Correction: High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice.

[This corrects the article DOI: 10.1371/journal.pone.0296006.].

Dectin-2 Deficiency Promotes Proinflammatory Cytokine Release From Macrophages and Impairs Insulin Secretion.

Pancreatic islet inflammation plays a crucial role in the etiology of type 2 diabetes (T2D). Macrophages residing in pancreatic islets have emerged as key players in islet inflammation. Macrophages express a plethora of innate immune receptors that bind to environmental and metabolic cues and integrate these signals to trigger an inflammatory response that contributes to the development of islet inflammation. One such receptor, Dectin-2, has been identified within pancreatic islets; however, its role in glucose metabolism remains largely unknown. Here we have demonstrated that mice lacking Dectin-2 exhibit local inflammation within islets, along with impaired insulin secretion and ß-cell dysfunction. Our findings indicate that these effects are mediated by proinflammatory cytokines, such as interleukin (IL)-1α and IL-6, which are secreted by macrophages that have acquired an inflammatory phenotype because of the loss of Dectin-2. This study provides novel insights into the mechanisms underlying the role of Dectin-2 in the development of islet inflammation.

High frequency of germline recombination in Nestin-Cre transgenic mice crossed with Glucagon-like peptide 1 receptor floxed mice.

The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods.

[A case of repeated severe hypoglycemia caused by accidental ingestion of sulfonylurea in an elderly patient].

An 81-year-old man was being treated with oral medication for chronic heart failure and epilepsy. He had no history of diabetes, cirrhosis, or gastric surgery. He was admitted to our hospital due to disturbance of consciousness. His blood glucose level was 6 mg/dl, with a relatively high insulin level (14.4 µU/ml). Computed tomography and a 48 h fasting test showed no signs of insulinoma. There were no signs of reactive hypoglycemia, insulin autoimmune syndrome, or adrenal insufficiency. His wife had been taking medication for diabetes, including sulfonylurea. She had dementia, and he managed her medication. Since his medication was found in his wife's medicine box, we considered the possibility that he might have taken sulfonylurea by mistake. We asked his daughter to manage their medicine. However, one month later, he was admitted to our hospital again with severe hypoglycemia. His wife's HbA1c value and estimated glomerular filtration rate were 6.9% and 30 ml/min/1.73 m2. We asked his wife's home doctor to stop sulfonylurea prescription, and the hypoglycemia did not recur, with his wife's level of HbA1c remaining stable.Elderly individuals and patients with an impaired renal function are prone to hypoglycemia from sulfonylurea. In elderly households, there is a possibility of accidental ingestion of oral hypoglycemic agents by other family members living with the patient. It is therefore necessary to understand and manage the medications of family members living together. It is also important to avoid prescribing medications with a high risk of hypoglycemia to elderly patients.

Non-alcoholic fatty liver disease in mice with hepatocyte-specific deletion of mitochondrial fission factor.

AIMS/HYPOTHESIS: Mitochondria are highly dynamic organelles continuously undergoing fission and fusion, referred to as mitochondrial dynamics, to adapt to nutritional demands. Evidence suggests that impaired mitochondrial dynamics leads to metabolic abnormalities such as non-alcoholic steatohepatitis (NASH) phenotypes. However, how mitochondrial dynamics are involved in the development of NASH is poorly understood. This study aimed to elucidate the role of mitochondrial fission factor (MFF) in the development of NASH. METHODS: We created mice with hepatocyte-specific deletion of MFF (MffLiKO). MffLiKO mice fed normal chow diet (NCD) or high-fat diet (HFD) were evaluated for metabolic variables and their livers were examined by histological analysis. To elucidate the mechanism of development of NASH, we examined the expression of genes related to endoplasmic reticulum (ER) stress and lipid metabolism, and the secretion of triacylglycerol (TG) using the liver and primary hepatocytes isolated from MffLiKO and control mice. RESULTS: MffLiKO mice showed aberrant mitochondrial morphologies with no obvious NASH phenotypes during NCD, while they developed full-blown NASH phenotypes in response to HFD. Expression of genes related to ER stress was markedly upregulated in the liver from MffLiKO mice. In addition, expression of genes related to hepatic TG secretion was downregulated, with reduced hepatic TG secretion in MffLiKO mice in vivo and in primary cultures of MFF-deficient hepatocytes in vitro. Furthermore, thapsigargin-induced ER stress suppressed TG secretion in primary hepatocytes isolated from control mice. CONCLUSIONS/INTERPRETATION: We demonstrated that ablation of MFF in liver provoked ER stress and reduced hepatic TG secretion in vivo and in vitro. Moreover, MffLiKO mice were more susceptible to HFD-induced NASH phenotype than control mice, partly because of ER stress-induced apoptosis of hepatocytes and suppression of TG secretion from hepatocytes. This study provides evidence for the role of mitochondrial fission in the development of NASH.

Nivolumab-induced thyroid dysfunction lacking antithyroid antibody is frequently evoked in Japanese patients with malignant melanoma.

BACKGROUND: Nivolumab, an anti-programmed cell death-1 monoclonal antibody, has improved the survival of patients with malignant melanoma. Despite its efficacy, nivolumab inconsistently induces thyroid dysfunction as an immune-related adverse event (irAE). This study aimed to evaluate nivolumab-induced thyroid dysfunction to determine the risks and mechanisms of thyroid irAEs. METHODS: After excluding 10 patients, data of 24 patients with malignant melanoma (aged 17-85 years; 54% female) were retrospectively analyzed. RESULTS: Thyroid irAEs were observed in seven patients (29%). Three patients had hypothyroidism after preceding transient thyrotoxicosis, and the other four patients had hypothyroidism without thyrotoxicosis. Levothyroxine-Na replacement was required in three patients. Antithyroid antibody (ATA) titer was elevated in one of four assessable patients. The average (±SD) time to onset of thyroid irAE was 33.6 (±21.9) weeks. The administration period of nivolumab was longer in patients with thyroid irAEs than in those without thyroid irAEs (P < 0.01). There were no significant differences between patients with and without thyroid irAEs regarding age, sex, tumor stage, response to nivolumab therapy, baseline thyroid function, antithyroid peroxidase antibody (anti-TPO Ab) and antithyroglobulin antibody (anti-Tg Ab). CONCLUSIONS: Thyroid dysfunction was a common irAE of nivolumab in malignant melanoma. Neither anti-TPO Ab nor anti-Tg Ab was associated with the risk for nivolumab-induced thyroid dysfunction. A conventional ATA-independent mechanism might be involved in thyroid irAEs. Further studies are required to clarify the mechanism and identify the predictive factors of thyroid irAEs.

Isolated ACTH deficiency probably induced by autoimmune-related mechanism evoked with nivolumab.

Nivolumab, an anti-programmed death-1 antibody, is a breakthrough treatment for several malignancies. Its specific adverse effects caused by autoimmunity are termed immune-related adverse events, which involve several endocrine dysfunctions. Herein, we report two cases of isolated adrenocorticotropic hormone (ACTH) deficiency induced by nivolumab for the treatment of metastatic malignant melanoma. Case 1 was a 39-year-old man and Case 2 was a 50-year-old woman, both of whom presented with progressive melanoma. After 13 courses of nivolumab administration, both cases were diagnosed with adrenal insufficiency. Despite their basal serum ACTH and cortisol levels being low with little response to corticotropin-releasing hormone loading, other anterior pituitary hormone levels were preserved. Based on these endocrinological data, isolated ACTH deficiency was diagnosed. Magnetic resonance imaging showed normal pituitary glands, excluding hypophysitis. Finally, hydrocortisone replacement enabled the patients to continue nivolumab treatment. Therefore, it is important to consider isolated ACTH syndrome as a possible and potentially severe immune-related adverse event of nivolumab, even when head magnetic resonance imaging of affected cases does not show enlargement. We should not misdiagnose hidden immune-related adverse events behind general complaints of malignancies such as general malaise and appetite loss, to allow successful treatment using this beneficial immune checkpoint inhibitor.

Disruption of mitochondrial fission in the liver protects mice from diet-induced obesity and metabolic deterioration.

AIM/HYPOTHESIS: Mitochondria and the endoplasmic reticulum (ER) physically interact by close structural juxtaposition, via the mitochondria-associated ER membrane. Inter-organelle communication between the ER and mitochondria has been shown to regulate energy metabolism and to be central to the modulation of various key processes such as ER stress. We aimed to clarify the role of mitochondrial fission in this communication. METHODS: We generated mice lacking the mitochondrial fission protein dynamin-related protein 1 (DRP1) in the liver (Drp1LiKO mice). RESULTS: Drp1LiKO mice showed decreased fat mass and were protected from high-fat diet (HFD)-induced obesity. Analysis of liver gene expression profiles demonstrated marked elevation of ER stress markers. In addition, we observed increased expression of the fibroblast growth factor 21 (FGF21) gene through induction of activating transcription factor 4, master regulator of the integrated stress response. CONCLUSIONS/INTERPRETATION: Disruption of mitochondrial fission in the liver provoked ER stress, while inducing the expression of FGF21 to increase energy expenditure and protect against HFD-induced obesity.

Dietary vitamin D3 improves postprandial hyperglycemia in aged mice.

Type 2 Diabetes is closely associated with our daily diets and has become a global health problem with an increasing number of patients. Recent observational and randomized studies on vitamin D3 suggested that higher plasma 25-hydroxyvitamin D3 [25(OH)D3] concentrations and more vitamin D3 intake are associated with lower risk of type 2 diabetes, which is characterized by postprandial hyperglycemia due to inappropriate glucose stimulated insulin secretion (GSIS) and its age-dependent increase of onset. However, rapid action of dietary vitamin D3 on the postprandial glucose profile has not been analyzed. When vitamin D3 is orally ingested in mice aged 12-14 weeks during an oral glucose tolerance test (OGTT), the serum glucose profile was not changed. In contrast, when OGTT was performed with old mice aged 30-34 weeks, the glucose profile was dramatically improved with increased insulin secretion, suggesting that orally ingested vitamin D3 potentiated GSIS in aged mice. Interestingly, there was also a significant increase in plasma GLP-1 in these aged mice. Our results suggest that orally ingested dietary vitamin D3 in aged mice improves glucose metabolism as a GLP-1 enhancer.