Sequence analysis of the putative regulatory region of rat alpha 2-macroglobulin gene

Rat alpha 2-macroglobulin (alpha 2M) is an acute-phase reactant, concentration of which in serum increases more than 100-fold in the course of inflammation. Glucocorticoid and some protein factors such as interleukin 1 (IL-1) have been known to be involved in the regulation of this plasma protein synthesis. To understand the regulatory mechanism of alpha 2M production at the molecular level, we isolated genomic DNA clones of rat alpha 2M gene and characterized the promoter region of the gene by comparing the nucleotide sequence with those of other acute-phase reactant genes. Several possible regulatory signals were identified. Particularly, a sequence (T/A)T(C/G)TGGGA(A/T) was found about at 170 bp upstream from a putative capping site, which was also found in the 5'flanking region of various acute-phase reactant genes.     

Chromatin structure and protein binding in the putative regulatory region of the c-myc gene in Burkitt lymphoma

A chromosomal myc gene displays one of three patterns of activity depending upon the arrangement of the gene and its allelic partner. In nonmalignant B cells both myc alleles are normally expressed. In Burkitt lymphoma cells carrying both a translocated and a nontranslocated myc allele, the translocated allele is inappropriately expressed, while the nontranslocated allele is virtually inactive. Here we examine the chromatin structure of these genes using DNAase I hypersensitivity in nonmalignant lymphoblastoid cells and in the Burkitt lymphoma, BL31 . Three hypersensitivity patterns emerge that correlate with the state of the gene and reveal sites associated with putative regulatory structures. One region is associated with the two myc promoters, one with a specific nuclear protein binding site, and one--which is markedly enhanced in the inactive germline gene in the Burkitt cell--with a putative negative control region. The perturbation of the normal pattern in this particular Burkitt cell may be due to the action of an immunoglobulin enhancer.     

Inhibition of gastric inhibitory polypeptide signaling prevents obesity

Secretion of gastric inhibitory polypeptide (GIP), a duodenal hormone, is primarily induced by absorption of ingested fat. Here we describe a novel pathway of obesity promotion via GIP. Wild-type mice fed a high-fat diet exhibited both hypersecretion of GIP and extreme visceral and subcutaneous fat deposition with insulin resistance. In contrast, mice lacking the GIP receptor (Gipr(-/-)) fed a high-fat diet were clearly protected from both the obesity and the insulin resistance. Moreover, double-homozygous mice (Gipr(-/-), Lep(ob)/Lep(ob)) generated by crossbreeding Gipr(-/-) and obese ob/ob (Lep(ob)/Lep(ob)) mice gained less weight and had lower adiposity than Lep(ob)/Lep(ob) mice. The Gipr(-/-) mice had a lower respiratory quotient and used fat as the preferred energy substrate, and were thus resistant to obesity. Therefore, GIP directly links overnutrition to obesity and it is a potential target for anti-obesity drugs.     

The insulinotropic action of gastric inhibitory polypeptide.

The effect of highly purified gastric inhibitory polypeptide (GIP) on immunoreactive insulin (IRI) secretion in the conscious fasted dog was investigated. Significant increases in IRI release were observed with intravenous administration of three different doses of GIP. These were accompanied by depression in fasting serum-glucose levels. Preliminary studies were undertaken to determine whether this insulinotropic action of GIP could be attributed to a particular segment of the GIP molecule. GIP fragments produced by cleavage with cyanogen bromide and trypsin showed no significant stimulation of IRI release. The possibility that GIP might itself enhance glucose uptake or potentiate insulin-induced glucose uptake was studied with the rat hemidiaphragm preparation. No such effect was observed. In the light of this and other recent work, it is concluded that GIP is a strong candidate for an active principle in the enteroinsular axis.     

Postinhibitory overshoot of insulin release by gastric inhibitory polypeptide

Following intravenous infusion of somatostatin $\text{in vivo}$ occasionally there is a large rebound overshoot of insulin release. An $\text{in vitro}$ model to simulate this phenomenon was made by perfusing rat pancreas with gastric inhibitory polypeptide (GIP) during simultaneous perfusion with somatostatin. Adding GIP (100 ng/ml) to the perfusate for 2 minutes beginning either 3 or 9 minutes before terminating the somatostatin perfusion produced a large overshoot in insulin release. The magnitude of overshoot was greater when medium contained 300 mg/dl glucose that when it contained 150 mg/dl glucose. Perfusion with GIP for 2 minutes beginning 9 minutes before increasing the glucose concentration of the medium from 30 to 300 mg/dl elicited a large increase in both the acute and second-phase release of insulin. These suggest that post-inhibitory overshoot of insulin release after somatostatin may be produces $\text{in vitro}$ by the suppressed action of stimulatory hormones such as GIP. Prior infusion with GIP can also potentiate glucose-stimulated insulin increase.     

Gastric inhibitory polypeptide does not inhibit gastric emptying in humans

The insulinotropic gut hormone gastric inhibitory polypeptide (GIP) has been demonstrated to inhibit gastric acid secretion and was proposed to possess "enterogastrone" activity. GIP effects on gastric emptying have not yet been studied. Fifteen healthy male volunteers (23.9 +/- 3.3 yr, body mass index 23.7 +/- 2.3 kg/m(2)) were studied with the intravenous infusion of GIP (2 pmol.kg(-1).min(-1)) or placebo, each administered to the volunteers on separate occasions from -30 to 360 min in the fasting state. At 0 min, a solid test meal (250 kcal containing [(13)C]sodium octanoate) was served. Gastric emptying was calculated from the (13)CO(2) exhalation rates in breath samples collected over 360 min. Venous blood was drawn in 30-min intervals for the determination of glucose, insulin, C-peptide, and GIP (total and intact). Statistical calculations were made by use of repeated-measures ANOVA and one-way ANOVA. During the infusion, GIP rose to steady-state concentrations of 159 +/- 15 pmol/l for total and 34 +/- 4 pmol/l for intact GIP (P < 0.0001). Meal ingestion further increased GIP concentrations in both groups, reaching peak levels of 265 +/- 20 and 82 +/- 9 pmol/l for total and 67 +/- 7 and 31 +/- 9 pmol/l for intact GIP during the administration of GIP and placebo, respectively (P < 0.0001). There were no differences in glucose, insulin, and C-peptide between the experiments with the infusion of GIP or placebo. Gastric half-emptying times were 120 +/- 9 and 120 +/- 18 min (P = 1.0, with GIP and placebo, respectively). The time pattern of gastric emptying was similar in the two groups (P = 0.98). Endogenous GIP secretion, as derived from the incremental area under the curve of plasma GIP concentrations in the placebo experiments, did not correlate to gastric half-emptying times (r(2) = 0.15, P = 0.15 for intact GIP; r(2) = 0.21, P = 0.086 for total GIP). We conclude that gastric emptying does not appear to be influenced by GIP. The secretion of GIP after meal ingestion is not suppressed by its exogenous administration. The lack of effect of GIP on gastric emptying underlines the differences between GIP and the second incretin glucagon-like peptide 1.     

Lack of inhibitory effect of alpha-human atrial natriuretic polypeptide on cortisol secretion in cultured adrenocortical adenoma cells from the patients with Cushing's syndrome

The effects of synthetic alpha-human atrial natriuretic polypeptide (alpha-hANP) on cortisol secretion by adrenocortical adenoma cells from patients with Cushing's syndrome (CS cells) in primary monolayer cultures, compared to cultured normal adrenal cells, were studied. alpha-hANP significantly inhibited cortisol secretion by human normal adrenal cells in culture, but had no direct effect on cortisol secretion from CS cells, in the presence or absence of 10(-8) M ACTH. alpha-hANP enhanced the accumulation of intracellular cyclic GMP in normal adrenal cells in culture, but not in CS cells. Visualization of [125I] iodo-alpha-hANP-specific binding sites by an in vitro receptor autoradiographic technique showed that these sites were lacking in adrenocortical adenoma tissues. These results suggest that the loss of alpha-hANP inhibitory effect on cortisol secretion in CS cells may be due to the absence of alpha-hANP receptor sites.     

Response of gastric inhibitory polypeptide to fat ingestion in normal dogs

Because of the differences in the data concerning the mechanism by which gastric inhibitory polypeptide (GIP) is secreted following fat ingestion, we were prompted to investigate the characteristics of the GIP response to the triacylglycerol components in normal dogs. Oral administration of glycerol (1 g/kg) slightly elevated the blood glucose levels but not the plasma GIP. Palmitate (1 g/kg) administration did not change the blood glucose whereas the plasma GIP was increased remarkably and remained elevated at 120 min. Oral administration of tricaprylin (2 g/kg) did not elicit any discernible changes in the blood glucose nor in the plasma GIP. Column chromatography of plasma obtained from a dog after palmitate ingestion revealed three GIP-immunoreactive peaks: one peak corresponding to the authentic GIP, one to the large molecular weight peak, and one to the small molecular weight peak. Similar results were obtained with the plasma collected after fat ingestion. From the present study, it would appear that hydrolysis of triacylglycerol plays an important role in GIP release from the intestine. Furthermore, it is concluded that the long chain fatty acids stimulate GIP release whereas the medium chain fatty acids do not.     

In depth analysis of the N-terminal bioactive domain of gastric inhibitory polypeptide

Gastric inhibitory polypeptide/glucose-dependent insulinotropic polypeptide (GIP) is an important gastrointestinal regulator of insulin release and glucose homeostasis following a meal. Strategies have been undertaken to delineate the bioactive domains of GIP with the intention of developing small molecular weight GIP mimetics. The molecular cloning of receptors for GIP and the related hormone GLP-1 (glucagon-like peptide-1) has allowed examination of the characteristics of incretin analogs in transfected cell models. The current report examines the N-terminal bioactive domain of GIP residing in residues 1-14 by alanine scanning mutagenesis and N-terminal substitution/modification. Further studies examined peptide chimeras of GIP and GLP-1 designed to localize bioactive determinants of the two hormones. The alanine scan of the GIP(1-14) sequence established that the peptide was extremely sensitive to structural perturbations. Only replacement of amino acids 2 and 13 with those found in glucagon failed to dramatically reduce receptor binding and activation. Of four GIP(1-14) peptides modified by the introduction of DP IV-resistant groups, a peptide with a reduced bond between Ala2 and Glu3 demonstrated improved receptor potency compared to native GIP(1-14). The peptide chimera studies supported recent results on the importance of a mid-region helix for bioactivity of GIP, and confirmed existence of two separable regions with independent intrinsic receptor binding and activation properties. Furthermore, peptide chimeras showed that binding of GLP-1 also involves both N- and C-terminal domains, but that it apparently contains only a single bioactive domain in its N-terminus. Together, these results should facilitate development of incretin based therapies using rational drug design for potential treatment of diabetes.     

Suppression of insulin receptor binding by prolonged enteral or parenteral nutrient infusion in the rat: role of gastric inhibitory polypeptide

In the rat, prolonged enteral or parenteral alimentation with a high-carbohydrate diet results in hyperinsulinemia, which is substantially greater with the parenteral route. Supplementing the parenteral infusate with porcine gastric inhibitory polypeptide (GIP) to approximate plasma immunoreactive GIP levels achieved with enteral feeding further increases steady-state plasma insulin and glucose concentrations, suggesting insulin resistance. We examined the effects of sustained hyperinsulinemia elicited by continuous nutrient infusion on insulin binding to isolated rat adipocytes and the modification of this response by GIP. Compared with a baseline group, both enterally and parenterally alimented groups showed decreased insulin receptor binding affinity. However, despite substantially different steady-state plasma insulin levels, insulin binding was similar with either infusion route. Factors other than plasma insulin concentration alone therefore contribute to insulin receptor down-regulation during prolonged enteral alimentation. Supplementing the parenteral infusate with exogenous GIP resulted in a further reduction in insulin receptor affinity. Thus, adaptation to continuous nutrient infusion is characterized by insulin receptor down-regulation regardless of the route of nutrient delivery. An additional suppression of insulin receptor binding may in part be responsible for the insulin resistance elicited by prolonged exogenous GIP administration.     

Neural-restrictive silencers in the regulatory mechanism of pituitary adenylate cyclase-activating polypeptide gene expression

Pituitary adenylate cyclase-activating polypeptide (PACAP) is known as a pleiotropic neuropeptide and is present abundantly in central nervous system. During a detailed analysis of the 5'-flanking region of the mouse PACAP gene, we found and characterized two negative regulatory elements, which are homologous to the neural-restrictive silencer element, and are termed neural-restrictive silencer-like elements 1 and 2 (NRSLE1 and NRSLE2). Their sequence and position were significantly conserved among mouse, human, and rat PACAP genes. In the electrophoretic mobility shift assay (EMSA) with nuclear extracts of Swiss-3T3 cells and individual oligonucleotide probes for NRSLE1 and NRSLE2, a specific complex was observed to have the same migration as compared with the NRSE probe of rat type II sodium channel gene (NaII). Furthermore, these complexes were efficiently competed by the unlabeled NaII probe. In the luciferase reporter assay, the reporter gene constructs containing NRSLEs, driven by heterologous SV40 promoter, exhibited repression of luciferase activity almost equal to basal level in Swiss-3T3 cells. In contrast, the repression was not observed in differentiated PC12 cells with NGF. These results suggested that the neural-restrictive silencer system might be involved in the regulatory mechanism of neuron-specific PACAP gene expression.