Insulin antibodies in insulin-dependent diabetics before insulin treatment.

A sensitive assay was used to measure the binding of iodine-125-labeled insulin in serum obtained from 112 newly diagnosed insulin-dependent diabetics before insulin treatment was initiated. Two groups of nondiabetics served as controls: children with a variety of diseases other than diabetes and nondiabetic siblings of insulin-dependent diabetics. Eighteen of the diabetics were found to have elevated binding and 36 were above the 95th percentile of control values. The insulin-binding protein is precipitated by antibody to human immunoglobulin G, has a displacement curve that is parallel and over the same concentration range as serum from long-standing insulin-dependent diabetics, and elutes from a Sephacryl S-300 column at the position of gamma globulin. These insulin antibodies are present in a large percentage of newly diagnosed, untreated diabetics and may be an immune marker of B-cell damage.

In vivo inhibition of glucagon secretion by paracrine beta cell activity in man.

The close anatomical relationships betaeen pancreatic alpha and beta cells makes possible their interaction at a local (paracrine) level. To demonstrate this in vivo, we have compared the acute glucagon response to intravenous arginine in the basal state and after beta cell suppression by infusions of insulin. The plasma glucose concentration was maintained by the glucose clamp technique. In six normal weight nondiabetics, infusion of insulin at 0.2 mU/kg per min (rate 1) raised the mean +/- SEM plasma insulin levels from 10 +/- 3 to 32 +/- 4 mU/liter and at 1 mU/kg per min (rate 2) raised plasma insulin to 84 +/- 8 mU/liter. This resulted in beta cell suppression, as shown by a diminution in the acute insulin response (incremental area under the insulin response curve, 0-10 min): basal = 283 +/- 61, 199 +/- 66 (rate 1) and 143 +/- 48 mU/liter per 10 min (rate 2) and a fall in prestimulus C-peptide from 1.05 +/- 0.17 to 0.66 +/- 0.15 and to 0.44 +/- 0.15 mM/liter (all P less than 0.01). This beta cell suppression was associated with increased glucagon responses to arginine: 573 +/- 75 (basal), 829 +/- 114 (rate 1), and 994 +/- 136 ng/liter per 10 min (rate 2) and increased peak glucagon responses 181 +/- 11 (basal), 214 +/- 16 (rate 1), and 259 +/- 29 ng/liter (rate 2) (all P less than 0.01). In all subjects, there was a proportional change between the rise in he acute glucagon response to arginine and the fall in the prearginine C-peptide level. To demonstrate that augmented glucagon response was due to betw cell suppression, and not to the metabolic effect of infused insulin, similar studies were performed in C-peptide-negative-diabetics. Their acute glucagon response to arginine was inhibited by the insulin infusion: 701 +/- 112 (basal), 427 +/- 33 (rate 1), and 293 +/- 67 ng/liter per 10 min (rate 2) as was their peak glucagon response: 268 +/- 69, 170 +/- 36, and 115 +/- 33 ng/liter (all P less than 0.01). Thus, hyperinsulinemia, within the physiological range achieved by insulin infusion, inhibits beta cell secretion which, via a paracrine mechanism, potentiates glucagon secretion.

Canine pancreatic polypeptide complementary deoxyribonucleic acid sequence: pancreatic polypeptide and insulin messenger ribonucleic acid distribution in the lobes of the pancreas.

The structure of the canine prepropancreatic polypeptide (preproPP) cDNA was determined. The nucleotide sequence conservation between human and canine preproPP is very high for the signal peptide (82%) and the region coding for the 36 amino acid pancreatic polypeptide (PP) (92%). The overall sequence homology for the C-terminal portion of proPP containing the icosapeptide and a C-terminal extension peptide is only 63% whereas the 3'-untranslated regions of human and canine PP mRNA share 73% homology after alignment for maximal homology. The only sequence conservation in icosapeptide is the region coding for the last 10 amino acids of the icosapeptide. Comparison of PP immunoactivity and PP mRNA concentrations in extracts of the developmentally distinct uncinate process and splenic lobes of the canine pancreas revealed the same ratio of mRNA concentrations (16 +/- 6.5) and PP peptide concentrations (18 +/- 7.0) in the uncinate process compared to the splenic lobe (n = 6). However, a similar comparison of insulin C-peptide (CP) immunoactivity and insulin mRNA concentration revealed a smaller ratio of CP immunoactivity (0.37 +/- 0.05) than insulin mRNA (0.58 +/- 0.10) between the same lobes (P less than 0.0074, n = 6). This increased steady state CP concentration relative to insulin mRNA in splenic lobe compared to the uncinate process was not observed for PP peptide and mRNA.

Molecular weight forms of adrenocorticotropic hormone secreted by primary cultures of mouse anterior pituitary.

Extracts of mouse anterior pituitary cells in monolayer culture were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis to separate the molecular weight forms of ACTH. The gels were sliced and each segment was eluted. The eluates were assayed for ACTH immunoactivity. Approximately 10% of the immunoactivity in the extracts was found to be present as 20,000--32,000 mol wt ACTH. The remainder of the immunoactivity was equally distributed between two forms of ACTH with apparent molecular weights of 11,800 and 4,500. This distribution is very similar to that found in extracts of mouse anterior pituitary. Mouse anterior pituitary cultures were incubated for 2 h in serum-free tissue culture medium. ACTH was concentrated from the medium and fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The medium was found to contain predominantly the 4,500 and 11,800 forms of ACTH. When vasopressin was added to these cultures (100 ng/ml), the rate of secretion of ACTH was more than doubled in serum free medium. Analysis of the medium from vasopressin-stimulated cultures showed that the 4,500 and 11,800 mol wt forms of ACTH were again the predominant forms present.

Reversal of dexamethasone inhibition of adrenocorticotropin release in a mouse pituitary tumor cell line either by growing cells in the absence of dexamethasone or by addition of hypothalamic extract.

Release of ACTH by a mouse pituitary tumor cell line )AtT-20/D-16v) is inhibited by 10(-8)-10(-6) M 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha, 21-trihydroxy-1,4-pregnadiene-3,20-dione (dexamethasone). Dexamethasone does not inhibit cell growth in this concentration range. Cortisol and corticosterone are almost as potent as dexamethasone in inhibiting ACTH release, whereas 17 beta-estradiol and testosterone have no effect. In rapidly growing cultures of tumor cells removal of dexamethasone leads to complete reversal of the inhibitory effect of the steroid on ACTH accumulation in culture medium within 4-5 days (3.5-4 generation times). The extent of reversal of the dexamethasone effect in slowly growing cultures (generation time 96-150 h) and in rapidly growing cultures (24-30 h) is proportional to the amount of growth that takes place in the absence of dexamethasone. Addition of hypothalamic extract to dexamethasone-treated cultures and to untreated cultures stimulates the release of ACTH 4- to 8-fold. The response occurs within 15 min after the addition of the extract and is dependent on the dose of the extract.

Sympathetic nerve stimulation versus pancreatic norepinephrine infusion in the dog: 2). Effects on basal release of somatostatin and pancreatic polypeptide.

We compared the effects of sympathetic nerve stimulation to that of pancreatic norepinephrine (NE) infusion on pancreatic somatostatin-like immunoreactivity (SLI) and pancreatic polypeptide (PP) secretion in the halothane-anesthetized dog. During electrical stimulation (8 Hz, 1 msc, 10 mA, n = 6) of the sympathetic nerves surrounding the pancreatic artery, the pancreatic SLI output decreased from 827 +/- 340 fmol/min to 231 +/- 47 fmol/min (delta = -596 +/- 217 fmol/min, P less than 0.05) after 5 min, and PP output decreased from 11,972 +/- 374 pg/min to 5,518 +/- 774 pg/min (delta = -6,454 +/- 1,932 pg/min, P less than 0.02) after 3 min of stimulation. Arterial SLI or PP levels did not change. Sympathetic nerve stimulation also decreased pancreatic blood flow and increased pancreatic venous NE levels. To determine whether the NE, released locally during sympathetic nerve stimulation, is responsible for this inhibition of pancreatic SLI and PP outputs, exogenous NE was infused into the pancreatic artery at three different dose levels. The low dose of 12 ng/min (n = 6) increased pancreatic venous NE levels like sympathetic nerve stimulation. The medium dose of 120 ng/min (n = 6) reproduced the nerve stimulation-induced decrease of pancreatic blood flow. The high dose of 1,200 ng/min (n = 6) markedly exceeded both. It was found that neither the low nor the medium infusion rates of NE significantly affected pancreatic SLI or PP output. In contrast, infusion of NE at the very high dose level of 1,200 ng/min decreased pancreatic SLI output from 850 +/- 165 fmol/min to 318 +/- 59 fmol/min after 5 min of infusion (delta = -532 +/- 143 fmol/min, P less than 0.01) and decreased PP secretion from 22,777 +/- 7,082 pg/min to 12,764 +/- 6,100 pg/min after 3 min of infusion (delta = -10,013 +/- 2,399 pg/min, P less than 0.01). During this high dose rate NE infusion, both the increment of pancreatic venous SPV levels of NE and the decrement of pancreatic blood flow markedly exceeded the effects produced by sympathetic nerve stimulation. We conclude from this study in dogs: that selective electrical stimulation of the sympathetic nerves entering the pancreas decreases blood flow and inhibits pancreatic SLI and PP secretion, that NE infused into the pancreatic artery at moderate rates reproduces the decrease in blood flow yet does not reproduce the inhibition of pancreatic SLI and PP secretion, and that extremely high concentrations of NE, which markedly restrict pancreatic blood flow, decrease both pancreatic SLI and PP outputs.(ABSTRACT TRUNCATED AT 400 WORDS)

Lobe-specific responses of cultured islet cells of canine pancreas to carbamylcholine, glucose, and pancreatic polypeptide.

Using cultured islet cells from both splenic lobe (SL) and uncinate process (UP) of the dog pancreas, we have measured responses of insulin, glucagon, and pancreatic polypeptide (PP) to glucose, carbamylcholine (carbachol), and exogenous PP. The results show that both insulin and PP cells of the two developmentally distinct lobes of the pancreas respond differentially to secretagogues. The results suggest that PP may play a role in regulation of islet cell secretion. Secretion of insulin by SL and UP cultures in response to 28 mM glucose in a 2h incubation was significantly greater, 2.9- (p less than 0.01) and 1.5-fold (p less than 0.01), respectively, than secretion by respective control cultures at 2.8 mM glucose. The difference in degree of stimulation between SL and UP was also significant (p less than 0.02). At 2.8 mM glucose, SL and UP cultures secreted 10% and 8.8%, respectively, of immunoreactive insulin (IRI) contents of the cultured cells (NS). Dose-response curves showed that for up to 8.5 mM glucose the degree of stimulation of SL was no greater than UP, but the UP response had nearly plateaued; whereas, the SL response continued to increase, such that SL was greater than UP at 16.7 mM glucose (p less than 0.01). Secretion of PP by SL and UP cultures in response to 5 microM carbachol and 2.8 mM glucose in a 2-h incubation was significantly greater, 2.2- (p less than 0.002) and 3.9-fold (p less than 0.002), respectively, than secretion by respective control cultures without carbachol. The difference in degree of stimulation between SL and UP was also significant (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Altered amidation of pancreatic polypeptide in cultured dog islet tissue.

Three forms of immunoreactive pancreatic polypeptide (PPI) were detected in extracts of cultured dog pancreatic PP cells: PPI of (1) larger apparent molecular weight than PP, (2) similar apparent molecular weight but different isoelectric point than PP, and (3) identical apparent molecular weight and isoelectric point with PP. Dog pancreatic endocrine cells in culture were labeled biosynthetically with tritiated amino acids, and extracted proteins were fractionated by sodium dodecyl sulfate gel electrophoresis. A total of 97% of the PPI migrated like PP itself while about 3% of the PPI migrated like proteins up to 7200 molecular weight. PPI migrating like PP was analyzed further by isoelectric focusing and was found to occur in a neutral form like PP and a more acidic form. Peptide mapping of neutral and acidic PPI forms showed that both were like PP with the exception that the C-terminal [3H]tyrosine-containing peptide was a peptide with a net negative charge of 1 arising from a peptide extension of one or a few amino acids. The acidic form of PP was also shown to occur in pancreas extracts. However, neutral PPI was 90% of the total PPI in the pancreas extracts while the converse was true of culture extracts. We conclude that culturing the PP cell affects the efficiency of the process of amidation, that acidic PP could be either biosynthetic precursor or end product, and that the existence of the larger PP form(s) signals (signal) the possible production of yet other peptides by the PP cell.

Halothane is less suppressive than pentobarbital on reflex and neural activation of pancreatic F-cell.

To determine the suitability of halothane anesthesia for studies of parasympathetic control of the endocrine pancreas in dogs, we assessed the effect of halothane on reflex, direct neural, and direct chemical activation of the parasympathetic input to the islet. Levels or output of pancreatic polypeptide (PP), an islet hormone under predominant cholinergic influence, were used as an indicator of the degree of parasympathetic activation and its potential suppression by anesthesia. Reflex stimulation of the parasympathetic nervous system by 2-deoxy-D-glucose (2-DG) in dogs anesthetized with halothane (0.8%) caused a fourfold increase in plasma PP levels, equivalent to the response in conscious dogs. In contrast, 2-DG at this dose and at a threefold higher dose did not alter PP levels in dogs anesthetized with pentobarbital (30 mg/kg iv), suggesting that halothane at this dose is not suppressive and that pentobarbital is very suppressive on reflex activation of the parasympathetic nerves to the pancreas. Bilateral electrical stimulation of the cervical vagi in halothane-anesthetized dogs elicited a sixfold increase in the pancreatic output of PP. The same stimulation caused only a twofold increase of PP output in pentobarbital-anesthetized dogs. These data suggests that halothane is also less inhibitory than pentobarbital on either peripheral neurotransmission or pancreatic F-cell responsiveness. The effect of direct activation of the F-cell by bethanechol did not differ between the two anesthesias. Therefore, the attenuated PP response to vagal stimulation in pentobarbital-anesthetized dogs is probably due to an action of pentobarbital on peripheral neurotransmission, perhaps at the intrapancreatic parasympathetic ganglia.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect and mechanism of vagal nerve stimulation on somatostatin secretion in dogs.

To investigate the effect of vagal nerve stimulation on the release of pancreatic somatostatin, we electrically stimulated (10 Hz, 5 ms, 13.5 mA, and 10 min) the thoracic vagi just below the heart in halothane anesthetized dogs (n = 15). The stimulation increased the pancreatic output of somatostatinlike immunoreactivity (SLI) (delta = +248 +/- 81 fmol/min, P less than 0.005; base-line levels = 455 +/- 150 fmol/min). min). Arterial plasma SLI levels increased as well (delta = +16 +/- 3 fmol/ml, P less than 0.001; base-line levels = 65 +/- 3 fmol/ml), reflecting stimulation of extrapancreatic SLI secretion. Significant vagal activation was verified by a fivefold increase of pancreatic output of pancreatic polypeptide (PP) (delta = +31.4 +/- 5.9 ng/min, P less than 0.001; base-line levels = 7.8 +/- 0.9 ng/min). Atropine pretreatment (n = 6) inhibited partially both the PP response (delta = +7.9 +/- 3.8 ng/min after atropine) and the pancreatic SLI response (delta = +92 +/- 29 fmol/min) to vagal nerve stimulation. However, atropine pretreatment did not modify the arterial SLI response (delta = +20 +/- 7 fmol/ml). Hexamethonium pretreatment (n = 9) completely abolished all three responses. We conclude that 1) electrical stimulation of the vagus stimulates pancreatic SLI, extrapancreatic SLI, and PP release in vivo in the dog; 2) both muscarinic and nonmuscarinic mechanisms mediate the PP and pancreatic SLI responses; 3) a nonmuscarinic mechanism mediates the extrapancreatic SLI response; and 4) all three responses are mediated via ganglionic nicotinic receptors.

Pentobarbital suppresses basal and reflexive pancreatic polypeptide release in dogs.

We sought to determine in overnight-fasted dogs whether basal or 2-deoxy-D-glucose (2-DG)-stimulated levels of pancreatic polypeptide (PP) could be reliably used as an index of cholinergic activity at the pancreas and thereby determine the effect of pentobarbital anesthesia on this cholinergic outflow. At low basal PP levels, either atropine or pentobarbital had a small effect on PP levels; at higher basal levels, both atropine and pentobarbital had a larger effect. Thus both drugs decreased PP in proportion to its initial basal level, suggesting that basal PP levels have a variable cholinergic component. Atropine abolished the PP response to intravenous 2-DG, confirming in our animal model that the PP response to neuroglucopenia is entirely cholinergically mediated. Pentobarbital also abolished the PP response to 2-DG, suggesting that anesthesia either suppresses cholinergic outflow to the pancreas or the response of the pancreatic F-cell to it. To test the latter hypothesis, the acetylcholine analogue bethanechol was administered before and during pentobarbital anesthesia. The PP response to direct cholinergic stimulation was not abolished by pentobarbital, although it was reduced modestly. We conclude that only part of the basal level of PP is under cholinergic control; all of the PP response to 2-DG is cholinergically mediated; pentobarbital anesthesia abolishes the cholinergic input to the pancreas; and if the endogenous cholinergic input influences certain pancreatic endocrine and exocrine responses, then its contribution would be seriously underestimated when studied in pentobarbital-anesthetized animals.

Postnatal development of intestinal secretin in rats and guinea pigs.

The amounts and concentrations of secretin in extracts of rat and guinea pig small intestine were measured with a porcine secretin radioimmunoassay. Immunoactivity in the extracts comigrated with porcine secretin in sodium dodecyl sulfate acrylamide gel electrophoresis. In the small intestines of adult rats and newborn and adult guinea pigs, there was a marked proximal-to-distal gradient in secretin concentration; newborn rats exhibited approximately equal proximal and distal secretin concentrations. Concentrations were as follows: most proximal newborn or adult guinea pig segments, 80 ng/mg prot; most distal newborn or adult guinea pig segments, 2 ng/mg prot; most proximal adult rat segments, 50 ng/mg prot; most distal adult rat segments, 8 ng/mg prot; and newborn rat intestine, 11 ng/mg prot. In either rats or guinea pigs, the secretin concentration in the proximal small intestine fell between days 2 and 6 postpartum to levels 3- and 10-fold below their respective newborn values. It is consistent with the relatively mature state of the newborn guinea pig digestive tract that it displays the proximal-to-distal gradient of secretin that is characteristic of both adult guinea pigs and rats. In the proximal intestine of both species, intestinal growth outpaces the accumulation of secretin.

A sensitive radioimmunoassay for human proinsulin, with sequential use of antisera to C-peptide and insulin.

In this assay for immunoreactive human proinsulin (IRPI), it first is separated from plasma by use of an antiserum to human C-peptide. An immunoprecipitate is then formed by using a precipitating antiserum and polyethylene glycol, after which IRPI is dissociated from the antiserum by incubation in warm HCl, pH 2.0. The resulting mixture is assayed for insulin immunoreactivity by a double-antibody tracer-competition method involving incubation for four days with a high-affinity anti-insulin antiserum. Human proinsulin of recombinant-DNA origin is used as the standard. Added C-peptide at supraphysiological concentrations did not interfere with or react in the assay. Human insulin cross reacted by 1.5%. The detection limit for IRPI (2 SD from zero-dose binding) is 3 pmol/L. Proinsulin conversion intermediates are measured nearly as well as intact proinsulin. IRPI concentrations in 10 nondiabetic human subjects averaged 12.0 (SEM 1.6) pmol/L. The ratio of proinsulin to immunoreactive insulin averaged 14.3 (SEM 2.2)%. After intravenous arginine, the increase in proinsulin was less than that of insulin, and it declined more slowly.

Disproportionate elevation of immunoreactive proinsulin in type 2 (non-insulin-dependent) diabetes mellitus and in experimental insulin resistance.

In this study, we found that the ratio of proinsulin to total immunoreactive insulin was much higher in 22 patients with Type 2 (non-insulin-dependent) diabetes mellitus than in 28 non-diabetic control subjects of similar age and adiposity (32 +/- 3 vs 15 +/- 1%, p less than 0.001). In addition, the arginine-induced acute proinsulin response to total immunoreactive insulin response ratio was greater in diabetic patients (n = 10) than in control subjects (n = 9) (8 +/- 2 vs 2 +/- 0.5%, p = 0.009), suggesting that increased islet secretion per se accounted for the increased ratio of proinsulin to immunoreactive insulin. One explanation for these findings is that increased demand for insulin in the presence of islet dysfunction leads to a greater proportion of proinsulin secreted from the B cell. We tested this hypothesis by comparing proinsulin secretion before and during dexamethasone-induced insulin resistance in diabetic patients and control subjects. Dexamethasone treatment (6 mg/day for 3 days) raised the proinsulin to immunoreactive insulin ratio in control subjects from 13 +/- 2 to 21 +/- 2% (p less than 0.0001) and in diabetic patients from 29 +/- 5 to 52 +/- 7% (p less than 0.001). Dexamethasone also raised the ratio of the acute proinsulin response to the acute immunoreactive insulin response in control subjects from 2 +/- 0.5 to 5 +/- 2% (p = 0.01) and in diabetic patients from 8 +/- 2 to 14 +/- 4% (p = NS), suggesting that the dexamethasone-induced increment in the basal ratio of proinsulin to immunoreactive insulin was also due to increased secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for noncholinergic ganglionic neural stimulation of B cell secretion.

Insulin levels increase after 2-deoxyglucose (2DG) administration in dogs. This observation is in contrast to the decrease in insulin level post-2DG in baboons and rabbits. To evaluate a possible neural mechanism mediating this increase in insulin level, we studied normal mongrel dogs with 2DG alone, 2DG during ganglionic blockade, beta-adrenergic blockade, and postganglionic parasympathetic blockade. There was an increase in plasma epinephrine, norepinephrine, pancreatic polypeptide, insulin, and glucose post-2DG alone. During ganglionic blockade, the increase in epinephrine, norepinephrine, and pancreatic polypeptide post-2DG was completely abolished, verifying ganglionic blockade of sympathetic and parasympathetic pathways, respectively. Despite this, ganglionic blockade failed to abolish the insulin rise after 2DG. Postganglionic parasympathetic blockade did not change the insulin rise after 2DG. However, beta-adrenergic blockade completely abolished the insulin rise after 2DG. The above data suggests that 1) the insulin rise post-2DG is beta-adrenergic but 2) the ganglionic neurotransmitter mediating the 2DG-induced insulin rise during ganglionic blockade is noncholinergic (possibly peptidergic).