Adaptation of Islets of Langerhans to Pregnancy ...

0013-7227/92/1303-1459$03.00/0 Endocrinology Copyright 0 1992 by The Endocrine

Adaptation Increased Correlates Secretion* JONATHAN Department Minneapolis,

Vol. 130, No. 3 Printed in U.S.A.

Societ,y

of Islets of Langerhans to Pregnancy: Islet Cell Proliferation and Insulin Secretion with the Onset of Placental Lactogen

A. PARSONS,

T. CLARK

Cell Biology and Neuroanatomy, Minnesota 55455

of

BRELJE, University

AND of

ROBERT

Minnesota

ABSTRACT. To elucidate the temporal profile of adaptive changes of the islets of Langerhans to the increased insulin demands of pregnancy, we have studied islet cell proliferation and insulin secretion during gestation in the rat. 5-Bromo-2’deoxyuridine incorporation into dividing islet cells was significantly (P < 0.05) increased over age-matched controls by day 10, rose continuously to a peak at day 14, and then returned to control levels by day 18. By day 20, cell division was significantly inhibited (P < 0.05). The pattern of changes in insulin secretory profiles observed with perfused pancreata of pregnant animals was similar to that obtained for islet cell proliferation. Both the threshold of glucose-stimulated insulin secretion and the amount of above threshold insulin secretion began to diverge from controls by day 10. By day 12, the glucose-stimulation threshold was significantly decreased from 5.7 mM glucose to 3.3 mM (P < 0.05), remained at this low level through day 15, and returned toward normal by day 20. Concomitant with the increased sensitivity of B cells to glucose, the above threshold insulin secretion was significantly increased by day 12 (P < 0.05), peaked at day 15, and returned to control levels by day 20. This insulin secretory data demonstrates that the increased sensitivity of B cells to glucose is an important component of the adaptation of islets

L. SORENSON

Medical

School,

during pregnancy to the increased demand for insulin at physiological concentrations of plasma glucose. To correlate the above changes in islet cell proliferation and insulin secretion with levels of placental iactogen (PL), serum lactogenic hormone activity was measured by Nb2 lymphoma cell replication assays. This analysis revealed the expected biphasic pattern: a midpregnancy peak at day 12, followed by a nadir at day 14, and then continuously elevated levels until term. The bioassay data agreed with the known secretory profiles of rat (r) PL-I (midpregnancy) and rPL-II (late pregnancy). Our results provide the first systematic evaluation of changes in islet function during pregnancy in the rat. In addition, they provide evidence that rPL-I may be the critical hormonal signal which triggers the primary adaptive changes in islet function characteristic of pregnancy. The return to normal values of insulin secretion and inhibition of cell division observed at day 20 in the presence of high concentrations of rPL-II suggests that other inhibitory influences become dominant in the later stages of rat pregnancy. These observations suggest that complex interactions exist among lactogens and other pregnancy hormones in the regulation of islet function during late pregnancy. (Endocrinology

I

N ORDER to accommodate to the increased demand for insulin that occurs during pregnancy, it is essential that the islets of Langerhans undergo major structural and functional changes. The inability of the maternal islets to respond to the increased demand for insulin can lead to the development of gestational diabetes, a condition which if untreated is threatening to the well being of both the mother and the fetus (1). Adaptive changes that occur during normal pregnancy include: 1) enhanced glucose-stimulated insulin secretion and a decreased glucose stimulation threshold (2,3); 2) enhanced insulin synthesis (4, 5); 3) enhanced B cell proliferation

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and islet volume (6-8); 4) enhanced gap-junctional coupling among B cells (9); and 5) enhanced glucose oxidation and CAMP metabolism (10). In previous in vivo and in vitro studies, we have demonstrated that lactogenic hormones induce all of the known changes in islet function that are characteristic of pregnancy (3,9, 11, 12). Based upon these studies, we have proposed that lactogenic activity is responsible for the elevated islet function observed during pregnancy. In contrast to human pregnancy, where both PRL and placental lactogen (PL) levels increase throughout (13), rat pregnancy is characterized by suppression of pituitary PRL levels with the onset of PL secretion (14). Accordingly, the rat provides a unique opportunity to address the question whether adaptive changes in islets correlate with the onset of PL secretion. In the current study, we report the first examination

Received July 26, 1991. Address correspondence and reprint requests to: Dr. J. A. Parsons, University of Minnesota, Department of Cell Biology and Neuroanatomy, 4-135 Jackson Hall, 321 Church Street SE. Minneapolis, Minnesota 55455. * This work was supported by NIH Grant DK-33655. 1459

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of the temporal sequence of adaptive changes in islets during pregnancy in the rat. We evaluated serum lactogenie activity, islet cell proliferation, B cell sensitivity to glucose, and the extent of glucose-stimulated insulin secretion observed from early to late pregnancy. These results support our hypothesis that PLs have a prominent role in the adaptation of islet B cells to the increased demands of pregnancy.

Materials

and Methods

Animals

Timed pregnant (day zero taken as first day females were sperm positive) and age-matched virgin adult Sprague-Dawley rats were obtained from Holtzman Laboratory Animals (Madison, WI). All animals received food and water ad libitum until killed. Twenty-four hours before use in islet cell proliferation studies, the rats were lightly anesthetized with ether and were implanted sc in the dorsal scapular region with eight 50 mg pellets of the thymidine analog 5-bromo-2’-deoxyuridine (BrdU, Boehringer Mannheim Corp. Indianapolis, IN). In all cases, the pellets were completely dissolved after 24 h. We have previously demonstrated that treatment of islets in vitro with BrdU followed by immunohistochemistry for the incorporated analog is an effective method for assessing islet cell division (12). Islet isolation

Islets were isolated from pentobarbital-anesthetized pregnant and age-matched control rats by pancreatic distention with a collagenase solution followed by in vitro stationary digestion (15). Islets were purified from pancreatic digests on a discontinuous dextran gradient formed from 27%, 23%, and 11% solutions of 60,000-90,000 mol wt dextran (Sigma, St. Louis, MO) in Hank’s saline containing 15 mM HEPES buffer (pH 7.2) by centrifugation at 50 X g for 5 min followed by 400 x g for 15 min. Islets and acinar debris from the 23%-11% interface were rinsed three times in RPM1 1640 culture medium containing 10% horse serum and placed in a petri dish containing the same culture medium. The islets were then hand picked using a Pasteur pipette, washed in PBS and transferred to 12 x 75 mm plastic culture tubes (Becton Dickinson Labware, Lincoln Park, NJ) previously modified by replacing their rounded base with fine mesh surgical screen. These islet incubation chambers were used for subsequent steps by introduction into culture tubes of slightly greater diameter containing the appropriate reagents or rinses. BrdU

immunocytochemistry

Islets isolated from BrdU-implanted rats were immunostained as previously described (12). In brief, islets were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 30 min at room temperature. Excess fixative was removed by several rinses of PBS before islet DNA was denatured by acid hydrolysis in 2 M HCl for 1 h at room temperature. After several brief rinses with PBS, the islets were incubated in PBS

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for an additional hour at room temperature to neutralize any residual acid. All remaining steps were accomplished at 4 C. Islets were incubated for 18 h on a rotating table with a mouse monoclonal anti-BrdU antibody (Becton-Dickinson, Mountain View, CA) diluted 1:25 in PBS containing 0.3% Triton X-100 (PBS/T). Islets were rinsed with Sorensen’s phosphate buffer containing 0.1% Triton X-100 (SPB/T, 4 X 30 min) and were then incubated with fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin (Jackson Laboratories, West Grove, PA) diluted 1:25 in PBS/T for 18 h on a rotating table. After rinsing (4 x 30 min with SPB/T), islets were mounted in a glycerol-based medium containing the antifade agent p-phenylenediamine (16) and glass beads (50-100 pm maximum diameter) to support the cover slips. The slides were coded and submitted for evaluation in a blind study. Islet cell proliferation was determined by direct observation with conventional epifluorescence microscopy (Olympus BH -2, Lake Success,NY). For each islet, the intense positive and low background staining allowed the number of BrdU-labeled nuclei/islet to be rapidly determined while focusing through the islet. A minimum of 45 islets were evaluated per animal but in most cases, 100-200 islets were scored for each. Insulin

secretion

Insulin secretion and threshold for glucose-stimulated insulin secretion were measured by use of the isolated, perfused pancreas as previously reported (3). In brief, cardiac blood samples and pancreata were obtained from pentobarbital-anesthetized pregnant and age-matched control rats. Pairs of organs were then perfused at a rate of 2 ml/min in parallel via the celiac trunk in 37 C water-jacketed chambers. The perfusion medium consisted of Krebs-Ringer bicarbonate balanced salt solution (pH 7.2) containing 15 mM HEPES, 3.8% dextran, 0.25% BSA (Intergen, Purchase, NY), 10 mg/dl soybean trypsin inhibitor (Sigma, type l-s), and 1.67 mM glucose. Medium was gassed continuously with 95% O,-5% CO, and maintained at 37 C. After an initial equilibration of about 20 min duration, fractions were collected every 2 min and baseline insulin secretion established by another 20 min infusion of medium with 1.67 mM glucose. After this basal period, the pancreata were switched to a continuous glucose gradient of 1.67 mM to 16.7 mM for an additional 100 min of perfusion to determine the threshold for glucose-stimulated insulin secretion. At the end of the glucose gradient, the organs were perfused with 15 mg/ dl Neutral Red dye in Hanks balanced salt solution to gain a qualitative assessment of the extent of perfusion, Experiments in which no tissue staining was observed were not analyzed further. The glucose concentration of each fraction of perfusate was determined by use of a Beckman Glucose Analyzer II, and insulin concentrations were determined by RIA as reported previously (17). The results were graphed as the amount of insulin secreted as a function of glucose concentration. The threshold of glucose-stimulated insulin secretion was determined as previously described (18). Secretion threshold was defined as the glucose concentration of the first occurrence of five consecutive points with at least four points with secretion in excess of the average baseline secretion observed for each pancreas.


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Lactogenic hormone bioassay

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In order to determine circulating levels of lactogenic hormones in pregnant rats, blood samples obtained at the time of pancreas perfusion were evaluated by Nb2 rat lymphoma cell replication assaysas previously described (19,20). In brief, cells were maintained as suspension cultures in Fischer’s medium (FM) for leukemic mouse cells (Gibco, Grand Island, NY) containing 10% fetal calf serum and 10% horse serum (HS). Sixteen to 18 h before assay, cells were synchronized in Go/G1 by incubation in FM containing only 10% HS. Samples and ovine PRL (oPRL) standards (NIADDK oPRL-18, AFP82773, 5-2000 pg/well) were diluted with FM and assayed in duplicate at multiple dose levels in 2 ml medium containing 2 X 10’ cells/ml. Tissue culture plates (24 well, 2.5 ml capacity, Flow Labs, McLean VA) were incubated for 72 h at 37 C in a humidified atmosphere of 95% air-5% CO,. Numbers of cells were enumerated electronically (Coulter Electronics, Hialeah, FL) and were corrected for the 10% HS zero-oPRL controls. The EDso was 59 f 1 pg/well (n = 11) for the assays used in these studies.

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Statistical analysis

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Results were evaluated by analysis of variance followed by Neuman-Keuls multiple range tests. Differences were considered significant when P levels were ~0.05.

Results Serum lactogenic hormone levels

From the very low levels observed on day 6 of pregnancy, a biphasic pattern of serum lactogenic activity was observed during the rest of gestation. Lactogenic activity was significantly elevated by day 10, further increased on day 12, significantly reduced on day 14 when compared to days 12 and 18, and then showed a marked elevation later in pregnancy (Fig. 1). These changes are temporally similar to those observed for the two PLs during rat pregnancy (21). Maternal plasma levels of rat (r) PL-I are known to peak at midpregnancy and become nondetectable by day 15; while rPL-II is first detectable on day 12 and increases until term (22, 23). Since the Nb2 lymphoma cell assay cannot discriminate between the two PLs, the biphasic pattern we observed is in agreement with the known profiles of rPLI and rPL-II levels. Islet cell proliferation

Evaluation of BrdU labeling of islet cell nuclei during pregnancy in the rat revealed a pronounced wave of increased islet cell proliferation starting at midpregnancy (Fig. 2). On day 6, levels of incorporation were not different from controls. By day 10, a significant 3-fold elevation was observed which subsequently peaked on day 14 with a dramatic lo-fold increase in BrdU-labeled cells. The number of BrdU-labeled cells decreased from

(9) (11)

0 Cont

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10 12 14 18 Days Pregnancy

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FIG. 1. Serum lactogenic hormone levels during pregnancy in the rat determined by Nb2 lymphoma cell replication assays. The bars represent the mean f SEM for (n) animals of serum lactogenic activity measured relative to oPRL standards. Significant elevation was fit detected on day 10 (P < 0.05). Levels at day 14 are significantly less that at days 12 and 18, but significantly greater than control (P
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cance of both of these mechanisms in meeting the increased demand for insulin during pregnancy is highlighted by an evaluation of the ratio of insulin secretion by pancreata of pregnant rats to controls across the perfusate glucose concentration gradient (Fig. 7). This analysis demonstrates that t,he secretory capacity of rat islets that have adapted to pregnancy is such that they can secrete up to lo-fold more insulin in the presence of normal blood glucose concentrations. The importance of this increased sensitivity of B cells to glucose is critical because if the threshold were to remain unchanged, then the enhanced above threshold secretion would only result in a small increase in circulating insulin. Also, the sustained hyperglycemia necessary to meet the increased demands for insulin if increased sensitivity of B cells to glucose did not occur could have detrimental effects on the developing fetus. The temporal changes of threshold and secretion were similar to that observed for islet cell proliferation, and again correlate very well with the profile of rPL-I serum concentrations. Peak secretion and minimal threshold occurred on day 15 and then returned to normal levels by day 20. These results suggest that counterregulatory influences are competing with lactogenic effects on islets during the later stages of pregnancy. That this actually occurs is indicated by the elevated PL levels maintained until term and by our previous in uiuo and in uitro studies with PRL which have shown that changes in insulin secretion can be maintained for extended periods (3, 11, 12). Although there are limited data that B cell division is greater at midpregnancy than at term (28, 29), the ac-

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FIG;. 7. Ratio 01 insulin secretion by perfused pancreata of pregnant rats compared to controls. The amount of insulin secreted by experimental pancrrata at each glucose concentration was divided by control secretion at the same glucose concentration and the results plotted as a family ol’cur~es. In panel A. the L erlical line represents the nonfasting plasma glucose cwncentration of rats during the later stages of pregnancy. Panel 13 shows the increment in insulin secretion by pregnant rats over wntrclls itl nr)rlno#lvct’lnia.


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celerated return of both BrdU incorporation and insulin secretion to control levels was unexpected. Even though there are structural differences between rPL-I and rPLII (26), it is unlikely that these differences are sufficient to explain our observations. Both hormones demonstrate potent Nb2 lymphoma cell-replicating activity as shown by the profile of bioassay results. PRL receptors have been demonstrated on B cells of normal rats (30, 31), and PRL, PL, and GH receptors have been detected on rat insulinoma cells (32). Thus, another possibility to explain return toward normalcy is that there is downregulation of B cell lactogen receptors in the presence of sustained high concentrations of PLs. Further studies will be necessary to determine the plausibility of this mechanism. However, our previous in uiuo and in uitro studies (3, 9, 11, 12) showing enhanced B cell function in the face of sustained high concentrations of lactogens for prolonged periods of time lessen the attractiveness of a receptor down-regulation explanation. An alternative mechanism to account for the return of islet B cell division and insulin secretion to control levels in the face of high concentrations of rPL-II is the presence of high levels of steroids during the later states of pregnancy (33, 34). In the rat, progesterone is elevated during pregnancy and peaks on days 14-18. Estrogens are also elevated and reach major peaks on days 18-21. In several studies, using a variety of conditions, it can concluded that estrogens and progesterone enhance glucose-stimulated insulin secretion (28,35-39) and progesterone inhibits [3H]thymidine incorporation into B cells (10, 35). In experiments in which pregnancy steroids were tested in combination with human PL, different results were obtained. For example, PL plus progesterone treatment resulted in a decreased islet function in comparison to progesterone treatment alone (35). Using a treatment protocol in which a combination of PL, estrogens, and progesterone were examined, islet function was decreased compared to control islets (36). Because of the diverse nature of the experimental protocols and the use of heterologous human PL, it is difficult to draw firm conclusions from the above on the effects of hormones of pregnancy on islets. However, the available evidence indicates that there are complex interrelationships among PLs and pregnancy steroids which may explain the shift of islet function back toward normal at the end of pregnancy in the rat. In summary, this study used the incorporation of BrdU to assess islet cell proliferation and pancreas perfusions to evaluate insulin secretion throughout pregnancy in the rat. In both cases, these adaptive changes in islet function during gestation were phasic. The temporal sequence of these changes suggests that rPL-I may be responsible for the initiation of increased BrdU incorporation and enhanced insulin secretion. The inhibition

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of islet cell division and return of insulin secretion to normal levels near term suggests that counterregulatory influences become dominant during the later stages of pregnancy. Acknowledgments We thank Celest Roth, Jane Wobken, and John Dalle for excellent technical assistance. We are grateful to the National Hormone and Pituitary Program of the NIDDK (University of Maryland School of Medicine) for the gift of oPRL used in these studies.

References 1. Jovanovic-Peterson L, Peterson CM 1991 Pregnancy and the endocrine pancreas. In: Samols E (ed) The Endocrine Pancreas. Raven Press, New York, pp 229-252 2. Green IC, Taylor KW 1972 Effects of pregnancy in the rat on the size and insulin secretory response of the islets of Langerhans. J Endocrinol54:317-325 3. Sorenson RL, Parsons JA 1985 insulin secretion in mammosomatotropic tumor-bearing and pregnant rats: a role for lactogens. Diabetes 34:337-341 4. Bone AJ, Taylor KW 1976 Metabolic adaptation to pregnancy shown bv increased biosynthesis of insulin in islets of Langerhans isolated from pregnant rats. Nature 262:501-502 SL. Montaaue W. Tavlor 5. Green IC. Howell _ KW 1973 Reaulation of insulin release from isolatld islets of Langerhans of the rat in pregnancy. Biochem J 134:481-487 6. Hellman B 1960 The islets of Langerhans in the rat during pregnancy and lactation, with special reference to the changes in the B/A cell ratio. Acta Obstet Gynecol Stand 39:331-342 7. Van Assche FA 1974 Quantitative morphologic and histoenzymatic study of the endocrine pancreas in nonpregnant and pregnant rats. Am J Obstet Gynecol 11839-41 8. Aerts L, Van Assche FA 1975 Ultrastructural changes of the endocrine pancreas in pregnant rats. Diabetologica 11:285-289 9. Sheridan JD, Anaya P, Parsons JA, Sorenson RL 1988 Increased dye coupling in pancreatic islets from rats in late-term pregnancy. Diabetes 37:908-911 10. Green IC, Perrin D, Howell SL 1978 Insulin release in isolated islets of Langerhans of pregnant rats: relationship between glucose metabolism and cyclic AMP. Horm Metab Res 10~32-35 11. Brelje TC, Allaire P, Hegre 0, Sorenson RL 1989 Effect of prolactin versus growth hormone on islet function and the importance of using homologous mammosomatotropic hormones. Endocrinology 125:2392-2399 TC, Sorenson RL 1991 Role of prolactin versus growth 12. Brelje hormone on Islet B-cell proliferation in vitro: implications for pregnancy. Endocrinology 128:45-57 13. Handwerger S, Freemark M 1987 Role of placental lactogen and prolactin in human pregnancy. Adv Exp Med Biol219:399-420 14. Neil1 J 1974 Prolactin: its secretion and control. In: Knohil E, Sawyer WH (eds) Handbook of Physiology. American Physiology Society, Washington DC, vol4:469-488 15. Gotah M, Maki T, Satomi S, Porter J, Bonner-Weir S, O’Hara CJ, Monaco AP 1987 Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase-injection. Transplantation 43:725-730 16. Johnson GD. Davidson RS. McNamee KC. Russell G. Goodwin D. Holborow EJ 1982 Fading’of immunofluorescence during microscopy: a study of the phenomenon and its remedy. d Immunol Methods 55:231-242 17. Morgan CR, Lazarow A 1963 Immunoassay for insulin; two antibody system. Diabetes 12:115 18 Brelje TC, Sorenson RL 1988 Nutrient and hormonal regulation of the threshold of glucose-stimulated insulin secretion in isolated rat pancreata. Endocrinology 123:1582-1590 19 Tanaka, T, Shiu RPC, Gout PW, Beer CT, Nobel RL, Friesen HG


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1980 A new sensitive and specific bioassay for lactogenic hormones: measurement of prolactin and growth hormone in human serum. J Clin Endocrinol Metab 51:1058-1063 Parsons JA, Peterson EK, Hartfel MA 1984 Effects of cysteamine on pituitary, MtTW15 tumor and serum prolactin levels measured by rat lymphoma cell bioassay and radioimmunoassay. Endocrinology 114:1812-1817 Ogren L, Talamantes F 1988 Prolactins of pregnancy and their cellular source. Int Rev Cytol 112:1-65 Robertson MC, Friesen HG 1981 Two forms of rat placental lactogen revealed by radioimmunoassay. Endocrinology 108:23882390 Robertson MC, Gillespie B, Friesen HG 1982 Characterization of the two forms of rat placental lactogen (rPL):rPL-I and rPL-II. Endocrinology 111:1862-1866 Ogren L, Southard JN, Colosi P, Linzer DIH, Talamantes F 1989 Mouse placental lactogen-I: RIA and gestational profile in materal serum. Endocrinology-125:2253-2257Katsuva A. Kalra SP. Fawcett CP. Krulich L. McCann SM 1972 The effect of stress and nembutal on plasma levels of gonadotropina and prolactin in ovariectomized rats. Endocrinology 90:707-715 Wuttke W 1973 Failure to induce pseudopregnancy in Na-pentobarbital-anesthetized rats: effects on serum prolactin and LH. Endocrinology 92:1280-1282 Swenne I 1983 Effects of aging on the regenerative capacity of the pancreatic B-cell of the rat. Diabetes 32:14-19 Green IC, Se% SE, Perrin D, Howell SL 1981 Cell replication in the islets of Langerhans of adult rats: effects of pregnancy, ovariectomy and treatment with steroid hormones. J Endocrinol 88:219-224 Teitelman G, Alpert S, Hanahan D 1988 Proliferation, senescence, and neoplastic progression of B cells in hyperplasic pancreatic

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islets. Cell 5297-105 30. Tesone M, Oliveria-Filho R. Charreau EH 1980 Prolactin bindina in rat Langerhans islets. J Recept Res 1:355-372 31. Polak M. Scharfmann R. Ban E. Haour F. Pastel-Vinav MC. Cxernichow P 1990 Demonstration of lactogenic receptors-in rat endocrine pancreata by quantitative autoradiography. Diabetes 39:1045-1049 32. Moldrup A, Billestrup N, Neilsen JH 1990 Rat insulinoma cells express both a 115-kDa growth hormone receptor and a 95-kDa prolactin receptor structurally related to the hepatic receptors. J Biol Chem 2658686-8690 33. Shaika AA 1971 Estrone and estradiol levels in the ovarian venous blood from rats during the estrous cycle and pregnancy. Biol Reprod 5:297-307 34. Bartholomeusz RK, Bruce NW, Martin CE, Hartmann PE 1976 Serial measurement of arterial plasma progesterone levels throughout gestation and parturition in individual rats. Acta Endocrinol (Copenh) 82:436-443 35. Nielsen JH, Nielsen V, Pedersen LM, Deckert T 1986 Effects of pregnancy hormones on pancreatic islets in organ culture. Acta Endocrinol (Copenh) 111:336-341 36. Hazer D. Geore RH. Leitner JW. Beck P 1972 Insulin secretion and content in-isolated rat pancreatic islets following treatment with gestational hormones. Endocrinology 91:977-981 37. Neilsen JH 1984 Direct effect of gonadal and contraceptive steroids on insulin release from mouse pancreatic islets in organ culture. Acta Endocrinol (Copenhl 105245-250 38. Howell SL, Tyhurst M, Green IC 1977 Direct effects of progeeterone on rat islets of Langerhans in vivo and in tissue culture. Diabetologia 13:579-583 39. Costrini NV, Kalkhoff RK 1971 Relative effects of pregnancy, e&radio1 and progesterone on plasma insulin and pancreatic islet insulin secretion. J Clin Invest 50:992-999
