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The recent identification in Heloderma horridum venom of exendin-3, a new member of the glucagon superfamily that acts as a pancreatic secretagogue, prompted a search for a similar peptide in Heloderma suspectum venom. An amino acid sequencing assay for peptides containing an amino-terminal histidine residue (His1) was used to isolate a 39-amino acid peptide, exendin-4, from H. suspectum venom. Exendin-4 differs from exendin-3 by two amino acid substitutions, Gly2-Glu3 in place of Ser2-Asp3, but is otherwise identical. The structural differences make exendin-4 distinct from exendin-3 in its bioactivity. In dispersed acini from guinea pig pancreas, natural and synthetic exendin-4 stimulate a monophasic increase in cAMP beginning at 100 pM that plateaus at 10 nM. The exendin-4-induced increase in cAMP is inhibited progressively by increasing concentrations of the exendin receptor antagonist, exendin-(9-39) amide. Unlike exendin-3, exendin-4 does not stimulate a second rise in acinar cAMP at concentrations greater than 100 nM, does not stimulate amylase release, and does not inhibit the binding of radiolabeled vasoactive intestinal peptide to acini. This indicates that in dispersed pancreatic acini, exendin-4 interacts only with the recently described exendin receptor.

Evolutionary history suggests that the marsupials entered South America from North America about 75 million years ago and subsequently dispersed into Australia before the separation between South America and Antarctica-Australia. A question of interest is whether marsupial peptides resemble the corresponding peptides of Old or New World mammals. Previous studies had shown that "little" gastrin of the North American marsupial, the opossum, is identical in length to that of the New World mammals, the guinea pig and chinchilla. In this report, we demonstrate that opossum cholecystokinin octapeptide, like that of the Australian marsupials, the Eastern quoll and the Tamar wallaby, is identical to the cholecystokinin octapeptide of Old World mammals and differs from that of the guinea pig and chinchilla. However, opossum vasoactive intestinal polypeptide differs from the usual Old World mammalian vasoactive intestinal polypeptide in five sites: [sequence; see text].

Exendin-3 and exendin-4 are biologically active peptides isolated from venoms of the Gila monster lizards, H. horridum and H. suspectum, respectively. They were isolated using a chemical assay which detects peptides with amino-terminal histidine residues. Both are 39 amino acid peptides containing an amino-terminal histidine and a carboxyl-terminal serine amide and are members of the glucagon superfamily of peptide hormones. When tested in a dispersed pancreatic acinar cell assay, exendin-3 stimulates amylase release and with increasing concentrations causes a biphasic increase in cellular cAMP. In contrast, exendin-4 at concentrations up to 1 microM does not stimulate amylase release and produces a monophasic increase in cellular cAMP despite differing from exendin-3 by only two amino acid substitutions at positions 2 and 3 from the N-terminus. Endogenous Mammalian Analog to Exendins? The differences in biological activities can be explained by the observation that exendin-3 interacts with VIP receptors to stimulate amylase release, whereas exendin-4 does not. Both exendin-3 and exendin-4 interact with a putative exendin receptor on pancreatic acinar cells. The presence of this receptor was determined and defined by the ability of a specific inhibitor, exendin(9-39) amide, to abolish the increase in cAMP observed with 0.1-3 nM exendin-3 or exendin-4. The presence of the exendin receptor, although functionally undefined at the present time, predicts the existence of an endogenous mammalian analog to the exendin peptides.

The amino acid sequences of the gastroenteropancreatic peptides of Old World mammals are generally well-conserved. However, only the glucagons and vasoactive intestinal polypeptides (VIP) have been shown to be identical among the species studied to date. Rhesus monkey (Macaca mulatta) insulin has been shown to be identical with human insulin. The question addressed in this study is whether other gastroenteropancreatic peptides are identical to the human peptides. Purification and sequencing of glucagon, pancreatic polypeptide, VIP and insulin confirmed their identity with the corresponding human peptides. However, the 17 amino acid monkey gastrin is identical to dog gastrin and differs from human gastrin by substitution of methionine for leucine at position 5 from the N-terminus and alanine for glutamic acid in position 10. If additional rhesus monkey tissues become available, it would be of interest to determine whether other gastrointestinal peptides also differ from the corresponding human peptides.

It was reported two decades ago that insulin was not detectable in the glucose-stimulated state in Saimiri sciurea, the New World squirrel monkey, by a radioimmunoassay system developed with guinea pig anti-pork insulin antibody and labeled pork insulin. With the same system, reasonable levels were observed in rhesus monkeys and chimpanzees. This suggested that New World monkeys, like the New World hystricomorph rodents such as the guinea pig and the coypu, might have insulins whose sequences differ markedly from those of Old World mammals. In this report we describe the purification and amino acid sequences of squirrel monkey insulin and glucagon. We demonstrate that the substitutions at B29, B27, A2, A4, and A17 of squirrel monkey insulin are identical with those previously found in another New World primate, the owl monkey (Aotus trivirgatus). The immunologic cross-reactivity of this insulin in our immunoassay system is only a few percent of that of human insulin. Squirrel monkey glucagon is identical with the usual glucagon found in Old World mammals, which predicts that the glucagons of other New World monkeys would not differ from the usual Old World mammalian glucagon. It appears that the peptides of the New World monkeys have diverged less from those of the Old World mammals than have those of the New World hystricomorph rodents. The striking improvements in peptide purification and sequencing have the potential for adding new information concerning the evolutionary divergence of species.

An amino-terminal histidyl structure (His1) is characteristic of most peptides in the glucagon superfamily. An assay for His1 peptides performed by amino-terminal amino acid sequencing was used to screen venom from the Gila monster lizard, Heloderma horridum. Two His1 peptides were identified: helospectin and a new His1 peptide that has been named exendin-3 to indicate that it is the third peptide to be found in an exocrine secretion of Heloderma lizards which has endocrine activity, the first two being helospectin (exendin-1) and helodermin (exendin-2). In the lot of H. horridum venom tested, exendin-3 was 5-10-fold more abundant in molar concentration than helospectin. The structure of exendin-3 was analyzed by amino acid sequencing and mass spectrometry. Exendin-3 is a 39-amino acid peptide with a mass of 4200. It contains a carboxyl-terminal amide and has a strong homology with secretin at its amino-terminal 12 amino acids. The complete structure of exendin-3 is His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala- Val-Arg - Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro- Ser- amide. It is 32 and 26% homologous with helospectin and helodermin, respectively. It has greatest homology with glucagon (48%) and human glucagon-like peptide-1 (50%). Exendin-3 (3 microM) stimulated increases in cellular cAMP and amylase release from dispersed guinea pig pancreatic acini.

Molecular cloning of cholecystokinin (CCK) mRNA from porcine brain and gut has demonstrated that CCK is synthesized as an identical precursor in both tissues. The sequence for porcine CCK-58 predicted from CCK cDNA was identical with the amino acid sequence of the peptide purified from different lots of animals. However one group did report that there were differences in the N-terminus of CCK-58 purified from the intestines of two different lots of mongrel dogs. In the current report it is demonstrated that the amino acid sequences of CCK-58 purified separately from three bovine brains are identical through the first 19 N-terminal amino acid residues. The peptides were sequenced for ten additional steps and were shown to be identical with the previously reported sequences for the N-terminus of CCK-39. The N-terminus of bovine CCK-58 has the following sequence: AVPRVDDEPRAQLGALLAR.

Glucagon was purified from chinchilla pancreas and its biological activity determined. It was isolated using a chemical assay to identify peptides with a histidyl residue at the N-terminus. Chinchilla glucagon has the amino acid sequence HSQGTFTSDYSKHLDSRYAQEFVQWLMNT. It differs from the usual mammalian glucagon by amino acid substitutions at positions 13, 18 and 21 from the N-terminus. Despite these sequence changes, its biological activity is conserved. Chinchilla glucagon has approximately the same potency as pig glucagon in stimulating liver membrane adenyl cyclase activity. Pancreatic polypeptide was also purified from chinchilla pancreas based on its Ala1 signal and has the sequence APLEPVYPGDNATPEQMAQYAAEMRRYINMLTRPRY#.

Seasonal changes in pancreatic insulin and glucagon were investigated in the little brown bat (Myotis lucifugus). Pancreatic protein content increased 21% from the nonhibernating state in June compared to the nonhibernating or early hibernating state in late October, and then decreased 50% between October and the months of deep hibernation. The content of insulin and glucagon increased from June to October, and both hormones further increased during hibernation, when a fall in pancreatic protein content occurred. The concentration of both insulin and glucagon increased from October to February. Of note, the insulin concentration progressively increased during deep hibernation from February to April, whereas the glucagon concentration remained constantly elevated throughout this time interval. The persistent elevation of pancreatic glucagon during hibernation may be related to its role in counterregulation and carbohydrate homeostasis during fasting. The elevation of pancreatic insulin, and its further increase during hibernation, is less clear but may be hypothesized to provide a rapidly releasable storage pool required for immediate secretion during arousal, possibly to prevent a hyperosmolar state.

1. "Little" gastrins from most mammalian species are 17 amino acid peptides and the precursor "big" gastrins are 34 amino acid peptides. 2. "Little" gastrins of the New World hystricomorphs, guinea-pig and chinchilla, are 16 amino acid peptides due to deletion of a glutamic acid in the region 6-9 from their NH2-terminus and the corresponding "big" gastrins are 33 amino acid peptides. 3. Antral gastrins from the opossum, a New World marsupial, have a glutamic acid deletion in the same region as the hystricomorph gastrins. 4. Opossum "big" gastrin is a 33 amino acid peptide with the following sequence: less than ELGPQDLPYLTADLSKKQGPWLEEEEAYGWMDF#.

Insulin is readily concentrated from 10 to 50 ml of urine with better than 75% recovery using octadecylsilyl (ODS) silica columns (C18Sep-Pak cartridge) and can then be measured by radioimmunoassay. Fractionation on Sephadex G50 gel filtration reveals that the apparent immunoreactivity corresponds for the most part to 6000 dalton insulin. Renal clearance of insulin in 5 normal subjects does not appear to differ in the fasted or fed state and ranged from 0.34 to 0.58 ml/min with an average of 0.44 +/- 0.10 (S.D.) ml/min. Increased urinary insulin output was observed following feeding and fell during prolonged fasting. Insulin output in urine from 7 non-diabetic subjects ranged from 11 to 39 mU/24 hr, averaging 25 +/- 10 mU/24 hr. In normal subjects without renal disease a single determination of renal insulin clearance and a timed urinary insulin output appear to be sufficient for determination of mean plasma insulin during that time period. Concentration of urine using this methodology could provide the material for HPLC screening for abnormal insulins and for their subsequent purification to determine the site of change in amino acid sequence.

The concentrations of bioamines and their metabolites have been determined in March and April during arousal from hibernation in the cerebral cortex of the little brown bat (Myotis lucifugus). The patterns during arousal for dopamine and serotonin (5-HT) were similar with a significant fall in concentrations by 1 h of arousal, and an inverse relationship with their respective metabolites, 3,4-dihydroxyphenylacetic acid and 5-hydroxyindolacetic acid (5-HIAA). This suggests an acute release and metabolism of these bioamines with onset of arousal. During arousal, cerebral cortical concentrations of norepinephrine (NE) were not significantly changed. Levels of homovanillic acid were markedly depressed during hibernation and rose acutely with arousal. After arousal and 4 days of feeding in April, there was an increase in all bioamines and their metabolites studied except for NE. Of note is the marked decrease in the hibernating level of 5-HT and increase in its metabolite 5-HIAA from March to April, which may herald the natural termination of hibernation. Our results suggest that the brain of hibernators undergoes complex changes in the modulation of neurotransmitter systems which are consistent with both down- and up-regulation of neuronal activity in the maintenance of hibernation and the initiation of the arousal process.

We studied 43 women in their third trimester of pregnancy whose fetuses were at significant risk of intrauterine growth retardation. To define metabolic subtypes for intrauterine growth retardation, a 100 gm glucose load was administered after an overnight fast. Twenty-seven women were normotensive and 16 had hypertension. The glucose tolerance of the hypertensive group was essentially the same as that of the normotensive group. However, 8 of the 16 women with hypertension had marked hyperinsulinemia in response to an oral glucose load. Of the five women with hypertension who gave birth to offspring of low birth weight, three had hyperinsulinemia.

Pancreatic hormones have been purified from the opossum, a New World marsupial. Opossum insulin contains a Leu substitution at the N-terminus of the B-chain in place of the Phe that is generally present in mammalian insulins. In addition, there are two other amino acid substitutions in the opossum insulin A-chain (positions 8 and 18) compared to pig insulin. Opossum glucagon is identical to chicken glucagon with both differing from the usual mammalian glucagon by Ser in place of Asn at its penultimate C-terminal position. Opossum PP differs from the porcine peptide in only 3 sites (position 3, 19 and 30).

Glucagon is a 29 amino acid peptide that is generally highly conserved. Among mammalian glucagons the only one that has been shown to differ significantly is that of the guinea pig which differs from the others in 5 of the 9 COOH-terminus amino acids. The amino acid content and partial sequencing of chicken glucagon had been reported earlier. This report describes the purification and complete amino acid sequencing of chicken glucagon and demonstrates that it differs from the usual mammalian glucagon by the replacement of asparagine at position 28 with serine. Chicken glucagon is indistinguishable from porcine glucagon in the rat liver receptor assay system.

Cholecystokinin octapeptides (CCK8s) have been purified from methanol extracts of two brains from each of two Australian marsupials, Tammar Wallaby and Eastern Quoll, containing 3 nmol and 2 nmol of the peptides, respectively. Immunoreactive CCK was concentrated on QMA SepPak cartridges and purified by two successive HPLC steps on Nova C18 radial-pak cartridges. The sequence of each of the peptides is identical with that previously reported for Old World mammals (DYMGWMDF). This is in contrast to the previously reported sequence for CCK8 from the South American hystricomorphs, guinea pig and chinchilla, which differs in a substitution of valine for methionine in position 3 from the NH2-terminus. Although evolutionary history suggests that marsupials migrated from South America into Australia before the two continents separated, this peptide resembles that found in Old World mammals rather than that of South American hystricomorphs. Such molecular data are useful in assessing phylogenetic relationships among taxa.

Previous studies have shown that pig intestine contains a 69 amino acid glucagon (glicentin) as well as a 37 amino acid glucagon (oxyntomodulin). In pig pancreas the 29 amino acid glucagon predominates. Since glucagon is thought to be expressed from a single gene in mammals, these differences in molecular forms indicate differential posttranslational processing of the glucagon precursor by different tissues. In the current study glucagon immunoreactivity (IR) was separately purified from dog pancreas, stomach mucosa and ileum mucosa. Purification and sequence analysis of the different tissue glucagons show that dog pancreas and stomach mucosa contain glucagon-29 while ileum mucosa contains glucagon-37 and glucagon-69. The latter is the major form present with glucagon-37 accounting for only 10-20% of the total ileum glucagon content. The N-terminal 32 amino acid portion of dog glucagon-69 differs at 6 sites from pig glucagon-69: RSLQDTEEKSRSFSAPQTEPLNDLDQMNEDKR... The C-terminal glucagon-37 is identical to pig oxyntomodulin.

The rat has been a useful model for studying neuronal and metabolic abnormalities associated with fetal and neonatal hypothyroidism produced by treatment of the mother with antithyroid medication. The neonates are then maintained on this medication via the mother's milk until weaning and subsequently through the drinking water. We have determined the concentrations and contents of immunoreactive cholecystokinin (CCK) and vasoactive intestinal peptide (VIP) in the brain and gut of groups of rats exposed to antithyroid medication from day 16 of gestation. The neonates were sacrificed at 2, 4, 8 and 12 weeks. Compared to controls total body weight was greatly reduced in methimazole (MMI)-treated rats, all of whom were hypothyroid as evidenced by marked reduction of T4 and increase in TSH. Discontinuation of MMI-treatment after 8 weeks resulted in normalization of T4 and TSH and a dramatic weight gain but at 12 weeks the brain weights of the MMI-treated rats were reduced by 17% and the brain contents, of CCK and VIP were similarly reduced. Tissue weights throughout the gut were 1/2 or less than those of control rats. Since VIP but not CCK concentrations in the gut of MMI-treated animals were significantly greater than those of the control animals, it would appear that there was greater loss of mucosal tissue with its endocrine content of CCK than of neuronal tissue with its greater content of VIP.(ABSTRACT TRUNCATED AT 250 WORDS)

Patients with mild or early non-insulin-dependent diabetes mellitus often display a delay in insulin response followed by late hyperinsulinemia during oral glucose tolerance testing. Those patients with long-standing disease or elevations of fasting plasma glucose in excess of 140 mg/dl are generally hypoinsulinemic in response to an oral glucose tolerance test. Diabetic patients who do not have an acute response to intravenous glucose may have normal responses to intravenous tolbutamide or intravenous arginine, suggesting that delayed responsiveness to glucose is not due to decreased pancreatic insulin content. An association between hyperinsulinemia and hypertension has been suggested by recent studies from several laboratories. In a homogeneous population of men who suffered traumatic bilateral above-the-knee amputation in the Vietnam War with subsequent development of obesity, it was shown that there was strong correlation between hypertension and hyperinsulinemia during oral glucose tolerance testing despite only mild glucose intolerance. In addition, a subset of hypertensive women who were in their third trimester of pregnancy were markedly hyperinsulinemic during oral glucose tolerance testing in the absence of any abnormalities of glucose tolerance. Thus, the relationship between hyperinsulinemia and hypertension, and the possible reasons for this relationship, are fields of active investigation at present.