SenNet recommendations for detecting senescent cells in different tissues.

Once considered a tissue culture-specific phenomenon, cellular senescence has now been linked to various biological processes with both beneficial and detrimental roles in humans, rodents and other species. Much of our understanding of senescent cell biology still originates from tissue culture studies, where each cell in the culture is driven to an irreversible cell cycle arrest. By contrast, in tissues, these cells are relatively rare and difficult to characterize, and it is now established that fully differentiated, postmitotic cells can also acquire a senescence phenotype. The SenNet Biomarkers Working Group was formed to provide recommendations for the use of cellular senescence markers to identify and characterize senescent cells in tissues. Here, we provide recommendations for detecting senescent cells in different tissues based on a comprehensive analysis of existing literature reporting senescence markers in 14 tissues in mice and humans. We discuss some of the recent advances in detecting and characterizing cellular senescence, including molecular senescence signatures and morphological features, and the use of circulating markers. We aim for this work to be a valuable resource for both seasoned investigators in senescence-related studies and newcomers to the field.

Kinase signalling in excitatory neurons regulates sleep quantity and depth.

Progress has been made in the elucidation of sleep and wakefulness regulation at the neurocircuit level1,2. However, the intracellular signalling pathways that regulate sleep and the neuron groups in which these intracellular mechanisms work remain largely unknown. Here, using a forward genetics approach in mice, we identify histone deacetylase 4 (HDAC4) as a sleep-regulating molecule. Haploinsufficiency of Hdac4, a substrate of salt-inducible kinase 3 (SIK3)3, increased sleep. By contrast, mice that lacked SIK3 or its upstream kinase LKB1 in neurons or with a Hdac4S245A mutation that confers resistance to phosphorylation by SIK3 showed decreased sleep. These findings indicate that LKB1-SIK3-HDAC4 constitute a signalling cascade that regulates sleep and wakefulness. We also performed targeted manipulation of SIK3 and HDAC4 in specific neurons and brain regions. This showed that SIK3 signalling in excitatory neurons located in the cerebral cortex and the hypothalamus positively regulates EEG delta power during non-rapid eye movement sleep (NREMS) and NREMS amount, respectively. A subset of transcripts biased towards synaptic functions was commonly regulated in cortical glutamatergic neurons through the expression of a gain-of-function allele of Sik3 and through sleep deprivation. These findings suggest that NREMS quantity and depth are regulated by distinct groups of excitatory neurons through common intracellular signals. This study provides a basis for linking intracellular events and circuit-level mechanisms that control NREMS.

SIK3-HDAC4 in the suprachiasmatic nucleus regulates the timing of arousal at the dark onset and circadian period in mice.

Mammals exhibit circadian cycles of sleep and wakefulness under the control of the suprachiasmatic nucleus (SCN), such as the strong arousal phase-locked to the beginning of the dark phase in laboratory mice. Here, we demonstrate that salt-inducible kinase 3 (SIK3) deficiency in gamma-aminobutyric acid (GABA)-ergic neurons or neuromedin S (NMS)-producing neurons delayed the arousal peak phase and lengthened the behavioral circadian cycle under both 12-h light:12-h dark condition (LD) and constant dark condition (DD) without changing daily sleep amounts. In contrast, the induction of a gain-of-function mutant allele of Sik3 in GABAergic neurons exhibited advanced activity onset and a shorter circadian period. Loss of SIK3 in arginine vasopressin (AVP)-producing neurons lengthened the circadian cycle, but the arousal peak phase was similar to that in control mice. Heterozygous deficiency of histone deacetylase (HDAC) 4, a SIK3 substrate, shortened the circadian cycle, whereas mice with HDAC4 S245A, which is resistant to phosphorylation by SIK3, delayed the arousal peak phase. Phase-delayed core clock gene expressions were detected in the liver of mice lacking SIK3 in GABAergic neurons. These results suggest that the SIK3-HDAC4 pathway regulates the circadian period length and the timing of arousal through NMS-positive neurons in the SCN.

Induction of Mutant Sik3Sleepy Allele in Neurons in Late Infancy Increases Sleep Need.

Sleep is regulated in a homeostatic manner. Sleep deprivation increases sleep need, which is compensated mainly by increased EEG δ power during non-rapid eye movement sleep (NREMS) and, to a lesser extent, by increased sleep amount. Although genetic factors determine the constitutive level of sleep need and sleep amount in mice and humans, the molecular entity behind sleep need remains unknown. Recently, we found that a gain-of-function Sleepy (Slp) mutation in the salt-inducible kinase 3 (Sik3) gene, which produces the mutant SIK3(SLP) protein, leads to an increase in NREMS EEG δ power and sleep amount. Since Sik3Slp mice express SIK3(SLP) in various types of cells in the brain as well as multiple peripheral tissues from the embryonic stage, the cell type and developmental stage responsible for the sleep phenotype in Sik3Slp mice remain to be elucidated. Here, we generated two mouse lines, synapsin1CreERT2 and Sik3ex13flox mice, which enable inducible Cre-mediated, conditional expression of SIK3(SLP) in neurons on tamoxifen administration. Administration of tamoxifen to synapsin1CreERT2 mice during late infancy resulted in higher recombination efficiency than administration during adolescence. SIK3(SLP) expression after late infancy increased NREMS and NREMS δ power in male synapsin1CreERT2; Sik3ex13flox/+ mice. The expression of SIK3(SLP) after adolescence led to a higher NREMS δ power without a significant change in NREMS amounts. Thus, neuron-specific expression of SIK3(SLP) after late infancy is sufficient to increase sleep.SIGNIFICANCE STATEMENT The propensity to accumulate sleep need during wakefulness and to dissipate it during sleep underlies the homeostatic regulation of sleep. However, little is known about the developmental stage and cell types involved in determining the homeostatic regulation of sleep. Here, we show that Sik3Slp allele induction in mature neurons in late infancy is sufficient to increase non-rapid eye movement sleep amount and non-rapid eye movement sleep δ power. SIK3 signaling in neurons constitutes an intracellular mechanism to increase sleep.

Carbonic anhydrase 8 (CAR8) negatively regulates GLP-1 secretion from enteroendocrine cells in response to long-chain fatty acids.

Glucagon-like peptide-1 (GLP-1) is an incretin secreted from enteroendocrine preproglucagon (PPG)-expressing cells (traditionally known as L cells) in response to luminal nutrients that potentiates insulin secretion. Augmentation of endogenous GLP-1 secretion might well represent a novel therapeutic target for diabetes treatment in addition to the incretin-associated drugs currently in use. In this study, we found that PPG cells substantially express carbonic anhydrase 8 (CAR8), which has been reported to inhibit inositol 1,4,5-trisphosphate (IP3) binding to the IP3 receptor and subsequent Ca2+ efflux from the endoplasmic reticulum in neuronal cells. In vitro experiments using STC-1 cells demonstrated that Car8 knockdown increases long-chain fatty acid (LCFA)-stimulated GLP-1 secretion. This effect was reduced in the presence of phospholipase C (PLC) inhibitor; in addition, Car8 knockdown increased the intracellular Ca2+ elevation caused by α-linolenic acid, indicating that CAR8 exerts its effect on GLP-1 secretion via the PLC/IP3/Ca2+ pathway. Car8wdl null mutant mice showed significant increase in GLP-1 response to oral corn oil administration compared with that in wild-type littermates, with no significant change in intestinal GLP-1 content. These results demonstrate that CAR8 negatively regulates GLP-1 secretion from PPG cells in response to LCFAs, suggesting the possibility of augmentation of postprandial GLP-1 secretion by CAR8 inhibition.NEW & NOTEWORTHY This study focused on the physiological significance of carbonic anhydrase 8 (CAR8) in GLP-1 secretion from enteroendocrine preproglucagon (PPG)-expressing cells. We found an inhibitory role of CAR8 in LCFA-induced GLP-1 secretion in vitro and in vivo, suggesting a novel therapeutic approach to diabetes and obesity through augmentation of postprandial GLP-1 secretion by CAR8 inhibition.

Biochemical Methanol Gas Sensor (MeOH Bio-Sniffer) for Non-Invasive Assessment of Intestinal Flora from Breath Methanol.

Methanol (MeOH) in exhaled breath has potential for non-invasive assessment of intestinal flora. In this study, we have developed a biochemical gas sensor (bio-sniffer) for MeOH in the gas phase using fluorometry and a cascade reaction with two enzymes, alcohol oxidase (AOD) and formaldehyde dehydrogenase (FALDH). In the cascade reaction, oxidation of MeOH was initially catalyzed by AOD to produce formaldehyde, and then this formaldehyde was successively oxidized via FALDH catalysis together with reduction of oxidized form of ß-nicotinamide adenine dinucleotide (NAD+). As a result of the cascade reaction, reduced form of NAD (NADH) was produced, and MeOH vapor was measured by detecting autofluorescence of NADH. In the development of the MeOH bio-sniffer, three conditions were optimized: selecting a suitable FALDH for better discrimination of MeOH from ethanol in the cascade reaction; buffer pH that maximizes the cascade reaction; and materials and methods to prevent leaking of NAD+ solution from an AOD-FALDH membrane. The dynamic range of the constructed MeOH bio-sniffer was 0.32-20 ppm, which encompassed the MeOH concentration in exhaled breath of healthy people. The measurement of exhaled breath of a healthy subject showed a similar sensorgram to the standard MeOH vapor. These results suggest that the MeOH bio-sniffer exploiting the cascade reaction will become a powerful tool for the non-invasive intestinal flora testing.

Vasodilator-stimulated phosphoprotein (VASP) is recruited into dendritic spines via G-actin-dependent mechanism and contributes to spine enlargement and stabilization.

Actin organization and dynamics are modulated by diverse actin regulators during dendritic spine development. To understand the molecular network that regulates actin organization and spine morphology, it is important to investigate dynamic redistribution of actin regulators during spine development. One of the actin regulators, vasodilator-stimulated phosphoprotein (VASP), has multiple functions in actin regulation and is known to regulate spine morphology. However, dynamics and temporal regulation of VASP during spine development have not been clarified. In this study, we performed time-lapse imaging of mouse hippocampal dissociated neurons to analyse the change in localization of VASP during spine development. We found that accumulation of VASP within spines precedes the start of persistent F-actin increase, which are temporally coupled with spine enlargement. Using domain deletion or mutation constructs of VASP, we revealed that the interaction with G-actin is important for the preceding accumulation of VASP. Furthermore, we showed that accumulation of VASP contributes to actin enrichment within spines and stabilization of spine morphology by dominant negative experiments. These data suggest that G-actin-dependent VASP recruitment has dual functions in spine development, enlargement and stabilization, through the interaction with actin and other cytoskeletal regulators.

Medium-chain triglyceride diet stimulates less GIP secretion and suppresses body weight and fat mass gain compared with long-chain triglyceride diet.

Gastric inhibitory polypeptide (GIP) is an incretin secreted from enteroendocrine K cells and potentiates insulin secretion from pancreatic ß-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.

Transcriptional factor Pdx1 is involved in age-related GIP hypersecretion in mice.

Fat accumulation with aging is a serious problem; glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) is an incretin that plays an important role in fat accumulation. GIP receptor knockout mice show reduced fat mass and improved insulin sensitivity associated with aging. Therefore, GIP is involved in fat accumulation and insulin resistance with aging. However, age-related changes of GIP secretion remain unclear. The present study aimed to elucidate age-related changes of GIP secretion and enteroendocrine K cells using GIP reporter [GIP-green fluorescent protein (GFP) knock-in heterozygous (GIPgfp/+)] mice. Aged 1-yr-old GIPgfp/+ mice exhibited a phenotype of fat accumulation, insulin resistance, and GIP hypersecretion compared with young (3-4 mo old) GIPgfp/+ mice. In aged mice, K-cell number in the small intestine and the mRNA expression levels of GIP and transcriptional factor pancreatic and duodenal homeobox-1 (Pdx1) in K cells were increased. K-cell number, GIP mRNA expression and content in small intestine, and GIP secretion were decreased after posteriori suppression of Pdx1 using intestine-specific gene transfer. Thus, Pdx1 positively regulates GIP mRNA and K-cell number in small intestine. Increased Pdx1 expression might be involved in GIP hypersecretion with aging. NEW & NOTEWORTHY Age-related changes of glucose-dependent insulinotropic polypeptide/gastric inhibitory polypeptide (GIP) secretion and K cells were investigated. We found that K-cell number and GIP and pancreatic and duodenal homeobox-1 (Pdx1) expression in K cells were increased in aged mice, which showed greater GIP secretion compared with young mice. In addition, we have succeeded in posteriori suppression of Pdx1 in small intestine using the method of intestine-specific gene transfer, and showed that K-cell number, GIP expression, and GIP secretion were decreased in the Pdx1-knockdown intestine.

Diverse metabolic effects of O-GlcNAcylation in the pancreas but limited effects in insulin-sensitive organs in mice.

AIMS/HYPOTHESIS: O-GlcNAcylation is characterised by the addition of N-acetylglucosamine to various proteins by O-GlcNAc transferase (OGT) and serves in sensing intracellular nutrients by modulating various cellular processes. Although it has been speculated that O-GlcNAcylation is associated with glucose metabolism, its exact role in whole body glucose metabolism has not been fully elucidated. Here, we investigated whether loss of O-GlcNAcylation globally and in specific organs affected glucose metabolism in mammals under physiological conditions. METHODS: Tamoxifen-inducible global Ogt-knockout (Ogt-KO) mice were generated by crossbreeding Ogt-flox mice with R26-Cre-ERT2 mice. Liver, skeletal muscle, adipose tissue and pancreatic beta cell-specific Ogt-KO mice were generated by crossbreeding Ogt-flox mice with Alb-Cre, Mlc1f-Cre, Adipoq-Cre and Pdx1 PB-CreER™ mice, respectively. Glucose metabolism was evaluated by i.p. glucose and insulin tolerance tests. RESULTS: Tamoxifen-inducible global Ogt-KO mice exhibited a lethal phenotype from 4 weeks post injection, suggesting that O-GlcNAcylation is essential for survival in adult mice. Tissue-specific Ogt deletion from insulin-sensitive organs, including liver, skeletal muscle and adipose tissue, had little impact on glucose metabolism under physiological conditions. However, pancreatic beta cell-specific Ogt-KO mice displayed transient hypoglycaemia (Ogt-flox 5.46 ± 0.41 vs Ogt-ßKO 3.88 ± 0.26 mmol/l) associated with about twofold higher insulin secretion and accelerated adiposity, followed by subsequent hyperglycaemia (Ogt-flox 6.34 ± 0.32 vs Ogt-ßKO 26.4 ± 2.37 mmol/l) with insulin depletion accompanied by beta cell apoptosis. CONCLUSIONS/INTERPRETATION: These findings suggest that O-GlcNAcylation has little effect on glucose metabolism in insulin-sensitive tissues but plays a crucial role in pancreatic beta cell function and survival under physiological conditions. Our results provide novel insight into O-GlcNAc biology and physiology in glucose metabolism.

Fatty acid-binding protein 5 regulates diet-induced obesity via GIP secretion from enteroendocrine K cells in response to fat ingestion.

Gastric inhibitory polypeptide (GIP) is an incretin released from enteroendocrine K cells in response to nutrient intake, especially fat. GIP is one of the contributing factors inducing fat accumulation that results in obesity. A recent study shows that fatty acid-binding protein 5 (FABP5) is expressed in murine K cells and is involved in fat-induced GIP secretion. We investigated the mechanism of fat-induced GIP secretion and the impact of FABP5-related GIP response on diet-induced obesity (DIO). Single oral administration of glucose and fat resulted in a 40% reduction of GIP response to fat but not to glucose in whole body FABP5-knockout (FABP5(-/-)) mice, with no change in K cell count or GIP content in K cells. In an ex vivo experiment using isolated upper small intestine, oleic acid induced only a slight increase in GIP release, which was markedly enhanced by coadministration of bile and oleic acid together with attenuated GIP response in the FABP5(-/-) sample. FABP5(-/-) mice exhibited a 24% reduction in body weight gain and body fat mass under a high-fat diet compared with wild-type (FABP5(+/+)) mice; the difference was not observed between GIP-GFP homozygous knock-in (GIP(gfp/gfp))-FABP5(+/+) mice and GIP(gfp/gfp)-FABP5(-/-) mice, in which GIP is genetically deleted. These results demonstrate that bile efficiently amplifies fat-induced GIP secretion and that FABP5 contributes to the development of DIO in a GIP-dependent manner.

Neuronal subtype-specific transcriptomic changes in the cerebral neocortex associated with sleep pressure.

Sleep is homeostatically regulated by sleep pressure, which increases during wakefulness and dissipates during sleep. Recent studies have suggested that the cerebral neocortex, a six-layered structure composed of various layer- and projection-specific neuronal subtypes, is involved in the representation of sleep pressure governed by transcriptional regulation. Here, we examined the transcriptomic changes in neuronal subtypes in the neocortex upon increased sleep pressure using single-nucleus RNA sequencing datasets and predicted the putative intracellular and intercellular molecules involved in transcriptome alterations. We revealed that sleep deprivation (SD) had the greatest effect on the transcriptome of layer 2 and 3 intratelencephalic (L2/3 IT) neurons among the neocortical glutamatergic neuronal subtypes. The expression of mutant SIK3 (SLP), which is known to increase sleep pressure, also induced profound changes in the transcriptome of L2/3 IT neurons. We identified Junb as a candidate transcription factor involved in the alteration of the L2/3 IT neuronal transcriptome by SD and SIK3 (SLP) expression. Finally, we inferred putative intercellular ligands, including BDNF, LSAMP, and PRNP, which may be involved in SD-induced alteration of the transcriptome of L2/3 IT neurons. We suggest that the transcriptome of L2/3 IT neurons is most impacted by increased sleep pressure among neocortical glutamatergic neuronal subtypes and identify putative molecules involved in such transcriptional alterations.

Crucial role of TFAP2B in the nervous system for regulating NREM sleep.

The AP-2 transcription factors are crucial for regulating sleep in both vertebrate and invertebrate animals. In mice, loss of function of the transcription factor AP-2ß (TFAP2B) reduces non-rapid eye movement (NREM) sleep. When and where TFAP2B functions, however, is unclear. Here, we used the Cre-loxP system to generate mice in which Tfap2b was specifically deleted in the nervous system during development and mice in which neuronal Tfap2b was specifically deleted postnatally. Both types of mice exhibited reduced NREM sleep, but the nervous system-specific deletion of Tfap2b resulted in more severe sleep phenotypes accompanied by defective light entrainment of the circadian clock and stereotypic jumping behavior. These findings indicate that TFAP2B in postnatal neurons functions at least partly in sleep regulation and imply that TFAP2B also functions either at earlier stages or in additional cell types within the nervous system.

Regulation of Cellular Senescence in Type 2 Diabetes Mellitus: From Mechanisms to Clinical Applications.

Cellular senescence is accelerated by hyperglycemia through multiple pathways. Therefore, senescence is an important cellular mechanism to consider in the pathophysiology of type 2 diabetes mellitus (T2DM) and an additional therapeutic target. The use of drugs that remove senescent cells has led to improvements in blood glucose levels and diabetic complications in animal studies. Although the removal of senescent cells is a promising approach for the treatment of T2DM, two main challenges limit its clinical application: the molecular basis of cellular senescence in each organ is yet to be understood, and the specific effect of removing senescent cells in each organ has to be determined. This review aims to discuss future applications of targeting senescence as a therapeutic option in T2DM and elucidate the characteristics of cellular senescence and senescence-associated secretory phenotype in the tissues important for regulating glucose levels: pancreas, liver, adipocytes, and skeletal muscle.

Exploratory study on glycemic control improvement for patients with diabetes mellitus by appropriate re-education on insulin self-injection technique during COVID-19 pandemic.

AIMS: To conduct a study on glycemic control improvement by appropriate re-education on the self-injection technique (SIT) in patients with diabetes mellitus undergoing insulin therapy. METHODS: Patients who received appropriate SIT and were treated with insulin for more than a year were re-educated. For the observation period of six months, the subjects' SIT was checked, and hemoglobin A1c (HbA1c) levels were measured at each visit. HbA1c levels, insulin doses, and behavioral changes in SIT were investigated at baseline and at the end of the observation period. RESULTS: In the per-protocol set population, the HbA1c level decreased by 0.2 % (2.0 mmol/mol) on average, showing a significant difference (p = 0.009). No significant difference was observed in the proportion of subjects with decreased HbA1c levels, changes in total daily insulin doses, or blood glucose levels. Four of the six SIT items covered by re-education were improved. CONCLUSIONS: Providing re-education on insulin SIT was considered effective in reducing HbA1c levels and improving adherence to proper SIT.

Decreased IGF1R attenuates senescence and improves function in pancreatic ß-cells.

Introduction: The enhanced ß-cell senescence that accompanies insulin resistance and aging contributes to cellular dysfunction and loss of transcriptional identity leading to type 2 diabetes (T2D). While senescence is among the 12 recognized hallmarks of aging, its relation to other hallmarks including altered nutrient sensing (insulin/IGF1 pathway) in ß-cells is not fully understood. We previously reported that an increased expression of IGF1R in mouse and human ß-cells is a marker of older ß-cells; however, its contribution to age-related dysfunction and cellular senescence remains to be determined. Methods: In this study, we explored the direct role of IGF1R in ß-cell function and senescence using two independent mouse models with decreased IGF1/IGF1R signaling: a) Ames Dwarf mice (Dwarf +/+), which lack growth hormone and therefore have reduced circulating levels of IGF1, and b) inducible ß-cell-specific IGF1R knockdown (ßIgf1rKD) mice. Results: Compared to Dwarf+/- mice, Dwarf+/+ mice had lower body and pancreas weight, lower circulating IGF1 and insulin levels, and lower IGF1R and p21Cip1 protein expression in ß-cells, suggesting the suppression of senescence. Adult ßIgf1rKD mice showed improved glucose clearance and glucose-induced insulin secretion, accompanied by decreased p21Cip1 protein expression in ß-cells. RNA-Seq of islets isolated from these ßIgf1rKD mice revealed the restoration of three signaling pathways known to be downregulated by aging: sulfide oxidation, autophagy, and mTOR signaling. Additionally, deletion of IGF1R in mouse ß-cells increased transcription of genes important for maintaining ß-cell identity and function, such as Mafa, Nkx6.1, and Kcnj11, while decreasing senescence-related genes, such as Cdkn2a, Il1b, and Serpine 1. Decreased senescence and improved insulin-secretory function of ß-cells were also evident when the ßIgf1rKD mice were fed a high-fat diet (HFD; 60% kcal from fat, for 5 weeks). Discussion: These results suggest that IGF1R signaling plays a causal role in aging-induced ß-cell dysfunction. Our data also demonstrate a relationship between decreased IGF1R signaling and suppressed cellular senescence in pancreatic ß-cells. Future studies can further our understanding of the interaction between senescence and aging, developing interventions that restore ß-cell function and identity, therefore preventing the progression to T2D.

Plant sterol hyperabsorption caused by uncontrolled diabetes in a patient with a heterozygous ABCG5 variant.

Plant sterol intake is widely recommended for patients with cardiovascular risk factors based on the inhibitory effect on intestinal cholesterol absorption. Although plant sterols, once absorbed, can promote atherosclerosis, their intake is believed to be safe because of poor absorption, except in rare hyperabsorbers with homozygous ABCG5/8 mutations. We report a case of new-onset type 1 diabetes accompanied by hypercholesterolemia. At the initial presentation with diabetic ketoacidosis, the patient showed marked hypercholesterolemia. Whole-exome sequencing revealed a heterozygous pathogenic variant in ABCG5 (p.R419H). The initial serum plant sterol levels were markedly high (sitosterol 32.5 µg/mL, campesterol 66.0 µg/mL), close to the range observed in patients with homozygous ABCG5/8 mutations, which were largely reduced by insulin treatment without ezetimibe. The addition of ezetimibe normalized plant sterol levels. These findings provide the first evidence that uncontrolled diabetes plays a causal role in the pathogenesis of phytosterolemia.

Sporadic Pseudohypoparathyroidism Type 1B in Monozygotic Twins: Insights Into the Pathogenesis of Methylation Defects.

CONTEXT: Sporadic pseudohypoparathyroidism type 1B (sporPHP1B) is an imprinting disease without a defined genetic cause, characterized by broad methylation changes in differentially methylated regions (DMRs) of the GNAS gene. OBJECTIVE: This work aims to provide insights into the causative event leading to the GNAS methylation defects through comprehensive molecular genetic analyses of a pair of female monozygotic twins concordant for sporPHP1B who were conceived naturally, that is, without assisted reproductive techniques. METHODS: Using the leukocyte genome of the twins and family members, we performed targeted bisulfite sequencing, methylation-sensitive restriction enzyme (MSRE)-quantitative polymerase chain reaction (qPCR), whole-genome sequencing (WGS), high-density single-nucleotide polymorphism (SNP) array, and Sanger sequencing. RESULTS: Methylation analyses by targeted bisulfite sequencing and MSRE-qPCR revealed almost complete losses of methylation at the GNAS AS, XL, and A/B DMRs and a gain of methylation at the NESP55 DMR in the twins, but not in other family members. Except for the GNAS locus, we did not find apparent methylation defects at other imprinted genome loci of the twins. WGS, SNP array, and Sanger sequencing did not detect the previously described genetic defects associated with familial PHP1B. Sanger sequencing also ruled out any novel genetic alterations in the entire NESP55/AS region. However, the analysis of 28 consecutive SNPs could not exclude the possibility of paternal heterodisomy in a span of 22 kb comprising exon NESP55 and AS exon 5. CONCLUSION: Our comprehensive analysis of a pair of monozygotic twins with sporPHP1B ruled out all previously described genetic causes. Twin concordance indicates that the causative event was an imprinting error earlier than the timing of monozygotic twinning.

Exploring a Suitable Marker of Glycemic Response to Dulaglutide in Patients with Type 2 Diabetes: A Retrospective Study.

INTRODUCTION: Previous studies suggested that ß-cell function markers such as fasting and postprandial serum C-peptide and C-peptide increment (FCPR, PCPR, and ΔCPR, respectively) may be useful in estimating glycemic response to glucagon-like peptide-1 receptor agonists. However, it remains elusive whether baseline glycemic control confounds these markers. Here we aimed to identify the least confounded ß-cell function markers and investigate whether these markers could predict glycemic response to dulaglutide. METHODS: We evaluated FCPR, PCPR, and ΔCPR levels in patients with type 2 diabetes who initiated dulaglutide treatment after a standardized meal tolerance test (MTT). We first investigated the confounding effects of baseline HbA1c on ß-cell function markers using Pearson's correlation test. Then, we evaluated the association between each ß-cell function marker and glycemic response (HbA1c change 0-6 months) to dulaglutide using generalized linear model and logistic regression analysis with adjustment for baseline HbA1c. RESULTS: In 141 patients, baseline HbA1c was significantly inversely correlated with PCPR and ΔCPR (P < 0.01 for both) but not with FCPR (r = 0.02; P = 0.853), suggesting that FCPR was the marker least confounded by baseline glycemic control. Of all patients, 59 continued dulaglutide for at least 6 months without initiating any additional glucose-lowering medications. Mean ± SE HbA1c change 0-6 months was - 1.16 ± 0.17% (P < 0.001 vs. baseline). The ß-cell function markers were significantly associated with HbA1c change 0-6 months in the generalized linear model. FCPR was also a significant predictor for achieving a reduction in HbA1c of at least 1% (P = 0.044) with an area under the receiver operating characteristic curve of 0.83 (sensitivity = 0.81 and specificity = 0.79). CONCLUSION: Fasting and meal-induced C-peptide levels are associated with glycemic response to dulaglutide, among which FCPR is least confounded by baseline glycemic control, suggesting its utility as a marker for glycemic response to dulaglutide.

First Japanese Family With PDX1-MODY (MODY4): A Novel PDX1 Frameshift Mutation, Clinical Characteristics, and Implications.

CONTEXT: The PDX1 gene encodes pancreatic and duodenal homeobox, a critical transcription factor for pancreatic ß-cell differentiation and maintenance of mature ß-cells. Heterozygous loss-of-function mutations cause PDX1-MODY (MODY4). CASE DESCRIPTION: Our patient is an 18-year-old lean man who developed diabetes at 16 years of age. Given his early-onset age and leanness, we performed genetic testing. Targeted next-generation sequencing and subsequent Sanger sequencing detected a novel heterozygous frameshift mutation (NM_00209.4:c.218delT. NP_000200.1: p.Leu73Profs*50) in the PDX1 transactivation domain that resulted in loss-of-function and was validated by an in vitro functional study. The proband and his 56-year-old father, who had the same mutation, both showed markedly reduced insulin and gastric inhibitory polypeptide (GIP) secretion compared with the dizygotic twin sister, who was negative for the mutation and had normal glucose tolerance. The proband responded well to sitagliptin, suggesting its utility as a treatment option. Notably, the proband and his father showed intriguing phenotypic differences: the proband had been lean for his entire life but developed early-onset diabetes requiring an antihyperglycemic agent. In contrast, his father was overweight, developed diabetes much later in life, and did not require medication, suggesting the oligogenic nature of PDX1-MODY. A review of all reported cases of PDX1-MODY also showed heterogeneous phenotypes regarding onset age, obesity, and treatment, even in the presence of the same mutation. CONCLUSIONS: We identified the first Japanese family with PDX1-MODY. The similarities and differences found among the cases highlight the wide phenotypic spectrum of PDX1-MODY.