetic rats showed improved body (plasma glucose, plasma insulin levels, body weight) and renal parameters (kidney weight, kidney to body weight ratio, creatinine clearance, urinary albumin excretion) in comparison with the diabetic controls. Our results suggest that yacon water extract produces an increase in plasma insulin concentration.

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats.

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.

Changes in liver gangliosides in streptozotocin-induced diabetic rats.

Diabetes mellitus is associated with various structural and functional liver abnormalities that affect the glycogen and lipid metabolisms. The effects of streptozotocin-induced diabetes and of insulin supplementation to Sprague-Dawley diabetic rats on ganglioside patterns in liver were determined. Diabetic livers showed a tendency to hepatomegaly 3 weeks after STZ-induction of diabetes. The concentration of total gangliosides in diabetic and non-diabetic livers was similar, but the concentration of total gangliosides in the liver of insulin-stabilized rats was slightly increased. Bidimensional TLC chromatographic analysis of gangliosides isolated from normal diabetic and insulin-stabilized diabetic livers showed quantitative and qualitative changes. In comparison with normal controls, the densitometric analyses of diabetic liver ganglioside patterns had increased amounts of GM3, GM1, GD1b, and GT1b gangliosides, while GM2 could not be detected. The hepatic ganglioside pattern of insulin-stabilized diabetic rats was partially restored, resembling the profile of normal rats. The activity of GalNAcT, GalT-2 and SialT-4 transferases was measured in liver microsomal fractions of the different groups of animals. Diabetic rats showed an increased activity of GalNAcT and a decrease in the activity of GalT-2 and SialT-4 compared with the controls. The enzymatic activities found in insulin-treated rats showed a tendency to return to the values observed in normal control animals. The results evidenced that streptozotocin-induced diabetes affects the liver ganglioside pattern and the ganglioside synthesis enzyme activity. The alterations found in ganglioside metabolism could represent one of the earliest changes associated with the diabetic pathology.

Inhibition of the synthesis of glycosphingolipid by a ceramide analogue (PPMP) in the gastrulation of Bufo arenarum.

In the present study the role of glycosphingolipids (GSL) in amphibian development was investigated. We analysed the de novo synthesis of neutral GSL and gangliosides through the initial stages of Bufo arenarum embryo development and their participation during gastrulation using 1-phenyl-2-palmitoyl-3-morpholino-1-propanol (PPMP), a potent inhibitor of glucosylceramide synthase. Ganglioside synthesis began at the blastula stage and reached a maximum during gastrulation (stages 10-12) while neutral GSL synthesis showed a slight gradual increase, the former being quantitatively more significant than the latter. Ganglioside synthesis was reduced by 90% while neutral GSL synthesis was inhibited by 65% when embryos at blastula stage were cultured for 24 h in 20 microM PPMP. The depletion of GSL from amphibian embryos induced an abnormal gastrulation in a dose-dependent manner. We found that PPMP had a pronounced effect on development since no embryos exhibited normal gastrulation; their developmental rate either slowed down or, more often, became totally arrested. Morphological analysis of arrested embryos revealed inhibition of the gastrulation morphogenetic movements. Analysis of mesodermal cell morphology in those embryos showed a severe decrease in the number and complexity of cellular extensions such as filopodia and lamellipodia. Mesodermal cells isolated from PPMP-treated embryos had very low adhesion percentages. Our results suggest that glycosphingolipids participate in Bufo arenarum gastrulation, probably through their involvement in cell adhesion events.

Effect of gap junction uncoupling in full-grown Bufo arenarum ovarian follicles: participation of cAMP in meiotic arrest.

The aim of the present study was to determine the presence of the connexins Cx43, Cx32 and Cx26 in Bufo arenarum ovarian follicles during the breeding season as well as to analyse the possible alterations in the meiotic process when connexins are blocked by specific antibodies. Western blot analysis revealed that the Cx43 and Cx32 proteins were present but not Cx26. We demonstrated that the anti-Cx43 and anti-Cx32 antibodies produced the uncoupling of the gap junctions. When these junctions are blocked the maturation process is triggered in the oocytes. We determined that dbcAMP exerts an inhibitory effect on the maturation induced by the uncoupling of the gap junctions when the oocytes are injected or pretreated with this metabolite. We propose the idea that cAMP is the regulatory molecule in meiotic arrest in this amphibian species.

Participation of the GM1 ganglioside in the gastrulation of anuran amphibian Bufo arenarum.

In the present paper we established the ganglioside composition of the blastula and gastrula stages of the anuran amphibian Bufo arenarum, two relevant stages characterized by dynamic changes in morphology and cellular rearrangements. Densitometric studies evidenced that GD1a and GT1b were the more abundant gangliosides of the blastula embryos whereas GM1 and GM2 were the predominant species in gastrula embryos. Analysis of ganglioside abundance indicates that the "a" and "b" synthesis pathways perform similar biosynthetic activities in the blastula stage, in contrast to the gastrula stage in which a marked predominance of the "a" pathway occurred. The spatio-temporal expression of GM1 and of polygangliotetraosyl ceramides (pGTC) was investigated by wholemount immunocytochemistry using cholera toxin B subunit (CTB) and an affinity purified human anti-GM1 antibody. The pGTC were detected as GM1 after treatment with neuraminidase. Blastomeres from the inner surface of the blastocoelic roof (BCR) of blastula embryos were GM1 and pGTC positive. At midgastrula stage, embryos showed an increased labeling on the inner surface of BCR. To establish whether the GM1 ganglioside was involved in the gastrulation processes, CTB, anti-GM1 antibodies and anti-GM1 Fab' fragments were microinjected into the blastocoel cavity of blastula embryos. Treatment with the probes blocked gastrulation. Scanning electron microscopy analysis of blocked embryos revealed that mesodermal cell migration, radial interdigitation, and convergent extension movements were affected. The blocking of gastrulation was correlated with the absence of fibronectin and EP3/EP4 on the inner surface of blastocoelic roof of CTB- or anti-GM1 treated embryos. Results show that the GM1 ganglioside is differentially expressed by embryonic cells and participates in the morphogenetic processes of amphibian gastrulation. J. Exp. Zool. 286:457-472, 2000.

Comparative study of vitellogenesis in the anuran amphibians Ceratophrys cranwelli (Leptodactilidae) and Bufo arenarum (Bufonidae).

The present study analyses, by transmission electron microscopy, vitellogenesis in two anuran amphibian families: Leptodactilidae (Ceratophrys cranwelli) and Bufonidae (Bufo arenarum). These differ in the type of stimulus that sets off their reproductive period, pluvial changes being the trigger in C. cranwelli and temperature increase in B. arenarum. We found that vitellogenesis follows an endocytic pathway that involves membranous structures (coated pits, coated vesicles, endosomes and multivesicular bodies). This process results in a fully grown yolk platelet of similar structure in both species. Despite the above similarity, a distinctive feature in B. arenarum was that the multivesicular bodies exhibited condensed proteins together with lipid droplets, the latter remaining as such even in the primordial yolk platelet. In C. cranwelli, however, lipids droplets were only found attached to the primordial yolk platelet. The coexistence of lipid droplets together with proteins in the nascent precursor yolk platelets observed in B. arenarum is similar to that found in B. marinus. This fact might constitute a characteristic feature of the Bufonidae family.

Evidence for the presence and participation of 85-75 KDa extracellular matrix components in cell interactions of Bufo arenarum gastrulation.

We studied the presence and distribution of the extracellular materials (ECM), obtained by mild embryonic dissociation through nondenaturing and denaturing PAGE, immunoblotting and immunocytochemical wholemount in the gastrulation of anuran amphibian Bufo arenarum. The SDS-PAGE, under reducing conditions, revealed the protein profile of the ECM which comprised six bands. The Western immunoblotting effected with antibodies against fibronectins (FN) of Xenopus laevis, Ambystoma mexicanum and Bufo arenarum revealed that the 210 and 190 KDa bands (EP1-EP2) present in the ECM were identified as FN. Polyclonal antibodies against the 85-75 KDa polypeptides (EP3-EP4) were obtained and used throughout this study. The distribution of FN and EP3-EP4 was comparatively studied in the blastocoelic roof (BCR) of stage 10.5 Bufo arenarum, Xenopus laevis and Ambystoma mexicanum embryos. In the anurans, FN appeared as a network of fine fibrils apparently oriented at random, while in Ambystoma, FN appeared as a complex anastomosing network of oriented fibrils. EP3-EP4 were found in Bufo and in Xenopus both in the intercellular contact zones and in the cellular periphery. No linear arrangements of these proteins were observed. Few, if any, EP3-EP4 were found on the BCR of Ambystoma mexicanum. At stage 11, EP3-EP4, which showed a dramatic increase at the chordomesoderm-neuroectoderm junction in Bufo arenarum embryos, appeared as an amorphous material. For the purpose of analyzing the role of EP3-EP4 during Bufo arenarum gastrulation, anti-EP3-EP4 antibodies and anti-EP3-EP4 Fab fragments were microinjected into the blastocoel cavity of stage 9 embryos, an event that cause severe alterations in the gastrulation process. Convergent extension of the dorsal marginal zone and the epiboly of the BCR were the most strongly affected events. Results show that EP3-EP4 are required for normal Bufo arenarum gastrulation.

Amphibian Bufo arenarum vitronectin-like protein: its localization during oogenesis.

In the present study, we analyzed the localization of vitronectin-like protein in oocytes during oogenesis as well as in the serum and liver tissue of the amphibian Bufo arenarum. Vitronectin-like protein was purified from serum by heparin-affinity chromatography and showed to have the two biological properties in common with most animal vitronectins (VN): heparin binding activity and an RGD-dependent cell-spreading activity. SDS-PAGE of vitronectin-like protein revealed that it consists of two bands of 64 kDa and 72 kDa, while immunoblotting analyses showed that this protein strongly cross-reacts with two monoclonal antibodies against human VN. No immunofluorescent staining of vitronectin-like protein was observed in previtellogenic oocytes (stages I and II). In vitellogenic oocytes (stages III, IV and V) fluorescence was observed in the cortical cytoplasm localized in yolk platelets, extending concomitantly with the vitellogenic process. When we examined the yolk platelet formation pathway by immunoelectron microscopy, gold particles indicated that vitronectin-like protein was located on the yolk platelet precursors: multivesicular bodies and primordial yolk platelets. Gold particles also were seen sparsely distributed in all oocyte investing layers. The mean serum vitronectin-like protein concentration in amphibian animals was 127.8 +/- 11.6 micrograms/ml in adult males and 181.5 +/- 14.3 micrograms/ml in adult females. Serum vitronectin-like protein of males and females was susceptible to hormonal stimulation (17-beta estradiol). These results suggest that vitronectin-like protein is stored in the yolk platelets and may be involved in the later events of amphibian development.

Vitelline envelope formation during oogenesis in Bufo arenarum.

The formation of vitelline envelope (VE) during the oogenesis of Bufo arenarum (Amphibia Anura) is described. At the stage of early vitellogenesis, the first structures appear: the number of oocyte microvilli increases, and many cross sections of them are observed between the follicle cells and the oocyte. A filamentous material is observed inside the follicle cells and between the follicle cells and the oocyte. Multivesicular bodies are also found in the follicle cells, and in the perivitelline space. The micrographs also suggest the participation of the oocyte in the process of VE formation: large vesicles are present in the cortex of the oocyte, filled with an amorphous material of low and uniform electron density. Some of them are in the process of releasing their content to the perivitelline space. Many vesicles (probably resulting from microvilli fragmentation) are also observed in the perivitelline space. During late vitellogenesis the VE is a continuous structure between the layer of follicle cells and the oocyte. The filamentous material is aggregated in bundles, forming a net, and the spherical components are now either included in the orifices of the net, or free near the oocyte's surface. At the end of oogenesis, when the VE is completely formed, it is difficult to distinguish its components independently. Immunolocalization with antibodies against VE, show a positive reaction in follicle cells and oocytes in previtellogenic and full grown ovarian follicles. This analysis suggests that both the oocyte and the follicle cells are directly involved in the synthesis and secretion of the components of the vitelline envelope in Bufo arenarum.

Vitelline envelope formation during oogenesis in Bufo arenarum

The formation of vitelline envelope (VE) during the oogenesis of Bufo arenarum (Amphibia Anura) is described. At the stage of early vitellogenesis, the first structures appear: the number of oocyte microvilli increases, and many cross sections of them are observed between the follicle cells and the oocyte. A filamentous material is observed inside the follicle cells and between the follicle cells and the oocyte. Multivesicular bodies are also found in the follicle cells, and in the perivitelline space. The micrographs also suggest the participation of the oocyte in the process of VE formation: large vesicles are present in the cortex of the oocyte, filled with an amorphous material of low and uniform electron density. Some of them are in the process of releasing their content to the perivitelline space. Many vesicles (probably resulting from microvilli fragmentation) are also observed in the perivitelline space. During late vitellogenesis the VE is a continuous structure between the layer of follicle cells and the oocyte. The filamentous material is aggregated in bundles, forming a net, and the spherical components are now either included in the orifices of the net, or free near the oocyte's surface. At the end of oogenesis, when the VE is completely formed, it is difficult to distinguish its components independently. Immunolocalization with antibodies against VE, show a positive reaction in follicle cells and oocytes in previtellogenic and full grown ovarian follicles. This analysis suggests that both the oocyte and the follicle cells are directly involved in the synthesis and secretion of the components of the vitelline envelope in Bufo arenarum.

Vitelline envelope formation during oogenesis in Bufo arenarum

The formation of vitelline envelope (VE) during the oogenesis of Bufo arenarum (Amphibia Anura) is described. At the stage of early vitellogenesis, the first structures appear: the number of oocyte microvilli increases, and many cross sections of them are observed between the follicle cells and the oocyte. A filamentous material is observed inside the follicle cells and between the follicle cells and the oocyte. Multivesicular bodies are also found in the follicle cells, and in the perivitelline space. The micrographs also suggest the participation of the oocyte in the process of VE formation: large vesicles are present in the cortex of the oocyte, filled with an amorphous material of low and uniform electron density. Some of them are in the process of releasing their content to the perivitelline space. Many vesicles (probably resulting from microvilli fragmentation) are also observed in the perivitelline space. During late vitellogenesis the VE is a continuous structure between the layer of follicle cells and the oocyte. The filamentous material is aggregated in bundles, forming a net, and the spherical components are now either included in the orifices of the net, or free near the oocytes surface. At the end of oogenesis, when the VE is completely formed, it is difficult to distinguish its components independently. Immunolocalization with antibodies against VE, show a positive reaction in follicle cells and oocytes in previtellogenic and full grown ovarian follicles. This analysis suggests that both the oocyte and the follicle cells are directly involved in the synthesis and secretion of the components of the vitelline envelope in Bufo arenarum.(AU)

Alteration of morphogenetic cell interactions by 1-beta-D-arabinofuranosylcytosine in Bufo arenarum embryo.

Amphibian gastrulation was used as a model system to study the action of the nucleoside 1-beta-D-arabinofuranosylcytosine (Ara-C) on the early events of amphibian morphogenesis. Ara-C inhibits both glycoprotein and glycolipid synthesis and interferes with DNA synthesis. Thus, it is useful to investigate the importance of the cell surface and the nucleous during Bufo arenarum morphogenesis. Living embryos were incubated with Ara-C at blastula and gastrula stages. Treated-embryos undergo abnormal gastrulation, most of the embryos exogastrulate, although some do not gastrulate at all. This antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. We have proven that Area-C disturbs the typical intercellular organization and inhibits the radial intercalation of the blastocoelic roof. The mesodermal migration is the most affected morphogenetic process. The results described in this paper demonstrate that the timing of gastrulation movements strongly involves the participation of surface and extracellular molecules in cell recognition and cell interaction but does not involve a significant increase in cell division rate and can also occur in the absence of the cell division.

Alteration of morphogenetic cell interactions by 1-beta-D-arabinofuranosylcytosine in Bufo arenarum embryo.

Amphibian gastrulation was used as a model system to study the action of the nucleoside 1-beta-D-arabinofuranosylcytosine (Ara-C) on the early events of amphibian morphogenesis. Ara-C inhibits both glycoprotein and glycolipid synthesis and interferes with DNA synthesis. Thus, it is useful to investigate the importance of the cell surface and the nucleous during Bufo arenarum morphogenesis. Living embryos were incubated with Ara-C at blastula and gastrula stages. Treated-embryos undergo abnormal gastrulation, most of the embryos exogastrulate, although some do not gastrulate at all. This antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. We have proven that Area-C disturbs the typical intercellular organization and inhibits the radial intercalation of the blastocoelic roof. The mesodermal migration is the most affected morphogenetic process. The results described in this paper demonstrate that the timing of gastrulation movements strongly involves the participation of surface and extracellular molecules in cell recognition and cell interaction but does not involve a significant increase in cell division rate and can also occur in the absence of the cell division.