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Tissue optical clearing permits detailed evaluation of organ three-dimensional (3-D) structure as well as that of individual cells by tissue staining and autofluorescence. In this study, we evaluated intestinal morphology, intestinal epithelial cells (IECs), and enteroendocrine cells, such as incretin-producing cells, in reporter mice by intestinal 3-D imaging. 3-D intestinal imaging of reporter mice using optical tissue clearing enabled us to evaluate both detailed intestinal morphologies and cell numbers, villus length and crypt depth in the same samples. In disease mouse model of lipopolysaccharide (LPS)-injected mice, the results of 3-D imaging using tissue optical clearing in this study was consistent with those of 2-D imaging in previous reports and could added the new data of intestinal morphology. In analysis of incretin-producing cells of reporter mice, we could elucidate the number, the percentage, and the localization of incretin-producing cells in intestine and the difference of those between L cells and K cells. Thus, we established a novel method of intestinal analysis using tissue optical clearing and 3-D imaging. 3-D evaluation of intestine enabled us to clarify not only detailed intestinal morphology but also the precise number and localization of IECs and incretin-producing cells in the same samples.

The involvement of a gut-bone axis in controlling bone physiology has been long suspected, although the exact mechanisms are unclear. We explored whether glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine K cells were involved in this process. The bone phenotype of transgenic mouse models lacking GIP secretion (GIP-GFP-KI) or enteroendocrine K cells (GIP-DT) was investigated. Mice deficient in GIP secretion exhibited lower bone strength, trabecular bone mass, trabecular number, and cortical thickness, notably due to higher bone resorption. Alterations of microstructure, modifications of bone compositional parameters, represented by lower collagen cross-linking, were also apparent. None of these alterations were observed in GIP-DT mice lacking enteroendocrine K cells, suggesting that another K-cell secretory product acts to counteract GIP action. To assess this, stable analogues of the known K-cell peptide hormones, xenin and GIP, were administered to mature NIH Swiss male mice. Both were capable of modulating bone strength mostly by altering bone microstructure, bone gene expression, and bone compositional parameters. However, the two molecules exhibited opposite actions on bone physiology, with evidence that xenin effects are mediated indirectly, possibly via neural networks. Our data highlight a previously unknown interaction between GIP and xenin, which both moderate gut-bone connectivity. © 2020 American Society for Bone and Mineral Research.

Objective Glucose-dependent insulinotropic polypeptide (GIP) is released during meals and promotes nutrient uptake and storage. GIP receptor knockout mice are protected from diet induced weight gain and thus GIP antagonists have been proposed as a treatment for obesity. In this study, we assessed the role of GIP in hyperphagia induced obesity and metabolic abnormalities in leptin deficient (Lepob/ob) mice. Methods We crossbred GIP-GFP knock-in homozygous mice (GIPgfp/gfp) that have complete GIP knockout, and mice heterozygous for the ob mutation (Lepob/+) mice to generate Lepob/+/GIP+/+, Lepob/ob/GIP+/+, and Lepob/ob/GIPgfp/gfp mice. Male animals were weighed weekly and both oral glucose and insulin tolerance testing were performed to assess glucose homeostasis and circulating profiles of GIP and insulin. Body composition was evaluated by computerized tomography (CT) scan and analyses of indirect calorimetry and locomotor activity were performed. Results Postprandial GIP levels were markedly elevated in Lepob/ob/GIP+/+ mice compared to Lepob/+/GIP+/+ controls and were undetectable in Lepob/ob/GIPgfp/gfp mice. Insulin levels were equivalently elevated in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to controls at 8 weeks of age but the hyperinsulinemia was marginally reduced in Lepob/ob/GIPgfp/gfp by 21 weeks, in association with amelioration of glucose intolerance. Both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice remained equivalently insulin resistant. Body weight gain and subcutaneous and visceral fat volume of both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice were significantly higher than that of Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. Locomotor activity and energy expenditure were decreased in both Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice compared to control Lepob/+/GIP+/+ mice, while no significant differences were seen between Lepob/ob/GIP+/+ and Lepob/ob/GIPgfp/gfp mice. There was no significant difference in fat oxidation among the three groups. Fat content in liver was significantly lower in Lepob/ob/GIPgfp/gfp compared to Lepob/ob/GIP+/+ mice, while that of control Lepob/+/GIP+/+ mice was the lowest. Conclusions Our results indicate that GIP knockout does not prevent excess weight gain and metabolic derangement in hyperphagic leptin deficient mice., Highlights • Complete absence of GIP does not prevent hyperinsulinemia and insulin resistance from developing in Lepob/ob mice. • GIP deficiency does not alleviate excess weight gain and fat accumulation in Lepob/ob mice. • Endogenous GIP is not involved in the development of obesity in mice with complete absence of leptin.

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin secreted from enteroendocine K cells after nutrient ingestion. Fat strongly induces GIP secretion, and GIP hypersecretion is involved in high-fat diet-induced obesity and insulin resistance. Aging also induces GIP hypersecretion, but its effect on body weight gain and insulin sensitivity remains unclear. In the present study, we investigated the effect of GIP on age-related body weight gain and insulin resistance using GIP-knockout homozygous (GIP−/−) and heterozygous (GIP+/−) mice, which have entirely absent and 50% reduced GIP secretion compared to wild-type (WT) mice, respectively. Under 12% fat-containing normal diet feeding condition, body weight was significantly lower in GIP−/− mice compared to that in WT and GIP+/− mice from 38 weeks of age, while there was no significant difference between WT and GIP+/− mice. Visceral and s.c. fat mass were also significantly lower in GIP−/− mice compared to those in WT and GIP+/− mice. During oral glucose tolerance test, blood glucose levels did not differ among the three groups. Insulin levels were significantly lower in GIP−/− mice than those in WT and GIP+/− mice. During insulin tolerance test, GIP−/− mice showed higher insulin sensitivity than that of WT and GIP+/− mice. Adiponectin mRNA levels were increased and leptin mRNA levels tended to be decreased in adipose tissue of GIP−/− mice. These results demonstrate that GIP is involved in age-related obesity and insulin resistance and that inhibition of GIP secretion alleviates age-related fat mass gain and insulin resistance under carbohydrate-based diet feeding condition.

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells and potentiates insulin secretion from pancreatic β-cells. GIP also enhances long-chain triglyceride (LCT) diet-induced obesity and insulin resistance. Long-term intake of medium-chain triglyceride (MCT) diet is known to induce less body weight and fat mass gain than that of LCT diet. However, the effect of MCT diet feeding on GIP secretion and the effect of GIP on body weight and fat mass under MCT diet-feeding condition are unknown. In this study, we evaluated the effect of single MCT oil administration on GIP secretion and compared the effect of long-term MCT and LCT diet on body weight and fat mass gain in wild-type (WT) and GIP-knockout (GIP KO) mice. Single administration of LCT oil induced GIP secretion but that of MCT oil did not in WT mice. Long-term intake of LCT diet induced GIP hypersecretion and significant body weight and fat mass gain compared with that of control fat (CF) diet in WT mice. In contrast, MCT diet did not induce GIP hypersecretion, and MCT diet-fed mice showed smaller increase in body weight and fat mass gain compared with CF diet-fed mice. In GIP KO mice, body weight and fat mass were markedly attenuated in LCT diet-fed mice but not in MCT diet-fed mice. Our results suggest that long-term intake of MCT diet stimulates less GIP secretion and suppresses body weight and fat mass gain compared with that of LCT diet.

Given the established roles of glucose‐dependent insulinotropic polypeptide (GIP) in promoting fat storage and bone formation, we assessed the contribution of GIP to obesity and osteopenia in ovariectomized mice with a gene encoding green fluorescent protein (GFP) inserted into the GIP locus, in which GIP was either reduced (GIPgfp/+) or absent (GIPgfp/gfp). In GIPgfp/gfp mice, weight gain, subcutaneous and visceral fat mass were reduced, and glucose intolerance was improved compared with wild‐type mice with the same magnitude of insulin responses. Cancellous bone mineral density and bone cortical thickness were reduced in GIPgfp/gfp mice compared with wild‐type mice. In GIPgfp/+ mice, weight gain, glucose intolerance and cancellous bone mineral density were not different from that of wild‐type mice. These results indicate that the total elimination of GIP ameliorates weight gain and adiposity in ovariectomized mice, but it enhances osteopenia, particularly in cancellous bone by partly suppressing bone formation.

To elucidate the clinical implication of ghrelin, we have been trying to generate variable models of transgenic (Tg) mice overexpressing ghrelin. We generated Tg mice overexpressing des-acyl ghrelin in a wide variety of tissues under the control of β-actin promoter. While plasma des-acyl ghrelin level in the Tg mice was 44-fold greater than that of control mice, there was no differences in the plasma ghrelin level between des-acyl ghrelin Tg and the control mice. The des-acyl ghrelin Tg mice exhibited the lower body weight and the shorter body length due to modulation of GH-IGF-1 axis. We tried to generate Tg mice expressing a ghrelin analog, which possessed ghrelin-like activity (Trp3-ghrelin Tg mice). The plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was approximately 85-fold higher than plasma ghrelin (acylated ghrelin) concentration seen in the control mice. Because Trp3-ghrelin is approximately 24-fold less potent than ghrelin, the plasma Trp3-ghrelin concentration in Trp3-ghrelin Tg mice was calculated to have approximately 3.5-fold biological activity greater than that of ghrelin (acylated ghrelin) in the control mice. Trp3-ghrelin Tg mice did not show any phenotypes except for reduced insulin sensitivity in 1-year old. After the identification of ghrelin O-acyltransferase (GOAT), we generated doubly Tg mice overexpressing both mouse des-acyl ghrelin and mouse GOAT in the liver by cross-mating the two kinds of Tg mice. The plasma ghrelin concentration of doubly Tg mice was approximately 2-fold higher than that of the control mice. No apparent phenotypic changes in body weight and food intake were observed in doubly Tg mice. Further studies are ongoing in our laboratory to generate Tg mice with the increased plasma ghrelin level to a greater extent. The better understanding of physiological and pathophysiological significance of ghrelin from experiments using an excellent animal model may provide a new therapeutic approach for human diseases.

Free fatty acid receptors GPR120 and GPR40 are involved in the secretion of gut hormones. GPR120 and GPR40 are expressed in enteroendocrine K cells, and their activation induces the secretion of the incretin glucose-dependent insulinotropic polypeptide (GIP). However, the role of these receptors in fat-induced GIP secretion in vivo and the associated mechanisms are unclear. In this study, we investigated corn oil-induced GIP secretion in GPR120-knockout (GPR120-/-) and GPR40-knockout (GPR40-/-) mice. Oil-induced GIP secretion was reduced by 50% and 80% in GPR120-/- and GPR40-/- mice, respectively, compared with wild-type mice. This was not associated with a significant difference in K-cell number or GIP content in K cells, nor messenger RNA levels of the lipid receptor GPR119, nor bile acid receptors TGR5 and farnesoid X receptor. GPR120-/- and GPR40-/- mice also exhibited substantially decreased levels of cholecystokinin (CCK), a hormone from I cells that promotes bile and pancreatic lipase secretion, and this decrease was associated with impaired gallbladder contraction. Notably, treatment with a CCK analog resulted in recovery of oil-induced GIP secretion in GPR120-/- mice but not in GPR40-/- mice. These results indicate that corn oil-induced GIP secretion from K cells involves both GPR120 and GPR40 signaling pathways, and GPR120-induced GIP secretion is indirectly mediated by CCK.

ObjectiveDopamine agonists normalize prolactin (PRL) levels and reduce tumour size in responsive prolactinoma. However, several cases have shown resistance to dopamine agonists upon initial treatment. Infrequently, prolactinoma initially responds, but then becomes refractory to prolonged treatment (secondary resistance). We investigated the possible mechanisms of resistance to dopamine agonists.Subjects and methodsTwelve cases of prolactinoma were surgically resected and classified according to the responsiveness of PRL levels and tumour size to dopamine agonists: good responders (n=5), poor responders (n=5), or secondary resistance (n=2). We examined the expression of dopamine D2receptor (D2R) isoform (short: D2S and long: D2L) mRNA and protein. We investigated DNA methylation patterns in the promoter region of theDRD2gene.ResultsThe predominant D2R isoform expressed in prolactinoma was D2L. Levels of D2L mRNA were significantly lower in secondary resistance and poor responders than in good responders. Expression of D2R protein was variable among cases. Almost no CpG sites of theDRD2gene promoter region were methylated.ConclusionResistance of prolactinoma to dopamine agonists is correlated with a reduction in D2L isoform mRNA levels. Silencing of theDRD2gene by methylation in the promoter region is unlikely to play a role in dopamine agonist resistance in prolactinoma.

Heterozygous germline mutation of the tumor suppressor gene MEN1 is responsible for multiple endocrine neoplasia type 1 (MEN1), a familial cancer syndrome characterized by pituitary, parathyroid and enteropancreatic tumors. Various mutations have been identified throughout the entire gene region in patients with MEN1 and its incomplete forms often manifested as familial isolated hyperparathyroidism and apparently sporadic parathyroid tumor. Mutation analysis of the MEN1 gene is a powerful tool for the early diagnosis of MEN1; however, the clinical significance of the identified mutations is not always obvious. In this study, a previously unreported missense MEN1 mutation, c.824G>T was identified in a patient with primary hyperparathyroidism and evaluated for its pathogenicity. This mutation was predicted to generate a putative missense menin protein, R275M. A stability test of the menin protein demonstrated that the stability of R275M mutant was reduced only slightly as compared with wild type menin, and therefore could not preclude the possibility that it was a rare benign polymorphism. However, further analysis of leukocyte mRNA and minigene experiments indicated that the mutant c.824G>T allele gives rise to abnormally spliced menin mRNA, and thereby confirmed that c.824G>T mutation is causative for MEN1. Thus, leukocyte mRNA analysis has been demonstrated useful to identify a splicing mutation of the MEN1 gene.

Objective β-adrenergic antagonists (β-blockers) are often used to attenuate the hyperadrenergic symptoms of Graves' disease (GD), including palpitation. Although β-blockers reduce the heart rate, cardiac output and oxygen consumption, no firm evidence exists regarding the effects of combined therapy with β-blockers and anti-thyroid drugs. The objective is to elucidate the effects of β-blockers on anti-thyroid drug therapy in GD. Methods Patients newly diagnosed with mild GD were randomly assigned to receive methimazole with or without β-blockers in a prospective multi-center survey. The heart rate and thyroid function were measured and the quality of life was assessed using original and SF-36 questionnaires at 0 and 4 weeks. Results A total of 28 patients were enrolled in the study. Fourteen patients (one man, 13 women) were randomly assigned to the group treated with β-blockers and 14 patients (one man, 13 women) were randomly assigned to the group not treated with β-blockers. Although no significant differences in the improvement of thyroid function were observed between the two groups, the heart rates improved more significantly in the group treated with β-blockers. Specific symptoms, such as easy fatigability and shortness of breath, also improved more significantly with the β-blocker treatment. In addition, 'physical functioning' assessed with the SF-36 questionnaires significantly improved only in the group treated with β-blockers. Conclusion Although β-blockers may not reinforce the effects of anti-thyroid drugs on thyroid function, at least during the course of one month, they are effective in reducing heart rates and ameliorating specific symptoms in patients with mild GD.

Germline mutations of the tumor suppressor gene MEN1 are found not only in typical multiple endocrine neoplasia type 1 (MEN1) but also in its incomplete forms such as familial isolated hyperparathyroidism (FIHP) and apparently sporadic parathyroid tumor (ASPT). No definitive genotype-phenotype correlation has been established between these clinical forms and MEN1 gene mutations. We previously demonstrated that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. To examine whether the intracellular stability of mutant menin is correlated with clinical phenotypes, we developed a method of evaluating menin stability and examined 20 mutants associated with typical MEN1 (17 missense, two in-frame deletion, one nonsense) and 21 mutants associated with FIHP or ASPT (19 missense, two in-frame deletion). All tested mutants associated with typical MEN1 showed reduced stability. Some missense and in-frame deletion mutants (G28A, R171W, T197I, E255K, E274A, Y353del and E366D) associated with FIHP or ASPT were almost as stable as or only slightly less stable than wild-type menin, while others were as unstable as those associated with typical MEN1. Some stable mutants exhibited substantial biological activities when tested by JunD-dependent transactivation assay. These findings suggest that certain missense and in-frame mutations are fairly stable and retain intrinsic biological activity, and might be specifically associated with incomplete clinical phenotypes. The menin stability test will provide useful information for the management of patients carrying germline MEN1 mutations especially when they have missense or in-frame variants of ambiguous clinical significance.

Germline MEN1 mutation analysis is a powerful tool for an early diagnosis of multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant familial cancer syndrome characterized by the parathyroid, pituitary and gastroenteropancreatic endocrine tumors. However, the clinical significance of MEN1 gene variants, especially missense and in-frame mutations as well as some splicing mutations, is not always obvious. We have previously shown that mutant menin proteins associated with MEN1 are rapidly degraded by the ubiquitin-proteasome pathway. We also demonstrated by a fluorescent immunocytochemical stability test that the stability of missense and in-frame deletion mutants varies widely but that unstable mutants were found only in MEN1 and related disorders and not in normal polymorphisms. In the present study, we evaluated by this stability test the pathogenicity of a novel MEN1 missense mutation, c.1118C>T, encoding a P373L mutant menin, identified in a suspected MEN1 patient. The results demonstrated that the mutant menin is highly unstable, indicating that this mutation is causative for MEN1. These findings encouraged us to proceed with presymptomatic genetic screening for this mutation among the family members, which resulted in the identification of asymptomatic mutation carriers. Thus, the information from the menin stability test was useful for genetic diagnosis and counseling of MEN1 in the case with a previously unreported MEN1 missense mutation.

Menin is lost by the sequential inactivation of both MEN1 alleles in subsets of non-hereditary endocrine tumors as well as those associated with multiple endocrine neoplasia type 1 (MEN1), an autosomal dominant hereditary cancer syndrome characterized by multiple tumors including parathyroid, pituitary and enteropancreatic endocrine tumors. Loss of menin has been reported to be associated with lowered caspase 8 expression and resistance to apoptosis in murine fibroblasts and in pancreatic islet tumors arising in heterozygous MEN1 gene knockout mice, the animal model of the human MEN1 syndrome. We confirmed by menin-knockdown experiments with specific siRNA that menin is crucial for caspase 8 expression in human culture cells while overexpression of menin did not increase caspase 8 protein over basal levels. We then examined expression of menin, caspase 8 and cyclin-dependent kinase inhibitors p27(Kip1) and p15(Ink4b) by Western blotting in human parathyroid tumors surgically resected from patients with MEN1 and those with non-hereditary primary hyperparathyroidism. The menin and p27(Kip1) expression levels were correlated with MEN1 mutation status that was confirmed by DNA analysis. The caspase 8 and p15(Ink4b) protein levels were variable among tumors, and were not correlated with menin protein levels. These findings suggest that human endocrine tumors lacking menin may not always exhibit lowered caspase 8 expression and hence may not be resistant to apoptosis-inducing therapy.

To evaluate the prevalence of dyslipidemia in the population of Hashimoto thyroiditis, we reviewed medical records on the consecutive 1181 cases with adult Hashimoto thyroiditis and 830 cases were adopted for the study. First, the serum TSH level increased and serum free T4 level decreased, slightly but significantly, with increasing age. There were significant positive correlations between serum TSH levels and lipid parameters such as total cholesterol (TC), triglyceride (TG), HDL-cholesterol (HDL-C), LDL-cholesterol (LDL-C), non-HDL-C and LDL-C/HDL-C ratio (L/H). In contrast, there were significant negative correlations between serum free T4 levels and all of these lipid parameters. According to the thyroid function, the cases were classified into 4 groups such as thyrotoxicosis (TT), euthyroidism (EU), subclinical hypothyroidism (SH) and overt hypothyroidism (OH). TC, HDL-C, non-HDL-C and LDL-C of TT were significantly lower than those in EU. In contrast, TC, TG, non-HDL-C, LDL-C, L/H and age of OH were significantly higher than those in EU. Interestingly, LDL-C and L/H of SH were significantly higher compared with EU. Thirty-two of SH patients were treated with small doses of levothyroxine and the effects on the lipid profile were examined. The TC, non-HDL-C, LDL-C and L/H were significantly decreased after treatment. In conclusion, the prevalence of dyslipidemia increases along with hypofunction of the thyroid and T4 replacement therapy may improve lipid profile in the cases of SH with Hashimoto thyroiditis.