Effect of GIP on the secretion of insulin and somatostatin and the accumulation of cyclic AMP in vitro in the rat

The effects of gastric inhibitory polypeptide (GIP) on insulin secretion as well as on the intra-islet accumulation of [3H]cyclic AMP were investigated in isolated pancreatic islets of the rat. In the presence of 6.7 mmol/l of glucose, 3.0 and 30 nmol/l of GIP induced both insulin and [3H]cyclic AMP responses, while lower and higher concentrations of the peptide were ineffective. A coupling of the two parameters was also found with regard to interaction between glucose and GIP. Thus while 30 nmol/l of GIP was stimulatory together with 6.7, 16.7 or 33.3 mmol/l of glucose, the peptide stimulated neither insulin release, nor the accumulation of [3H]cyclic AMP in the presence of a low concentration of glucose (3.3 mmol/l). The concomittant release of insulin and somatostatin was studied in the perfused pancreas in order to assess a possible influence by somatostatin on the dose-response pattern for GIP-induced insulin release. In this preparation 1.0 to 10 nmol/l of GIP stimulated insulin and somatostatin secretion; however while these concentrations were equipotent on insulin release, 10 nmol/l of GIP stimulated somatostatin release more than 1 nmol/l, indicating differences in dose-response curves for the GIP-induced stimulation of the two hormones. It is concluded that 1) modulation of GIP-induced insulin release is coupled to changes in cyclc AMP response in the islet, 2) GIP-induced somatostatin secretion may influence the concomittant insulin response.     

Regulation of amino acid uptake and deoxyribonucleic acid synthesis in isolated human fetal fibroblasts and myoblasts: effect of human placental lactogen, somatomedin-C, multiplication-stimulating activity, and insulin

We compared the abilities of human placental lactogen (hPL), somatomedin-C/insulin-like growth factor I (SM-C/IGF-I), multiplication-stimulating activity (MSA), and insulin to induce a rapid anabolic event, the uptake of the nonmetabolizable amino acid [3H]alpha-aminoisobutyric acid ([3H] AIB) or the more long term action of increasing [3H]thymidine incorporation, as a measure of DNA synthesis, in isolated human fetal fibroblasts and myoblasts. Myoblasts were derived from skeletal muscle and fibroblasts from skin explants removed from human fetuses delivered between 12 and 19 weeks gestation after prostaglandin-induced abortion. Each of the four peptides caused a dose-dependent increase in [3H]AIB uptake by both fibroblasts and myoblasts, with mean half-maximal concentrations (ED50) ranging from 0.9-1.9 nM. The concentration of each peptide required to stimulate [3H]thymidine uptake was significantly greater, with the exception of insulin, which was inactive. For myoblast cultures, the mean ED50 values were: hPL, 7.9 nM; SM-C/IGF-I, 2.0 nM; and MSA, 2.2 nM. For fibroblast cultures, the mean ED50 values were: hPL, 2.3 nM; SM-C/IGF-I, 3.3 nM; and MSA, 4.3 nM. Insulin did not stimulate [3H]thymidine incorporation into either cell type at concentrations up to 6.9 nM. Incubation in the presence of monoclonal antibody against SM-C/IGF-I abolished the ability of SM-C/IGF-I to stimulate either [3H]thymidine or [3H]AIB uptake into fetal fibroblasts. The antibody substantially inhibited the incorporation of [3H]thymidine by these cells in response to hPL, but was less effective in blocking hPL-stimulated [3H]AIB uptake. It did not inhibit the uptake of either radioisotope in response to MSA or [3H]AIB uptake in response to insulin. The actions of SM-C/IGF-I and hPL on thymidine incorporation were additive at submaximal concentrations, but not so at maximal individual concentrations. Their actions on AIB uptake were additive at both submaximal and maximal concentrations. The results suggest that hPL as well as the SMs may contribute to the growth stimulus in human fetal connective tissues. Since incubation with SM-C/IGF-I antibody reduced the mitogenic response of fetal cells to hPL, the actions on DNA synthesis may be partially mediated by local release of SM. However, the similar ED50 values with which these peptides stimulated [3H]AIB uptake during a short incubation, and their additive effects at maximal individual concentrations, suggest that hPL may also have direct actions.     

Increased thymidine incorporation into fetal rat cartilage in vitro in the presence of human somatomedin, epidermal growth factor and other growth factors

The incorporation of [3H]thymidine by rat costal cartilage in vitro was studied at different fetal and postnatal ages and the effect of partially purified human somatomedin, mouse epidermal growth factor, platelet secretion products, insulin and growth hormone on thymidine uptake by fetal cartilage was examined. Thymidine uptake in plasma-free medium was many times greater in late fetal life than after birth. The incorporation of [3H]thymidine into costal cartilage from 21-day fetuses was significantly (P less than 0.05) increased above control values in the presence of 10 micrograms somatomedin/1, and when cartilage was incubated in medium containing somatomedin and diluted human plasma there was a synergistic action. Epidermal growth factor at a concentration of 1 ng/l was a potent stimulator of thymidine uptake. Secretion products from human platelets after their aggregation by thrombin stimulated [3H]thymidine uptake at a concentration of 2% (v/v), but were inhibitory at high concentrations. High concentrations of platelet secretion products stimulated the incorporation of [35S]sulphate by cartilage. A pharmacological concentration of 10 mu. insulin/ml stimulated [3H]thymidine uptake, but not concentrations of 1 or 100 mu./ml. Growth hormone had no effect. The results showed that fetal cartilage had a greater endogenous mitogenic activity than postnatal cartilage. While somatomedins may be important in the regulation of fetal body growth, other protein growth factors also stimulate fetal skeletal tissues.     

Mitogenic actions of insulin on fetal and neonatal rat cells in vitro

Insulin has been implicated in the regulation of fetal growth. The aim of these studies was to determine if insulin has a direct mitogenic effect on fetal and neonatal rat cells in vitro. Myoblasts and fibroblasts were isolated from skeletal muscle and grown until myotube formation began or until fibroblasts were confluent. The cultures were then incubated in the presence of insulin (10(-5)-10(-1) units/ml) and its effects were measured by the cellular incorporation of [3H]-thymidine. Myoblasts from fetuses of 21 days of gestation showed a marked, linear dose-response to insulin, significant increases over control values being observed at 2 X 10(-5) units/ml or 2 X 10(-4) units/ml in five out of seven experiments. Neither myoblasts from 19-day fetuses or neonates nor fibroblasts from animals of any of the three ages showed a significant thymidine uptake response to insulin. Myoblasts released immunoreactive somatomedin-C-like activity into the culture medium, but this was not related to fetal age nor to the presence of insulin in the culture medium. The results suggest that insulin may have a direct role in fetal muscle growth.     

Metformin enhances certain insulin actions in cultured rat hepatoma cells

Summary The effect of the oral antidiabetic agent metformin on insulin regulation of glycogen metabolism, tyrosine-aminotransferase activity, and [1-¹⁴C]aminoisobutyric acid uptake was studied in H4IIE cultured rat hepatoma cells. Metformin enhanced both basal (from 0.213±0.016 to 0.262±0.024 nmol/mg protein,p<0.01) and insulin stimulated [³H] glucose incorporation into glycogen in a time-dependent and dose-dependent manner. A small effect of metformin was seen at 1 μmol/l, and its greatest effects were obtained at 10 μmol/l. At the same concentrations, metformin did not influence basal tyrosine-aminotransferase activity but it potentiated insulin stimulated tyrosine-aminotransferase activity (+ 29.2±1.4%,p<0.01) and prevented the loss of tyrosine-aminotransferase responsiveness to insulin in H4IIE cells desensitised by a previous exposure to insulin. In contrast, metformin had no effect on basal or insulin-stimulated [1-¹⁴C]aminoisobutyric acid uptake. Over the concentrations of metformin that enhanced insulin action in H4IIE cells, the drug had no significant effect on insulin binding to its receptor. These studies suggest, therefore, that metformin may influence cellular metabolism by potentiating certain insulin actions through mechanisms that may be beyond insulin receptor binding.     

Metabolic effects of growth factors and polycyclic aromatic hydrocarbons on cultured human placental cells of early and late gestation

The metabolic effects of epidermal growth factor (EGF), insulin, insulin-like growth factor-I (IGF-I), and IGF-II were determined on human placental cells in monolayer culture obtained from early gestation (less than 20 weeks) and late gestation (38-42 weeks). Parameters studied were uptake of aminoisobutyric acid (AIB), uptake of 3-O-methylglucose and [3H]thymidine incorporation into cell protein. Since benzo[alpha]pyrene (BP) inhibits EGF binding and autophosphorylation in cultured human placental cells, particularly in early gestation, we also studied the effect of benzo[alpha]pyrene and other polycyclic aromatic hydrocarbons (PAHs) on EGF-mediated AIB uptake. The metabolic effects of EGF, insulin, and the IGFs in cultured human placental cells varied with gestational age and the growth factor studied. All three classes of growth factors stimulated AIB uptake in both early and late gestation at concentrations from 10-100 micrograms/L, well within a physiological range. However, insulin stimulation of AIB uptake was maximal at a high concentration (200 micrograms/L) in both early and late gestation cells, suggesting an action via type 1 IGF receptors rather than via insulin receptors. EGF stimulated 3-O-methylglucose uptake only in term placental cells. No significant stimulation of [3H]thymidine incorporation by any of the growth factors tested was seen with either early or late gestation cells. The effect of PAHs on AIB uptake by cultured placental cells was variable. BP alone stimulated AIB uptake by both very early and late gestation cells and enhanced EGF-stimulated AIB uptake. alpha-naphthoflavone alone inhibited AIB uptake at all gestational ages and inhibited EGF-stimulated AIB uptake. beta-Naphthoflavone and 3-methylcholanthrene minimally inhibited AIB uptake by early gestation cells and did not modify EGF-stimulated uptake at any gestational period. Our prior results demonstrated that BP more significantly inhibited EGF than IGF or insulin receptor binding as well as autophosphorylation in early gestation placenta, and that BP was the most potent of the PAHs tested. Thus, the direct effect of the PAHs on placental EGF receptors and amino acid transport may indicate altered function of EGF in the regulation of placental growth in smoking mothers that is developmentally regulated.     

Studies on the mechanisms of somatostatin action on insulin release. IV. effect of somatostatin on cyclic AMP levels and phosphodiesterase activity in isolated rat pancreatic islets.

The effects of somatostatin on insulin release and cyclic AMP metabolism were studied in collagenase-isolated islets of Langerhans from the rat. Ceoncentrations from 500 to 2000 ng/ml significantly inhibited glucose stimulated insulin release, while 100 and 200 ng/ml were ineffective. Somatostatin (2000 ng/ml) inhibited insulin release and [3H]-cyclic AMP accumulation induced by 16.7 mM glucose after 10 and 30 min of incubation. In dose-response studies, the inhibition by somatostatin of the effect of glucose on [3H]cyclic AMP and insulin release could be overcome by a high concentration of the hexose (44.9 mM), suggesting competitive inhibition. In the absence of glucose, somatostatin inhibited [3H]cyclic AMP accumulation induced by the phosphodiesterase inhibitor, IBMX, while no inhibition was seen, again in the absence of hexose, when the [3H]cyclic AMP levels had been raised by the adenyl cyclase stimulator, cholera toxin. Somatostatin did not affect phosphodiesterase activity when added to islet homogenates, but preincubation of the islets with the peptide before homogenization decreased the activity by about 30%. It is suggested that somatostatin-induced inhibition of insulin release is, at least partially, mediated by cyclic AMP, probably through an action on islet adenyl cyclase.     

Direct effects of protirelin (TRH) on cultured porcine thyrocytes.

In several cases of thyroid adenoma in which no TSH was detectable in the serum, a paradoxical decrease of PB123I and its conversion rate Q (PB123I/serum total radioactivity) was observed after infusion of protirelin (TRH). The possibile direct effect of TRH on thyrocytes was therefore studied in vitro. Porcine thyroid cells were cultured in NCTC-135 medium supplemented with a serum substitute and insulin. TRH and some of its analogs enhanced the incorporation of [3H]thymidine into DNA and the uptake of radioiodide into thyrocytes. In the presence of TSH, TRH did not stimulate radioiodine uptake, whereas incorporation of [3H]thymidine into DNA was not antagonized by TSH. Thus, in this culture system, TRH had a direct effect on both growth and metabolism of thyrocytes. TSH can modulate these effects.     

Fetal rat myoblasts release both rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity in vitro: partial characterization and biological activity of myoblast-derived SM-C/IGF I

The relative release of rat somatomedin-C (SM-C)/insulin-like growth factor I (IGF I) and multiplication-stimulating activity (MSA) immunoreactivity and bioactivity from isolated fetal rat myoblasts was assessed by a partial characterization of the SM peptides present in concentrated myoblast-conditioned culture medium (MCM). The SM bioactivity of MCM, measured by [3H]thymidine or [35S]sulfate uptake by fetal rat cartilage explants, eluted with an apparent size of 50-80K on Sephadex G-200 at pH 7.5, and was associated with SM-C/IGF I immunoreactivity. Chromatography of MCM on Bio-Gel P-10 or Sephadex G-75 at acidic pH resulted in a peak of SM bioactivity associated with both SM-C/IGF I and MSA immunoreactivity in the 6-9K region. SM-binding activity, measured by competition with activated charcoal for [125I]SM-C or MSA, eluted in the void volume. When these fractions were incubated with [125I]SM-C and chromatographed on Sephadex G-200 at neutral pH, a heterogeneous pattern of binding proteins was seen, with a major component of 50-80K. After chromatofocusing of proteins in the 6-9K region from Bio-Gel P-10, three peaks of SM bioactivity were recovered, each associated with SM-C immunoreactivity, with pI values of 8.5, 7.1, and 6.5. Although both the basic and neutral peaks enhanced [3H]thymidine uptake by growth-restricted fetal rat myoblasts in vitro, only the bioactivity of the former could be blocked by incubation with a monoclonal antibody to human SM-C. Both human SM-C/IGF I and MSA purified from Buffalo rat liver cell-conditioned medium enhanced thymidine incorporation by growth-restricted fetal rat myoblasts. The results suggest that unlike reports of other fetal rat tissues, fetal rat myoblasts released approximately equal amounts of rat SM-C/IGF I and MSA during culture. The myoblast-derived SM-C/IGF I was biologically active on the cell type of origin and may play a paracrine role in muscle development.     

Growth hormone-releasing factor stimulates proliferation of somatotrophs in vitro.

The mitogenic effect of the hypothalamic peptides growth hormone-releasing factor (GRF) and somatostatin on cultured growth hormone (GH)-producing cells (somatotrophs) was studied. Using autoradiographic detection of [3H]thymidine uptake and immunocytochemical identification of GH-producing cells, we show that 5 nM GRF causes a 20-fold increase in the percentage of somatotrophs labeled with [3H]thymidine. The total number of somatotrophs in GRF-treated cultures was increased by 60%. Somatostatin had no measurable effect on the labeling index by itself, but it partly inhibited the GRF-induced increase in both the labeling index and the total number of cells. Forskolin caused an increase in both the percentage of somatotrophs with a [3H]thymidine-labeled nucleus and the somatotroph number similar to that caused by GRF. GH secretion as well as cellular GH content in the GRF- or forskolin-treated cells increased with culture time over the entire period, whereas secretion and content of GH gradually decreased in control or somatostatin-treated cultures during the entire culture period. These data suggest that GRF and somatostatin regulate the mitotic activity of GH-producing cells and that the effect of GRF is possibly mediated by cyclic AMP.     

T3 stimulation of 2-deoxy-D-glucose uptake in cultured chick embryo carcass derived cells, requires neosynthesis of proteins

T3 stimulated, in a dose dependent manner, the uptake of 2-deoxy-D-glucose (2-DOG) into cultured chick embryo carcass derived cells. A significant increase in sugar uptake was seen already at a T3 concentration of 1 pmol/l. The stimulation of 2-DOG uptake increased with time during the 6 h of exposure to T3. The hormone also stimulated, within 45 min, the incorporation of [3H]leucine and [3H]uridine into the trichloroacetic acid precipitable material of these cells. Actinomycin-D (100 micrograms/l) and cycloheximide (1 mg/l) each were capable of blocking the stimulatory effect of 10(-8) mol/l T3 on sugar uptake and on uridine and leucine incorporation. Thus, T3 in these cultured chick embryo cells stimulated sugar transport through processes dependent on neosynthesis of proteins. In this respect the effect of T3 is different from that seen in cultured chick embryo cardiomyocytes.     

Effects of 3-isobutyl-1-methylxanthine on neonatal pancreatic islets maintained in tissue culture

The phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) promoted the formation of monolayers in cultured pancreatic islets isolated from neonatal rats. Immunofluorescence with specific antisera to insulin and glucagon revealed B-cells and A-cells in these monolayers. Glucose-mediated insulin release was increased by raising the glucose concentration from 5 to 10 mmoles/l. Addition of IBMX (0.1 mmoles/l) to medium containing 10 moles/l glucose produced a further increase in insulin release. Recovery of total insulin, i.e. intracellular insulin plus insulin secreted, was also increased by approximately 50% after 8 days of culture. The B-cells showed a marked biosynthetic response to an acute glucose challenge after prior culture with 10 mmoles/l glucose. Although both unstimulated (1.5 mmoles/l glucose) and stimulated rates (1.5 mmoles/l glucose) of [3H]leucine incorporation into (pro)insulin were significantly higher following culture in 10 mmoles/l glucose plus IBMX (0.1 mmoles/l) than after prior culture with 10 mmoles/l glucose alone, the percentage of (pro)insulin synthesized in relation to total protein synthesis was only increased at the low concentration of glucose. These studies demonstrate that monolayer cultures of neonatal B-cells can be readily produced by IBMX and maintained in a functional state, as defined by their secretory and biosynthetic response. It is suggested that the phosphodiesterase inhibitor exerts a sensitizing effect on the responsiveness of the B-cell to glucose. Moreover, the culture system employed in the present study may prove to be useful for further studies of various agents affecting the B-cell function.     

Metabolic actions of insulin-like growth factor I in cultured glomerular mesangial cells

Glomerular mesangial cells in culture have been reported to possess a considerable number of receptors specific to insulin-like growth factor I (IGF-I), with very small number of receptors specific to insulin. To explore acute metabolic effects of IGF-I on mesangial cells, uptake of glucose and amino acid was measured in the presence of IGF-I or insulin. IGF-I stimulated D-[U-14C]glucose incorporation, 2-deoxy[1-3H]glucose uptake and alpha-[methyl-3H]aminoisobutyric acid (AIB) uptake into cultured mesangial cells by 139.8% +/- 2.1%, 116.6% +/- 1.7%, and 214.9% +/- 12.8% (percent of basal), respectively. Similar maximal stimulation was also induced by insulin, while the ED50 of IGF-I to stimulate these uptake systems (9.98 +/- 2.36, 3.45 +/- 1.86, and 3.35 +/- 0.40 ng/mL, respectively) was significantly lower than that of insulin (120.8 +/- 28.5, 61.8 +/- 7.7, and 76.3 +/- 17.5, respectively). These results indicate that, in cultured glomerular mesangial cells, IGF-I induces acute metabolic effects, possibly through its own receptors.     

Angiotensin II type-1 receptor blocker valsartan enhances insulin sensitivity in skeletal muscles of diabetic mice

Angiotensin II has been shown to contribute to the pathogenesis of insulin resistance; however, the mechanism is not well understood. The present study was undertaken to investigate the potential effect of an angiotensin II type-1 (AT1) receptor blocker, valsartan, to improve insulin resistance and to explore the signaling basis of cross-talk of the AT1 receptor- and insulin-mediated signaling in type 2 diabetic KK-Ay mice. Treatment of KK-Ay mice with valsartan at a dose of 1 mg/kg per day, which did not influence systolic blood pressure, significantly increased insulin-mediated 2-[3H]deoxy-d-glucose (2-[3H]DG) uptake into skeletal muscle and attenuated the increase in plasma glucose concentration after a glucose load and plasma concentrations of glucose and insulin. In contrast, insulin-mediated 2-[3H]DG uptake into skeletal muscle was not influenced in AT2 receptor null mice, and an AT2 receptor blocker, PD123319, did not affect 2-[3H]DG uptake and superoxide production in skeletal muscle of KK-Ay mice. Moreover, we observed that valsartan treatment exaggerated the insulin-induced phosphorylation of IRS-1, the association of IRS-1 with the p85 regulatory subunit of phosphoinositide 3 kinase (PI 3-K), PI 3-K activity, and translocation of GLUT4 to the plasma membrane. It also reduced tumor necrosis factor-alpha (TNF-alpha) expression and superoxide production in skeletal muscle of KK-Ay mice. Specific AT1 receptor blockade increases insulin sensitivity and glucose uptake in skeletal muscle of KK-Ay mice via stimulating the insulin signaling cascade and consequent enhancement of GLUT4 translocation to the plasma membrane.     

Evidence that somatostatin inhibits proinsulin synthesis and insulin release by isolated pancreatic islets of the rat

Conflicting reports on the direction and magnitude of the effect of somatostatin on (pro)insulin synthesis prompted our investigation. Two assays for proinsulin synthesis were designed in which [4,5-3H]-L-leucine incorporation into proinsulin was normalized on the basis of postincubation insulin levels rather than on the number of islets incubated. Somatostatin at a concentration of 10 micrograms/ml inhibited 300 mg/dl glucose-stimulated proinsulin synthesis by 25% from 448 +/- 25 dpm/microunits insulin to 336 +/- 25 dpm/microunits insulin (disintegrations per minute in the proinsulin peak per microunit extractable insulin) (p less than 0.05). Glucagon (10 micrograms/ml) reversed the inhibitory effect of somatostatin on proinsulin synthesis from 336 +/- 25 dpm/microunits insulin to 480 +/- 44 dpm/microunits insulin (p less than 0.02). Somatostatin (10 micrograms/ml) had no significant effect on proinsulin synthesis in the presence of 70 mg/dl or 150 mg/dl glucose. Insulin release in 300 mg/dl glucose was inhibited 38% by 10 micrograms/ml somatostatin from 3.05 +/- 0.40 mU medium/mU tissue to 1.90 +/- 0.10 mU medium/mU tissue (p less than 0.01) over a 45-minute incubation period. These data suggest that somatostatin may act on glucose signal transduction on a level at which both insulin synthesis and secretion are affected. Further, the results are consistent with the hypothesis that cyclic AMP participates in mediating somatostatin effects on B-cell metabolism.     

Insulin-like growth factor (IGF)-II and insulin, but not IGF-I, are mitogenic for fetal rat adrenal cells in vitro

The agents controlling growth of the fetal adrenal gland are poorly defined. The purpose of the present study was to determine the factors required to promote proliferation of adrenal cells obtained from fetal rats at 20 days of gestation and grown in monolayer cell culture under serum-free conditions. Insulin stimulated [3H] thymidine incorporation into DNA at all concentrations (0.01-10 mg/l) tested. Rat insulin-like growth factor (IGF)-II in the presence of insulin promoted a dose-dependent increase in mitogenic activity, with a half-maximal concentration of approximately 1 microgram/l; IGF-II had no effect in the absence of insulin. There was no significant effect of IGF-I on mitogenic activity in the presence or absence of insulin. Adrenal cell DNA synthesis was not stimulated by ACTH, several ACTH-related peptides, a variety of known growth factors, several steroids, or conditioned medium from fetal adrenal cells. We conclude that in the presence of insulin, IGF-II is a specific growth factor for the fetal rat adrenal. We also suggest that rat fetal adrenal cells, in common with other epithelial cell types under serum-free culture conditions, may respond to this progression factor without an obligatory requirement for an initial exposure to competence factors.     

Uptake of nicotinamide by rat pancreatic beta cells with regard to streptozotocin action.

Exposure of rat pancreatic beta cells in monolayer culture to 2 mmol streptozotocin (STZ)/l for 1 h followed by thorough washing inhibited their uptake of [14C]nicotinamide and [3H]2-deoxyglucose [( 3H]2-DG) to about 50% and also reduced the intracellular ATP concentration to 50% of that in control cells. These changes were not due to a lethal cytotoxic effect of STZ, because cell viability, as estimated by succinic dehydrogenase activity, was 90% of that of control cells. Oligomycin and carbonylcyanide-m-chlorophenylhydrazone (CCCP), an uncoupler of oxidative phosphorylation, caused a dose-dependent decrease in intracellular ATP concentration while maintaining high cell viability. These ATP-depleted cells showed a decrease in insulin release and an inhibition of the uptake of [14C]nicotinamide and [3H]2-DG in a dose-dependent manner. Therefore oligomycin and CCCP reproduced the same effects as those found in beta cells treated with STZ. These results suggest that the uptake of nicotinamide and 2-DG by beta cells might be regulated by their intracellular ATP concentration. The decreased uptake of nicotinamide in ATP-depleted beta cells caused by STZ might explain the lack of protective effect of nicotinamide against STZ cytotoxicity when administered after the latter. Furthermore, the radiotracer experiments demonstrated that the transport of nicotinamide by intact beta cells was inhibited in a dose-dependent manner by 2-DG and vice versa, i.e. the transport of 2-DG was inhibited by nicotinamide. These findings suggest the existence of a common transport mechanism in beta cells responsible for the uptake of nicotinamide and 2-DG, the transport of which is known to occur by facilitated diffusion.     

Temporal sequence of prolactin actions on phospholipid biosynthesis in mouse mammary gland explants

Studies were carried out to determine the effect of PRL on the metabolic fate of 32PO4 in cultured mammary gland explants derived from 12 to 14-day pregnant mice. Explants were initially cultured for 24-36 h with 10(-7) M cortisol and 1 microgram/ml insulin. PRL (1 microgram/ml) was then added to certain of the cultures and incubation continued for 2-24 h. Tissues were pulse labeled with 5 microCi/ml 32PO4 during the final 2 h of culture. Tissues were fractionated by the method of Bligh-Dyer. Radioactivity was determined in the organic fraction (containing phospholipids), the aqueous fraction, and an insoluble fraction containing macromolecules. In all these fractions, PRL effected a greater than 2-fold increase in radioactivity content; the onset of the PRL responses was 8-12 h after PRL exposure and PRL effected responses at concentrations of 2.5 ng/ml and above. The enhanced rate of 32P incorporation in the macromolecular fraction was found to occur in both the RNA and phosphoproteins in that fraction. As determined by TLC, PRL was also found to increase 32P incorporation into all phospholipid fractions. This was confirmed by observing that [3H]inositol and [3H]choline incorporation into their respective phospholipids was also increased by PRL; the time sequence of response was similar to that when 32PO4 incorporation was determined. The magnitude of the PRL stimulation of 32PO4 incorporation, however, was about 2-fold higher than either the [3H]inositol or [3H]choline incorporation. The magnified response when 32PO4 was employed may reflect a PRL effect on phosphate uptake into the mammary cells; this is supported by the fact that the radioactive content of the aqueous fraction was significantly elevated in the Bligh-Dyer extract. The effect of PRL on phospholipid synthesis probably reflects the initiation of the packaging process involved in the assimilation of milk products.     

Uptake of (125I) iododeoxyuridine in cultured rat and human prostate: effects of insulin and testosterone.

DNA synthetic activity was monitored in rat and human prostate using [125I] iododeoxyuridine ([125I]UdR). Fresh prostate tissue from 6-week-old rats showed higher incorporation of [125I]UdR than that from 12- or 26-week-old rats. During culture for up to 6 days in the absence of hormones, the incorporation of [125I]UdR fell to a low level for all three age groups. Stimulatory effects were seen when rat prostates were cultured in the presence of insulin (3 mug/ml) and testosterone (10(-7) mol/l), the incorporation on day 4 of culture being commensurate with that of fresh prostrate of the corresponding age. Thus the magnitude of the response was higher for the 6-week-old prostate than that for the other two age groups. A similar age-related pattern of androgen stimulation was observed in experiments in which immature and adult castrated rats were injected daily with testosterone and the freshly removed prostates were incubated with [125I]UdR. Although insulin, by itself, had a stimulatory effect on [125I]UdR incorporation in cultured prostate, the magnitude of the response did not differ in the 6- and 26-week-old prostate tissue. Maximal stimulation was obtained using 25 mug insulin/ml. Tissue from a benigh prostatic hyperplasia was also responsive to insulin in culture but it differed from rat prostate in that increased proliferative activity occurred even in the absence of hormone stimulation. This spontaneous surge in activity during culture tended to mask the stimulatory effects of insulin and testosterone at concentrations of 3 mug/ml and 10(-7) mol/l respectively.     

Effects of tris(hydroxymethyl)aminomethane on biosynthesis and release of insulin in the pancreatic Langerhans islets

Tris(hydroxymethyl)aminomethane (Tris) has been shown to inhibit selectively the Golgi apparatus and Golgi-endoplasmic reticulum-lysosomal system (GERL system) of several kinds of cells including pancreatic B cells. This study was designed to assess the effect of Tris on insulin, glucagon and somatostatin release and insulin synthesis in pancreatic B cells by using isolated rat pancreatic islets. Tris suppressed glucose-induced insulin release, whereas it did not affect the glucagon and somatostatin release. Furthermore, the incorporation of [3H]leucine into the insulin fraction was suppressed by 10 mM Tris, but the sum of the radioactivity of both proinsulin and insulin fraction were not influenced. The present study suggests that the Golgi apparatus and GERL system may play a role in insulin secretion and biosynthesis in pancreatic B cells.