Hidetaka Suga

Self-formation of functional adenohypophysis in three-dimensional culture

The adenohypophysis (anterior pituitary) is a major centre for systemic hormones. At present, no efficient stem-cell culture for its generation is available, partly because of insufficient knowledge about how the pituitary primordium (Rathke’s pouch) is induced in the embryonic head ectoderm. Here we report efficient self-formation of three-dimensional adenohypophysis tissues in an aggregate culture of mouse embryonic stem (ES) cells. ES cells were stimulated to differentiate into non-neural head ectoderm and hypothalamic neuroectoderm in adjacent layers within the aggregate, and treated with hedgehog signalling. Self-organization of Rathke’s-pouch-like three-dimensional structures occurred at the interface of these two epithelia, as seen in vivo, and various endocrine cells including corticotrophs and somatotrophs were subsequently produced. The corticotrophs efficiently secreted adrenocorticotropic hormone in response to corticotrophin releasing hormone and, when grafted in vivo, these cells rescued the systemic glucocorticoid level in hypopituitary mice. Thus, functional anterior pituitary tissue self-forms in ES cell culture, recapitulating local tissue interactions.

In vitro organogenesis in three dimensions: self-organising stem cells

Organ formation during embryogenesis is a complex process that involves various local cell-cell interactions at the molecular and mechanical levels. Despite this complexity, organogenesis can be modelled in vitro. In this article, we focus on two recent examples in which embryonic stem cells can self-organise into three-dimensional structures – the optic cup and the pituitary epithelium; and one case of self-organising adult stem cells – the gut epithelium. We summarise how these approaches have revealed intrinsic programs that drive locally autonomous modes of organogenesis and homeostasis. We also attempt to interpret the results of previous in vivo studies of retinal development in light of the self-organising nature of the retina.

Diabetes mellitus after transplant: relationship to pretransplant glucose metabolism and tacrolimus or cyclosporine A-based therapy.

Objective. The purpose of this study was to identify pretransplantation and posttransplantation indicators for the development of diabetes mellitus in the first 2 months after renal transplantation and to examine the influence of a cyclosporine A (CsA)-based versus a tacrolimus-based immunosuppressive regimen on these risk factors. Methods. Key variables associated with the development of posttransplant diabetes mellitus (PTDM) in the first 2 months after transplantation were assessed in 48 patients who underwent living-related renal transplantation and who were treated with a CsA-based or a tacrolimus-based immunosuppressive regimen. The insulinogenic index (I Index) and glucose infusion rate (GIR) were measures of insulin secretion and insulin sensitivity, respectively. Results. Eight patients developed PTDM. I Index (odds ratio, 0.000384) and GIR (odds ratio, 0.349) were significant risk factors for PTDM development. The cumulative steroid dose had a borderline association. PTDM developed in 4 of 28 CsA-treated patients and in 4 of 20 tacrolimus-treated patients. CsA therapy increased the mean I Index from 0.713±0.071 preoperatively to 1.130±0.140 postoperatively (P <0.01), whereas in tacrolimus-treated patients, I Index remained unchanged (1.09±0.264 preoperatively and 0.949±0.296 postoperatively; P =not significant). Age, duration of pretransplant dialysis, and body mass index did not predict PTDM development. All eight patients with PTDM had hypertension. Conclusions. Pre- and posttransplant abnormalities of insulin secretion and sensitivity are significant predictors of PTDM. Corticosteroid cumulative dose may affect the incidence of PTDM during the first 2 months after transplantation. CsA treatment increases insulin secretion in patients with a high pretransplant risk of PTDM.

Functional anterior pituitary generated in self-organizing culture of human embryonic stem cells

Anterior pituitary is critical for endocrine systems. Its hormonal responses to positive and negative regulators are indispensable for homeostasis. For this reason, generating human anterior pituitary tissue that retains regulatory hormonal control in vitro is an important step for the development of cell transplantation therapy for pituitary diseases. Here we achieve this by recapitulating mouse pituitary development using human embryonic stem cells. We find that anterior pituitary self-forms in vitro following the co-induction of hypothalamic and oral ectoderm. The juxtaposition of these tissues facilitated the formation of pituitary placode, which subsequently differentiated into pituitary hormone-producing cells. They responded normally to both releasing and feedback signals. In addition, after transplantation into hypopituitary mice, the in vitro-generated corticotrophs rescued physical activity levels and survival of the hosts. Thus, we report a useful methodology for the production of regulator-responsive human pituitary tissue that may benefit future studies in regenerative medicine.

Lack of alpha2-antiplasmin improves cutaneous wound healing via over-released vascular endothelial growth factor-induced angiogenesis in wound lesions.

Summary.  Background: The fibrinolytic system is supposed to play an important role in the degradation of extracellular matrices for physiological and pathological tissue remodeling; however, the detailed mechanism regarding how this system affects cutaneous wound healing remains to be clarified. Methods and results: We performed experimental cutaneous wounding in mice with a deficiency of α2‐antiplasmin (α2AP), which is a potent and specific plasmin inhibitor. We found that an accelerated wound closure was observed in α2AP‐deficient (α2AP−/−) mice in comparison with wild type (WT) mice. Moreover, we observed that a greater increase of angiogenesis occurred in the process of wound healing in α2AP−/− mice than in the WT mice. Intriguingly, mRNA expression of vascular endothelial growth factor (VEGF), which is the best characterized positive regulator of angiogenesis, in wound lesions was found to show a greater increase in the early phase of the healing process in α2AP−/− mice than in WT mice. In addition, the amount of released‐VEGF from the explanted fibroblasts of α2AP−/− mice increased dramatically more than in the WT mice. Finally, the intra‐jugular administration of anti‐VEGF antibody clearly suppressed the increased angiogenesis and accelerated wound closure in the wound lesion of α2AP−/− mice. Conclusion: The lack of α2AP markedly causes an over‐release of VEGF from the fibroblasts in cutaneous wound lesions, thereby inducing angiogenesis around the area, and thus resulting in an accelerated‐wound closure. Conclusions: This is the first report to describe the crucial role that α2AP plays following angiogenesis in the process of wound healing. Our results provide new insight into the role of α2AP on cutaneous wound healing.

GABA Type B Receptor Signaling in Proopiomelanocortin Neurons Protects Against Obesity, Insulin Resistance, and Hypothalamic Inflammation in Male Mice on a High-Fat Diet

There is evidence suggesting that the GABA system in the arcuate nucleus, where orexigenic neuropeptide Y and agouti-related peptide as well as anorexigenic proopiomelanocortin (POMC) are expressed, plays an important role in energy balance. In this study, we generated POMC-specific GABAB receptor-deficient [knock-out (KO)] mice. Male KO mice on a high-fat diet (HFD) showed mild increases in body weight (BW) at the age of 9 weeks compared to wild-type (WT) mice, and the differences remained significant until 16 weeks old. However, there was no difference in BW in females between genotypes. While food intake was similar between genotypes, oxygen consumption was significantly decreased in the male KO mice. The insulin tolerance test revealed that the male KO mice were less insulin sensitive compared to WT mice at the age of 8 weeks, when there was no significant difference in BW between genotypes. Despite increased BW, POMC mRNA expression in the arcuate nucleus was significantly decreased in the KO mice compared to WT mice at the age of 16 weeks. Furthermore, the expression of TNFα as well as IL-6, proinflammatory markers in the hypothalamus, was significantly increased in the KO mice on a HFD compared to WT mice. This demonstrates that the deletion of GABAB receptors in POMC neurons in the male mice on a HFD results in obesity, insulin resistance, and hypothalamic inflammation. Furthermore, the decreased POMC expression in the obese KO mice suggests that the regulation of POMC expression through GABAB receptors is essential for proper energy balance.

Biochemical roles of the oligosaccharide chains in thyrostimulin, a heterodimeric hormone of glycoprotein hormone subunits alpha2 (GPA2) and beta5 (GPB5)

Thyrostimulin is a heterodimeric hormone composed of GPA2 and GPB5, and shares the thyroid-stimulating hormone receptor (TSHR). Thyrostimulin has three N-linked oligosaccharide chains, two in GPA2 and one in GPB5. The roles of these N-linked oligosaccharides in secretion, heterodimer formation and signal transduction were analyzed. Recombinant GPA2s lacking either of the two oligosaccharides were obtained from conditioned medium, whereas dual site-disrupted GPA2 and the GPB5 mutant were not expressed in either the conditioned medium or cell lysate. The binding between GPA2 and GPB5 was weaker than that between TSH subunits GPA1 and TSH beta. Neither of the oligosaccharides in GPA2 had significant effects on heterodimerization. Disruption of either of the oligosaccharides in GPA2 significantly decreased receptor activation, suggesting their critical role in receptor activation.

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

Rabphilin-3A as a Targeted Autoantigen in Lymphocytic Infundibulo-neurohypophysitis

CONTEXT Central diabetes insipidus (CDI) can be caused by several diseases, but in about half of the patients the etiological diagnosis remains unknown. Lymphocytic infundibulo-neurohypophysitis (LINH) is an increasingly recognized entity among cases of idiopathic CDI; however, the differential diagnosis from other pituitary diseases including tumors can be difficult because of similar clinical and radiological manifestations. The definite diagnosis of LINH requires invasive pituitary biopsy. OBJECTIVE The study was designed to identify the autoantigen(s) in LINH and thus develop a diagnostic test based on serum autoantibodies. DESIGN Rat posterior pituitary lysate was immunoprecipitated with IgGs purified from the sera of patients with LINH or control subjects. The immunoprecipitates were subjected to liquid chromatography-tandem mass spectrometry to screen for pituitary autoantigens of LINH. Subsequently, we made recombinant proteins of candidate autoantigens and analyzed autoantibodies in serum by Western blotting. RESULTS Rabphilin-3A proved to be the most diagnostically useful autoantigen. Anti-rabphilin-3A antibodies were detected in 22 of the 29 (76%) patients (including 4 of the 4 biopsy-proven samples) with LINH and 2 of 18 (11.1%) patients with biopsy-proven lymphocytic adeno-hypophysitis. In contrast, these antibodies were absent in patients with biopsy-proven sellar/suprasellar masses without lymphocytic hypophysitis (n = 34), including 18 patients with CDI. Rabphilin-3A was expressed in posterior pituitary and hypothalamic vasopressin neurons but not anterior pituitary. CONCLUSIONS These results suggest that rabphilin-3A is a major autoantigen in LINH. Autoantibodies to rabphilin-3A may serve as a biomarker for the diagnosis of LINH and be useful for the differential diagnosis in patients with CDI.

Vasopressin phosphorylates HSP27 in aortic smooth muscle cells

Administration of arginine vasopressin (AVP) time‐dependently induced the phosphorylation of heat shock protein 27 (HSP27) at Ser‐15 and Ser‐85 in smooth muscle of aorta in vivo. The AVP‐induced phosphorylation of HSP27 at Ser‐15 and Ser‐85 was inhibited by a V1a receptor antagonist but not by a V2 receptor antagonist. In cultured aortic smooth muscle A10 cells, AVP markedly stimulated the phosphorylation of HSP27 at Ser‐15 and Ser‐85. The AVP‐induced phosphorylation of HSP27 was attenuated by SB203580 and PD169316, inhibitors of p38 mitogen‐activated protein (MAP) kinase, but not by PD98059, a MEK inhibitor. These results strongly suggest that AVP phosphorylates HSP27 via p38 MAP kinase in aortic smooth muscle cells.