Enteroinsular hormones in two siblings with Donohue syndrome and complete leptin deficiency.

The main biochemical hallmark of the rare and lethal condition of Donohue syndrome (DS) is hyperinsulinemia. The roles of the gut and other pancreatic hormones involved in glucose metabolism, satiety and energy expenditure have not been previously reported in DS. Two siblings with genetically confirmed DS and extremely low weight underwent a mixed meal (MM) test where pancreatic hormones insulin, C-peptide, glucagon, active amylin, pancreatic polypeptide (PP) as well as gut hormones active glucagon-like peptide 1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), ghrelin, peptide YY (PYY) and leptin were analyzed using a Multiplex assay. Results were compared to those of 2 pediatric controls. As expected, concentrations of insulin, C-peptide and amylin were very high in DS cases. The serum glucagon concentration was undetectable at the time of hypoglycemia. GIPs concentrations were lower in the DS, however, this was not mimicked by the other incretin, GLP-1. Ghrelin concentrations were mainly undetectable (<13.7 pg/mL) in all participants. DS cases had higher PYY and dampened PP concentrations. Leptin levels remained completely undetectable (<137.0 pg/mL). Patients with DS have extremely high amylin levels, completely undetectable serum glucagon and leptin levels with abnormal satiety regulating hormone PP with a relatively normal ghrelin response during a MM test. The low serum GIP might be acting as physiological brake on insulin secretion. The undetectable serum leptin levels suggest the potential of using leptin analogues as therapy for DS patients.

Involvement of guanylin and GC-C in rat mesenteric macrophages in resistance to a high-fat diet.

A high-fat diet (HFD) is a well-known contributing factor in the development of obesity. Most rats fed HFDs become obese. Those that avoid obesity when fed HFDs are considered diet resistant (DR). We performed a microarray screen to identify genes specific to the mesenteric fat of DR rats and revealed high expression of guanylin and guanylyl cyclase C (GC-C) in some subjects. Our histologic studies revealed that the cellular source of guanylin and GC-C is macrophages. Therefore, we developed double-transgenic (Tg) rats overexpressing guanylin and GC-C in macrophages and found that they were resistant to the effects of HFDs. In the mesenteric fat of HFD-fed Tg rats, Fas and perilipin mRNAs were downregulated, and those of genes involved in fatty acid oxidation were upregulated, compared with the levels in HFD-fed wild-type rats. In vitro studies demonstrated that lipid accumulation was markedly inhibited in adipocytes cocultured with macrophages expressing guanylin and GC-C and that this inhibition was reduced after treatment with guanylin- and GC-C-specific siRNAs. Our results suggest that the macrophagic guanylin-GC-C system contributes to the altered expression of genes involved in lipid metabolism, leading to resistance to obesity.

Loss-of-function mutation in the prokineticin 2 gene causes Kallmann syndrome and normosmic idiopathic hypogonadotropic hypogonadism.

Gonadotropin-releasing hormone (GnRH) deficiency in the human presents either as normosmic idiopathic hypogonadotropic hypogonadism (nIHH) or with anosmia [Kallmann syndrome (KS)]. To date, several loci have been identified to cause these disorders, but only 30% of cases exhibit mutations in known genes. Recently, murine studies have demonstrated a critical role of the prokineticin pathway in olfactory bulb morphogenesis and GnRH secretion. Therefore, we hypothesize that mutations in prokineticin 2 (PROK2) underlie some cases of KS in humans and that animals deficient in Prok2 would be hypogonadotropic. One hundred IHH probands (50 nIHH and 50 KS) with no known mutations were examined for mutations in the PROK2 gene. Mutant PROK2s were examined in functional studies, and the reproductive phenotype of the Prok2(-/-) mice was also investigated. Two brothers with KS and their sister with nIHH harbored a homozygous deletion in the PROK2 gene (p.[I55fsX1]+[I55fsX1]). Another asymptomatic brother was heterozygous for the deletion, whereas both parents (deceased) had normal reproductive histories. The identified deletion results in a truncated PROK2 protein of 27 amino acids (rather than 81 in its mature form) that lacks bioactivity. In addition, Prok2(-/-) mice with olfactory bulb defects exhibited disrupted GnRH neuron migration, resulting in a dramatic decrease in GnRH neuron population in the hypothalamus as well as hypogonadotropic hypogonadism. Homozygous loss-of-function PROK2 mutations cause both KS and nIHH.

Altered circadian and homeostatic sleep regulation in prokineticin 2-deficient mice.

STUDY OBJECTIVES: Sleep is regulated by circadian and homeostatic processes. Recent studies with mutant mice have indicated that circadian-related genes regulate sleep amount, as well as the timing of sleep. Thus a direct link between circadian and homeostatic regulation of sleep may exist, at least at the molecular level. Prokineticin 2 (PK2), which oscillates daily with high amplitude in the suprachiasmatic nuclei (SCN), has been postulated to be an SCN output molecule. In particular, mice lacking the PK2 gene (PK2-/-) have been shown to display significantly reduced rhythmicity for a variety of circadian physiological and behavioral parameters. We investigated the role of PK2 in sleep regulation. DESIGN: EEG/EMG sleep-wake patterns were recorded in PK2-/- mice and their wild-type littermate controls under baseline and challenged conditions. MEASUREMENTS AND RESULTS: PK2-/- mice exhibited reduced total sleep time under entrained light-dark and constant darkness conditions. The reduced sleep time in PK2-/- mice occurred predominantly during the light period and was entirely due to a decrease in non-rapid eye movement (NREM) sleep time. However, PK2-/- mice showed increased rapid eye movement (REM) sleep time in both light and dark periods. After sleep deprivation, compensatory rebound in NREM sleep, REM sleep, and EEG delta power was attenuated in PK2-/- mice. In addition, PK2-/- mice had an impaired response to sleep disturbance caused by cage change in the light phase. CONCLUSIONS: These results indicate that PK2 plays roles in both circadian and homeostatic regulation of sleep. PK2 may also be involved in maintaining the awake state in the presence of behavioral challenges.

Attenuated circadian rhythms in mice lacking the prokineticin 2 gene.

Circadian clocks drive daily rhythms in virtually all organisms. In mammals, the suprachiasmatic nucleus (SCN) is recognized as the master clock that synchronizes central and peripheral oscillators to evoke circadian rhythms of diverse physiology and behavior. How the timing information is transmitted from the SCN clock to generate overt circadian rhythms is essentially unknown. Prokineticin 2 (PK2), a clock-controlled gene that encodes a secreted protein, has been indicated as a candidate SCN clock output signal that regulates circadian locomotor rhythm. Here we report the generation and analysis of PK2-null mice. The reduction of locomotor rhythms in PK2-null mice was apparent in both hybrid and inbred genetic backgrounds. PK2-null mice also displayed significantly reduced rhythmicity for a variety of other physiological and behavioral parameters, including sleep-wake cycle, body temperature, circulating glucocorticoid and glucose levels, as well as the expression of peripheral clock genes. In addition, PK2-null mice showed accelerated acquisition of food anticipatory activity during a daytime food restriction. We conclude that PK2, acting as a SCN output factor, is important for the maintenance of robust circadian rhythms.

Impaired pain sensation in mice lacking prokineticin 2.

Prokineticins (PKs), consisting of PK1 and PK2, are a pair of newly identified regulatory peptides. Two closely related G-protein coupled receptors, PKR1 and PKR2, mediate the signaling of PKs. PKs/PKRs participate in the regulation of diverse biological processes, ranging from development to adult physiology. A number of studies have indicated the involvement of PKs/PKRs in nociception. Here we show that PK2 is a sensitizer for nociception. Intraplantar injection of recombinant PK2 resulted in a strong and localized hyperalgesia with reduced thresholds to nociceptive stimuli. PK2 mobilizes calcium in dissociated dorsal root ganglion (DRG) neurons. Mice lacking the PK2 gene displayed strong reduction in nociception induced by thermal and chemical stimuli, including capsaicin. However, PK2 mutant mice showed no difference in inflammatory response to capsaicin. As the majority of PK2-responsive DRG neurons also expressed transient receptor potential vanilloid (TRPV1) and exhibited sensitivity to capsaicin, TRPV1 is likely a significant downstream molecule of PK2 signaling. Taken together, these results reveal that PK2 sensitize nociception without affecting inflammation.

Autoantibodies to gut hormone secreting cells as markers of peptide deficiency.

Autoantibodies reacting with endocrine cells in the gastrointestinal mucosa were found by indirect immunofluorescence in 22 out of 268 sera (8.2%) obtained from patients with coeliac disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, and from subjects without bowel disease. A double immunofluorescence technique showed that the autoantibodies reacted with cells secreting gastric inhibitory polypeptide (glucose dependent insulinotropic polypeptide, GIP), secretin, somatostatin or enteroglucagon. Most sera contained antibodies against more than one cell type. Neither the presence of a particular antibody nor the pattern of antibody combinations appeared to be specific for any diagnostic category. The mean plasma GIP concentrations, however, both fasting and two hours after a test meal, were significantly lower in subjects with GIP cell autoantibodies. Thus gut hormone cell autoantibodies may be markers of impaired hormone secretion.

Influence of gastric inhibitory polypeptide antiserum on glucose-induced insulin secretion in rats.

The action of gastric inhibitory polypeptide (GIP) antiserum on glucose tolerance and insulin secretion after an intraduodenal glucose load (600 mg/kg) was examined in anesthetized rats. In control experiments the insulin secretion was nearly doubled when glucose was administered intraduodenally, as compared to an iv glucose load to simulate the blood glucose curve after the intraduodenal glucose administration. After injection of GIP antiserum, the glucose curve resulting from the intraduodenal glucose load was slightly elevated and the insulin response was significantly reduced. No free GIP could be measured in the plasma of antibody-treated rats. However, the GIP antiserum did not offset the incretin effect of the intraduodenal glucose load completely. In control experiments the same amount of GIP antibody completely blocked the insulinotropic effect of exogenous porcine GIP (0.6 microgram/kg . h). In nonanesthetized rats serial oral glucose tolerance tests were performed for 14 days after injection of the GIP antiserum. Despite the blockage of endogenous GIP, the glucose tolerance did not change significantly in the antibody-treated group of rats as compared to a control group. These data indicate that GIP is not the exclusive incretin and that additional gut factors with insulinotropic activity exist.

Lack of a gastrointestinal mediator of insulin action in maturity-onset diabetes.

It is suggested that hepatic uptake of orally ingested glucose depends not only on insulin secretion but also on the release of a gastrointestinal factor which mediates insulin action on the liver. In maturity-onset diabetes characterised by hyperinsulinaemia and insulin resistance, deficiency of this gastrointestinal factor may be the primary pathogenetic event leading to postprandial hyperglycaemia. Postprandial hyperglycaemia brings about an increase in insulin secretion; and hyperinsulinaemia, in turn, results in decreased binding of insulin to its receptor and in peripheral (extrahepatic) resistance to insulin.

Gastrointestinal hormones.

The availability of pure intestinal hormones and the development of radioimmunoassays for their measurement has expedited research into many aspects of gastrointestinal endocrinology. A complex balance evidently exists between the different intestinal hormones and also the rest of the endocrine system. Polyendocrinopathies have been described, and, so far, two diseases due to intestinal hormone excess (Zollinger-Ellison syndrome and the syndrome of watery diarrhea, hypokalemia and achlorhydria) elucidated. It seems likely that many more gastrointestinal endocrine diseases await discovery.