Michiyo Yamamoto

Mice Deficient for Glucagon Gene-Derived Peptides Display Normoglycemia and Hyperplasia of Islet α-Cells But Not of Intestinal L-Cells

Multiple bioactive peptides, including glucagon, glucagon-like peptide-1 (GLP-1), and GLP-2, are derived from the glucagon gene (Gcg). In the present study, we disrupted Gcg by introduction of GFP cDNA and established a knock-in mouse line. Gcg(gfp/gfp) mice that lack most, if not all, of Gcg-derived peptides were born in an expected Mendelian ratio without gross abnormalities. Gcg(gfp/gfp) mice showed lower blood glucose levels at 2 wk of age, but those in adult Gcg(gfp/gfp) mice were not significantly different from those in Gcg(+/+) and Gcg(gfp/+) mice, even after starvation for 16 h. Serum insulin levels in Gcg(gfp/gfp) mice were lower than in Gcg(+/+) and Gcg(gfp/+) on ad libitum feeding, but no significant differences were observed on starvation. Islet alpha-cells and intestinal L-cells were readily visualized in Gcg(gfp/gfp) and Gcg(gfp/+) mice under fluorescence. The Gcg(gfp/gfp) postnatally developed hyperplasia of islet alpha-cells, whereas the population of intestinal L-cells was not increased. In the Gcg(gfp/gfp), expression of Aristaless-related homeobox (Arx) was markedly increased in pancreas but not in intestine and suggested involvement of Arx in differential regulation of proliferation of Gcg-expressing cells. These results illustrated that Gcg-derived peptides are dispensable for survival and maintaining normoglycemia in adult mice and that Gcg-derived peptides differentially regulate proliferation/differentiation of alpha-cells and L-cells. The present model is useful for analyzing glucose/energy metabolism in the absence of Gcg-derived peptides. It is useful also for analysis of the development, differentiation, and function of Gcg-expressing cells, because such cells are readily visualized by fluorescence in this model.

Remodeling of Hepatic Metabolism and Hyperaminoacidemia in Mice Deficient in Proglucagon-Derived Peptides

Glucagon is believed to be one of the most important peptides for upregulating blood glucose levels. However, homozygous glucagon–green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp: GCGKO) are normoglycemic despite the absence of proglucagon-derived peptides, including glucagon. To characterize metabolism in the GCGKO mice, we analyzed gene expression and metabolome in the liver. The expression of genes encoding rate-limiting enzymes for gluconeogenesis was only marginally altered. On the other hand, genes encoding enzymes involved in conversion of amino acids to metabolites available for the tricarboxylic acid cycle and/or gluconeogenesis showed lower expression in the GCGKO liver. The expression of genes involved in the metabolism of fatty acids and nicotinamide was also altered. Concentrations of the metabolites in the GCGKO liver were altered in manners concordant with alteration in the gene expression patterns, and the plasma concentrations of amino acids were elevated in the GCGKO mice. The insulin concentration in serum and phosphorylation of Akt protein kinase in liver were reduced in GCGKO mice. These results indicated that proglucagon-derived peptides should play important roles in regulating various metabolic pathways, especially that of amino acids. Serum insulin concentration is lowered to compensate the impacts of absent proglucagon-derived peptide on glucose metabolism. On the other hand, impacts on other metabolic pathways are only partially compensated by reduced insulin action.

Ectopic Expression of GIP in Pancreatic β-Cells Maintains Enhanced Insulin Secretion in Mice With Complete Absence of Proglucagon-Derived Peptides

Glucagon and glucagon-like peptide-1 (GLP-1) are produced in pancreatic α-cells and enteroendocrine L-cells, respectively, in a tissue-specific manner from the same precursor, proglucagon, that is encoded by glucagon gene (Gcg), and play critical roles in glucose homeostasis. Here, we studied glucose homeostasis and β-cell function of Gcg-deficient mice that are homozygous for a Gcg-GFP knock-in allele (Gcggfp/gfp). The Gcggfp/gfp mice displayed improved glucose tolerance and enhanced insulin secretion, as assessed by both oral glucose tolerance test (OGTT) and intraperitoneal glucose tolerance test (IPGTT). Responses of glucose-dependent insulinotropic polypeptide (GIP) to both oral and intraperitoneal glucose loads were unexpectedly enhanced in Gcggfp/gfp mice, and immunohistochemistry localized GIP to pancreatic β-cells of Gcggfp/gfp mice. Furthermore, secretion of GIP in response to glucose was detected in isolated islets of Gcggfp/gfp mice. Blockade of GIP action in vitro and in vivo by cAMP antagonism and genetic deletion of the GIP receptor, respectively, almost completely abrogated enhanced insulin secretion in Gcggfp/gfp mice. These results indicate that ectopic GIP expression in β-cells maintains insulin secretion in the absence of proglucagon-derived peptides (PGDPs), revealing a novel compensatory mechanism for sustaining incretin hormone action in islets.

Growth hormone releasing hormone receptor (GHRH-R) gene mutation in indian children with familial isolated growth hormone deficiency : A study from western India

BACKGROUND Various mutations of the growth hormone releasing hormone receptor (GHRH-R) gene have been recently described to cause familial isolated growth hormone (GH) deficiency (FIGHD), with the GHRH-R nonsense mutation E72X reported in patients with FIGHD from South Asia. The molecular genetic basis of FIGHD in Indian children is not known. OBJECTIVE To look for the GHRH-R E72X non-sense mutation in our patients with FIGHD and describe its clinical phenotype. PATIENTS AND METHOD A total of 31 patients from 22 families diagnosed 4-20 years previously, 20 patients with familial IGHD-IB from 11 families and 11 patients with non-familial isolated GH deficiency (NFIGHD) (phenotypes IGHD-IB in eight patients and -IA in three) were included. Twenty-eight of 31 patients with IGHD-IB came from two states of Western India, 27 of them Hindus from 18 families (three consanguineous) and one from an inbred Moslem kindred. RESULTS Twenty-two of the patients (71%) (18 FIGHD and four NFIGHD) had a homozygous G-->T transversion in exon 3, with this GHRH-R gene mutation E72X in 90% (18/20) of patients with FIGHD, 36% (4/11) of NFIGHD, altogether 78% (22/28) with phenotype IB. One parent pair with IGHD had homozygous E72X mutation, the rest were heterozygous carriers. Two siblings with IGHD due to homozygous E72X mutation were also heterozygous carriers for GH-1 gene 6.7 kb deletion, inherited from their mother, heterozygous for both GH-1 and GHRH-R mutations. Initial chronological age was 10.89 +/- 3.69 years, bone age 6.4 +/- 3.4 years, and mean height SDS was -5.83 +/- 1.41. The clinical phenotype, with sharp features, lean habitus, lack of frontal bossing or hypoglycemia, was characteristic. The mean peak GH was 1.25 +/- 0.75 ng/ml, IGF-I and IGFBP-3 below -2 SDS with no response to GHRH in those tested. MRI (n = 10) showed pituitary hypoplasia, mean vertical height 2.61 +/- 0.76 mm. Among the other 7/11 NFIGHD patients, four with phenotype IB were negative for genotypes tested in this study; of three patients with phenotype IA, two had the GH-1 gene 6.7 kb deletion, and one was a compound heterozygote with 6.7 and 7.6 kb deletions. CONCLUSIONS The majority of patients with FIGHD from different communities belonged to non-consanguineous Hindu families from Western India. The GHRH-R gene E72X mutation was found in 71% of this series, in 90% of FIGHD, 36% of NFIGHD, and in 78% with phenotype IB. The characteristic phenotype helped in suspecting this mutation. GHRH-R gene mutations may be the most reasonable candidate for IGHD-IB with the E72X mutation predominating in the Indian subcontinent. More extensive studies need to be undertaken.

A Japanese family with autosomal dominant growth hormone deficiency.

Abstract We report a 1-year-old Japanese boy and his father with isolated growth hormone deficiency II. In both cases, a G → A transition of the first base of the donor splice site of intron 3 of the growth hormone-1 gene was detected. All unaffected family members were homozygous normal. Conclusion This is the fourth reported case of autosomal isolated growth hormone deficiency II with a G → A transition. The CG dinucleotide at the exon 3-intron 3 junction of the growth hormone-1 gene appears to be a hot spot for point mutations.

Fertility and Pregnancy-Associated ß-Cell Proliferation in Mice Deficient in Proglucagon-Derived Peptides

Proglucagon, which is encoded by the glucagon gene (Gcg), is the precursor of several peptide hormones, including glucagon and glucagon-like peptide 1 (GLP-1). Whereas glucagon stimulates hepatic glycogenolysis and gluconeogenesis, GLP-1 stimulates insulin secretion to lower blood glucose and also supports ß-cell proliferation and protection from apoptotic stimuli. Pregnancy is a strong inducer of change in islet function, however the roles of proglucagon-derived peptides in pregnancy are only partially understood. In the present study, we analyzed fertility and pregnancy-associated changes in homozygous glucagon-green fluorescent protein (gfp) knock-in mice (Gcggfp/gfp), which lack all the peptides derived from proglucagon. Female Gcggfp/gfp mice could deliver and raise Gcggfp/gfp pups to weaning and Gcggfp/gfp pups from Gcggfp/gfp dams were viable and fertile. Pregnancy induced ß-cell proliferation in Gcggfp/gfp mice as well as in control mice. However, serum insulin levels in pregnant Gcggfp/gfp females were lower than those in control pregnant females under ad libitum feeding, and blood glucose levels in pregnant Gcggfp/gfp females were higher after gestational day 12. Gcggfp/gfp females showed a decreased pregnancy rate and smaller litter size. The rate of successful breeding was significantly lower in Gcggfp/gfp females and was not improved by experience of breeding. Taken together, proglucagon-derived peptides are not required for pregnancy-associated ß-cell proliferation, however, are required for regulation of blood glucose levels and normal reproductive capacity. Gcggfp/gfp mice may serve as a novel model to analyze the effect of mild hyperglycemia during late gestational periods.

Isolated growth hormone deficiency in two siblings because of paternal mosaicism for a mutation in the GH1 gene

Context  Mutations in the GH1 gene have been identified in patients with isolated growth hormone deficiency (IGHD). Mutations causing aberrant splicing of exon 3 of GH1 that have been identified in IGHD are inherited in an autosomal dominant manner, whereas other mutations in GH1 that have been identified in IGHD are inherited in an autosomal recessive manner.

Transgenic mice expressing a mutant human GH gene causing type II IGHD

We identified several mutations in the intron 3 of human growth hormone gene I (hGH-I) in patients with isolated GH deficiency (IGHD) type II characterized by an autosomal dominant trait. The mutations result in exon 3 skipping and generation of 17 Kd mutant GH. To elucidate how the mutation causes dominant trait, transgenic mice expressing a mutant hGH gene (the first guanine to adenine transversion in intron 3: GH-I; IVS3+1: G-A) were produced in C57BL/6 strain. Genotypes of mice were identified by PCR-amplified products of tail snip DNAs. Delivery of the mutant hGH transgene into 76 fertilized eggs resulted in production of two male heterozygous transgenic mice (hGH + / - , the zero filial generation, F0). Since the mating of the transgenic mice with the same strain was unsuccessful, they were outcrossed with CD-I (ICR) strain. Only one mouse gave birth, producing 4 male and 7 female (Fl) harboring the mutant hGH gene in one allele (hGH + / - ). F1 mice were mated again with the wild type ICR strain, generating 82 hGH + / - mice (F2: 51 males and 31 females). To study whether somatotrophs in F2 mice express the mutant hGH gene, RNA extracted from the pituitary was subjected to RT-PCR. It was demonstrated that the F2, hGH + / - mice express the mutant hGH gene, lacking exon 3. Thus, these heterozygous mice were sib-mated to generate homozygous mice (F3). The mating resulted in 27% hGH - / - , 64% hGH + / - and 9% hGH + / + mice, indicating that the transgene was carried stably to the descendants and did not interfere with the reproduction. These mice will be a valuable model to study how type II IGHD develops during the course of development.