Dopamine and norepinephrine stimulate somatostatin release by median eminence fragments invitro

The effect of catecholamines on somatostatin release by median eminence (ME) fragments was evaluated using an $\text{in}$$\text{vitro}$ incubation system. Adult male rats were used as tissue donors. Somatostatin release was readily detected during short-term incubations (10 and 30 minutes). Dopamine (DA) significantly stimulated somatostatin release during a 30 minute incubation period at the two doses tested (0.6 and 6 μM). Under similar conditions, norepinephrine (NE) stimulated somatostatin release only at the 6 μM dose. Using a shorter incubation period (10 min) and a 6 μM dose, only DA stimulated somatostatin release. The effects of DA and NE were specifically blocked by the $\text{in}$$\text{vitro}$ addition of pimozide or phentolamine, respectively, suggesting that dopaminergic and noradrenergic receptors may be present in the somatostatinergic terminals of the ME. The results indicate that both DA and NE may be involved in the regulation of somatostatin secretion.     

Insulin binding in cultured Chinese hamster kidney epithelial cells the effects of glucose concentration in the medium and tunicamycin

An epithelial cell line established from a Chinese hamster kidney, CHK-AC_E, was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by 18 successive passages in medium containing 100 and 400 mg/dl glucose, respectively. Binding of CHK-AC_E-100 and CHK-AC_E-400 cells to ¹²⁵I-labeled insulin showed similar pH and time dependency; ¹²⁵I-labeled insulin binding as a function of insulin concentration differed in the two sublines, however. Degradation of ¹²⁵I-labeled insulin, as determined by its ability to bind insulin antibody and cells, was more extensive when preincubated with CHK_AC_E-400 cells than with CHK-AC_E-100 cells. When CHK-AC_E-100 cells were grown in 400 mg/dl glucose for six passages, these cells showed more insulin binding sites than cells grown parallel in 100 mg/dl glucose; whereas CHK-AC_E-400 cells grown in 100 mg/dl glucose for six passages showed fewer insulin binding sites than those grown parallel in 400 mg/dl glucose. A slight increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio was observed in both sublines when grown in 400 mg/dl glucose as compared to 100 mg/dl glucose, indicating attenuated negative cooperativity of the binding sites in cells grown in 400 mg/dl glucose. Tunicamycin, at concentrations from 0.016 to 0.125 μg/ml, showed no direct effect on the assay of ¹²⁵I-labeled insulin binding to CHK-AC_E-100 cells; exposure of CHK-AC_E-100 cells to tunicamycin, at concentrations from 0.01 to 0.2 μg/ml, for 24 h caused a dose-dependent decrease in insulin binding capacity and an increase in $\text{K}{}_{\mathrm{<mtext>f</mtext>}}\text{/}\text{K}{}_{\mathrm{<mtext>e</mtext>}}$ ratio. These data indicate that the number of insulin binding sites in the cultured Chinese hamster kidney epithelial cells increased with high glucose concentrations in the culture medium, whereas tunicamycin, an inhibitor of protein glycosylation, lowered the number of insulin binding sites.     

Increased somatostatin release from pancreases of alloxan diabetic rats perfused in vitro.

Insulin, glucagon, and somatostatin release $\text{in vitro}$ from perfused pancreases of normal and alloxan-diabetic rats were compared. Insulin and glucagon responses to arginine were decreased in the diabetic group whereas both basal and arginine-stimulated somatostatin release was increased. These results suggest that alterations in pancreatic D cell $\text{function}$ as well as in D cell $\text{mass}$ may contribute to the abnormal insulin and glucagon secretion found in alloxan diabetes.     

Synthesis and biological activity of glycosylated analogs of somatostatin

The synthesis by solid-phase methodology of two glycosylated analogs of somatostatin [Glc-Asn⁵]-SS and [NAcGlc-Asn⁵]-SS is described. These two analogs have been biologically tested on the secretion of pituitary growth hormone, pancreatic glucagon and insulin. The results show that glycosylation of somatostatin on the Asn⁵ residue decreases by a hundred fold the inhibition activity on GH release when tested $\text{in}$$\text{vitro}$. $\text{In}$$\text{vivo}$, since the activity is similar to somatostatin the carbohydrates are probably removed by some enzymatic reaction and thus liberate the full activity of somatostatin.     

Acid glycohydrolase in Chinese hamster with spontaneous diabetes VII. The lack of short-term glucose-effect in cultured kidney cells

An epithelial cell line, designated CHK-AC_E, was established from the kidney of a spontaneously diabetic Chinese hamster from the highly inbred AC line. CHK-AC_E was separated into two sublines, CHK-AC_E-100 and CHK-AC_E-400, by successive passages in 100 and 400 mg/dl glucose respectively. Extra- and intracellular activities of $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase were measured in these cultures after exposure to varying concentrations of glucose (100, 200, 300 and 400 mg/dl) for one passage and 10% heated fetal calf serum for 6.5 h before enzyme measurements were taken; no apparent dependence on medium-glucose concentration was found. In serum-free medium, the time-dependent release of both $\text{N-}\text{acetyl}\text{-β-}\text{D}\text{-}\text{glucosaminidase}$ and β-D-galactosidase was sustained for up to 24 h; no significant difference in their activities was found between CHK-AC_E-100 cultures grown in 100 and 400 mg/dl glucose for one passage.     

Effects of cholecystokinin, secretin, and pancreatic polypeptide on secretion of gastric inhibitory polypeptide, insulin, and glucagon

Although the capacity of food components to cause more insulin secretion when given orally than when given intravenously is related significantly to increased plasma concentration of gastric inhibitory polypeptide (GIP), stimulated only by the oral route, questions arise as to what extent other gastrointestinal hormones modify insulin secretion either directly or by influencing the secretion of GIP. The triacontatriapeptide form of cholecystokinin (CCK₃₃), infused in dose gradients intravenously in dogs increases insulin secretion, and comparably to equimolar doses of the carboxy-terminal octapeptide of cholecystokin (CCK₈); neither compound changes fasting plasma levels of GIP or glucose. Glucagon was increased only by the largest dose of CCK₈ (0.27 ug/kg). Unlike the situation with GIP, it is not necessary to increase the plasma glucose above fasting level to obtain the insulin-releasing action of CCK. When glucose is infused intravenously (2 g in 0.5 min) at the beginning of a 15-minute infusion of CCK₈ (10 ng/kg/min), the amount of insulin release is greater than is produced by CCK₈ or glucose alone. In the same type of experiment, the infusion of GIP, in equimolar amounts as CCK₈, plus glucose causes no more insulin secretion than is stimulated by glucose alone. Secretin has only a small stimulating action on insulin release, and pancreatic polypeptide (PP) has no effect. Neither secretin nor PP affects GIP secretion, whether either is given alone, or together, or with CCK₈. Either secretin or CCK₈ inhibits oral glucose-stimulated increase in plasma GIP. These inhibitory effects are probably very much related to the hormone-induced decrease in gastric emptying, but changes in somatostatin secretion and other hormones possibly exert contributory actions. In conclusion, GIP in certain dose ranges has been reported to cause major increase in insulin secretion, but we showed that the insulin-releasing action of a small dose of glucose (2 g) infused intravenously was not augmented by GIP (44.5 ng/kg/min), although it was significantly increased by an equimolar dose of CCK₈. When plasma glucose was maintained at a fasting level, gradient equimolar dosages of CCK₈ and CCK₃₃ had comparable insulin-releasing action; GIP had no effect.     

Somatostatin analogs which inhibit glucagon and growth hormone more than insulin release

Three analogs of somatostatin, [$\text{D}$-Cys¹⁴] -, [Ala², $\text{D}$-Cys¹⁴] - and [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, were synthesized by the solid phase method, characterized by several means, and tested for their effects on the release of insulin, glucagon, and growth hormone. The peptides sharply suppressed the release of growth hormone $\text{in vitro}$ and glucagon $\text{in vivo}$, but had less effect on insulin secretion $\text{in vivo}$. These analogs, particularly [$\text{D}$-Trp⁸, $\text{D}$-Cys¹⁴] - somatostatin, could possibly be useful for the treatment of diabetes mellitus.     

Phe-met-arg-phe-amide (FMRF-NH2) inhibits insulin and somatostatin secretion and anti-FMRF-NH2 sera detects pancreatic polypeptide cells in the rat islet

FMRF-NH2-like immunoreactivity was localized in the pancreatic polypeptide containing cells of the rat islet. FMRF-NH2 was investigated with regard to its effect on insulin, somatostatin and glucagon secretion from the isolated perfused rat pancreas. FMRF-NH2 (1 microM) significantly inhibited glucose stimulated (300 mg/dl) insulin release (p less than 0.005) and somatostatin release (p less than 0.01) from the isolated perfused pancreas. FMRF-NH2 (1 and 10 microM) was without effect on glucagon secretion, either in low glucose (50 mg/dl), high glucose (300 mg/dl), or during arginine stimulation (5 mM). These findings indicate that these FMRF-NH2 antisera recognize a substance in the pancreatic polypeptide cells of the islet which may be capable of modulating islet beta and D cell activity.     

Dimethylurea: A radical scavenger that protects isolated pancreatic islets from the effects of alloxan and dihydroxyfumarate exposure

The hydroxyl radical scavenger dimethylurea was tested $\text{in vitro}$ for possible effectiveness in protecting insulin secreting cells from the deleterious effects of alloxan and dihydroxyfumarate. A five min exposure of isolated rat pancreatic islets to alloxan (0.15 and 0.20 mg/ml) caused a concentration dependent decrease in subsequent glucose-stimulated insulin release. The presence of 40 mM dimethylurea during alloxan exposure attenuated or eliminated the inhibition of insulin release caused by alloxan. Exposure of islets to autooxidizing dihydroxyfumarate, a known generator of hydroxyl free radicals, also caused an inhibition of glucose-stimulated insulin release. This effect was also eliminated when dimethylurea was present during the exposure period. These results support the concept that alloxan produces its insulin inhibitory effects $\text{in vitro}$ via the generation of hydroxyl free radicals.     

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [¹⁴C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [¹⁴C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.     

An absolute requirement for insulin in the control of hepatic glycogenesis by glucose

Livers from normal, adrenalectomized, and diabetic rats were perfused $\text{in}$$\text{vitro}$ in order to investigate the mode of action of insulin in the control of glycogenesis by glucose. Control of glycogen synthase and phosphorylase by glucose is completely lost in livers from 2 and 6 day alloxan diabetic rats. Three hour treatment of normal rats with anti-insulin serum results in a decrease in the effect of glucose on hepatic glycogenesis. Glucose infusion into isolated perfused livers from fed normal and adrenalectomized rats promotes an increase in glycogen synthase activation and phosphorylase inactivation. These data clearly demonstrate that the presence of insulin rather than glucocorticoids is an absolute requirement in the control of hepatic glycogen synthesis by glucose.     

The effect of somatostatin on release and insulinotropic action of gastric inhibitory polypeptide.

Studies were carried out in conscious dogs in which the effect of intravenous somatostatin on immunoreactive gastric inhibitory polypeptide (IR-GIP) release was investigated. In addition, the inhibitory action of somatostatin on the insulin response to pure porcine GIP was assessed. Intravenous administration of somatostatin resulted in a delayed IR-GIP and immunoreactive insulin (IRI) response to oral glucose. Somatostatin also delayed the IR-GIP response to the ingestion of fat. In both types of experiments, initial depression of IRI levels was followed by a sharp rise in IRI release. Intravenous infusion of somatostatin produced 80% inhibition of the IRI response to pure porcine GIP. It was concluded that somatostatin inhibits the physiological release of IR-GIP and the insulinotropic action of exogenous porcine GIP.     

The synthesis and biological activity of Ala3,14-somatostatin

The tetradecapeptide H-Ala-Gly-Ala-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Ala-OH (Ala³, 14-somatostatin) an analog of the somatotropin release inhibiting factor (somatostatin SRIF) was synthesized by solid phase peptide methods. It shows somatotropin release inhibiting activity $\text{in vitro}$ at 5 μg/ml concentration.     

Pentagastrin and adenosine 3',5'-cyclic monophosphate stimulated secretion of somatostatin from perfused rat gastric mucosa.

Immunoreactive somatostatin is secreted by rat gastric mucosa perifused $\text{in vitro}$. Somatostatin release is stimulated by pentagastrin and cyclic AMP with theophylline. These results suggest that gastric mucosal somatostatin may have a paracrine action as feedback inhibitor of gastrin secretion.     

Prostaglandin levels in preovulatory follicles from rabbit ovaries perfused invitro

Prostaglandin (PG) levels in follicular fluid from preovulatory follicles of rabbit ovaries perfused $\text{in}$$\text{vitro}$ were measured in order to compare PG changes in this model system with those that occur $\text{in}$$\text{vivo}$ and in isolated, LH-treated follicles $\text{in}$$\text{barvitro}$. One ovary from each rabbit was perfused without further treatment (control). The other ovary was exposed to LH (0.1 or 1 ug/ml) beginning 1 hour (h) after initiation of perfusion. Samples of perfusion medium were taken at frequent intervals for measurement of PGE, PGF, progesterone and estradiol 17β. The perfusions were terminated when the first ovulation occurred or appeared imminent as judged by changes in the size and shape of the follicles. Follicular fluid was then rapidly aspirated from all large follicles on both ovaries for PGE and PGF measurement.Ovulations occurred only in the LH-treated ovaries. Progesterone and estradiol levels were significantly elevated in the perfusion medium within 1 h of LH treatment in comparison to controls. PG levels in perfusion medium from the control and LH-treated ovaries were not different throughout perfusion and increased in both groups. In contrast, PG levels measured in follicular fluid from LH-treated ovaries were 4- to 5-fold greater than in fluid from control ovaries. It is concluded that ovulation induced by LH in this experimental model is accompanied by an increase in follicular PG levels similar to that seen in other $\text{in}$$\text{vivo}$ and $\text{in}$$\text{vitro}$ models. This difference in follicular PG levels between the LH-treated and control ovaries is, however, not reflected in the perfusion medium.     

Increased glucose production following neurotensin administration

Neurotensin (NT), a recently isolated extract from bovine hypothalami, has been shown to have a hyperglycemic effect when injected into fed rats. This hyperglycemia has been attributed entirely to glycogenolysis, but no evidence is available regarding the effect of NT on the rate of glucose production and uptake. We have therefore used the primed-constant infusion of 6-³H-glucose technique to evaluate the effect of an intravenous injection of NT (1.2 nmole/kg) on glucose production $\text{in vivo}$ in 48-h starvèd, conscious rats (n=10). NT induced a progressive rise in plasma glucose concentration from the control value of 101 ± 2.3 to 162.2 ± 10.8 mg/dl at 30 min. Glucose production was significantly (p<.05) elevated 35–40% throughout the first 30 minutes after NT, while the rate of disappearance of glucose was slightly, but not significantly, elevated. An average of 38.2 mg/animal of glucose was required to account for the average increase in glucose production above the basal rate during the 30 minutes following NT. This requirement exceeded the total amount of glycogen (27.9 mg.) found in the livers of 48 hour starved rats. We therefore concluded that the rise in blood glucose concentration after NT injection was entirely due to an increased rate of glucose production, and that at least part of the increased glucose production represented an elevated rate of gluconeogenesis.     

Cyclic AMP-dependent stimulation of somatostatin secretion by isolated rat islets of Langerhans.

Immunoreactive somatostatin is released from islets of Langerhans, isolated from rat pancreas by collagenase digestion, when incubated in an $\text{in vitro}$ system. The rate of somatostatin secretion is independent of extracellular glucose concentration, but is stimulated by addition of 8-Br-cyclic AMP or theophylline.     

Influence of caloric intake on gastric inhibitory polypeptide,VIP and gastrin release in man

Blood glucose, gastric inhibitory polypeptide (GIP), vasoactive intestinal polypeptide (VIP) and gastrin secretions were measured over a three-hour period following the ingestion by normal subjects of a mixed meal with two different caloric levels (1055 Kcal and 1192 Kcal). No VIP secretion was observed after either meal. Gastrin release was not modified by the increase of caloric intake (mainly carbohydrates and lipids), whereas GIP secretion was significantly more important after the meal with the highest caloric value (peak at 30 mn: $\text{499.5±250.4}$ vs. $\text{273.4±128.7}\text{pg/ml}$ and integrated response $\text{53.3±20.5}$ vs. $\text{28.2±9.9}\text{ng}\text{×}\text{ml}{}^{\mathrm{?1}}\text{×180}\text{min}{}^{\mathrm{?1}}\text{?p<0.05}$). This difference could not be attributed to glucose since the blood glucose levels were not significantly different. It is more probably related to the total amount of ingested food. This suggests the existence of rapid mechanisms of adaptation to the incoming load of the GIP-producing cells.     

Studies on the in vitro effects of insulin on glycogen synthesis and ultrastructure in isolated rat liver hepatocytes

Rat liver hepatocytes were isolated by collagenase $\text{in vitro}$ perfusion technique and effect of insulin on glycogen synthesis and ultra-structure was studied. Addition of insulin stimulated glycogen synthesis and maintained better cellular structure. Synthesis of glycogen was linear in isolated hepatocytes when incubated with various concentrations of glucose (0–800 mg%) reaching initial levels. Concanavaline A inhibited epinephrine stimulated glycogenolysis but had no effect on glucagon stimulated glycogenolysis. These studies indicate that insulin is required for glycogen synthesis and for maintaining hepatocytes ultrastructure. Furthermore, isolated hepatocytes retain various receptors and that different hormones utilize different receptor sites.