Evaluating the intensity of the 'prompt' 140.5 keV γ-ray of 99Mo using a 4παß(LS)-γ(HPGe) measurement system.

The absolute intensity for the 'prompt' 140.5 keV gamma-ray of 99Mo was evaluated using the ß-γ coincidence technique. A liquid sample of 99Mo was prepared from a99Mo/99mTc generator and measured in a 4παß(LS)-γ(HPGe) system that comprises a Liquid Scintillator (LS) detector and a High-Purity Germanium (HPGe) detector. The sample was introduced into scintillation fluid embedded in a photo-reflector assembly that provides almost 100% efficiency for detecting ß particles (in the energy range of intreset). The combination of the HPGe and the LS detectors provided a highly effective rejection mechanism for non-coincident events. Thereby, the distinction between the detected 140.5 keV events originating from decays of 99mTc (IT) and those from transitions bypassing the metastable state could be obtained and the 'prompt' intensity was evaluated directly. The system was calibrated for detecting ß particles and γ-rays using radioactive sources of known activities and having identical geometry as the sample containing 99Mo. The absolute intensity of the 'prompt' 140.5 keV was found to be (5.21 ± 0.02stat±0.16sys)%, in good agreement with results from more recently reported works.

Antidiabetic effect of novel modulating peptides of G-protein-coupled kinase in experimental models of diabetes.

AIMS/HYPOTHESIS: G-protein-coupled receptor kinases (GRKs) play a key role in agonist-induced desensitisation of G-protein-coupled receptors (GPCRs) that are involved in metabolic regulation and glucose homeostasis. Our aim was to examine whether small peptides derived from the catalytic domain of GRK2 and -3 would ameliorate Type 2 diabetes in three separate animal models of diabetes. METHODS: Synthetic peptides derived from a kinase-substrate interaction site in GRK2/3 were initially screened for their effect on in vitro melanogenesis, a GRK-mediated process. The most effective peptides were administered intraperitoneally, utilising a variety of dosing regimens, to Psammomys obesus gerbils, Zucker diabetic fatty (ZDF) rats, or db/db mice. The metabolic effects of these peptides were assessed by measuring fasting and fed blood glucose levels and glucose tolerance. RESULTS: Two peptides, KRX-683(107) and KRX-683(124), significantly reduced fed-state blood glucose levels in the diabetic Psammomys obesus. In animals treated with KRX-683(124) at a dose of 12.5 mg/kg weekly for 7 weeks, ten of eleven treated animals responded with mean blood glucose significantly lower than controls (4.7+/-0.4 vs 16.8+/-0.8 mmol/l, p

Induction of polycystic ovary by testosterone in immature female rats: Modulation of apoptosis and attenuation of glucose/insulin ratio.

Polycystic ovarian syndrome is seen in 5% of fertile aged women. However, there is no satisfactory PCOS model in experimental animals. To induce polycystic ovary phenotype in immature female rats, Wistar rats 21 days of age were injected daily with testosterone propionate 1 mg/100 g body weight dissolved in propylene glycol or propylene glycol for up to 35 days. Seven days of injection with testosterone (T) resulted in the appearance of large cystic follicles and a dramatic accumulation of multi-layer preantral follicles. At 42 days of age puberty in control animals was evident by the appearance of corpora lutea. In contrast in T treated animals no corpora lutea formation was seen even at the age of 56 days. Progesterone in the control animals was elevated at the age of 42 days in contrast with the T treated animals in which progesterone remained low (20% of control). While during 14 days of T injection most of the follicles did not have progressive apoptosis, at 21-35 days of injection (42-56 days of age) the vast majority of follicles became apoptotic. Progressive degeneration of oocytes was evident in T treated animals reaching 70-85% of total oocytes at 21-35 days of T injection compared to 30-40% in control animals. Western blot analysis of ovarian homogenates revealed gradual decrease in Bcl-2 content, evident at 28 and 35 days of T injection compared to control animals. Interestingly, the fasting glucose/insulin ratio was dramatically reduced in T treated animals following 14 days of testosterone treatment compared to controls. Our data suggest that T injection to immature female rats can induce polycystic ovaries, block ovulation and attenuate progesterone production. Moreover, normal/low glucose and high insulin blood levels in the testosterone treated rats raises the possibility that elevated androgens can lead to insulin resistance in this experimental PCOS model.

Gonadotropin-induced gene regulation in human granulosa cells obtained from IVF patients: modulation of genes coding for growth factors and their receptors and genes involved in cancer and other diseases.

Gonadotropins play a crucial role in ovarian homeostasis and fertilization. However, hypergonadotropin stimulation has been thought to increase the risk for ovarian cancer. Moreover, some correlation between high levels of gonadotropins in the circulation and Alzheimer's disease has been implicated, with no clear evidence on the molecular mechanism involved. Using DNA microarray technology and RNA from gonadotropin-stimulated human granulosa cells, which comprise the main bulk of the ovarian follicular somatic cells, we discovered that stimulation of cells with saturating doses of gonadotropins gives rise to the expression of genes coding for presenilin 1 and 2, along with the up-regulation of genes involved in steroidogenesis such as StAR, cytochrome P450scc enzyme system and aromatase. Moreover, gonadotropin stimulation in these cells dramatically elevates activity of genes coding for epiregulin and amphiregulin, which can bind and activate the EGF receptor and ERB4. These gene products may elevate the risk for ovarian, breast, endometrial and other non-gynecological cancers. Gene transcripts for oncogenes and tumor markers such as pleiomorphic adenoma gene-like 1 (Plagl1) tumor antigen (L6) and claudin 3 were markedly elevated following LH and FSH stimulation. In parallel, downregulation in ovarian cancer 1 (DOC1) and suppression of tumorigenicity (ST5) genes was observed, suggesting a potential increase for cancer development. In contrast, increase in tumor rejection antigen (gp96) 1 and decrease in connective tissue growth factor (CTGF), transforming growth factor-beta 1 induced transcript 1 (TGFB1Il), pim-1 oncogene (PIM1), v-maf musculoaponeurotic fibrosarcoma oncogene homologue (MAF) and CD24 antigen may be associated with a decreased risk for specific cancers. In conclusion, gonadotropin stimulation may modulate specific sets of gene transcripts that may either elevate or reduce the risk for specific diseases.

Gonadotrophin-induced gene regulation in human granulosa cells obtained from IVF patients. Modulation of steroidogenic genes, cytoskeletal genes and genes coding for apoptotic signalling and protein kinases.

Gonadotrophins exert a major effect on ovarian development and on the control of fertilization. By stimulating cells with forskolin (FK), it is possible to study which genes are activated by gonadotrophins via the cAMP cascade, and which by alternative pathways. Using RNA isolated from stimulated cells, we found that 59% of the total genes modulated by LH were also modulated by FK, while 69% of the genes modulated exclusively by FSH were also modulated by FK. Gene transcripts involved in steroidogenesis/progesterone production were highly elevated, while 17beta-hydroxysteroid dehydrogenase was down-regulated. This suggests that a decrease in the conversion of androstenedione to testosterone and estrone to estradiol occurs during luteinization. Down-regulation of genes coding for actin cytoskeleton proteins and cytokeratin 18 was observed in response to gonadotrophin and cAMP stimulation. Several of the genes coding for the microtubule network were also modulated, implying that rearrangement of the cytoskeletal proteins permits better coupling between organelles involved in steroidogenesis. A dramatic change in gene transcripts coding for signalling enzymes was observed following LH stimulation. This includes the down-regulation of adenylyl cyclase 7 and 9, elevation of cAMP-dependent phosphodiesterase, and the up-regulation of a negative regulator of G-protein signalling (RGS16) that may negate gonadotrophin signalling via guanine nucleotide binding proteins. Thus luteinized cells, despite increased gene transcripts to LH/chorionic gonadotrophin (CG) receptors, respond inefficiently to gonadotrophin stimulation, due to attenuation of signal transduction in the cAMP cascade at multiple steps. Novel genes involved in the regulation of apoptosis were found for the first time to be up-regulated by gonadotrophin stimulation, including: BAX inhibitor-1, granulysin and apoptosis repressor with caspase recruitment domain (ARC). These proteins may be involved in a unique alternative pathway of ovarian cell death. Such a pathway could temporarily preserve the mitochondria and progesterone production during the initial stages of granulosa cell apoptosis.

Activation of multiple signal transduction pathways by glucocorticoids: protection of ovarian follicular cells against apoptosis.

We have recently demonstrated that glucocorticoids protect against serum-deprivation, cAMP-, TNFalpha-, and p53-induced apoptosis in ovarian follicular cells involved in up-regulation of Bcl-2. We demonstrated that dexamethasone, which enhances steroidogenesis by up-regulation of the p450scc enzyme system, stimulates the MAPK cascade by phosphorylation of ERK1, ERK2 as well as by Akt phosphorylation within 1-5min with no effect on p38 MAPK phosphorylation. Moreover, glucocorticoids enhance expression of connexin 43, formation of gap junctions, expression of cadherins, and formation of adherence junctions within 24h of hormone stimulation of ovarian granulosa cells. It is suggested that the protective effects of glucocorticoids against apoptosis are mediated by both genomic and non-genomic mechanisms. Moreover, for the first time we show that protein phosphorylation, cell-cell contact, and intracellular communication are important mediators in glucocorticoid protection against apoptosis in ovarian follicular cells.

Alternative pathways of ovarian apoptosis: death for life.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few, but healthy embryos. Degeneration of the old corpora lutea in each estrus/menstrual cycle by programmed cell death is essential for maintaining the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, play an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows for systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. We have discovered that during initial stages of granulosa cell apoptosis progesterone production does not decrease. In contrast, we found that it is elevated for up to 24hr following the onset of the apoptotic stimuli exerted by starvation, cAMP, p53 or tumor necrosis factor alpha stimulation, before total cell collapse. These observations raise the possibility for an alternative unique apoptotic pathway, one that does not involve mitochondrial cytochrome C release associated with the destruction of mitochondrial structure and steroidogenic function. Using mRNA from apoptotic cells and Affymetrix DNA microarray we discovered that Granzyme B, a protease that normally resides in T cytotoxic lymphocytes and natural killer cells of the immune system is expressed and activated in granulosa cells, thereby allowing the apoptotic signals to bypass mitochondrial signals for apoptosis, which can preserve their steroidogenic activity until complete cell destruction. This unique apoptotic pathway assures the cyclicity of estradiol and progesterone release in the estrus/menstrus cycle even during the initial stage of apoptosis.

Pleiotropic anti-apoptotic activity of glucocorticoids in ovarian follicular cells.

Glucocorticoids (GC) such as hydrocortisone and dexamethasone (DEX) protect steroidogenic granulosa cells against apoptosis induced by serum deprivation, cAMP, tumor necrosis factor alpha stimulation or p53 activation. The protective effects were evident both in primary rat and human granulosa cells, which comprise the main population of the ovarian follicular cells, as well as in steroidogenic granulosa cell lines established in our laboratory. A correlation between the expression of Bcl-2 protein and protection against apoptosis induced by DEX was found in granulosa cell lines expressing various levels of Bcl-2. Incubation with DEX leads to development of a rigid network of actin cytoskeleton and increased incidence of adherence and gap junctions. Higher content of connexin 43 and total cadherins were found in GC stimulated cells compared to non-stimulated, suggesting that cell contact and intracellular communication contribute to the DEX induced resistance to apoptotic signals. Activation by DEX of MAPK and Akt/PKB but not p38 supported the view of a pleiotropic action of GC against apoptotic signals. Granzyme B, a protease characteristic for induction of apoptosis by T-cytotoxic lymphocytes and natural killer cells, was expressed and augmented during stimulation of apoptosis in the granulosa cells, and its synthesis and activation was blocked by DEX. It is concluded that GC exerted their anti-apoptotic effects in granulosa cells by multiple characteristic pathways. Moreover, the presence of endogenous granzyme B in granulosa cells suggest a novel intrinsic alternative apoptotic pathway that was earlier reported to be mediated uniquely by T-cytotoxic lymphocytes and natural killer cells. The anti-apoptotic effect of GC may play an important role in the healing process of the ovulatory follicle subsequent to follicular rupture and its rapid conversion to an active corpus luteum.

Placental apoptosis in discordant twins.

OBJECTIVE: To investigate placental apoptosis in discordant dichorial twins. METHODS: Placental samples were obtained from 7 third-trimester suitable twins. Discordancy was defined as a >25 per cent difference in newborn birth weight. Light microscopy using hematoxylin and eosin (H&E)-stained paraffin slides and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labelling (TUNEL) methods were used to confirm the incidence of apoptosis. Investigators were blinded to pregnancy outcome. RESULTS: Both methods revealed that the incidence of apoptosis in the placentas of the smaller fetuses was significantly higher than in placentas of the larger fetuses. The incidence of TUNEL-positive cells in the former was 1.4+/-0.26 per cent: this was significantly higher than the incidence of apoptosis in the placental specimens of the latter (0.9+/-0.07 per cent, P< 0.02 Wilcoxon rank test). The same results were obtained with H&E: the incidence of apoptosis detected in placentas from the former was 1.07+/-0.1 per cent compared to 0.72+/-0.08 per cent in those of the latter (P< 0.02 Wilcoxon rank test). CONCLUSIONS: Despite similar environment conditions, placental apoptosis is increased in the smaller fetus and thus might play a role in discordancy between twins. Since increased placental apoptosis has also been found in singleton intrauterine growth restriction, this supports the hypothesis that the smaller twin is selectively growth restricted.

Establishment of FSH-responsive cell lines by transfection of pre-ovulatory human granulosa cells with mutated p53 (p53val135) and Ha-ras genes.

Human granulosa cells were immortalized by transfection of the primary cells with a mutated p53 gene in combination with the Harvey-ras oncogene, yielding established cell lines designated HGP53. Here we report that forskolin, 8-Br-cAMP and FSH modulate cell growth and steroidogenesis in HGP53 cells. Low concentrations of 8-Br-cAMP or FSH stimulated cell proliferation, while higher doses attenuated cell proliferation. Progesterone production was already evident at an FSH concentration of 0.3 mIU/ml and was maximally stimulated (50-135-fold) at 50 mIU/ml of FSH. Expression levels of steroidogenic acute regulatory protein (StAR), adrenodoxin and cytochrome P450scc were enhanced 64-, 48- and 3.1-fold respectively by FSH stimulation. Dexamethasone enhanced FSH/cAMP-induced steroidogenesis and this effect involved a marked elevation in the intracellular level of adrenodoxin and P450scc, concomitantly with a marked decrease in StAR. Conversely, basic fibroblast growth factor attenuated FSH-stimulated progesterone production, and this effect involved reductions in adrenodoxin, P450scc and StAR levels. These data suggest that the rate of steroidogenesis may be determined by the ratio of StAR and P450scc, rather than by the level of each protein alone. Whereas FSH at a low dose slightly reduced apoptosis induced by serum withdrawal from HGP53 cells, higher doses enhanced it. Dexamethasone dramatically attenuated FSH- or forskolin-enhanced apoptosis. In conclusion, FSH-dependent mechanisms of differentiation, luteinization and apoptosis can be preserved in human granulosa cells immortalized by mutated p53. Moreover, this system lends itself to studies on cross-talk between the endocrine and paracrine factors that control these processes.

Glucocorticoids protect against apoptosis induced by serum deprivation, cyclic adenosine 3',5'-monophosphate and p53 activation in immortalized human granulosa cells: involvement of Bcl-2.

Glucocorticoid hormones are known to enhance gonadotropin/cAMP-induced steroidogenesis in rat and human granulosa cells. As glucocorticoids induce apoptosis in numerous cell types, we investigated the role of glucocorticoids in the control of apoptosis in immortalized human granulosa cells (HO-23) transfected with a temperature-sensitive mutant of p53 (Val(135)). When HO-23 were incubated with forskolin in the presence or absence of dexamethasone (Dex) at 32 or 37 C, progesterone production was higher by 4- and 8-fold in the presence of Dex at 37 or 32 C, respectively (P: < 0. 01). The expression of adrenodoxin (ADX), which is an intrinsic part of the cytochrome P450 side-chain cleavage enzyme system, remained the same in the presence or absence of Dex in forskolin-stimulated cells. Dex reduced apoptosis (to 33% of control) in cultures after activation of p53 by shifting the temperature from 37 to 32 C. Moreover, Dex suppressed apoptosis induced by serum deprivation (to 40% of control) or forskolin stimulation (to 28% and 40% at 37 and at 32 C, respectively). The protective effect of Dex on cAMP-, p53-, and serum deprivation-induced apoptosis was confirmed by both 4',6-diamido-2-phenylindole hydrochloride DNA staining and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling with an ED(50) of 7 nM Dex. Hydrocortisone showed a similar antiapoptotic effect. The protective effect of glucocorticoids against apoptosis was completely abolished by RU486 when cells were coincubated with 10 nM Dex and 10-100 nM RU486. The protection against apoptosis by glucocorticoid involved a sharp elevation in intracellular levels of Bcl-2 (3-7.6 fold; P: < 0.01). In contrast to the effect of Dex in the prevention of apoptosis in HO-23 granulosa cells, Dex dramatically stimulated apoptosis by 3-fold in LTR-6 myeloid leukemia cells expressing the same temperature-sensitive mutant (Val(135) p53) and the same amount of glucocorticoid receptor-alpha. Forskolin did not stimulate apoptosis when incubated with these cells. However, it augmented by 1.2-fold the p53-induced apoptosis in cells shifted from 37 to 32 C. Dex further enhanced apoptosis by 1.9-fold in p53-activated cultures (32 C). Incubation of the cells with Dex dramatically reduced Bcl-2 levels to 15% of control at 37 C (P: < 0.01) or 32 C in the presence or absence of forskolin (P: < 0.01). Our data suggest that glucocorticoids exert a protective effect against induced apoptosis in immortalized granulosa cells and a stimulatory effect on apoptosis in myeloid leukemia cells. Moreover, modulation of Bcl-2 levels plays an important role in mediating the glucocorticoid effect on cell survival. The opposite effect of glucocorticoids on Bcl-2 levels in the two cell lines may be due to the different ontogeneses of the two cell types: epithelial for granulosa cells vs. mesenchymal for myeloid cells studied in the present work.

Crosstalk Among Multiple Signaling Pathways Controlling Ovarian Cell Death.

Ovarian cell death is an essential process for the homeostasis of ovarian function in human and other mammalian species. It ensures the selection of the dominant follicle and the demise of excess follicles. In turn, this process minimizes the possibility of multiple embryo development during pregnancy and assures the development of few but healthy embryos. Degeneration of the old corpora lutea in each estrous/menstrual cycle by programmed cell death is essential to maintain the normal cyclicity of ovarian steroidogenesis. Although there are multiple pathways that can determine cell death or survival, crosstalk among endocrine, paracrine and autocrine factors, as well as among protooncogenes, tumor suppressor genes, survival genes and death genes, plays an important role in determining the fate of ovarian somatic and germ cells. The establishment of immortalized rat and human steroidogenic granulosa cell lines and the investigation of pure populations of primary granulosa cells allows systematic studies of the mechanisms that control steroidogenesis and apoptosis of granulosa cells. These cells are the most abundant type of somatic follicular cell. Moreover, crosstalk between p53 and extracellular matrix components such as laminin, fibronectin and basic fibroblast growth factor, between cAMP- and p53-generated signals and between steroid hormones and Bcl-2, can explain some of the fine tuning that controls ovarian steroidogenesis and apoptosis. Further study of the mechanisms of ovarian cell death will lead to a better understanding of the processes involved and permit the formulation of novel strategies for the treatment of ovarian malfunctions, such as polycystic ovarian syndrome and ovarian cancer.

Irreversibility of nutritionally induced NIDDM in Psammomys obesus is related to beta-cell apoptosis.

Psammomys lapses into fully fledged diabetes when maintained on a high-energy diet. Progression to diabetes has been classified into stage A of normoglycemia and normoinsulinemia (<120 mg/ml and 100 mU/L, respectively); stage B of hyperinsulinemia (100-300 mU/L) with marked insulin resistance in the face of normoglycemia; stage C of pronounced hyperinsulinemia with hyperglycemia < or =500 mg/ml; stage D at 6-10 weeks after stage C, featuring further hyperglycemia and loss of insulin. Insulin resistance expressed in Psammomys at stages B and C was demonstrated by nonsuppression of the hepatic gluconeogenesis enzyme phosphoenolpyruvate carboxykinase by the endogenous hyperinsulinemia and by the reduced capacity of insulin to activate muscle and liver tyrosine kinase of the insulin receptor. Diabetes at stage C, but not at stage D, was fully reversed to stage A by restricting the food ration of animals by half (from 14 to 7 g/day) for 10-14 days. We examined islet beta cells of Psammomys in the four stages of progression to diabetes by staining for insulin as well as for apoptosis by the terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) and visualizing the biotin-labeled cleavage sites. Psammomys in stage A had insulin-laden beta cells. In stage B, a hypertrophy and partial insulin depletion of beta cells was evident with negative TUNEL staining. In stage C, beta cells were markedly depleted of insulin, and their number within the islets decreased, but the TUNEL staining was virtually negative. In stage D, beta cells were markedly diminished within the islets, almost void of insulin, showing distinct TUNEL staining of beta cells. These results indicate that prolonged exposure of islets to in vivo hyperglycemia with beta-cell overtaxation induces nuclear disintegration with irreversible damage to the insulin-secretion apparatus. This precludes the return to normalcy by restricting the food intake of Psammomys. The appearance of cells with TUNEL-positive staining may serve as a marker of impending irreversibility of nutritionally induced diabetes.

Immunocytochemical investigation of insulin secretion by pancreatic beta-cells in control and diabetic Psammomys obesus.

Hyperproinsulinemia is a characteristic feature of non-insulin-dependent diabetes mellitus (NIDDM) caused by pancreatic beta-cell dysfunction through a secretion-related alteration or impaired proinsulin processing. We have investigated the insulin processing and secretion in Psammomys obesus fed with low- and high-energy diets, which represent a model for diet-induced NIDDM. With a high-energy diet the animals develop hyperglycemia and hyperinsulinemia, whereas those maintained on a low-energy diet remain normoglycemic. Although a large amount of insulin immunoreactivity was detected in beta-cells of the normoglycemic compared to hyperglycemic animals, in situ hybridization for insulin mRNA demonstrated a particularly high signal in the beta-cells of the hyperglycemic animals. By electron microscopy, the beta-cells of normoglycemic animals displayed large accumulations of secretory granules, whereas those of the hyperglycemic animals contained very few granules and large deposits of glycogen. These results reflect a secretory resting condition for the cells of the normoglycemic animals in contrast to stimulated synthetic and secretory activities in the cells of the hyperglycemic ones. Using colloidal gold immunocytochemistry at the electron microscopic level, we have examined subcellular proinsulin processing in relation to the convertases PC1 and PC2. Immunolabeling of proinsulin, insulin, C-peptide, PC1, and PC2 in different cell compartments involved in beta-cell secretion were evaluated. Both PC1 and PC2 antigenic sites were detected in beta-cells of hyperglycemic Psammomys, but their labeling intensity was weak compared to the cells of normoglycemic animals. In both groups of animals, higher levels of PC2 were found in the Golgi apparatus than in the immature granules. Major decreases in proinsulin, insulin, PC1, and PC2 immunoreactivity were recorded in beta-cells of the hyperglycemic Psammomys. In addition, all these antigenic sites were detected in lysosome-like structures, revealing a major degradation process. These results suggest that the insulin-secreting cells in hyperglycemic Psammomys obesus are in a chronic secretory state during which impaired processing of proinsulin appears to take place.

Biochemical and morpho-cytochemical evidence for the intestinal absorption of insulin in control and diabetic rats. Comparison between the effectiveness of duodenal and colon mucosa.

A combined biochemical and morpho-cytochemical investigation was carried out in order to assess insulin absorption by the duodenal and colon epithelium. Insulin was introduced in the lumen of the rat duodenum or colon in combination with sodium cholate and aprotinin. Blood analysis made at several time points has demonstrated a rapid increase in circulating levels of insulin followed by significant and consistent decreases in blood glucose. This indicates that biologically active insulin is absorbed by the intestinal mucosa and transferred to the circulation. Because of the initial high blood glucose levels, the lowering of the glycaemic values was more significant in diabetic animals. Also, levels of circulating insulin remained higher for longer time when the administration was performed in the colon. The integrity of the intestinal wall after insulin administration, evaluated morphologically, was retained. Application of protein A-gold immunocytochemistry has established the pathway for insulin absorption. In both duodenal and colon epithelial cells the labelling for insulin was detected in the endosomal compartment, in the Golgi apparatus and in association with the baso-lateral plasma membrane interdigitations. Some labelling was also present in the interstitial space and in capillary endothelial plasmalemmal vesicles. Insulin introduced in the lumen of the rat duodenum and colon appears thus to be rapidly internalized by the epithelial cells and transferred through a transcytotic pathway to the interstitial space from which it reaches the blood circulation. This exogenous insulin then induces significant decreases in plasma glucose levels which lasts for several hours. The results obtained support the possibility for the clinical development of an oral preparation of insulin.

Morpho-cytochemical and biochemical evidence for insulin absorption by the rat ileal epithelium.

In order to investigate the mechanism through which insulin is absorbed by the intestinal epithelium and transferred to the circulation where it exercises its biological activity of lowering blood glucose levels, a combined biochemical morpho-cytochemical study was undertaken on rat ileal tissue, in vivo. Insulin was introduced into the lumen of the ileum in combination with sodium cholate and aprotinin and allowed to be absorbed for various periods of time. Analysis of blood samples from the inferior vena cava, at different time points has demonstrated an increase in plasma insulin followed by a decrease in blood glucose levels. The ileal tissues were studied at different time points after the introduction of the insulin, by applying the protein A-gold immunocytochemical technique. Insulin antigenic sites were detected with high resolution, at various levels of the enterocytes but were absent from goblet cells. At 2 to 5 min, the labelling was mainly associated with the microvilli and endocytotic vesicles in the apical portion of the epithelial cells. Some gold particles were in contact with the lateral membranes. At 10 min, the labelling was found at the level of the trans-side of the Golgi apparatus and mainly along the baso-lateral membranes of the epithelial cells. Labelling was also detected in the interstitial space. The control experiments have demonstrated the specificity of the labelling and confirmed the nature of the insulin molecules detected. Furthermore, the morphological study has confirmed that exposure of the tissue to the insulin-cholate-aprotinin solution does not affect the integrity of the epithelium while promoting insulin absorption.(ABSTRACT TRUNCATED AT 250 WORDS)

Windowless cell system for luminescence studies on liquids under excitation in the VUV.

A windowless cell system is described for luminescence studies on liquid organic solutions under excitation in the vacuum ultraviolet. Operation conditions for the cell are given as well as the excitation spectrum of a liquid solution of 0.2-g/liter 2,5-diphenyloxazole in n-hexane between 6 and 21 eV.