Effect of dietary protein on the capacity of urea synthesis in rats

The in vivo capacity of urea nitrogen synthesis (CUNS) during alanine stimulation was measured within the blood amino acid concentration interval 7.3-11.6 mmol/l, where urea synthesis is at maximum and independent of substrate concentration. Three groups of rats were fed for 14 days, either a low protein diet (8%), a normal diet (17%), or a high protein diet (53%). Diet protein modified both CUNS and plasma glucagon concentration. CUNS was 5.86 +/- 2.93, 7.43 +/- 2.16, and 19.31 +/- 4.32 mumol/(min.100 g BW) (mean +/- SD, N = 6), respectively. The corresponding plasma glucagon concentrations after alanine stimulation were 222 +/- 400, 633 +/- 229, and 1700 +/- 627 ng/l, respectively. The in vivo kinetics of urea production is regulated by dietary protein, possibly via glucagon. This implies that the liver plays an active part in adaptation of whole body nitrogen homeostasis to dietary changes.     

In vivo quantification of receptor-mediated uptake of asialoglycoproteins by rat liver

The in vivo kinetics of hepatic clearance of 125I-asialo-orosomucoid and 125I-asialofetuin was determined with a portal vein injection technique in barbiturate-anesthetized rats. Nonlinear regression analyses of saturation data gave the following parameters for asialo-orosomucoid, Km = 0.26 +/- 0.06 mg/ml, Vmax = 320 +/- 70 micrograms/min/g, and for asialofetuin, Km = 0.32 +/- 0.07 mg/ml, Vmax = 240 +/- 40 micrograms/min/g. Unlabeled asialofetuin inhibited the clearance of 125I-asialo-orosomucoid with a Ki = 0.25 +/- 0.04 mg/ml. Based on a model assuming that in vivo receptor concentration much greater than receptor KD, then the maximal binding capacity of the external surface of liver cells in vivo for asialo-orosomucoid is 2Km or 520 micrograms/ml or 52 micrograms/g of liver, assuming the liver interstitial space is 0.1 ml/g. Our estimate of in vivo binding capacity approximates in vitro estimates of total hepatic binding capacity, but is 10-fold greater than in vitro estimates of binding capacity on the external surface of liver cells. These results suggest the large majority of asialoglycoprotein receptors are located on the external surface of liver cells. The saturability of 125I-asialo-orosomucoid clearance was also demonstrated with a portal vein double bolus technique, wherein the portal injection of 20-1000 micrograms of unlabeled asialo-orosomucoid was followed 30 s later by the portal injection of tracer. Maximal inhibition of uptake was obtained with a portal vein injection of greater than or equal to 500 micrograms of asialo-orosomucoid. The specific extraction of the 125I-asialo-orosomucoid, which was near zero shortly after a 400-micrograms loading dose, gradually increased toward normal levels with a t1/2 of 21 min. This t1/2 may represent the in vivo rate of receptor recycling, since the gradual increase in unoccupied receptor sites is consistent with the model of receptor binding, internalization, and recycling.     

Effect of Hyperammonemia and Methionine Sulfoximine on the Kinetic Parameters of Blood-Brain Transport of Leucine and Phenylalanine

The activity of the blood-brain neutral amino acid transport system is increased in rats infused with ammonium salts or rendered hyperammonemic by a portacaval anastomosis. This effect may be due to a direct action of ammonia or to some metabolic consequence of high ammonia levels, such as increased brain glutamine synthesis. To test these possibilities we evaluated the kinetic parameters of blood-brain transport of leucine and phenylalanine in control rats, in rats after continuous 24 h infusion of ammonium salts (NH4+ = 2.5 mmol X kg-1 X h-1), and in rats treated with methionine sulfoximine, an inhibitor of glutamine synthetase, before infusion of ammonium salts. In ammonia-infused rats without methionine sulfoximine treatment, the KD and Vmax of phenylalanine transport were increased, respectively, about 170% and 80% compared to controls, whereas the Km and Vmax of leucine transport were increased, respectively, about 100% and 200%. Electron microscopy demonstrated marked swelling of astrocytic processes around brain capillaries of ammonia-infused rats; however, capillary permeability to horseradish peroxidase apparently was not increased by ammonia infusion. Administration of methionine sulfoximine before ammonia infusion inhibited glutamine synthesis and prevented the changes in transport of leucine and phenylalanine, but apparently did not reverse the perivascular swelling. These results suggest that the ammonia-induced increase in the activity of transport of large neutral amino acids across the blood-brain barrier requires glutamine synthesis in brain, and is not a direct effect of ammonia.     

Kinetics of glucose decarboxylation in the substrate-limited isolated perfused kidney.

Rat kidneys were perfused with a cell-free perfusate containing substrate-free albumin, different glucose concentrations (0.20-5.0 mmol/l), and uniformly labeled 14C-glucose. The rate of glucose decarboxylation (Qox), as a function of [glucose]p, displayed saturation kinetics [Vmax = 0.35 mumol/(g.min); Km = 0.87 mmol/l]; saturation occurred at [glucose]p = 1.0-2.0 mmol/l. Although the presence of as low as 0.2 mmol/l of glucose significantly increased fractional sodium reabsorption (%TNa), there was no correlation between [glucose]p or Qox and % TNa. However, free water clearance (CH2O or CH2O/GFR) was directly proportional to [glucose]p and independent of Qox. We conclude that (1) in the absence of other substrates, renal glucose Qox saturates at hypoglycemic levels of glucose and (2) glucose plays an important role in the generation of solute-free water, a role that is unrelated to glucose Qox.     

High capacity xylose transport in Candida intermedia PYCC 4715

Xylose-utilising yeasts were screened to identify strains with high xylose transport capacity. Among the fastest-growing strains in xylose medium, Candida intermedia PYCC 4715 showed the highest xylose transport capacity. Maximal specific growth rate was the same in glucose and xylose media (mu(max)=0.5 h-1, 30 degrees C). Xylose transport showed biphasic kinetics when cells were grown in either xylose- or glucose-limited culture. The high-affinity xylose/proton symport system (Km = 0.2 mM, Vmax = 7.5 mmol h-1 g-1) was more repressed by glucose than by xylose. The less specific low-affinity transport system (K = 50 mM, Vmax = 11 mmol h-1 g-1) appeared to operate through a facilitated-diffusion mechanism and was expressed constitutively. Inhibition experiments showed that glucose is a substrate of both xylose transport systems.     

The effect of glucagon administration on protein synthesis in skeletal muscles, heart and liver in vivo.

Infusion of glucagon (0.5 mg/h per 100 g body wt.) into fed rats for 6 h inhibited protein synthesis in skeletal muscle, but not in heart. The order of sensitivity of three muscles was plantaris greater than gastrocnemius greater than soleus. Treatment with glucagon for periods of 1 h or less had no effect. Liver protein synthesis was inhibited by glucagon treatment for 10 min, but stimulated after 6 h. The effect of glucagon on muscle was not secondary to impaired food absorption or to depletion of amino acids by increased gluconeogenesis, since the inhibition of protein synthesis was observed in postabsorptive and amino acid-infused rats. The failure of glucagon to inhibit muscle protein synthesis after 1 h may have been caused by the increase in plasma insulin that occurred at this time, since an inhibition was detected in insulin-treated diabetic rats. The lowest infusion rate that gave a significant decrease in muscle protein synthesis was 6 micrograms/h per 100 g body wt., despite a small increase in plasma insulin. This gave plasma glucagon concentrations in the high pathophysiological range, suggesting that glucagon may be significant in the pathogenesis of muscle wasting in metabolic stresses such as diabetes and starvation.     

Hyaluronate and type III procollagen peptide concentrations in bronchoalveolar lavage fluid as markers of disease activity in farmer's lung.

Ten patients were studied during an acute episode of farmer''s lung. Prominent findings were an impaired diffusion capacity (on average only 51% of predicted) and substantially increased amounts of hyaluronate and type III procollagen peptide recovered during bronchoalveolar lavage; mean concentrations of these constituents in lavage fluid were 547 (range 137-1125) and 9.7 (2.8-19.4) micrograms/l, respectively. In bronchoalveolar lavage fluid from healthy controls (n = 21) hyaluronate concentrations were less than 15 micrograms/l and procollagen peptide concentrations less than 0.2 micrograms/l. Lavage fluid concentrations of these potential markers of fibroblast activation declined during the recovery phase of farmer''s lung; four to 10 weeks after admission (n = 7) mean concentrations of hyaluronate and procollagen peptide were 154 (range 38-650) and 4.4 (0.6-15.8) micrograms/l, respectively. At clinical remission six to 14 months after admission concentrations of these markers had returned almost to normal, though slightly increased concentrations were still evident in about half the patients (n = 7). At that time lung volumes were normal but diffusion capacity remained slightly subnormal. It was concluded that in farmer''s lung release of hyaluronate and type III procollagen peptide reflects activity of the disease. Increased synthesis of these connective tissue components continuing in a patient avoiding mouldy plant material may signal an increased risk of developing fibrotic lung disease. The abnormal accumulation of hyaluronate in the smaller airways in acute farmer''s lung may be expected to immobilize water and thereby provide a possible mechanism of the interstitial inflammatory lung oedema with associated impaired gas diffusion. This hypothesis is supported by the relation found between hyaluronate in lavage fluid and reduced diffusion capacity.     

Ramipril inhibition of rabbit (Oryctolagus cuniculus) small intestinal brush border membrane angiotensin converting enzyme

1. Rabbit small intestinal brush border membranes possessed prominent angiotensin converting enzyme (ACE) activity. 2. Intestinal ACE was located on the lumen surface, as verified by ACE co-enrichment with brush border membrane marker enzymes. 3. Hydrolysis kinetics of rabbit intestinal ACE were comparable to the lung, utilizing the substrate (N-[3-(2-furyl)acryloyl]-L-phenylalanylglycylglycine; the Vmax = 543 +/- 51 mumol/min/g and Km = 0.62 +/- 0.09 mmol/l. 4. Intestinal brush border ACE activity was strongly inhibited by the antihypertensive drug Ramipril, which yielded an IC50 value of 5 nmol/l; the ACE activity remained completely inhibited during 15 days after a single dose of 10 mumol/l Ramipril.     

Effects of 3-isobutyl-1-methylxanthine on secretory response, cAMP accumulation and DNA synthesis of islets from postnatal and adult Wistar rats

A possible role for cyclic adenosine-3'-5'-monophosphate (cAMP) in islet cell replication was examined in collagenase-isolated pancreatic islets from Wistar rats of different age and different metabolic state (non-pregnant, pregnant, days 15.5-17.5). Islets obtained from pregnant rats released significantly more insulin in response to 10 mmol/l glucose (culture for 24 h) and their DNA synthesis (incorporation of [3H]thymidine into islet DNA) was doubled compared to islets from non-pregnant controls. Islets obtained from 4-6 days old rats showed a maximal stimulation of DNA synthesis after exposure to 0.1 mmol/l IBMX (3-isobutyl-1-methylxanthine) whereas the cAMP accumulation and the insulin biosynthesis measured in a subsequent short-term incubation were dose-dependent stimulated up to 1.0 mmol/l IBMX. In islets of 12 days old rats or 3 months old rats, however, IBMX did not stimulate DNA synthesis or insulin release measured during culture, although the cAMP content per islet was significantly enhanced after culture in the presence of IBMX.     

Hypoinsulinemic and hyperglycemic effects of beta-endorphin in rabbits

The present study was designed to investigate the in vivo effects of beta-endorphin on plasma levels of glucagon, insulin and glucose in rabbits, and to elucidate some of the mechanisms involved. beta-Endorphin (50 micrograms) injected intravenously into fasted rabbits, decreased plasma levels of insulin (-4.5 +/- 1.3 microU/ml, P less than 0.05) and increased plasma levels of glucose (+2.7 +/- 0.4 mmol/l, P less than 0.05). Similar hypoinsulinemic and hyperglycemic effects were observed for 25 and 2.5 micrograms beta-endorphin in fasted and 50 and 0.5 micrograms beta-endorphin in fed rabbits. beta-Endorphin produced slight and transient increases in plasma levels of glucagon at the highest dose in fed rabbits, only (+80 +/- 9 pg/ml, P less than 0.05). The beta-endorphin-induced hypoinsulinemia was not inhibited by phentolamine, yohimbine, propranolol or atropine, which is in consistency with a direct inhibitory effect of beta-endorphin on the beta-cell in rabbits. The beta-endorphin-induced hyperglycemia was reduced by naloxone (+0.8 +/- 0.1 mmol/l) but not by N-methyl-naloxone (ORG 10908) a peripheral opiate receptor blocking drug (+2.2 +/- 0.2 mmol/l), suggesting a central nervous action on opiate receptors. This central action of beta-endorphin was probably not mediated by catecholamine release or other stimulation of adrenergic or muscarinic receptors, since the beta-endorphin-induced hyperglycemia was not inhibited by phentolamine, yohimbine, propranolol or atropine. These results suggest that the beta-endorphin-induced hyperglycemia was caused, at least in part, by a peripheral inhibition of insulin release and a central stimulation on glucoregulation.     

Assimilation of alpha-glutamyl-peptides by human erythrocytes. A possible means of glutamate supply for glutathione synthesis.

Human erythrocytes are essentially impermeable to glutamate and yet there is a continual requirement for the amino acid for glutathione synthesis. In addition, the intracellular glutamate concentration is approximately five times that of plasma. We present evidence that glutamate enters the red cell as small peptides which are rapidly hydrolysed by cytoplasmic peptidase(s) and that with the estimated physiological levels of plasma glutamyl-peptides the rate of inward flux would be adequate to maintain the glutamate pool at its observed level. Experimentally, we used 1H spin-echo n.m.r. spectroscopy to follow peptide hydrolysis, since peptide spectra are different from those of the free amino acids and the spin-echo sequence enables the monitoring of reactions in concentrated lysates and whole cell suspensions. Thus, the system was studied under near-physiological conditions. Weighted non-linear regression analysis of progress curves using the integrated Michaelis-Menten equation was used to obtain estimates of Km and Vmax. for the hydrolysis of alpha-L-glutamyl-L-alanine and L-alanyl-alpha-L-glutamate in lysates and whole cell suspensions; the values for lysates were Km = 3.60 +/- 0.29 and 5.4 +/- 0.4 mmol/l and Vmax. = 120 +/- 4 and 46.7 +/- 1.7 mmol/h per 1 of packed cells respectively. In whole cell suspensions the rate of peptide hydrolysis was much slower and dominated by the transmembrane flux-rate. The estimates of the steady-state kinetic parameters for the transport were Kt = 2.35 +/- 0.41 and 11.2 +/- 1.0 mmol/l and Vmax. = 3.26 +/- 0.13 and 19.7 +/- 0.7 mmol/h per 1 of packed cells respectively for the previously mentioned peptides. Using the n.m.r. procedure we failed to detect any glutaminase activity in whole cells or lysates; thus, we exclude the possibility that glutamate gains entry to the cell as glutamine which is subsequently hydrolysed by glutaminase.     

Blood glucose control and glomerular capillary basement membrane thickening in experimental diabetes.

Glomerular capillary basement membrane thickness (BMT) was measured in 23 rats which had had streptozocin-induced diabetes for 14 months and in 12 age-matched controls. Diabetic rats were randomly allocated to different groups, either receiving no treatment or treated with a low carbohydrate diet or insulin, or both. Control rats were randomly allocated to a normal or low carbohydrate diet. Among the diabetic rats mean plasma glucose concentrations for the groups ranged from 27-4 mmol/l (494 mg/100 ml) in the untreated rats to 9-8 mmol/l (177 mg/100 ml) in those receiving both a low carbohydrate diet and insulin. A highly significant positive relation was found between BMT and plasma glucose concentration for individual rats. When BMT was corrected for body weight a similar relation was observed.     

Effects of adrenalectomy on urea synthesis in rats

The effect of depletion of glucocorticoids on the dynamics of hepatic amino-N conversion was examined 2 and 7 days after adrenalectomy in a total of 22 rats substituted by adrenaline. The capacity of urea synthesis was studied by infusion of alanine under steady state conditions with arterial concentrations of alanine between 7.3 and 11.6 mmol/l. The animals were nephrectomized and the capacity was calculated as accumulation of urea in total body water corrected for intestinal hydrolysis. Adrenalectomy reduced the capacity of urea synthesis to 55% of the capacity for control rats and reduced the alanine metabolic rate to 60%. In control rats the urea synthesis exceeded the alanine infusion by indicating an extrahepatic tissue release of amino acids. This difference disappeared after adrenalectomy. The body weight and food intake did not change during the study period. Thus lack of glucocorticoids influences the in vivo nitrogen economy both by decreasing the liver function as to conversion of amino-nitrogen and by decreasing release of tissue amino-nitrogen.     

Impaired pancreatic polypeptide response to insulin hypoglycemia in obese subjects

We have studied the effect of insulin hypoglycemia on the secretion of pancreatic polypeptide (PP) in 14 obese subjects with normal glucose tolerance and in 6 normal controls. Infusion of insulin 0.1 U/kg/h in controls and 0.12 U/kg/h in the obese, for one hour, produced a progressive hypoglycemia, similar in both groups (nadir 2 mmol/l at 50 min). The secretion of PP was less in obese subjects than in controls (peak 116 mmol/l vs 184 pmol/l, P less than 0.01) (integrated secretion sigma delta PP 288 vs 472 pmol/l, P less than 0.01) and was also delayed in the obese subjects beginning at 50 min instead of 40 min. The secretion of glucagon and of C-peptide were not different in the two groups, but the integrated response of ACTH was higher in the obese (sigma delta ACTH 52 pmol/l vs 25 pmol/l, P less than 0.01). The secretory response of growth hormone (STH) was smaller in the obese group (peak 8.6 +/- 1.28 vs 21.4 +/- 6.4 ng/ml, P less than 0.01). The reduced secretion of PP in obese subjects could be due to impaired sensitivity to hypoglycemia of the central control mechanism for PP release. The similarity of the reductions in the secretion of both PP and STH support this hypothesis, although a reduction in the secretory capacity of pancreatic PP cells cannot be excluded.     

Control of hepatic nitrogen metabolism and glutathione release by cell volume regulatory mechanisms

1. Urea synthesis was studied in isolated perfused rat liver during cell volume regulatory ion fluxes following exposure of the liver to anisotonic perfusion media. Lowering of the osmolarity in influent perfusate from 305 mOsm/l to 225 mOsm/l (by decreasing influent [NaCl] by 40 mmol/l) led to an inhibition of urea synthesis from NH4Cl (0.5 mmol/l) by about 60% and a decrease of hepatic oxygen uptake by 0.43 +/- 0.03 mumol g-1 min-1 [from 3.09 +/- 0.13 mumol g-1 min-1 to 2.66 +/- 0.12 mumol g-1 min-1 (n = 9)]. The effects on urea synthesis and oxygen uptake were observed throughout hypotonic exposure (225 mOsm/l). They persisted although volume regulatory K+ efflux from the liver was complete within 8 min and were fully reversible upon reexposure to normotonic perfusion media (305 mOsm/l). A 42% inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was also observed when the perfusion medium was supplemented with glucose (5 mmol/l). Urea synthesis was inhibited by only 10-20% in livers from fed rats, and was even stimulated in those from starved rats when an amino acid mixture (twice the physiological concentration) plus NH4Cl (0.2 mmol/l) was infused. 2. The inhibition of urea synthesis from NH4Cl (0.5 mmol/l) during hypotonicity was accompanied by a threefold increase of citrulline tissue levels, a 50-70% decrease of the tissue contents of glutamate, aspartate, citrate and malate, whereas 2-oxoglutarate, ATP and ornithine tissue levels, and the [3H]inulin extracellular space remained almost unaltered. Further, hypotonic exposure stimulated hepatic glutathione (GSH) release with a time course roughly paralleling volume regulatory K+ efflux. NH4Cl stimulated lactate release from the liver during hypotonic but not during normotonic perfusion. In the absence of NH4Cl, hypotonicity did not significantly affect the lactate/pyruvate ratio in effluent perfusate. With NH4Cl (0.5 mmol/l) present, the lactate/pyruvate ratio increased from 4.3 to 8.2 in hypotonicity, whereas simultaneously the 3-hydroxybutyrate/acetoacetate ratio slightly, but significantly decreased. 3. Addition of lactate (2.1 mmol/l) and pyruvate (0.3 mmol/l) to influent perfusate did not affect urea synthesis in normotonic perfusions, but completely prevented the inhibition of urea synthesis from NH4Cl (0.5 mmol/l) induced by hypotonicity. Restoration of urea production in hypotonic perfusions by addition of lactate and pyruvate was largely abolished in the presence of 2-cyanocinnamate (0.5 mmol/l). Addition of 3-hydroxybutyrate (0.5 mmol/l), but not of acetoacetate (0.5 mmol/l) largely reversed the hypotonicity-induced inhibition of urea synthesis from NH4Cl.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of highly potent octapeptide analogs of somatostatin on growth hormone, insulin and glucagon release

Biological activities of highly potent octapeptide analogs of somatostatin (SS), D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Trp-NH2 (RC-160) and D-Phe-Cys-Tyr-D-Trp-Lys-Val-Cys-Thr-NH2 (RC-121), were investigated in male rats. When analog RC-160 was administered to rats in which serum growth hormone (GH) levels were elevated by pentobarbital anesthesia, a dose-related inhibition of GH was obtained at dose range of 0.1 to 2.5 micrograms/kg. The time course of GH inhibition by RC-160, RC-121 and SS-14 was studied in rats treated with phenobarbital, morphine and chlorpromazine. Analogs RC-160 and RC-121 induced a prolonged inhibition of GH levels, in contrast to SS-14, whose effect was short-lived. The analogs suppressed the GH level for more than 2 hr, the peak inhibition being seen 30 to 60 min after the injection. The effects of analogs RC-160 and RC-121 on insulin secretion were observed in rats, in which insulin levels had been elevated by intravenous administration of glucose (500 mg/rat). Administration of RC-160 suppressed insulin secretion, dose-dependently, maximum but not complete inhibition being achieved at a dose of 100 micrograms/kg. In this model, RC-160 and RC-121, in doses of 30 micrograms/kg, induced a similar inhibition of insulin release as 200 micrograms/kg of SS-14, whose action of SS-14 was transient. The effect of analog RC-160 on glucagon release was studied in rats with glucagon levels elevated by hypoglycemia. RC-160 suppressed the secretion of glucagon, the inhibition being dose-dependent in the range of 0.1 to 2 micrograms/kg. Doses of 2 and 10 micrograms/kg of this analog completely suppressed the hypoglycemia-induced glucagon release. These results indicate that analogs RC-160 and RC-121 possess prolonged and enhanced biological activities, the former analog showing a high selectivity in inhibiting GH and glucagon release in vivo as compared with that of insulin secretion.     

Leukotriene and purinergic receptors are involved in the hyperpolarizing effect of glucagon in liver cells

The pancreatic hormone glucagon hyperpolarizes the liver cell membrane. In the present study, we investigated the cellular signalling pathway of glucagon-induced hyperpolarization of liver cells by using the conventional microelectrode method. The membrane potential was recorded in superficial liver cells of superfused mouse liver slices. In the presence of the K+ channel blockers tetraethylammonium (TEA, 1 mmol/l) and Ba2+ (BaCl2, 5 mmol/l) and the blocker of the Na+/K+ ATPase, ouabain (1 mmol/l), no glucagon-induced hyperpolarization was observed confirming previous findings. The hyperpolarizing effect of glucagon was abolished by the leukotriene B4 receptor antagonist CP 195543 (0.1 mmol/l) and the purinergic receptor antagonist PPADS (5 micromol/l). ATPgammaS (10 micromol/l), a non-hydrolyzable ATP analogue, induced a hyperpolarization of the liver cell membrane similar to glucagon. U 73122 (1 micromol/l), a blocker of phospholipase C, prevented both the glucagon- and ATPgammaS-induced hyperpolarization. These findings suggest that glucagon affects the hepatic membrane potential partly by inducing the formation and release of leukotrienes and release of ATP acting on purinergic receptors of the liver cell membrane.     

A proton nuclear magnetic resonance study of gamma-glutamyl-amino acid cyclotransferase in human erythrocytes

Spin-echo NMR spectroscopy was shown to be a reliable technique for the monitoring of the in situ cleavage of gamma-Glu-Ala by gamma-glutamyl-amino acid cyclotransferase in whole erythrocytes and hemolysates. Of particular importance was the difference in chemical shifts between peptide resonances and those of the constituent amino acids. Using lysates of varying dilution, it was shown that the specific activity of the enzyme was not concentration-dependent, thus suggesting a lack of cytosolic low-molecular-weight-effectors or enzyme dissociation. Furthermore, the initial velocities of the reaction as a function of substrate concentration obeyed Michaelis-Menten kinetics with a Km = 2.0 +/- 0.3 mmol/l and Vmax = 137 +/- 7 mmol/h/l of cell water in 1H2O medium. Similar analysis in 2H2O medium revealed a solvent kinetic isotope effect of 1.9 +/- 0.4 at low substrate concentrations. The implications of this observation for the mechanism of the reaction are discussed. Cleavage of the peptide by a suspension of intact erythrocytes was at a rate 300 times less than the corresponding lysate flux, thus indicating the rate limitation by transport in the coupled system.     

Hormone-independent activation of EGP during hypoglycemia is absent in type 1 diabetes mellitus

It has been suggested that insulin-induced suppression of endogenous glucose production (EGP) may be counteracted independently of increased epinephrine (Epi) or glucagon during moderate hypoglycemia. We examined EGP in nondiabetic (n = 12) and type 1 diabetic (DM1, n = 8) subjects while lowering plasma glucose (PG) from clamped euglycemia (5.6 mmol/l) to values just above the threshold for Epi and glucagon secretion (3.9 mmol/l). Individualized doses of insulin were infused to maintain euglycemia during pancreatic clamps by use of somatostatin (250 microg/h), glucagon (1.0 ng. kg(-1). min(-1)), and growth hormone (GH) (3.0 ng. kg(-1). min(-1)) infusions without need for exogenous glucose. Then, to achieve physiological hyperinsulinemia (HIns), insulin infusions were fixed at 20% above the rate previously determined for each subject. In nondiabetic subjects, PG was reduced from 5.4 +/- 0.1 mmol/l to 3.9 +/- 0.1 mmol/l in the experimental protocol, whereas it was held constant (5. 3 +/- 0.2 mmol/l and 5.5 mmol/l) in control studies. In the latter, EGP (estimated by [3-(3)H]glucose) fell to values 40% of basal (P < 0.01). In contrast, in the experimental protocol, at comparable HIns but with PG at 3.9 +/- 0.1 mmol/l, EGP was activated to values about twofold higher than in the euglycemic control (P < 0.01). In DM1 subjects, EGP failed to increase in the face of HIns and PG = 3.9 +/- 0.1 mmol/l. The decrease from basal EGP in DM1 subjects (4.4 +/- 1.0 micromol. kg(-1). min(-1)) was nearly twofold that in nondiabetics (2.5 +/- 0.8 micromol. kg(-1). min(-1), P < 0.02). When PG was lowered further to frank hypoglycemia ( approximately 3.1 mmol/l), the failure of EGP activation in DM1 subjects was even more profound but associated with a 50% lower plasma Epi response (P < 0. 02) compared with nondiabetics. We conclude that glucagon- or epinephrine-independent activation of EGP may accompany other counterregulatory mechanisms during mild hypoglycemia in humans and is impaired or absent in DM1.     

Galanin in the porcine pancreas.

Galanin, a 29 amino acid neuropeptide, was recently isolated from pig intestine. We studied the localization, nature and effect of galanin in pig pancreas. Galanin immunoreactive nerve fibers were regularly found in the pancreas. A peptide chromatographically similar to synthetic galanin was identified in pancreas extracts. The effect of galanin on the endocrine and exocrine secretion was studied in isolated pancreases, perfused with a synthetic medium containing 3.5, 5 or 8 mmol/l glucose and synthetic galanin (10(-10)-10(-8) mol/l). There was no effect on the basal exocrine secretion. The output of insulin, glucagon, somatostatin and pancreatic polypeptide (PP) was measured in the effluent. There was no effect on PP secretion. At a perfusate glucose concentration of 5 mmol/l, galanin at 10(-9) mol/l increased insulin secretion by 55 +/- 14% (mean +/- S.E.M., n = 5) of basal secretion, and at 10(-8) mol/l by 58 +/- 27% (n = 6). At 8 mmol/l glucose, insulin secretion increased by 25 +/- 10% (n = 6) and 62 +/- 17% (n = 8). At 5 mmol/l glucose glucagon secretion was increased by 15 +/- 3% (n = 5) by galanin at 10(-9) mol/l and by 29 +/- 11% (n = 5) by galanin at 10(-8) mol/l, and at 8 mmol/l glucose by 66 +/- 27% and 41 +/- 25%. Somatostatin secretion was inhibited to 72 +/- 2% (n = 5) of basal secretion by galanin at 10(-9) mol/l and to 65 +/- 7% (n = 7) at galanin at 10(-8) mol/l, both at 5 mmol/l glucose. At 8 mmol/l the figures were 83 +/- 6% and 70 +/- 10%. Insulin secretion in response to square wave increases in glucose concentration from 3.5 to 11 mmol/l (n = 5) increased 2-fold during simultaneous perfusion with galanin (10(-8) mol/l).