A specific plasma amino acid profile in the Insulin2Q104del Kuma mice at the diabetic state and reversal from hyperglycemia.

The metabolites in the plasma serve as potential biomarkers of disease. We previously established an early-onset diabetes mouse model, Ins2+/Q104del Kuma mice, under a severe immune-deficient (Rag-2/Jak3 double-deficient in BALB/c) background. Here, we revealed the differences in plasma amino acid profiles between Kuma and the wild-type mice. We observed an early reduction in glucogenic and ketogenic amino acids, a late increase in branched-chain amino acids (BCAAs) and succinyl CoA-related amino acids, and a trend of increasing ketogenic amino acids in Kuma mice than in the wild-type mice. Kuma mice exhibited hyperglucagonemia at high blood glucose, leading to perturbations in plasma amino acid profiles. The reversal of blood glucose by islet transplantation normalized the increases of the BCAAs and several aspects of the altered metabolic profiles in Kuma mice. Our results indicate that the Kuma mice are a unique animal model to study the link between plasma amino acid profile and the progression of diabetes for monitoring the therapeutic effects.

Selective proteasome degradation of C-terminally-truncated human WFS1 in pancreatic beta cells.

Wolfram syndrome is a monogenic disease mainly caused by mutations in the WFS1 gene. Mutations in the WFS1 gene give rise to diabetes. Here, we characterized mutant WFS1 proteins by studying the stability of full-length wild-type (WT) WFS1, a missense mutant P724L, and two C-terminally truncated mutants, W837X and Y652X. We compared their stability by overexpressing them in MIN6 and HEK293T cells. The C-terminally truncated mutants W837X and Y652X are degraded more rapidly than the missense P724L mutant or wild-type WFS1 in MIN6 cells. In contrast, Y652X is more stable than WT or other mutant WFS1 proteins in HEK293T. In conclusion, we found that C-terminally truncated WFS1 mutants are selectively degraded in a cell type-specific manner.

Dopamine Negatively Regulates Insulin Secretion Through Activation of D1-D2 Receptor Heteromer.

There is increasing evidence that dopamine (DA) functions as a negative regulator of glucose-stimulated insulin secretion; however, the underlying molecular mechanism remains unknown. Using total internal reflection fluorescence microscopy, we monitored insulin granule exocytosis in primary islet cells to dissect the effect of DA. We found that D1 receptor antagonists rescued the DA-mediated inhibition of glucose-stimulated calcium (Ca2+) flux, thereby suggesting a role of D1 in the DA-mediated inhibition of insulin secretion. Overexpression of D2, but not D1, alone exerted an inhibitory and toxic effect that abolished the glucose-stimulated Ca2+ influx and insulin secretion in ß-cells. Proximity ligation and Western blot assays revealed that D1 and D2 form heteromers in ß-cells. Treatment with a D1-D2 heteromer agonist, SKF83959, transiently inhibited glucose-induced Ca2+ influx and insulin granule exocytosis. Coexpression of D1 and D2 enabled ß-cells to bypass the toxic effect of D2 overexpression. DA transiently inhibited glucose-stimulated Ca2+ flux and insulin exocytosis by activating the D1-D2 heteromer. We conclude that D1 protects ß-cells from the harmful effects of DA by modulating D2 signaling. The finding will contribute to our understanding of the DA signaling in regulating insulin secretion and improve methods for preventing and treating diabetes.

Carborane as an Alternative Efficient Hydrophobic Tag for Protein Degradation.

Carboranes 1 and 2 were designed and synthesized for hydrophobic tag (HyT)-induced degradation of HaloTag fusion proteins. The levels of the hemagglutinin (HA)-HaloTag2-green fluorescent protein (EGFP) stably expressed in Flp-In 293 cells were significantly reduced by HyT13, HyT55, and carboranes 1 and 2, with expression levels of 49, 79, 43, and 65%, respectively, indicating that carborane is an alternative novel hydrophobic tag (HyT) for protein degradation under an intracellular environment. To clarify the mechanism of HyT-induced proteolysis, bovine serum albumin (BSA) was chosen as an extracellular protein and modified with maleimide-conjugated m-carborane (MIC). The measurement of the ζ-potentials and the lysine residue modification with fluorescein isothiocyanate (FITC) of BSA-MIC conjugates suggested that the conjugation of carborane induced the exposure of lysine residues on BSA, resulting in the degradation via ubiquitin E3 ligase-related proteasome pathways in the cell.

Dietary sodium chloride attenuates increased ß-cell mass to cause glucose intolerance in mice under a high-fat diet.

Excessive sodium salt (NaCl) or fat intake is associated with a variety of increased health risks. However, whether excessive NaCl intake accompanied by a high-fat diet (HFD) affects glucose metabolism has not been elucidated. In this study, C57BL/6J male mice were fed a normal chow diet (NCD), a NCD plus high-NaCl diet (NCD plus NaCl), a HFD, or a HFD plus high-NaCl diet (HFD plus NaCl) for 30 weeks. No significant differences in body weight gain, insulin sensitivity, and glucose tolerance were observed between NCD-fed and NCD plus NaCl-fed mice. In contrast, body and liver weights were decreased, but the weight of epididymal white adipose tissue was increased in HFD plus NaCl-fed compared to HFD-fed mice. HFD plus NaCl-fed mice had lower plasma glucose levels in an insulin tolerance test, and showed higher plasma glucose and lower plasma insulin levels in an intraperitoneal glucose tolerance test compared to HFD-fed mice. The ß-cell area and number of islets were decreased in HFD plus NaCl-fed compared to HFD-fed mice. Increased Ki67-positive ß-cells, and increased expression levels of Ki67, CyclinB1, and CyclinD1 mRNA in islets were observed in HFD-fed but not HFD plus NaCl-fed mice when compared to NCD-fed mice. Our data suggest that excessive NaCl intake accompanied by a HFD exacerbates glucose intolerance, with impairment in insulin secretion caused by the attenuation of expansion of ß-cell mass in the pancreas.

VMAT2 Safeguards ß-Cells Against Dopamine Cytotoxicity Under High-Fat Diet-Induced Stress.

Vesicular monoamine transporter 2 (VMAT2) uptakes cytoplasmic monoamines into vesicles for storage. VMAT2 plays a role in modulating insulin release by regulating dopamine levels in the pancreas, although the exact mechanism remains elusive. We found that VMAT2 expression in ß-cells specifically increases under high blood glucose conditions. The islets isolated from ß-cell-specific Vmat2 knockout (ßVmat2KO) mice show elevated insulin secretion levels in response to glucose stimulation. Under prolonged high-fat diet feedings, the ßVmat2KO mice exhibit impaired glucose and insulin tolerance and progressive ß-cell dysfunction. Here we demonstrate VMAT2 uptake of dopamine to protect dopamine from degradation by monoamine oxidase, thereby safeguarding ß-cells from excess reactive oxygen species (ROS) exposure. In the context of high demand for insulin secretion, the absence of VMAT2 leads to elevated ROS in ß-cells, which accelerates ß-cell dedifferentiation and ß-cell loss. Therefore, VMAT2 controls the amount of dopamine in ß-cells, thereby protecting pancreatic ß-cells from excessive oxidative stress.

Insulin2Q104del (Kuma) mutant mice develop diabetes with dominant inheritance.

Insulin gene mutations have been identified to cause monogenic diabetes, and most of which developed permanent neonatal diabetes at young ages before 6 months of age in humans. To establish an animal model of permanent diabetes, we performed genome editing using the CRISPR/Cas9 system. We generated a novel Kuma mutant mice with p.Q104del in the Insulin2 (Ins2) gene in a BRJ background that exhibits a severe immune deficiency. Kuma mutant mice are non-obese and developed hyperglycemia from 3 weeks after birth in both males and females, which are inherited in a dominant mode. Kuma mutant mice displayed reduced insulin protein levels from 3-weeks-old, which seem to be caused by the low stability of the mutant insulin protein. Kuma mutant showed a reduction in islet size and islet mass. Electron microscopic analysis revealed a marked decrease in the number and size of insulin granules in the beta-cells of the mutant mice. Hyperglycemia of the mutant can be rescued by insulin administration. Our results present a novel insulin mutation that causes permanent early-onset diabetes, which provides a model useful for islet transplantation studies.

Inhibition of Cdk5 Promotes ß-Cell Differentiation From Ductal Progenitors.

Inhibition of notch signaling is known to induce differentiation of endocrine cells in zebrafish and mouse. After performing an unbiased in vivo screen of ∼2,200 small molecules in zebrafish, we identified an inhibitor of Cdk5 (roscovitine), which potentiated the formation of ß-cells along the intrapancreatic duct during concurrent inhibition of notch signaling. We confirmed and characterized the effect with a more selective Cdk5 inhibitor, (R)-DRF053, which specifically increased the number of duct-derived ß-cells without affecting their proliferation. By duct-specific overexpression of the endogenous Cdk5 inhibitors Cdk5rap1 or Cdkal1 (which previously have been linked to diabetes in genome-wide association studies), as well as deleting cdk5, we validated the role of chemical Cdk5 inhibition in ß-cell differentiation by genetic means. Moreover, the cdk5 mutant zebrafish displayed an increased number of ß-cells independently of inhibition of notch signaling, in both the basal state and during ß-cell regeneration. Importantly, the effect of Cdk5 inhibition to promote ß-cell formation was conserved in mouse embryonic pancreatic explants, adult mice with pancreatic ductal ligation injury, and human induced pluripotent stem (iPS) cells. Thus, we have revealed a previously unknown role of Cdk5 as an endogenous suppressor of ß-cell differentiation and thereby further highlighted its importance in diabetes.

Dopamine D2 Receptor-Mediated Regulation of Pancreatic ß Cell Mass.

Understanding the molecular mechanisms that regulate ß cell mass and proliferation is important for the treatment of diabetes. Here, we identified domperidone (DPD), a dopamine D2 receptor (DRD2) antagonist that enhances ß cell mass. Over time, islet ß cell loss occurs in dissociation cultures, and this was inhibited by DPD. DPD increased proliferation and decreased apoptosis of ß cells through increasing intracellular cAMP. DPD prevented ß cell dedifferentiation, which together highly contributed to the increased ß cell mass. DRD2 knockdown phenocopied the effects of domperidone and increased the number of ß cells. Drd2 overexpression sensitized the dopamine responsiveness of ß cells and increased apoptosis. Further analysis revealed that the adenosine agonist 5'-N-ethylcarboxamidoadenosine, a previously identified promoter of ß cell proliferation, acted with DPD to increase the number of ß cells. In humans, dopamine also modulates ß cell mass through DRD2 and exerts an inhibitory effect on adenosine signaling.

Changes in expression of C2cd4c in pancreatic endocrine cells during pancreatic development.

C2cd4c, encoded by a gene belonging to the C2cd4 family, contains a C2 domain conserved across species and is localized to the cytoplasm. To examine the role of C2cd4c in the pancreas, we studied its localization and generated C2cd4c knockout (KO) mice. C2cd4c was expressed in pancreatic endocrine progenitors at early embryonic stages. When endocrine cells arise from their precursors, C2cd4c is gradually confined to the insulin- and pancreatic polypeptide-expressing cells of the endocrine. In the adult pancreas, C2cd4c is restricted to the beta cells. C2cd4c KO mice showed normal embryonic pancreatic development and adult pancreatic function. Thus, our results suggest that C2cd4c is dispensable for pancreatic development.

Pancreatic Differentiation from Murine Embryonic Stem Cells.

Pluripotent stem cells are considered as a cell source for replacement therapies for pancreatic beta cells and other organs.We identified tetrabenazine (TBZ), vesicular monoamine transporter 2 (VMAT2) inhibitor as a promoter of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Ngn3-positive endocrine progenitor cells. A cell-permeable cAMP analog, dBu-cAMP promotes beta cell maturation in late stage of differentiation. The induced beta cells can secrete insulin in a glucose-dependent manner.Our protocol consists of a three -step differentiation process. ES cell recapitulate embryonic developmental processes in vitro. Therefore, the ES cell differentiation system is a useful model for the understanding of molecular mechanism of beta-cell differentiation and are useful for application for future regenerative medicine.

Neural cells play an inhibitory role in pancreatic differentiation of pluripotent stem cells.

Pancreatic endocrine ß-cells derived from embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have received attention as screening systems for therapeutic drugs and as the basis for cell-based therapies. Here, we used a 12-day ß-cell differentiation protocol for mouse ES cells and obtained several hit compounds that promoted ß-cell differentiation. One of these compounds, mycophenolic acid (MPA), effectively promoted ES cell differentiation with a concomitant reduction of neuronal cells. The existence of neural cell-derived inhibitory humoral factors for ß-cell differentiation was suggested using a co-culture system. Based on gene array analysis, we focused on the Wnt/ß-catenin pathway and showed that the Wnt pathway inhibitor reversed MPA-induced ß-cell differentiation. Wnt pathway activation promoted ß-cell differentiation also in human iPS cells. Our results showed that Wnt signaling activation positively regulates ß-cell differentiation, and represent a downstream target of the neural inhibitory factor.

Generation of insulin-producing ß-like cells from human iPS cells in a defined and completely xeno-free culture system.

Human induced pluripotent stem (hiPS) cells are considered a potential source for the generation of insulin-producing pancreatic ß-cells because of their differentiation capacity. In this study, we have developed a five-step xeno-free culture system to efficiently differentiate hiPS cells into insulin-producing cells in vitro. We found that a high NOGGIN concentration is crucial for specifically inducing the differentiation first into pancreatic and duodenal homeobox-1 (PDX1)-positive pancreatic progenitors and then into neurogenin 3 (NGN3)-expressing pancreatic endocrine progenitors, while suppressing the differentiation into hepatic or intestinal cells. We also found that a combination of 3-isobutyl-1-methylxanthine (IBMX), exendin-4, and nicotinamide was important for the differentiation into insulin single-positive cells that expressed various pancreatic ß-cell markers. Most notably, the differentiated cells contained endogenous C-peptide pools that were released in response to various insulin secretagogues and high levels of glucose. Therefore, our results demonstrate the feasibility of generating hiPS-derived pancreatic ß-cells under xeno-free conditions and highlight their potential to treat patients with type 1 diabetes.

Beneficial effect of insulin treatment on islet transplantation outcomes in Akita mice.

Islet transplantation is a promising potential therapy for patients with type 1 diabetes. The outcome of islet transplantation depends on the transplantation of a sufficient amount of ß-cell mass. However, the initial loss of islets after transplantation is problematic. We hypothesized the hyperglycemic status of the recipient may negatively affect graft survival. Therefore, in the present study, we evaluated the effect of insulin treatment on islet transplantation involving a suboptimal amount of islets in Akita mice, which is a diabetes model mouse with an Insulin 2 gene missense mutation. Fifty islets were transplanted under the left kidney capsule of the recipient mouse with or without insulin treatment. For insulin treatment, sustained-release insulin implants were implanted subcutaneously into recipient mice 2 weeks before transplantation and maintained for 4 weeks. Islet transplantation without insulin treatment did not reverse hyperglycemia. In contrast, the group that received transplants in combination with insulin treatment exhibited improved fasting blood glucose levels until 18 weeks after transplantation, even after insulin treatment was discontinued. The group that underwent islet transplantation in combination with insulin treatment had better glucose tolerance than the group that did not undergo insulin treatment. Insulin treatment improved graft survival from the acute phase (i.e., 1 day after transplantation) to the chronic phase (i.e., 18 weeks after transplantation). Islet apoptosis increased with increasing glucose concentration in the medium or blood in both the in vitro culture and in vivo transplantation experiments. Expression profile analysis of grafts indicated that genes related to immune response, chemotaxis, and inflammatory response were specifically upregulated when islets were transplanted into mice with hyperglycemia compared to those with normoglycemia. Thus, the results demonstrate that insulin treatment protects islets from the initial rapid loss that is usually observed after transplantation and positively affects the outcome of islet transplantation in Akita mice.

VMAT2 identified as a regulator of late-stage ß-cell differentiation.

Cell replacement therapy for diabetes mellitus requires cost-effective generation of high-quality, insulin-producing, pancreatic ß cells from pluripotent stem cells. Development of this technique has been hampered by a lack of knowledge of the molecular mechanisms underlying ß-cell differentiation. The present study identified reserpine and tetrabenazine (TBZ), both vesicular monoamine transporter 2 (VMAT2) inhibitors, as promoters of late-stage differentiation of Pdx1-positive pancreatic progenitor cells into Neurog3 (referred to henceforth as Ngn3)-positive endocrine precursors. VMAT2-controlled monoamines, such as dopamine, histamine and serotonin, negatively regulated ß-cell differentiation. Reserpine or TBZ acted additively with dibutyryl adenosine 3',5'-cyclic AMP, a cell-permeable cAMP analog, to potentiate differentiation of embryonic stem (ES) cells into ß cells that exhibited glucose-stimulated insulin secretion. When ES cell-derived ß cells were transplanted into AKITA diabetic mice, the cells reversed hyperglycemia. Our protocol provides a basis for the understanding of ß-cell differentiation and its application to a cost-effective production of functional ß cells for cell therapy.

Secreted cerberus1 as a marker for quantification of definitive endoderm differentiation of the pluripotent stem cells.

To date, CXCR4 and E-cadherin double-positive cells detected by flow cytometry have been used to identify the differentiation of embryonic stem (ES) cells or induced pluripotent stem (iPS) cells into definitive endoderm (DE) lineages. Quantification of DE differentiation from ES/iPS cells by using flow cytometry is a multi-step procedure including dissociation of the cells, antibody reaction, and flow cytometry analysis. To establish a quick assay method for quantification of ES/iPS cell differentiation into the DE without dissociating the cells, we examined whether secreted Cerberus1 (Cer1) protein could be used as a marker. Cer1 is a secreted protein expressed first in the anterior visceral endoderm and then in the DE. The amount of Cer1 secreted correlated with the proportion of CXCR4+/E-Cadherin+ cells that differentiated from mouse ES cells. In addition, we found that human iPS cell-derived DE also expressed the secreted CER1 and that the expression level correlated with the proportion of SOX17+/FOXA2+ cells present. Taken together, these results show that Cer1 (or CER1) serves as a good marker for quantification of DE differentiation of mouse and human ES/iPS cells.

Recovery from diabetes in neonatal mice after a low-dose streptozotocin treatment.

Administration of streptozotocin (STZ) induces destruction of ß-cells and is widely used as an experimental animal model of type I diabetes. In neonatal rat, after low-doses of STZ-mediated destruction of ß-cells, ß-cells regeneration occurs and reversal of hyperglycemia was observed. However, in neonatal mice, ß-cell regeneration seems to occur much slowly compared to that observed in the rat. Here, we described the time dependent quantitative changes in ß-cell mass during a spontaneous slow recovery of diabetes induced in a low-dose STZ mice model. We then investigated the underlying mechanisms and analyzed the cell source for the recovery of ß-cells. We showed here that postnatal day 7 (P7) female mice treated with 50 mg/kg STZ underwent the destruction of a large proportion of ß-cells and developed hyperglycemia. The blood glucose increased gradually and reached a peak level at 500 mg/dl on day 35-50. This was followed by a spontaneous regeneration of ß-cells. A reversal of non-fasting blood glucose to the control value was observed within 150 days. However, the mice still showed impaired glucose tolerance on day 150 and day 220, although a significant improvement was observed on day 150. Quantification of the ß-cell mass revealed that the ß-cell mass increased significantly between day 100 and day 150. On day 150 and day 220, the ß-cell mass was approximately 23% and 48.5% of the control, respectively. Of the insulin-positive cells, 10% turned out to be PCNA-positive proliferating cells. Our results demonstrated that, ß-cell duplication is one of the cell sources for ß-cell regeneration.

Structural properties of silkworm small heat-shock proteins: sHSP19.9 and sHSP20.8.

sHSP20.8 and sHSP19.9 are silkworm small-heat shock proteins (sHSPs) comprising a number of polypeptides of molecular sizes of several tens of kilodaltons as subunits. The structural properties of sHSPs were investigated. sHSP19.9 was found to be aggregated by itself during incubation at 60 degrees C. Aggregation was suppressed in the presence of dithiothreitol and at high ionic strength. In contrast, sHSP20.8 was not aggregated. Aggregation of sHSP19.9 was partially suppressed by sHSP20.8 and in the presence of catalase as a target protein. Based on changes in small-angle X-ray scattering, it is possible that the molecular size of sHSP19.9 is larger than that of sHSP20.8, and that their molecular sizes increase with increasing temperature in a reversible, biphasic manner. sHSPs did not protect catalase from thermal inactivation, but protected it from precipitation by forming a soluble complex. sHSP20.8 and sHSP19.9 with dithiothreitol were stable against lyophilization, autoclaving at 120 degrees C, and boiling.

BCL6 canalizes Notch-dependent transcription, excluding Mastermind-like1 from selected target genes during left-right patterning.

Although the Notch signaling pathway is one of the most intensely studied intracellular signaling pathways, the mechanisms by which Notch signaling regulates transcription remain incompletely understood. Here, we report that B cell leukemia/lymphoma 6 (BCL6), a transcriptional repressor, is a Notch-associated factor. BCL6 is necessary to maintain the expression of Pitx2 in the left lateral plate mesoderm during the patterning of left-right asymmetry in Xenopus embryos. For this process, BCL6 forms a complex with BCL6 corepressor (BCoR) on the promoters of selected Notch target genes such as enhancer of split related 1. BCL6 also inhibits the transcription of these genes by competing for the Notch1 intracellular domain, preventing the coactivator Mastermind-like1 (MAM1) from binding. These results define a mechanism restricting Notch-activated transcription to cell-type-appropriate subsets of target genes, and elucidate its relevance in vivo during left-right asymmetric development.

Genes encoding small heat shock proteins of the silkworm, Bombyx mori.

Small heat shock protein (sHSP) is a family of ubiquitous polypeptides involved in a variety of physiological phenomena. From the silkworm, Bombyx mori, we isolated and sequenced the following cDNAs encoding sHSPs: shsp19.9, shsp20.1, shsp20.4, shsp20.8, shsp21.4, and shsp23.7. shsp21.4 was nearly twice as large in size as other shsps. The deduced amino acid sequence of sHSP21.4 was similar to that of Drosophila melanogaster CG14207-PA. Other sHSPs were highly similar to each other and, in a phylogenetic tree, formed a cluster including Plodia interpunctella alphaCP25. It was speculated that shsp21.4 has at least one intron in genome while other shsps do not. The transcripts of all shsps were subtle, but were constitutively detected in various tissues. Heat shock triggered a substantial increase in the transcripts other than shsp21.4. The B. mori sHSPs are perhaps classified into at least two groups: sHSP21.4 and others.