Supriya Ganguli

Adrenergic modulation of pancreatic hormone secretion in utero: studies in fetal sheep.

Summary: To assess the functional maturity of adrenergic modulation of plasma concentration of glucose, as well as immunoreactive glucagon (IRG) and immunoreactive insulin (IRI) secretion in utero, adrenergic agonists with or without β (propranolol) or α (phentolamine) antagonists were infused to the chronically catheterized sheep fetus (n = 35) late in the third trimester. Mean ± S.E. days at study was 129.5 ± 1.5; term is 150 days. In 9 separate studies at gestational age 129 ± 1 days, the infusion of saline for 3 hr was not associated with significant changes in the basal levels of glucose, IRG, or IRI.With epinephrine, 6 μg/min (n = 6) glucose rose from 16.7 ± 3.6 to 41.9 ± 9.7 mg/dl, IRG rose from 75 ± 8 to 219 ± 45 pg/ml, and IRI fell from 22.6 ± 1.7 to 12.7 ± 3.5 microunits/ml (P < 0.05 for each). Propranolol alone (n = 4) did not alter basal glucose or IRG but significantly suppressed IRI. Propranolol did, however, markedly attentuate the rise in glucose and IRG while exaggerating the fall in IRI during epinephrine infusion. Qualitatively similar but smaller responses were obtained with epinephrine, 0.4 μg/min (n = 10). Similarly, elevation of glucose and suppression of IRI was obtained with norepinephrine, 2 μg/min (n = 5), but IRG levels did not rise significantly. α-Adrenergic blockade alone augmented IRI from 18 ± 3 to 38 ± 5 microunits/ml without affecting glucose or IRG concentrations; during a blockade, norepinephrine infusion failed to induce the rise in glucose, IRG remained unchanged, and IRI remained elevated (n = 5). 2-Deoxy-D-glucose, 200 mg IV over 30 min, did not affect glucose, IRG, or IRI (n = 5). Thus, appropriate adrenergic modulation of plasma concentrations of glucose, and of IRG and IRI secretion is established in the third trimester.Speculation: Many of the metabolic adaptations that characterize the transition from intrauterine to extrauterine energy homeostasis are suggestive of a catecholamine effect. These changes include a surge in plasma immunoreactive glucagon concentrations, low immunoreactive insulin levels, an initial fall followed by stabilization of blood glucose, and a rapid rise in free fatty acid levels. An abrupt but brief increase in arterial plasma concentrations of epinephrine and norepinephrine also occurs at birth. In the present studies, infusion of epinephrine in utero simulated those changes that usually occur after birth, thereby suggesting that catecholamines are a major trigger for neonatal energy adaptations that involve insulin and glucagon secretion.

Bidirectional Placental Transfer of Glucose and Its Turnover in Fetal and Maternal Sheep

SUMMARY: Glucose biokinetics were assessed simultaneously in the pregnant ewe and its fetus by a primed constant infusion of 2-1H glucose and U-1 C glucose. Late in gestation fetal glucose turnover was 27.3 ± 3.7 mg/min; expressed in terms of fetal weight this is 6 to 10 mg/kg/min. In the fed state the results indicated that all of the fetal glucose turnover was derived from the mother via placental transfer and there was no evidence that the fetus was capable of glucose production. Maternal glucose turnover was 145.6 ± 9.3 mg/min (2.8 mg/kg/min). There was a significant amount of glucose (16.3 ± 2.3 mg/min) transferred from the fetus to the mother. This feto-maternal transfer of glucose accounted for 11% of the maternal glucose turnover and approximately 50% of the total glucose coming to the fetus from the mother. This study provides the first in vivo simultaneous quantification of the bidirectional glucose transfer across the placenta.Speculation: In the fed state all the fetal glucose is derived from the mother, and the fetus does not produce glucose. A large portion of the glucose coming from the mother to the fetus is returned to the mother, despite glucose concentrations that are threefold higher in the mother than fetus. Thus, the transplacental passage of glucose is bidirectional, this bidirectionality may be responsible for maintaining the concentration gradient that always keeps fetal glucose ooncentration at a lower level than that of the mother, and thereby facilitates fetal glucose supply.

Possible Dissociation Between Insulin Binding and Insulin Action in Isolated Fetal Rat Hepatocytes

To directly examine the relationship between insulin receptors and insulin action in fetal tissue, we compared insulin receptor characteristics and insulin-mediated 14C-glucose incorporation into glycogen, as well as glycogen synthase activity, in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of hepatocytes was documented by trypan blue exclusion (> 90%), time-dependent 14C-leucine incorporation into protein, and dose-related incorporation of glucose into glycogen. Percent specific binding of 125I-insulin per unit protein was significantly higher in F than A liver plasma membranes (32.2 ± 0.3 versus 18 ± 2.4; P < 0.01) and Scatchard plots revealed twice the number of receptors in F. Similarly, receptor number per cell surface area was threefold higher in F than in A (150 versus 50 sites/μm2). At a fixed medium glucose concentration of 11.2 mM, insulin stimulated 14C-glucose incorporation into glycogen in a dose-related manner in A with an apparent Km of 1.0 ng/ml and Vmax at 5–10 ng/ml corresponding to 30–40% of total receptor occupancy; no effect was obtained in F with insulin up to 100 ng/ml. Net glucose incorporation into glycogen (nmol/106 cells/h) increased progressively with increasing medium glucose concentrations ranging from 1.4 to 27.8 mM; incorporation by F was significantly greater than by A at each glucose concentration. However, whereas insulin at 100 ng/ml significantly augmented net glucose incorporation at each glucose concentration in A, no effect of insulin was apparent in F. Finally, insulin at 10 and 100 ng/ml significantly increased the active (a form) and total (a + b form) glycogen synthase activity in A; the ratio of active to inactive forms (% I) also increased significantly in A. In contrast, there was no demonstrable augmentation by insulin of glycogen synthase activity in F hepatocytes. Thus, insulin-mediated incorporation of glucose into glycogen and insulin-stimulated glycogen synthase activity was not demonstrable in fetal rat hepatocytes despite the presence of an increased number of insulin receptors.

Prostaglandin Synthesis Inhibitors Impair Hepatic Glucose Production in Response to Glucagon and Epinephrine Stimulation

To investigate whether inhibition of prostaglandin synthesis affects hormone-inducedglucose dynamics, we measured glucose turnover in response to glucagon alone (5 ng · kg−1 min−1) or combined with epinephrine (0.1 μg · kg−1 min−1) in conscious trained dogs (N = 6) on three separate occasions in each animal: (1) during a control saline infusion, (2) during infusion of indomethacin, and (3) during infusion of sodium sal icy late. Glucose production (Ra) and utilization (Rd) were determined by isotope dilution using the nonrecycling label 3-3H glucose. In controls, glucagon levels (IRG) rose from a basal of 44 ± 12 to 260 ± 40 pg/ml (mean ± SEM) during glucagon infusion; basal epinephrine levels (EPI) of 150 ± 20 pg/ml were unaffected by glucagon infusion but rose four- to fivefold during combined glucagon/epinephrine infusion. Plasma glucose rose transiently from 95 ± 1 to a peak of 136 ± 13 mg/dl after 20 min of glucagon; infusion of EPI resulted in a second glycemic response with a peak of 148 ± 9 mg/dl. Ra increased transiently from 2.9 ± 0.2 to a peak of 7.9 ± 1.4 mg ± 0.8 mg · kg−1 min−1 10 min after beginning EPI. With glucagon alone, Rd paralleled Ra but addition of EPI resulted in a relative fall in Rd. Insulin (IRI) rose from 9 ± 1 μU/ml to 29 ± 6 μU/ml with glucagon but IRI fell despite the second glycemic response during EPI. When either indomethacin or salicylate was infused, basal IRI, IRG, EPI, glucose, Ra and Rd were unaffected and were similar to controls. Although plasma levels of IRG and EPI during glucagon or glucagon plus epinephrine infusion were also similar to controls, the glycemic response was reduced (P < 0.05). This attenuation of glycemic response was due to a reduction of stimulated Ra (P < 0.05) and not to an increase in Rd. Changes in IRI paralleled the reduction in glycemic response. Thus, both indomethacin and salicylate blunt the glycemic response to glucagon and glucagon plus epinephrine by attenuating glucose production and not by enhancing glucose utilization or insulin secretion. These results with two prostaglandin synthesis inhibitors suggest that prostaglandins modulate the hepatic effects of glucagon and epinephrine.

Disappearance of Erythrocyte Insulin Receptors During Maturation in Sheep

We studied insulin receptor characteristics and glucose uptake in erythrocytes (RBCs) of sheep during maturation. RBC insulin receptors were characterized with respect to binding of monoiodinated 125I-insulin and by Scatchard analysis to determine binding sites, and their affinities. Glucose uptake by RBCs was assessed by incorporation of 14C-2-deoxy D-glucose in the presence of D-glucose. The percent specific 125I-insulin bound to RBCs from fetal sheep (4.81 ± 0.48 SE; N = 11) was significantly higher when compared with those from 1–7-day-old newborn lambs (3.41 ± 0.24, N = 7). Thereafter, insulin binding progressively decreased to 2.45 ± 0.46 at 8–14 days, 0.99 ± 0.08 at 15–21 days, and 0.41 ± 0.25 at 22–35 days. No specific insulin binding was observed in adult sheep RBCs. When individual percent specific 125I-insulin binding was plotted against age, there was a significant negative correlation (r = −0.79; P < 0.01). Both high (HA) and low (LA) affinity binding sites per erythrocyte were significantly higher in the fetus (HA, 2.2 ± 0.1; LA, 11.6 ± 0.4) compared with those in newborn lambs (HA, 1.3 ± 0.1; LA, 6.4 ± 0.2). Although the association constant (Ka) for HA sites was significantly lower (P < 0.01) for fetal RBCs (2.4 ± 0.1 × 109 M−1) compared with that for newborn RBCs (3.2 ± 0.1 × 109 M−1), no significant difference was observed for LA Ka in both groups. Because of low percent specific 125l-insulin binding after 1 wk of age, accurate competition curves could not be calculated. Glucose uptake by fetal sheep RBCs (53 ± 2 nmol/min/L × 109 cells, N = 6) was significantly higher than that from adult sheep (25 ± 7, N = 7). A negative correlation existed between RBC glucose uptake and the age of the animals. Although both glucose uptake and insulin receptors decrease postnatally, they appear to be independent phenomena, since insulin (120 μU/ml) did not augment glucose uptake in either fetal cells possessing insulin receptors or adult cells without suchreceptors. We conclude that disappearance of insulin receptors and decreased glucose uptake in sheep RBCs reflect ongoing and independent maturation of membrane function.

Hypothyroidism and Glucocorticoids Modulate the Development of Hepatic Insulin Receptors

Summary: We investigated the influence of hypothyroidism and of glucocorticoid treatment on the in utero and postnatal maturation of insulin receptors on partially purified liver plasma membranes of the rabbit. Treatment of pregnant does with propylthiouracil (PTU) 200 mg/day plus thyroxine (T4) 20 μg on alternate days resulted in fetal but not maternal hypothyroidism because PTU crosses the placenta whereas T4 does not (fetal free T4 undetectable compared to control of 0.21 ± 0.012 ng/dl; mean ± S.E.). At 28 days of gestation (term ∼31 days), insulin binding and receptor number of hypothyroid animals was only 50% of that found in controls (P < 0.01). Intramuscular injection of betamethasone (0.085 mg/kg) to mothers on days 25 and 26 of gestation suppressed fetal corticosterone concentration at day 28 of gestation from 10.5 ± 1.5 ng/ml (mean ± S.E.) in controls to 2.1 ± 0.28 ng/ml (P < 0.01); insulin binding and receptor number was ∼50% of controls (P < 0.01).Using the two-site receptor model, the decrease in receptor number induced by hypothyroidism and glucocorticoids was due solely to reduction in the number of low-affinity sites without any change in the high affinity sites or affinity constants of either receptor class. Ongoing treatment with PTU for 6 postnatal days produced persistant hypothyroidism (free T4 0.15 ± 0.1 ng/dl versus control 0.73 ± 0.1 ng/dl, P < 0.01) physical signs of immaturity, and prevented the normal postnatal maturation of insulin receptor characteristics. Insulin binding was reduced (P < 0.02) and the proportion of high and low affinity receptor sites remained similar to those of 28-day-old fetuses. In contrast, normal maturational changes in insulin receptor characteristics were evident on day 6 in the liver membranes of animals simultaneously receiving PTU and replacement T4 (free T4 0.63 ± 0.17 versus control 0.73 ± 0.1 ng/dl). Thus, the normal pattern of hepatic insulin receptor development can be modulated by hypothyroidism and glucocorticoid exposure.

Maternal Diabetes Does Not Alter Postnatal Development of the Hepatic Glucagon Receptor-Adenylate Cyclase System in the Rat

Summary: We examined the postnatal maturation of the glucagon receptor-adenylate cyclase system of liver plasma membrane from rats born to normal or streptozotocin-induced diabetic mothers and compared results to those of nonpregnant adult. Diabetes in mothers was confirmed by hyperglycemia (458 ± 41 versus 128 ± 13 mg/dl; mean ± S.E.) and relative hypoinsulinemia (4.3 ± 1.0 versus 7.4 ± 1.0 ng/ml) when compared to controls. Pups born to diabetic mothers (IDM) were hyperglycemic (59 ± 6 versus 23 ± 5 mg/dl) but not significantly hyperinsulinemic (4.3 ± 0.5 versus 3.5 ± 0.6 ng/ml) when compared to control pups on day 1; by day 7 and on day 21 glucose and insulin were similar in IDM and control pups. Binding of [125I]-glucagon to liver plasma membrane was markedly reduced in both IDM and control newborns corresponding to 20, 25, and 30% of normal adult values on days 1, 7, and 21 of life. Resolution of the curvilinear Scatchard plots of binding data by the two-site model (high affinity-low capacity; low affinity-high capacity) revealed a progressive increase in receptor number (X 1011/mg protein) of the high affinity sites from 0.9 on day 1 to 1.7 on day 7 and 2.2 on day 21; normal adult was 6.1. Neither the affinity constants of the high or low affinity components, nor the number of low affinity sites differed substantially between adult or newborns at any postnatal age. Basal cAMP production (pM/mg protein/10 min) was similar in adult and newborn. But cAMP production in response to 10–9 to 10–6 M glucagon was always greater in adult than newborn until day 21. Whereas adult liver plasma membrane increased cAMP production with 10–9 M glucagon, the minimum concentration of glucagon capable of consistently and significantly increasing cAMP in newborn on day 1 and 7 was 10–7 M. cAMP production above basal in response to glucagon correlated with glucagon binding and with the number of high affinity glucagon receptors. Adequacy of neonatal liver adenylate cyclase activity was demonstrated by the equivalent responses in cAMP in adult or newborn at any ages after stimulation with 15 mM sodium fluoride; thus, the reduction in number of glucagon receptors in the newborn was specifically responsible for decreased cAMP production after glucagon stimulation. No difference in this pattern of postnatal maturation of glucagon receptors-cAMP production was apparent in liver plasma membrane from controls or IDM pups.

Insulin Stimulates Amino Acid and Lipid Metabolism in Isolated Fetal Rat Hepatocytes

ABSTRACT. Although fetal hyperinsulinemia is associated with excessive deposition of glycogen in liver, both in vivo and in vitro studies show little effect of insulin on glycogen synthesis from glucose or the activity of the enzyme glycogen synthase in the fetus. To investigate whether lack of insulin effect extends to other fetal metabolic processes, we compared the influence of insulin on amino acid uptake (14C-α-aminoisobutyric acid) and lipid synthesis [14C-acetate) in freshly isolated hepatocytes from 21-day fetal (F) and adult (A) rats. Viability of F and A hepatocytes was documented by trypan blue exclusion (>90%). In A, insulin stimulated 14C-α-aminoisobutyric acid uptake in a dose dependent manner with an apparent Km at 2 ng/ml and a Vmax at 10 ng/ml. When corrected for cell surface area, F cells responded to insulin in a similar dose response manner, although absolute values per 1 x 106 cells always remained lower. In contrast, whereas A cells demonstrated a typical dose dependent response of 14C-acetate incorporation into lipid with a Km at 5 ng/ml and Vmax at 10 ng/ml of insulin, F cells remained totally unresponsive when the concentration of acetate was 5 mM or less. However, at higher medium acetate concentrations (15–30 mM) fetal responses were equal to or greater than that of adult, both basally and with insulin. These findings suggest differences in the maturation of insulin-mediated processes in fetal rat hepatocytes; effects on amino acid uptake appear earlier than those on lipid or glycogen synthesis.

INSULIN DOES NOT STIMULATE GLYCOGEN AND LIPID SYNTHESIS IN ISOLATED FETAL RAT HEPATOCYTES

We have previously reported differential maturation of insulin sensitivity for glucose metabolism and amino acid uptake in fetal rat hepatocytes. Compared to the adult, insulin binding to liver plasma membrane is double in the fetus (32 ± 0.3 vs. 18 ± 2.4% per 50 μgm membrane protein, p < 0.01) due to a 3 fold increase in receptor number per unit cell surface area. To study further the role of insulin in fetal metabolism, we examined two established insulin-mediated events in freshly isolated rat hepatocytes: glycogen synthase activity and 14C-acetate incorporation into lipid. Viability of both adult (A) and 21 day fetal (F) hepatocytes was documented throughout the experiments. The glucose-6-phosphate dependent form of glycogen synthase (D) was 2.5 fold higher in F (p < 0.01), while the independent form (I) was 50% lower in F compared to A when expressed per mg protein (p< 0.01) . In A, insulin at 10 and 100 ng/ml stimulated both the D and I forms (p < 0.05); %I increased with insulin (p < 0.05). No such augmentations occured in F. At concentrations varying from 0-30 mM, the incorporation of acetate into lipid was higher in A; at a fixed medium acetate (5mM), insulin at 100 ng/ml stimulated acetate incorporation in A (p < 0.025) but not in F. Thus whereas both insulin-mediated events are demonstrable in A they are not present in F despite higher insulin receptor numbers per cell surface area. These results further suggest a dissociation between insulin receptor binding and insulin mediated glucose or lipid metabolism in isolated fetal rat hepatocytes.

236 AN OVINE MODEL OP PETAL (F) HYPERINSULINEMIA — EFFECTS ON GLUCOSE (G) KINETICS

We used the chronically catheterized pregnant sheep in late gestation (130 ± 2D; term ∼150D) and simultaneous infusion of labeled G to mother (2-3H) and F (U-14C) to assess the effects of short term insulin (I) infusion on glucose kinetics across the placenta. After achieving steady state, I was infused to F at 0.05 U/kg/hr. (group A; n=10) or 0.1U/kg/hr (group B; n=4) for 3 hrs. F pH,pO2 and Hb did not change throughout. M glucose kinetics and plasma insulin (IRI) remained unchanged in all studies. In A, IRI rose from 18 ± 1 to 80-100 uU/ml after 60 min; glucagon levels were unchanged. Fetal G turnover (GT), production (Ra) and utilization (Rd) dropped from 31 ± 2 to 20 ± 2 mg/min at 3 hrs; no change in GT occurred until IRI was ∼100 uU/ml. Similarly fetal G fell by 25% after 2 hrs. In contrast, in B, F GT, Ra and Rd dropped by 50% within 15 min when IRI rose to 157 ± 5 although G levels remained unchanged. These effects were transient, so that by 90 min all had returned to pre-insulin values. The fall in Ra and Rd in the absence of a change in G concentration suggested a reduction of the glucose space. This was assessed by infusing U-14C inulin along with insulin; the radioactivity increased. In neither group was there any evidence of endogenous F Ra, all of F G being derived from M. Conclusions: (i) I at high normal concentrations reduces F glucose flux. (ii) At even higher doses, continuous I infusion exerts only a transient effect on GT. (iii) Failure to show an increase in F Rd may be a consequence of ignoring placental changes, since only M and F were sampled.