Novel time-resolved reporter mouse reveals spatial and transcriptional heterogeneity during alpha cell differentiation.

AIMS/HYPOTHESIS: Glucagon-expressing pancreatic alpha cells have attracted much attention for their plasticity to transdifferentiate into insulin-producing beta cells; however, it remains unclear precisely when, and from where, alpha cells emerge and what regulates alpha cell fate. We therefore explored the spatial and transcriptional heterogeneity of alpha cell differentiation using a novel time-resolved reporter system. METHODS: We established the mouse model, 'Gcg-Timer', in which newly generated alpha cells can be distinguished from more-differentiated cells by their fluorescence. Fluorescence imaging and transcriptome analysis were performed with Gcg-Timer mice during the embryonic and postnatal stages. RESULTS: Fluorescence imaging and flow cytometry demonstrated that green fluorescence-dominant cells were present in Gcg-Timer mice at the embryonic and neonatal stages but not after 1 week of age, suggesting that alpha cell neogenesis occurs during embryogenesis and early neonatal stages under physiological conditions. Transcriptome analysis of Gcg-Timer embryos revealed that the mRNAs related to angiogenesis were enriched in newly generated alpha cells. Histological analysis revealed that some alpha cells arise close to the pancreatic ducts, whereas the others arise away from the ducts and adjacent to the blood vessels. Notably, when the glucagon signal was suppressed by genetic ablation or by chemicals, such as neutralising glucagon antibody, green-dominant cells emerged again in adult mice. CONCLUSIONS/INTERPRETATION: Novel time-resolved analysis with Gcg-Timer reporter mice uncovered spatiotemporal features of alpha cell neogenesis that will enhance our understanding of cellular identity and plasticity within the islets. DATA AVAILABILITY: Raw and processed RNA sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE229090.

STAT3 suppression and ß-cell ablation enhance α-to-ß reprogramming mediated by Pdx1.

As diabetes results from the absolute or relative deficiency of insulin secretion from pancreatic ß cells, possible methods to efficiently generate surrogate ß cells have attracted a lot of efforts. To date, insulin-producing cells have been generated from various differentiated cell types in the pancreas, such as acinar cells and α cells, by inducing defined transcription factors, such as PDX1 and MAFA, yet it is still challenging as to how surrogate ß cells can be efficiently generated for establishing future regenerative therapies for diabetes. In this study, we demonstrated that the exogenous expression of PDX1 activated STAT3 in α cells in vitro, and STAT3-null PDX1-expressing α cells in vivo resulted in efficient induction of α-to-ß reprogramming, accompanied by the emergence of α-cell-derived insulin-producing cells with silenced glucagon expression. Whereas ß-cell ablation by alloxan administration significantly increased the number of α-cell-derived insulin-producing cells by PDX1, STAT3 suppression resulted in no further increase in ß-cell neogenesis after ß-cell ablation. Thus, STAT3 modulation and ß-cell ablation nonadditively enhance α-to-ß reprogramming induced by PDX1, which may lead to the establishment of cell therapies for curing diabetes.

Genetic ablation of p62/SQSTM1 demonstrates little effect on pancreatic ß-cell function under autophagy deficiency.

Autophagy is known to play an essential role in intracellular quality control through the degradation of damaged organelles and components. We previously demonstrated that ß-cell-specific autophagy deficient mice, which lack Atg7, exhibited impaired glucose tolerance, accompanied by the accumulation of sequestosome 1/p62 (hereafter referred to as p62). Whereas p62 has been reported to play essential roles in regulating cellular homeostasis in the liver and adipose tissue, we previously showed that ß-cell-specific p62 deficiency does not cause any apparent impairment in glucose metabolism. In the present study, we investigated the roles of p62 in ß cells under autophagy-deficient conditions, by simultaneously inactivating both Atg7 and p62 in a ß-cell specific manner. Whereas p62 accumulation was substantially reduced in the islets of Atg7 and p62 double-deficient mice, glucose tolerance and insulin secretion were comparable to Atg7 single-deficient mice. Taken together, these findings suggest that the p62 accumulation appears to have little effect on ß-cell function under conditions of autophagy inhibition.

Mizuo-Nakamura phenomenon in X-linked retinoschisis.

Purpose: To determine whether the Mizuo-Nakamura phenomenon, which is an important diagnostic sign of Oguchi's disease, also occurs in patients with genetically proven X-linked retinoschisis (XLRS). Methods: We examined three patients with a clinical and genetic diagnosis of XLRS and one patient who was clinically diagnosed with Oguchi's disease, with an emphasis on the Mizuo-Nakamura phenomenon. We obtained color fundus photographs, especially in the fully dark-adapted state, using the non-mydriatic mode on a digital retinal camera and infrared observation monitor to avoid the bleaching effects caused by the viewing light, which alters the fundus color in a short time. Results: The Mizuo-Nakamura phenomenon was observed in all patients with molecularly proven XLRS, similar to that in the patient with Oguchi's disease. The sets of photographs were obtained in the light- and dark-adapted states using our newly devised techniques needed to witness the Mizuo-Nakamura phenomenon. Conclusions and Importance: The Mizuo-Nakamura phenomenon was identified in three patients with genetically proven XLRS. To the best of our knowledge, this study provided the first genetic evidence of the Mizuo-Nakamura phenomenon in a patient with molecularly proven XLRS without the causative genetic abnormalities for Oguchi's disease. Our findings suggest that XLRS is responsible for the Mizuo-Nakamura phenomenon and its presence in XLRS is not a rare exception but may be a consistent manifestation of XLRS.

Cumulative autophagy insufficiency in mice leads to progression of ß-cell failure.

Autophagy is known to play a pivotal role in ß-cell function. While the lifelong inhibition of autophagy through Atg7 deletion in ß cells has been demonstrated to lead to impaired glucose tolerance together with ß-cell dysfunction, the temporal association between autophagy inhibition and ß-cell dysfunction remains unclear. To address such questions, inducible ß-cell-specific Atg7-knockout (ißAtg7KO) mice were generated, and autophagy inhibition was induced for two different time durations. Whereas 2 weeks of Atg7 ablation was sufficient to induce autophagy deficiency, confirmed by the accumulation of p62, ißAtg7KO mice exhibited normal glucose tolerance. In contrast, prolonged autophagy deficiency for 6 weeks resulted in glucose intolerance together with impaired insulin secretion. Direct mRNA sequencing and pathway analysis revealed that the gene set associated with insulin secretion was downregulated only after the 6-week prolonged autophagy inhibition. Furthermore, we identified a novel gene, Sprr1a, which was expressed at more than 50-fold higher levels during both the 2-week and 6-week autophagy inhibition. These findings suggest that autophagy insufficiency cumulatively leads to ß-cell failure after a certain interval, accompanied by stepwise alterations of gene expression patterns.

Spatial and transcriptional heterogeneity of pancreatic beta cell neogenesis revealed by a time-resolved reporter system.

AIMS/HYPOTHESIS: While pancreatic beta cells have been shown to originate from endocrine progenitors in ductal regions, it remains unclear precisely where beta cells emerge from and which transcripts define newborn beta cells. We therefore investigated characteristics of newborn beta cells extracted by a time-resolved reporter system. METHODS: We established a mouse model, 'Ins1-GFP; Timer', which provides spatial information during beta cell neogenesis with high temporal resolution. Single-cell RNA-sequencing (scRNA-seq) was performed on mouse beta cells sorted by fluorescent reporter to uncover transcriptomic profiles of newborn beta cells. scRNA-seq of human embryonic stem cell (hESC)-derived beta-like cells was also performed to compare newborn beta cell features between mouse and human. RESULTS: Fluorescence imaging of Ins1-GFP; Timer mouse pancreas successfully dissected newly generated beta cells as green fluorescence-dominant cells. This reporter system revealed that, as expected, some newborn beta cells arise close to the ducts (ßduct); unexpectedly, the others arise away from the ducts and adjacent to blood vessels (ßvessel). Single-cell transcriptomic analyses demonstrated five distinct populations among newborn beta cells, confirming spatial heterogeneity of beta cell neogenesis such as high probability of glucagon-positive ßduct, musculoaponeurotic fibrosarcoma oncogene family B (MafB)-positive ßduct and musculoaponeurotic fibrosarcoma oncogene family A (MafA)-positive ßvessel cells. Comparative analysis with scRNA-seq data of mouse newborn beta cells and hESC-derived beta-like cells uncovered transcriptional similarity between mouse and human beta cell neogenesis including microsomal glutathione S-transferase 1 (MGST1)- and synaptotagmin 13 (SYT13)-highly-expressing state. CONCLUSIONS/INTERPRETATION: The combination of time-resolved histological imaging with single-cell transcriptional mapping demonstrated novel features of spatial and transcriptional heterogeneity in beta cell neogenesis, which will lead to a better understanding of beta cell differentiation for future cell therapy. DATA AVAILABILITY: Raw and processed single-cell RNA-sequencing data for this study has been deposited in the Gene Expression Omnibus under accession number GSE155742.

A Pilot Study of Intervention With a Mobile Application Visualizing the Macronutrient Content for Type 2 Diabetes at a Japanese Center.

BACKGROUND: Estimating the nutritional content of food is essential for self-management in people with type 2 diabetes mellitus, but it is a difficult skill to learn. The aim of this study was to investigate how diabetes management was impacted by the ability of patients to search for items they ate from a database of 26,300 different foods, and to visualize nutritional intake using the Japanese mobile application (app) "Calomeal." METHODS: This was a single-arm, single-center, pilot study. Eighteen outpatients with type 2 diabetes mellitus used the "Calomeal" app for 3 months. The primary endpoint was change in hemoglobin A1c (HbA1c). Secondary endpoints were changes in body weight (BW), lipid parameters, and quality of life scores. RESULTS: The baseline characteristics of the study subjects were as follows: age: 53.4 ± 7.8 years; male/female ratio: 11/7; HbA1c: 7.9 (7.58 - 8.23)%; and body mass index (BMI): 25.17 (21.63 - 28.59) kg/m2. Significant reductions in HbA1c and BMI were observed over 3 months (HbA1c: 7.9 (7.58 - 8.23)% to 7.6 (7.3 - 8.23)%, P = 0.0410; BMI: 25.17 (21.63 - 28.59) to 24.54 (21.57 - 27.81) kg/m2, P = 0.0057). Reductions in HbA1c and BMI both correlated with decreased carbohydrate intake estimated by the mobile app. CONCLUSIONS: Japanese patients who used their smartphones to visualize their nutritional intake using the "Calomeal" app demonstrated improved short-term glycemic control and BMI. Although the validity of the results should be tested in future randomized controlled trials, this approach may be a clinical option for improving self-management in Japanese patients with type 2 diabetes mellitus.

Effect of real-life insulin pump with predictive low-glucose management use for 3 months: Analysis of the patients treated in a Japanese center.

AIMS/INTRODUCTION: In Japan, an insulin pump with predictive low-glucose management (PLGM) was launched in 2018. It automatically suspends insulin delivery when the sensor detects or predicts low glucose values. The aim of this study was to analyze the safety and efficacy of PLGM in patients treated in a Japanese center. MATERIALS AND METHODS: We carried out a retrospective observational analysis of 16 patients with type 1 diabetes mellitus and one patient after pancreatectomy. They switched from the MiniMed 620G device to the 640G device with PLGM. The primary outcome was the change in the percentage of time in hypoglycemia. The secondary outcome was the change in HbA1c (%) over a period of 3 months. We also explored the presence of "post-suspend hyperglycemia" with the 640G device. RESULTS: After changing to the 640G device, the percentage of time in hypoglycemia (glucose <50 mg/dL) significantly decreased from 0.39% (0-1.51%) to 0% (0-0.44%; P = 0.0407). The percentage of time in hyperglycemia (glucose >180 mg/dL) significantly increased from 25.53% (15.78-44.14%) to 32.9% (24.71-45.49%; P = 0.0373). HbA1c significantly increased from 7.6 ± 1.0% to 7.8 ± 1.1% (P = 0.0161). From 1.5 to 4.5 h after the resumption of insulin delivery, the percentage of time in hyperglycemia was 32.23% (24.2-53.75%), but it was significantly lower, 2.78% (0-21.6%), when patients manually restarted the pump within 30 min compared with automatic resumption 31.2% (20-61.66%; P = 0.0063). CONCLUSIONS: Predictive low-glucose management is an effective tool for reducing hypoglycemia, but possibly elicits "post-suspend hyperglycemia." This information is useful for achieving better blood glucose control in the patients treated with PLGM.

Biphasic changes in ß-cell mass around parturition are accompanied by increased serotonin production.

Pancreatic ß-cell mass is known to be considerably altered during pregnancy and after parturition in rodents and humans. While ß-cell mass increases during pregnancy and starts to return toward its original level after parturition, the cellular mechanisms by which ß-cell mass during this period is regulated remains unclear. To address this issue in mice, we quantified ß-cell mass and investigated the mechanisms underlying its regulation throughout the perinatal and postpartum period. The increased ß-cell size and proliferation during pregnancy were significantly reduced shortly after parturition, whereas there was no evidence of ß-cell reprogramming or increased apoptosis. Direct RNA sequencing of islets from pregnant and postpartum mice demonstrated dynamic changes in gene expression patterns, showing robust downregulation of cell cycle-related genes 1 day after parturition, and the reupregulation of serotonin metabolism-related genes at postpartum day 7. Serotonin synthesis was activated only in lactating females, accompanied by increased ß-cell mass. Taken together, these findings demonstrate that ß-cell mass is decreased shortly after parturition owing to reduced ß-cell size and proliferation, and is subsequently increased, in association with lactation and serotonin biosynthesis.

Acetaldehyde dehydrogenase 2 deficiency increases mitochondrial reactive oxygen species emission and induces mitochondrial protease Omi/HtrA2 in skeletal muscle.

Acetaldehyde dehydrogenase 2 (ALDH2) is an enzyme involved in redox homeostasis as well as the detoxification process in alcohol metabolism. Nearly 8% of the world's population have an inactivating mutation in the ALDH2 gene. However, the expression patterns and specific functions of ALDH2 in skeletal muscles are still unclear. Herein, we report that ALDH2 is expressed in skeletal muscle and is localized to the mitochondrial fraction. Oxidative muscles had a higher amount of ALDH2 protein than glycolytic muscles. We next comprehensively investigated whether ALDH2 knockout in mice induces mitochondrial adaptations in gastrocnemius muscle (for example, content, enzymatic activity, respiratory function, supercomplex formation, and functional networking). We found that ALDH2 deficiency resulted in partial mitochondrial dysfunction in gastrocnemius muscle because it increased mitochondrial reactive oxygen species (ROS) emission (2',7'-dichlorofluorescein and MitoSOX oxidation rate during respiration) and the frequency of regional mitochondrial depolarization. Moreover, we determined whether ALDH2 deficiency and the related mitochondrial dysfunction trigger mitochondrial stress and quality control responses in gastrocnemius muscle (for example, mitophagy markers, dynamics, and the unfolded protein response). We found that ALDH2 deficiency upregulated the mitochondrial serine protease Omi/HtrA2 (a marker of the activation of a branch of the mitochondrial unfolded protein response). In summary, ALDH2 deficiency leads to greater mitochondrial ROS production, but homeostasis can be maintained via an appropriate stress response.

Cast immobilization of hindlimb upregulates sarcolipin expression in atrophied skeletal muscles and increases thermogenesis in C57BL/6J mice.

Mechanical unloading impairs cytosolic calcium (Ca2+) homeostasis in skeletal muscles. In this study, we investigated whether sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) itself or one of the regulators of the Ca2+ SERCA pump, sarcolipin (SLN), is altered to deregulate Ca2+ homeostasis in cast immobilized, atrophied muscles. Hindlimb muscles of 8-wk-old male C57BL/6J mice were subjected to bilateral cast immobilization for 2 wk. Two-week-cast immobilization induced both body weight and skeletal muscle loss. Highly phosphorylated Ca2+/calmodulin-dependent protein kinase II in the atrophied muscles suggested that cytosolic Ca2+ concentration was elevated. Extremely high expression levels of SLN mRNA and protein were observed in the atrophied muscles. Upregulation of SLN at the transcriptional level was supported by low RCAN1 expression, which is a negative regulator of SLN. We treated C2C12 cells with dexamethasone to mimic muscle atrophy in vitro and showed a direct relationship between high SLN mRNA expression and low Ca2+ uptake by sarcoplasmic reticulum. Since SLN reportedly plays a role in nonshivering thermogenesis, we performed a cold tolerance test of the whole body. As a result, we found that mice with cast immobilization showed high cold tolerance, suggesting that cast immobilization promoted whole body thermogenesis. Although the activity level was decreased during cast immobilization without change in food intake, adipose tissue weights also decreased significantly after cast immobilization. Concomitantly, we conclude that cast immobilization of hindlimb increased thermogenesis in C57Bl/6J mice, probably via high expression of SLN.

Improved prediction of fellow-eye response in one-eye trials using multiple intraocular pressure measurements.

PURPOSE: To examine the effects of multiple intraocular pressure (IOP) measurements on the correlation of response to glaucoma medication between fellow eyes. METHODS: Latanoprost was applied to the first eye and then to both eyes of 16 patients with either primary open-angle glaucoma or ocular hypertension. IOP measurements were performed twice on different days for each of three periods: at baseline, during treatment of the first eye only and for both eyes. IOP decreases in the first and second eyes were evaluated with ∆IOP 1 (=IOP at baseline - IOP after treatment) and ∆IOP 2 (=∆IOP 1 - IOP fluctuation of the contralateral eye). The correlation of IOP decreases between the fellow eyes was analyzed by linear regression analysis using the Pearson correlation coefficient, r. RESULTS: No significant correlations of ∆IOP1 between fellow eyes were found regardless of the number of IOP measurements. ∆IOP2 was significantly correlated between fellow eyes using two post-treatment IOP measurements, but not with a single post-treatment IOP. CONCLUSIONS: Using multiple post-treatment IOP measurements may improve the prediction of a fellow eye's response to glaucoma medication in one-eye trials.

Unilateral choroidal excavation in the macula detected by spectral-domain optical coherence tomography.

PURPOSE: To report clinical findings of three patients with unilateral peculiar choroidal excavation in the macula detected by spectral-domain (SD) optical coherence tomography (OCT). METHODS: Three cases with unilateral choroidal excavation in the macula detected by SD OCT. Fluorescein angiography (FA), indocyanine green angiography (IA), ultrasonography, visual field tests and multifocal electroretinography (mfERG) were performed. RESULTS: Although all three patients complained of metamorphopsia, visual acuity and central visual field were normal in the affected eyes. SD OCT demonstrated choroidal excavation in the macula despite a normal foveal contour along the inner retinal surface. The excavation involved the outer retinal layers up to the external limiting membrane in cases 1 and 2, while only the retinal pigment epithelium was involved in case 3. The excavation corresponded to foveal pigment mottling in cases 1 and 2 and to a parafoveal yellowish fusiform lesion in case 3. The lesions appeared hypoautofluorescent and unremarkable in FA except for circumferential hyperfluorescence in case 3 and hypofluorescent in IA. B-scan ultrasonography was unremarkable. MfERG in cases 1 and 2 was normal. CONCLUSIONS: SD OCT demonstrated two types of choroidal excavation in the macula. More case accumulation and a longer follow-up will elucidate the pathogenesis and prognosis of the lesions.

Fundus autofluorescence and spectral-domain optical coherence tomography findings of leopard spots in nanophthalmic uveal effusion syndrome.

PURPOSE: To describe fundus autofluorescence (FAF) imaging and spectral domain optical coherence tomography (SD-OCT) findings of leopard spots in nanophthalmic uveal effusion syndrome. METHODS: A 34-year-old man with retinal detachment associated with nanophthalmic uveal effusion syndrome in the right eye underwent sclerotomy three times. After the final surgery, the subretinal fluid resolved gradually. Then, SD-OCT examination, FAF photography, fluorescein angiography (FA), and indocyanine green angiography (ICGA) were performed simultaneously with the spectralis Heidelberg retina angiograph + OCT system. RESULTS: SD-OCT revealed focal thickening of the retinal pigment epithelium (RPE) layer at the same locations as leopard spots, which appeared hypofluorescent on FA and ICGA. These spots showed hyperautofluorescence on FAF imaging. Six months later, focal thickening of the RPE layer became smaller on OCT and hyperautofluorescence was attenuated on FAF imaging. CONCLUSIONS: Simultaneous imaging of the fundus with multiple modalities including OCT, FAF, FA, and ICGA indicates that leopard spots in the fundus of uveal effusion syndrome may show hyperautofluorescence and correspond to focal thickening of the RPE layer by SD-OCT. This imaging method may help elucidate the pathology of various fundus lesions in vivo.