Biomedical subjects

Jun Nakae

Publications and source records attributed to Jun Nakae.

At least 19 recordsLinked to original sources

PubMed2006

Interaction of FoxO1 and TSC2 induces insulin resistance through activation of the mammalian target of rapamycin/p70 S6K pathway.

Both TSC2 (tuberin) and forkhead transcription factor FoxO1 are phosphorylated and inhibited by Akt and play important roles in insulin signaling. However, little is known about the relationship between TSC2 and FoxO1. Here we identified TSC2 as a FoxO1-binding protein by using a yeast two-hybrid screening with a murine islet cDNA library. Among FoxOs, only FoxO1 can be associated with TSC2. The physical association between the C terminus of TSC2 (amino acids 1280-1499) and FoxO1 degrades the TSC1-TSC2 complex and inhibits GTPase-activating protein activity of TSC2 toward Rheb. Overexpression of wild type FoxO1 enhances p70 S6K phosphorylation, whereas overexpression of TSC2 can reverse these effects. Knockdown of endogenous FOXO1 in human vascular endothelial cells decreased phosphorylation of p70 S6K. Prolonged overexpression of wild type FoxO1 enhanced phosphorylation of serine 307 of IRS1 and decreased phosphorylation of Akt and FoxO1 itself even in the presence of serum. These data suggest a novel mechanism by which FoxO1 regulates the insulin signaling pathway through negative regulation of TSC2 function.

Animals↗

PubMed2006

The LXXLL motif of murine forkhead transcription factor FoxO1 mediates Sirt1-dependent transcriptional activity.

The forkhead transcription factor FoxO1 has been identified as a negative regulator of insulin/IGF-1 signaling. Its function is inhibited by phosphorylation and nuclear exclusion through a PI3K-dependent pathway. However, the structure/function relationship of FoxO1 has not been elucidated completely. In this study, we carried out mutation analysis of the FoxO1 coactivator-interacting LXXLL motif (amino acids 459-463). Expression of a 3A/LXXAA mutant, in which 3 Akt phosphorylation sites (T24, S253, and S316) and 2 leucine residues in the LXXLL motif (L462 and L463) were replaced by alanine, decreased both Igfbp-1 and G6Pase promoter activity and endogenous Igfbp-1 and G6Pase gene expression in simian virus 40-transformed (SV40-transformed) hepatocytes. Importantly, mutagenesis of the LXXLL motif eliminated FoxO1 interaction with the nicotinamide adenine dinucleotide-dependent (NAD-dependent) deacetylase sirtuin 1 (Sirt1), sustained the acetylated state of FoxO1, and made FoxO1 nicotinamide and resveratrol insensitive, supporting a role for this motif in Sirt1 binding. Furthermore, intravenous administration of adenovirus encoding 3A/LXXAA FoxO1 into Lepr db/db mice decreased fasting blood glucose levels and improved glucose tolerance and was accompanied by reduced G6Pase and Igfbp-1 gene expression and increased hepatic glycogen content. In conclusion, the LXXLL motif of FoxO1 may have an important role for its transcriptional activity and Sirt1 binding and should be a target site for regulation of gene expression of FoxO1 target genes and glucose metabolism in vivo.

1-Methyl-3-isobutylxanthine↗

PubMed2004

Insulin/Foxo1 pathway regulates expression levels of adiponectin receptors and adiponectin sensitivity.

Adiponectin/Acrp30 is a hormone secreted by adipocytes, which acts as an antidiabetic and antiatherogenic adipokine. We reported previously that AdipoR1 and -R2 serve as receptors for adiponectin and mediate increased fatty acid oxidation and glucose uptake by adiponectin. In the present study, we examined the expression levels and roles of AdipoR1/R2 in several physiological and pathophysiological states such as fasting/refeeding, obesity, and insulin resistance. Here we show that the expression of AdipoR1/R2 in insulin target organs, such as skeletal muscle and liver, is significantly increased in fasted mice and decreased in refed mice. Insulin deficiency induced by streptozotocin increased and insulin replenishment reduced the expression of AdipoR1/R2 in vivo. Thus, the expression of AdipoR1/R2 appears to be inversely correlated with plasma insulin levels in vivo. Interestingly, the incubation of hepatocytes or myocytes with insulin reduced the expression of AdipoR1/R2 via the phosphoinositide 3-kinase/Foxo1-dependent pathway in vitro. Moreover, the expressions of AdipoR1/R2 in ob/ob mice were significantly decreased in skeletal muscle and adipose tissue, which was correlated with decreased adiponectin binding to membrane fractions of skeletal muscle and decreased AMP kinase activation by adiponectin. This adiponectin resistance in turn may play a role in worsening insulin resistance in ob/ob mice. In conclusion, the expression of AdipoR1/R2 appears to be inversely regulated by insulin in physiological and pathophysiological states such as fasting/refeeding, insulin deficiency, and hyper-insulinemia models via the insulin/phosphoinositide 3-kinase/Foxo1 pathway and is correlated with adiponectin sensitivity.

Adenoviridae↗

PubMed2004

Mosaic analysis of insulin receptor function.

Insulin promotes both metabolism and growth. However, it is unclear whether insulin-dependent growth is merely a result of its metabolic actions. Targeted ablation of insulin receptor (Insr) has not clarified this issue, because of early postnatal lethality. To examine this question, we generated mice with variable cellular mosaicism for null Insr alleles. Insr ablation in approximately 80% of cells caused extreme growth retardation, lipoatrophy, and hypoglycemia, a clinical constellation that resembles the human syndrome of leprechaunism. Insr ablation in 98% of cells, while resulting in similar growth retardation and lipoatrophy, caused diabetes without beta-cell hyperplasia. The growth retardation was associated with a greater than 60-fold increase in the expression of hepatic insulin-like growth factor binding protein-1. These findings indicate that insulin regulates growth independently of metabolism and that the number of insulin receptors is an important determinant of the specificity of insulin action.

Adipose Tissue↗

PubMed2004

Transgenic rescue of insulin receptor-deficient mice.

The role of different tissues in insulin action and their contribution to the pathogenesis of diabetes remain unclear. To examine this question, we have used genetic reconstitution experiments in mice. Genetic ablation of insulin receptors causes early postnatal death from diabetic ketoacidosis. We show that combined restoration of insulin receptor function in brain, liver, and pancreatic beta cells rescues insulin receptor knockout mice from neonatal death, prevents diabetes in a majority of animals, and normalizes adipose tissue content, lifespan, and reproductive function. In contrast, mice with insulin receptor expression limited to brain or liver and pancreatic beta cells are rescued from neonatal death, but develop lipoatrophic diabetes and die prematurely. These data indicate, surprisingly, that insulin receptor signaling in noncanonical insulin target tissues is sufficient to maintain fuel homeostasis and prevent diabetes.

Adipose Tissue↗

PubMed2003

Two heterozygous mutations of CLDN16 in a Japanese patient with FHHNC.

Familial hypomagnesemia with hypercalciuria and nephrocalcinosis (FHHNC, MIN 248250) is a rare autosomal recessive tubular disorder that eventually progresses to renal failure. However, the progression to end-stage renal failure can vary from patient to patient. A primary defect is related to impaired tubular resorption of magnesium and calcium in the thick ascending limb of Henle's loop. Recently, paracellin-1 was identified as a renal tight junction protein predominantly expressed in TAL. Mutations of its gene (CLDN16) have been shown to cause FHHNC. We describe a sporadic Japanese case of FHHNC. The male patient showed hematuria, hypercalciuria, and nephrocalcinosis at 5 years of age. Hypomagnesemia was also noticed at this time. As renal function gradually deteriorated, further evaluation was performed at 14 years of age and a diagnosis of FHHNC was made. Despite several medications (magnesium supplementation, citrate, and hydrochlorothiazide), he eventually progressed to renal insufficiency at 19 years of age. Analysis of the CLDN16 gene demonstrated two heterozygous mutations (R149Q and R216C). Mutations of the same amino acids have already been described in FHHNC and thus these mutations might be the cause of the disease in our patient. Hence, we confirm the genetic impairment of the CLDN16 gene in a Japanese patient with FHHNC.

Calcium↗

PubMed2003

Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors.

Insulin-like growth factors promote myoblast differentiation through phosphoinositol 3-kinase and Akt signaling. Akt substrates required for myogenic differentiation are unknown. Forkhead transcription factors of the forkhead box gene, group O (Foxo) subfamily are phosphorylated in an insulin-responsive manner by phosphatidylinositol 3-kinase-dependent kinases. Phosphorylation leads to nuclear exclusion and inactivation. We show that a constitutively active Foxo1 mutant inhibits differentiation of C2C12 cells and prevents myotube differentiation induced by constitutively active Akt. In contrast, a transcriptionally inactive mutant Foxo1 partially rescues inhibition of C2C12 differentiation mediated by wortmannin, but not by rapamycin, and is able to induce aggregation-independent myogenic conversion of teratocarcinoma cells. Inhibition of Foxo expression by siRNA resulted in more efficient differentiation, associated with increased myosin expression. These observations indicate that Foxo proteins are key effectors of Akt-dependent myogenesis.

Androstadienes↗

PubMed2003

Inhibition of Foxo1 function is associated with improved fasting glycemia in diabetic mice.

Excessive hepatic glucose production is a contributing factor to fasting hyperglycemia in diabetes. Insulin suppresses hepatic glucose production by inhibiting the expression of two gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G-6-Pase). The forkhead transcription factor Foxo1 has been implicated as a mediator of insulin action in regulating hepatic gluconeogenesis, and a Foxo1 mutant (Foxo1-Delta256), devoid of its carboxyl domain, has been shown to interfere with Foxo1 function and inhibit gluconeogenic gene expression in cultured cells. To study the effect of Foxo1-Delta256 on glucose metabolism in animals, the Foxo1-Delta256 cDNA was delivered to the livers of mice by adenovirus-mediated gene transfer. Hepatic Foxo1-Delta256 production resulted in inhibition of gluconeogenic activity, as evidenced by reduced PEPCK and G-6-Pase expression in the liver. Mice treated with the Foxo1-Delta256 vector exhibited significantly reduced blood glucose levels. In contrast, blood glucose levels in control vector-treated animals remained unchanged, which coincided with the lack of alterations in the expression levels of PEPCK and G-6-Pase. When tested in diabetic db/db mice, hepatic production of Foxo1-Delta256 was shown to reduce fasting hyperglycemia. Furthermore, we showed that hepatic Foxo1 expression was deregulated as a result of insulin resistance in diabetic mice and that Foxo1-Delta256 interfered with Foxo1 function via competitive binding to target promoters. These results demonstrated that functional inhibition of Foxo1, caused by hepatic expression of its mutant, is associated with reduced hepatic gluconeogenic activity and improved fasting glycemia in diabetic mice.

Animals↗

PubMed2003

Molecular pathogenesis of lipoid adrenal hyperplasia and adrenal hypoplasia congenita.

Congenital lipoid adrenal hyperplasia (lipoid CAH) is the most severe form of CAH in which the synthesis of all gonadal and adrenal cortical steroids is markedly impaired. Lipoid CAH may be caused by the defect in either the steroidogenic acute regulatory (StAR) protein or the P450scc. More than 34 different mutations in StAR gene have been identified. Clinically, most of the patients manifest adrenal insufficiency from 1 day to 2 months of age, but some patient show delayed onset of adrenal insufficiency. Affected 46, XY subjects do not show pubertal development, whereas affected 46, XX subjects undergo spontaneous feminization, breast development and cyclical vaginal bleeding at the usual age of puberty. X-linked adrenal hypoplasia congenital (AHC) is a rare congenital adrenal disorder characterized by severe adrenal insufficiency and hypogonadotropic hypogonadism. More than 80 different several intragenic mutations of DAX-1 have been identified. The failure of pubertal development may be caused by either abnormal hypothalamic or pituitary regulation of gonadotropin secretion. In addition, although the testicular steroidogenesis is largely intact, the functional maturity of Sertoli cells and also spermatogenesis are impaired. The type of mutation does not predict clinical phenotype. Thus, unified mechanism how DAX-1 gene defect gives rise to adrenal insufficiency, hypothalamic/pituitary hypogonadism and impaired spermatogenesis remains established.

Adrenal Gland Diseases↗

PubMed2003

The forkhead transcription factor Foxo1 regulates adipocyte differentiation.

An outstanding question in adipocyte biology is how hormonal cues are relayed to the nucleus to activate the transcriptional program that promotes adipogenesis. The forkhead transcription factor Foxo1 is regulated by insulin via Akt-dependent phosphorylation and nuclear exclusion. We show that Foxo1 is induced in the early stages of adipocyte differentiation but that its activation is delayed until the end of the clonal expansion phase. Constitutively active Foxo1 prevents the differentiation of preadipocytes, while dominant-negative Foxo1 restores adipocyte differentiation of fibroblasts from insulin receptor-deficient mice. Further, Foxo1 haploinsufficiency protects from diet-induced diabetes in mice. We propose that Foxo1 plays an important role in the integration of hormone-activated signaling pathways with the complex transcriptional cascade that promotes adipocyte differentiation.

3T3 Cells↗

PubMed2003

Sporadic heterozygous frameshift mutation of HESX1 causing pituitary and optic nerve hypoplasia and combined pituitary hormone deficiency in a Japanese patient.

HESX1/Hesx1 is a member of the paired-like class of homeobox genes and is essential for pituitary and forebrain development. Mice with a targeted homozygous deletion of the Hesx1 show severe central nervous system defects, absence of optic vesicles, and a very small anterior pituitary gland. This phenotype is similar to the abnormalities observed in the human disorder called septo-optic dysplasia, a syndromic form of congenital hypopituitarism. To date, four missense mutations in the human HESX1 have been described in individuals with phenotypes ranging from severe septo-optic dysplasia, relatively mild combined pituitary hormone deficiency (CPHD), to isolated GH deficiency. Here we report a Japanese patient with CPHD (GH, TSH, LH, FSH, and ACTH deficiency) due to a novel sporadic HESX1 mutation. Brain magnetic resonance imaging examination revealed hypoplastic anterior pituitary, ectopic posterior lobe, and left optic nerve hypoplasia. Molecular analysis identified the insertion of a heterozygous mutation (306/307ins AG) in the exon 2 of the HESX1. This mutation changes a reading frame and introduces a premature stop codon soon after the mutation site. Therefore, this mutation would be predicted to generate a protein lacking the carboxyl-terminal homebox domain (DNA-binding domain) and cause the disease. Family analysis demonstrated that neither of the patient's parents harbored this mutation, indicating that the mutation had arisen de novo. In conclusion, a de novo heterozygous frameshift mutation in exon 2 of the HESX1 causes severe CPHD with optic nerve hypoplasia in a human.

Asian People↗

PubMed2003

Two novel aquaporin-2 mutations in a sporadic Japanese patient with autosomal recessive nephrogenic diabetes insipidus.

We identified two novel mutations of the aquaporin-2 (AQP2) gene in a sporadic Japanese patient diagnosed with an autosomal recessive nephrogenic diabetes insipidus (NDI). The patient, a Japanese boy, was referred to our clinic at the age of 5 months because of unexplained recurrent fever. He was diagnosed with NDI by clinical, biochemical and endocrine findings. Molecular analysis demonstrated that he was a compound heterozygote for two mutations. One mutation consisted of a two base deletion in exon 1 (197, 198 delCA). This deletion caused a frameshift in the open reading frame, resulting in a premature stop codon 186 bases downstream in exon 1. The second mutation was a G to A transition of the terminal exon splice site (1502-1G-->A). To date, several mutations in the AQP2 gene have been described, however no splicing mutation in the AQP2 gene has been identified. The deletion mutation described in this case study was inherited patemally and the splicing site mutation was inherited maternally, indicating an autosomal recessive inheritance. In the present case study, we identified two new mutations in the AQP-2 gene. Previous studies have shown that there is no hot spot for mutations in the AQP-2 gene, and thus genetic analysis for individual patients is helpful for genetic counseling and early diagnosis.

Adenine↗

PubMed2002

Regulation of insulin action and pancreatic beta-cell function by mutated alleles of the gene encoding forkhead transcription factor Foxo1.

Type 2 diabetes results from impaired action and secretion of insulin. It is not known whether the two defects share a common pathogenesis. We show that haploinsufficiency of the Foxo1 gene, encoding a forkhead transcription factor (forkhead box transcription factor O1), restores insulin sensitivity and rescues the diabetic phenotype in insulin-resistant mice by reducing hepatic expression of glucogenetic genes and increasing adipocyte expression of insulin-sensitizing genes. Conversely, a gain-of-function Foxo1 mutation targeted to liver and pancreatic beta-cells results in diabetes arising from a combination of increased hepatic glucose production and impaired beta-cell compensation due to decreased Pdx1 expression. These data indicate that Foxo1 is a negative regulator of insulin sensitivity in liver, adipocytes and pancreatic beta-cells. Impaired insulin signaling to Foxo1 provides a unifying mechanism for the common metabolic abnormalities of type 2 diabetes.NOTE: In the AOP version of this article, the name of the fourth author was misspelled as W K Cavanee rather than the correct spelling: W K Cavenee. This has been corrected in the full-text online version of the article. The name will appear correctly in the print version.

Animals↗

PubMed2002

Effects of mutations in the insulin-like growth factor signaling system on embryonic pancreas development and beta-cell compensation to insulin resistance.

Insulin and insulin-like growth factors (IGF) play overlapping and complementary roles in pancreatic beta-cell function and peripheral metabolism. In this study, we have analyzed mice bearing loss-of-function mutations of the insulin/IGF signaling systems. Combined inactivation of insulin receptor (Insr) and Igf1 receptor (Igf1r), but not of either receptor alone, resulted in a 90% decrease in the size of the exocrine pancreas, because of decreased cellular proliferation. In contrast to the findings in the exocrine compartment, endocrine alpha- and beta-cell development was unperturbed. Combined ablation of Igf1 and Igf2, the ligands for these two receptors, resulted in an identical phenotype. We also examined the effect of heterozygous null Igf1r mutations on glucose homeostasis in adult mice. Igf1r haploinsufficiency did not affect insulin action and compensatory beta-cell growth in insulin-resistant mice with combined Insr and Igf1r heterozygous null mutations, resulting in a considerably milder phenotype than combined haploinsufficiency for Insr and its main signaling substrates, Irs1 and Irs2. We conclude that Igf1r and Insr are required for embryonic development of the exocrine but not of the endocrine pancreas and that defects of Igf1r do not alter glucose homeostasis as long as the insulin receptor system remains intact.

Animals↗

PubMed2002

Defective insulin secretion in pancreatic beta cells lacking type 1 IGF receptor.

Defective insulin secretion is a feature of type 2 diabetes that results from inadequate compensatory increase of beta cell mass and impaired glucose-dependent insulin release. beta cell proliferation and secretion are thought to be regulated by signaling through receptor tyrosine kinases. In this regard, we sought to examine the potential proliferative and/or antiapoptotic role of IGFs in beta cells by tissue-specific conditional mutagenesis ablating type 1 IGF receptor (IGF1R) signaling. Unexpectedly, lack of functional IGF1R did not affect beta cell mass, but resulted in age-dependent impairment of glucose tolerance, associated with a decrease of glucose- and arginine-dependent insulin release. These observations reveal a requirement of IGF1R-mediated signaling for insulin secretion.

Animals↗

PubMed2002

The forkhead transcription factor Foxo1 links insulin signaling to Pdx1 regulation of pancreatic beta cell growth.

Diabetes is caused by an absolute (type 1) or relative (type 2) deficiency of insulin-producing beta cells. The mechanisms governing replication of terminally differentiated beta cells and neogenesis from progenitor cells are unclear. Mice lacking insulin receptor substrate-2 (Irs2) develop beta cell failure, suggesting that insulin signaling is required to maintain an adequate beta cell mass. We report that haploinsufficiency for the forkhead transcription factor Foxo1 reverses beta cell failure in Irs2(-/-) mice through partial restoration of beta cell proliferation and increased expression of the pancreatic transcription factor pancreas/duodenum homeobox gene-1 (Pdx1). Foxo1 and Pdx1 exhibit mutually exclusive patterns of nuclear localization in beta cells, and constitutive nuclear expression of a mutant Foxo1 is associated with lack of Pdx1 expression. We show that Foxo1 acts as a repressor of Foxa2-dependent (Hnf-3beta-dependent) expression from the Pdx1 promoter. We propose that insulin/IGFs regulate beta cell proliferation by relieving Foxo1 inhibition of Pdx1 expression in a subset of cells embedded within pancreatic ducts.

Animals↗

PubMed2002

Hydrochlorothiazide effectively reduces urinary calcium excretion in two Japanese patients with gain-of-function mutations of the calcium-sensing receptor gene.

Gain-of-function mutations of the calcium-sensing receptor (CaR) gene cause autosomal dominant and/or sporadic hypocalcemia with hypercalciuria. Because treatment of the hypocalcemia with vitamin D and/or calcium in patients with such mutations results in increased hypercalciuria, nephrocalcinosis, and renal impairment, its use should be limited to alleviating the symptoms of symptomatic patients. Because thiazide diuretics have been successfully used to treat patients with hypercalciuria and hypoparathyroidism, they are theoretically useful in reducing urine calcium excretion and maintaining serum calcium levels in patients with gain-of-function mutations of the CaR gene. In this study, we report on the clinical course, molecular analysis, and effects of hydrochlorothiazide therapy in two Japanese patients with gain-of-function mutations of the CaR gene. Within a few weeks after birth, they developed generalized tonic seizures due to hypocalcemia (serum calcium values: 1.1 mmol/liter and 1.3 mmol/liter, respectively). Despite treatment with the standard dose of 1,25-dihydroxyvitamin D(3) in one patient and 1alpha-hydroxyvitamin D(3) in the other, acceptable serum calcium levels near the lower limit of normal were not established, and their urinary calcium excretion inappropriately increased. Addition of hydrochlorothiazide (1 mg/kg) reduced their urinary calcium excretion and maintained their serum calcium concentrations near the lower limit of normal, allowing the 1,25-dihydroxyvitamin D(3) and 1alpha-hydroxyvitamin D(3) doses to be reduced, and it alleviated their symptoms. A heterozygous missense mutation was identified in both patients. In one patient, the mutation was A843E in the seventh transmembrane domain of the CaR, and in the other it was L125P in the N-terminal extracellular domain. In vitro transient transfection of their mutant CaR cDNAs into HEK293 cells shifted the concentration-response curve of Ca(2+) to the left. In conclusion, two sporadic cases of hypercalciuric hypocalcemia were due to de novo gain-of-function mutations of the CaR gene. Hydrochlorothiazide with vitamin D(3) successfully reduced the patients' urinary calcium excretion and controlled their serum calcium concentrations and symptoms. Thiazide diuretics are effective in patients with gain-of function mutations of the CaR gene.

Adolescent↗

PubMed2002

Two novel mutations of thiazide-sensitive Na-Cl cotrans porter (TSC) gene in two sporadic Japanese patients with Gitelman syndrome.

Gitelman syndrome is a renal disorder characterized by hypokalemia, hypomagnesemia, metabolic alkalosis and hypocalciuria due to the defective tubular reabsorption of magnesium and potassium. This disease is caused by mutations of thiazide-sensitive Na-Cl cotransporter (TSC) gene. Gitelman syndrome is usually distinguished from Bartter syndrome by the presence of both hypomagnesemia and hypocalciuria. However, a phenotypic overlap is sometimes observed. We encountered two sporadic Japanese patients with Gitelman syndrome and analyzed their TSC gene. These patients were diagnosed as Gitelman syndrome by the typical clinical findings and biochemical abnormalities, such as mild muscular weakness, periodic paralysis, tetany, metabolic alkalosis, hypomagnesemia and hypocalciuria. In patient 1, a novel two base deletion (del TG at nucleotide 731 and 732) in exon 5 and a two base deletion (del TT at nucleotide 2543 and 2544) in exon 21 previously reported in a Japanese patient were identified. The patient 2 had a missense mutation (L623P), that was also identified in Japanese patients, and a novel in-frame 18 base insertion in exon 6 as a heterozygous state. Family analysis of two patients confirmed an autosomal recessive inheritance. In conclusion, we add two new mutations of the TSC gene in Japanese patients with Gitelman syndrome. Because the differential diagnosis between Bartter syndrome and Gitelman syndrome is sometimes difficult, molecular analysis would be a useful diagnostic tool, particularly in unusual cases with phenotypic overlapping.

Adolescent↗