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.

Macrophages rely on extracellular serine to suppress aberrant cytokine production.

A growing body of evidence indicates that cellular metabolism is involved in immune cell functions, including cytokine production. Serine is a nutritionally non-essential amino acid that can be generated by de novo synthesis and conversion from glycine. Serine contributes to various cellular responses, but the role in inflammatory responses remains poorly understood. Here, we show that macrophages rely on extracellular serine to suppress aberrant cytokine production. Depleting serine from the culture media reduced the cellular serine content in macrophages markedly, suggesting that macrophages depend largely on extracellular serine rather than cellular synthesis. Under serine deprivation, macrophages stimulated with lipopolysaccharide showed aberrant cytokine expression patterns, including a marked reduction of anti-inflammatory interleukin-10 expression and sustained expression of interleukine-6. Transcriptomic and metabolomics analyses revealed that serine deprivation causes mitochondrial dysfunction: reduction in the pyruvate content, the NADH/NAD+ ratio, the oxygen consumption rate, and the mitochondrial production of reactive oxygen species (ROS). We also found the role of mitochondrial ROS in appropriate cytokine production. Thus, our results indicate that cytokine production in macrophages is tightly regulated by the nutritional microenvironment.

Alu-Mediated MEN1 Gene Deletion and Loss of Heterozygosity in a Patient with Multiple Endocrine Neoplasia Type 1.

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disorder caused by mutations of the tumor suppressor gene MEN1. Most of the germline MEN1 gene mutations have been small mutations, and the whole gene deletion is rarely observed. In the present study, we revealed Alu retrotransposon-mediated de novo germline deletion of the whole MEN1 gene and somatic copy-neutral loss of heterozygosity (LOH) in a patient with MEN1. The patient is a 39-year-old woman who was referred to our department for the management of prolactinoma. She was also diagnosed with primary hyperparathyroidism and suspected of MEN1. Although nucleotide sequencing did not detect any MEN1 gene mutations, multiplex ligation-dependent probe amplification (MLPA) revealed a large germline deletion of the MEN1 gene. Subsequent quantitative polymerase chain reaction (qPCR)-based copy number mapping showed a monoallelic loss of approximately 18.5-kilobase region containing the whole MEN1 gene. Intriguingly, the 2 breakpoints were flanked by Alu repetitive elements, suggesting the contribution of Alu/Alu-mediated rearrangements (AAMR) to the whole MEN1 gene deletion. Furthermore, copy number mapping using MLPA and qPCR in combination with single nucleotide polymorphism analysis revealed copy-neutral LOH as a somatic event for parathyroid tumorigenesis. In conclusion, copy number mapping revealed a novel combination of Alu/Alu-mediated de novo germline deletion of the MEN1 gene and somatic copy-neutral LOH as a cytogenetic basis for the MEN1 pathogenesis. Moreover, subsequent in silico analysis highlighted the possible predisposition of the MEN1 gene to Alu retrotransposon-mediated genomic deletion.

Facilitating screening of Klinefelter syndrome among patients with diabetes.

Klinefelter syndrome (KS) is frequently complicated by diabetes. However, it is severely underdiagnosed due to a lack of reliable screening methods. We diagnosed two patients with KS at the Center for Diabetes and Endocrinology, Tazuke Kofukai Medical Research Institute Kitano Hospital, Osaka, Japan. By comparing the patients with 39 non-KS patients with diabetes, we propose a screening tool for KS in patients with diabetes.

The H3K9 methyltransferase Setdb1 regulates TLR4-mediated inflammatory responses in macrophages.

Proinflammatory cytokine production in macrophages involves multiple regulatory mechanisms, which are affected by environmental and intrinsic stress. In particular, accumulating evidence has suggested epigenetic control of macrophage differentiation and function mainly in vitro. SET domain, bifurcated 1 (Setdb1, also known as Eset) is a histone 3 lysine 9 (H3K9)-specific methyltransferase and is essential for early development of embryos. Here we demonstrate that Setdb1 in macrophages potently suppresses Toll-like receptor 4 (TLR4)-mediated expression of proinflammatory cytokines including interleukin-6 through its methyltransferase activity. As a molecular mechanism, Setdb1-deficiency decreases the basal H3K9 methylation levels and augments TLR4-mediated NF-κB recruitment on the proximal promoter region of interleukin-6, thereby accelerating interleukin-6 promoter activity. Moreover, macrophage-specific Setdb1-knockout mice exhibit higher serum interleukin-6 concentrations in response to lipopolysaccharide challenge and are more susceptible to endotoxin shock than wildtype mice. This study provides evidence that the H3K9 methyltransferase Setdb1 is a novel epigenetic regulator of proinflammatory cytokine expression in macrophages in vitro and in vivo. Our data will shed insight into the better understanding of how the immune system reacts to a variety of conditions.

Activating transcription factor 4 links metabolic stress to interleukin-6 expression in macrophages.

Chronic inflammation is a molecular element of the metabolic syndrome and type 2 diabetes. Saturated fatty acids (SFAs) are considered to be an important proinflammatory factor. However, it is still incompletely understood how SFAs induce proinflammatory cytokine expression. Hereby we report that activating transcription factor (ATF) 4, a transcription factor that is induced downstream of metabolic stresses including endoplasmic reticulum (ER) stress, plays critical roles in SFA-induced interleukin-6 (Il6) expression. DNA microarray analysis using primary macrophages revealed that the ATF4 pathway is activated by SFAs. Haploinsufficiency and short hairpin RNA-based knockdown of ATF4 in macrophages markedly inhibited SFA- and metabolic stress-induced Il6 expression. Conversely, pharmacological activation of the ATF4 pathway and overexpression of ATF4 resulted in enhanced Il6 expression. Moreover, ATF4 acts in synergy with the Toll-like receptor-4 signaling pathway, which is known to be activated by SFAs. At a molecular level, we found that ATF4 exerts its proinflammatory effects through at least two different mechanisms: ATF4 is involved in SFA-induced nuclear factor-κB activation; and ATF4 directly activates the Il6 promoter. These findings provide evidence suggesting that ATF4 links metabolic stress and Il6 expression in macrophages.