Nutrient-independent increases in proglucagon and ornithine decarboxylase messenger RNAs after jejunoileal resection.

To assess potential mediators of adaptive bowel growth, ileal proglucagon messenger RNA (mRNA) ornithine decarboxylase (ODC) mRNA, plasma enteroglucagons, and plasma glucagonlike peptide I (GLP-I) were analyzed in rats soon after jejunoileal resection or control transection. Analyses were performed before and after refeeding to establish whether responses are nutrient dependent. The elevation of ileal proglucagon and ODC mRNAs within 12 hours after resection and before refeeding shows a nutrient-independent component of the adaptive response. The onset of adaptive growth of the ileum required luminal nutrient but occurred very rapidly, within 4 hours of refeeding. The onset of adaptive growth was accompanied by transient elevation of ileal ODC mRNAs. Ileal proglucagon mRNA and plasma GLP-I levels were also elevated, and these increases were sustained up to 8 days after resection. These early and sustained increases in proglucagon mRNA and plasma GLP-I indicate that in addition to the enteroglucagons, other intestinal proglucagon-derived peptides must be considered as potential mediators of adaptive growth after jejunoileal resection.

Prothoracic gland synthesis of 3-dehydroecdysone and its hemolymph 3 beta-reductase mediated conversion to ecdysone in representative insects.

The prothoracic glands of a variety of insects were tested for their ability to synthesize ecdysteroids in vitro. More specifically, they were evaluated for their ability to produce 3-dehydroecdysone and ecdysone using both radioimmunoassay and reverse phase high performance liquid chromatography. Three categories of insect prothoracic glands were noted: a) those producing much more 3-dehydroecdysone than ecdysone; b) glands synthesizing almost equivalent amounts of each of these two ecdysteroids; c) prothoracic glands that yielded more ecdysone than 3-dehydroecdysone. In addition, the 3-oxoecdysteroid 3 beta-reductase activity of the hemolymph of these insects was evaluated for its ability to convert 3-dehydroecdysone to ecdysone. The lepidopteran species tested yielded the most potent enzyme activity, although activity was demonstrated in members of other orders. These data indicate that the dehydroecdysone-ecdysone axis is not restricted to moths and butterflies.

Molecular biology of gastrointestinal peptides and growth factors: relevance to intestinal adaptation.

New approaches towards understanding regulation of growth and adaptation of the small intestine are made possible by the isolation and characterization of genes and complementary DNAs (cDNAs) encoding gastrointestinal peptides, growth factors and their receptors. Nucleotide sequencing provides prerequisite structural information. Analyses of gene expression by quantitation and localization of mRNAs provide information about correlations between local alterations in peptide or receptor synthesis and intestinal growth. Analyses of intestinal growth in transgenic animals that overexpress or underexpress growth factor or receptor genes provides direct information about peptide effects on growth. Our recent studies with genes and cDNAs encoding proglucagon and the growth hormone dependent insulin-like growth factor 1 (IGF-I) represent examples of these approaches. Sequences of proglucagon and IGF-I cDNAs provide the primary structures of the peptide precursors. Analyses of proglucagon mRNA during adaptive growth after small bowel resection indicate that increases in proglucagon gene transcription or mRNA stability underly previously observed increases in serum enteroglucagons during adaptive growth. Analyses of IGF-I mRNAs in intestine indicate that small intestine expresses only a subset of the IGF-I mRNAs expressed in liver due to utilization of specific promotors and/or exon splicing mechanisms. Oligomers derived from the 3' end of the rat IGF-I gene detect a novel intestinal specific IGF-I related mRNA that shows an aboral decline in abundance from duodenum to colon and is upregulated in a number of situations of adaptive growth. Transgenic mice that overexpress growth hormone or IGF-I are under analysis to establish the effects of growth hormone and IGF-I on intestinal growth.

Regulation of the ecdysteroid titer of Manduca sexta: reappraisal of the role of the prothoracic glands.

It is generally accepted that the prothoracic glands of insects produce ecdysone, which is converted by a 20-monooxygenase in peripheral tissues to the major molting hormone, 20-hydroxyecdysone. Incubation in vitro of the prothoracic glands of larval or pupal Manduca sexta in the presence of a hemolymph protein fraction (HPF) increased the ecdysteroid content of the medium almost 8-fold. A comparable increase was noted when HPF was added to medium preconditioned with prothoracic glands but from which the glands had been removed. We used a differential RIA to show that a major product of the prothoracic glands in vitro cross-reacts with antiserum (20-hydroxyecdysone-2-succinylthyroglobulin amide; H-2) that retains affinity to ecdysteroids having a modified A ring. However, this product did not bind to antiserum (ecdysone-22-succinylthyroglobulin amide; H-22) that has affinity mainly for ecdysteroids modified at the side chain. We employed radiolabeled precursor studies with prothoracic glands in vitro and a combination of analytical techniques (NMR, CD, MS) to demonstrate that the major ecdysteroid release from the glands is a mixture of 2-dehydroecdysone and 3-dehydroecdysone (1:2), which is rapidly reduced to ecdysone in the presence of HPF. We postulate that the active component of HPF is 3 beta 3 beta (2 beta)-formin-3(2)-ketoecdysteroid reductase. These results may explain several anomalous observations pertaining to the molting of insect fragments in the absence of prothoracic glands and suggest a complex system for the control of insect molting and metamorphosis.

The release of the prothoracicotropic hormone in the tobacco hornworm, Manduca sexta, is controlled intrinsically by juvenile hormone.

Pupal development is elicited early in the last larval instar of the tobacco hornworm, Manduca sexta (Johannson), by a precise temporal and quantitative increase in the haemolymph titre of 20-hydroxyecdysone. This increase in the titre is referred to as the pupal commitment peak, and it occurs once the titre of juvenile hormone (JH) has dropped. If the haemolymph titre of JH remains elevated at this time due to topical application of the hormone or of its analogue ZR512, commitment is delayed or inhibited in a dose-dependent manner. This delay or inhibition is due to the curtailment of the commitment peak in the ecdysteroid titre, which results from a failure of the prothoracic glands (PG) to increase the synthesis/secretion of the premoulting hormone, ecdysone. Since the PG from ZR512- and JH 1-treated larvae are capable of being activated in vitro by the prothoracicotropic hormone (PTTH), the effect of JH on the PG does not involve suppression of gland sensitivity to PTTH. The locus of the JH effect was determined to be the brain-retrocerebral complexes (Br-CC-CA), on the basis of experiments which tested the effect of implanted Br-CC-CA from pre-commitment larvae treated with JH on the occurrence of pupal commitment in head-ligated larval hosts. The implanted, JH-treated Br-CC-CA exhibited a delayed release of PTTH, and the effect was at concentrations of JH that were physiological. These results argue that JH functions to control the time during the last larval instar when pupal commitment occurs by dictating when PTTH will undergo gated release.