Ampullary Neuroendocrine Neoplasm: Clinicopathological Characteristics and Novel Endoscopic Entity.

BACKGROUND: Neuroendocrine neoplasms of the ampulla of Vater (ampullary NEN) have features of both gastrointestinal and pancreato-biliary (PB) NEN. However, the limited number of studies examining ampullary NEN makes it difficult to clarify their unique characteristics. This study aimed to elucidate the clinical characteristics of ampullary NEN. METHODS: We enrolled 162 patients with PB-NEN diagnosed at Kyushu University Hospital between 2011 and 2020. Clinical features, pathological diagnoses, treatments, and prognoses were retrospectively analyzed. We also compared ampullary NEN with pancreatic NEN (PanNEN). RESULTS: We analyzed 10 ampullary NEN cases and 149 PanNEN cases. The ampullary NEN cases consisted of 4 cases of neuroendocrine tumor Grade 1 (NET G1), 1 NET G2 (Grade 2), and 5 neuroendocrine carcinomas (NECs). The incidences of NEC and cholangitis were significantly higher in ampullary NEN than in PanNEN. All ampullary NETs had a submucosal tumor-like appearance, as identified by endoscopic ultrasound-guided fine needle aspiration. We treated small NET G1 (<10 mm) with endoscopic papillectomy and large NET G1 with pancreaticoduodenectomy. There were no cases of recurrence after resection. All ampullary NECs presented with the characteristic endoscopic finding of a "crater sign" similar to deep-mining ulcers seen in gastric malignant lymphoma. Four cases underwent surgical resection, and 1 case was unresectable. Two patients who underwent multidisciplinary treatment were maintained without recurrence for over 2 years. CONCLUSIONS: Endoscopic findings showed identifiable distinctions between ampullary NETs and NECs.

Circulating CD8+CD122+ T cells as a prognostic indicator of pancreatic cancer.

PURPOSE: The distribution of tissue infiltrating lymphocytes has been shown to affect the prognosis of patients with pancreatic cancer in some previous studies. However, the role of peripheral lymphocytes in pancreatic cancer remains debated. The purpose of this study was to analyze the peripheral subtypes of T lymphocytes, and establish their association with the prognosis of patients with pancreatic cancer. METHODS: Blood and tissue samples were collected from patients with metastatic pancreatic cancer (n = 54), resectable pancreatic cancer (n = 12), and benign pancreatic cysts (n = 52) between April 2019 and January 2022 and analyzed. RESULTS: Patients with metastatic pancreatic cancer had a larger proportion of both tumor-suppressive and tumor-promoting cells than those with benign pancreatic cysts. In addition, the proportion of peripheral CD4+ T cells positively correlated with the survival of patients with metastatic pancreatic cancer, and the proportion of peripheral CD8+CD122+ T cells was associated with early mortality (< 90 days). After chemotherapy, CD8+CD122+ T cells decreased in patients who had a partial response or stable disease. Moreover, by analyzing resected specimens, we first proved that the existence of CD8+CD122+ T cells in a tumor microenvironment (TME) depends on their proportion in peripheral blood. CONCLUSION: Circulating CD8+CD122+ T cells can be a prognostic indicator in patients with pancreatic cancer.

Feasibility and Efficacy of Endoscopic Ultrasound-Guided Hepaticogastrostomy Without Dilation: A Propensity Score Matching Analysis.

BACKGROUND: Recently, endoscopic ultrasound-guided hepaticogastrostomy (EUS-HGS) procedures have been gradually established; nonetheless, some adverse events (AEs) have been reported. Dilation procedures using a non-cautery or cautery device increase the incidence of AEs in EUS-HGS. AIMS: We evaluated EUS-HGS procedures without dilation and the factors associated with dilation. METHODS: We enrolled 79 patients who underwent EUS-HGS between July 2015 and March 2021 at two centers, 72 of whom had technical success (72/79, 91%). During the EUS-HGS procedures, we defined patients without dilation procedures as the dilation (-) group. We divided the patients into two groups: the dilation (+) (35 patients) and dilation (-) (37 patients) groups. We performed a propensity score matching analysis to adjust for confounding bias between the two groups. Multivariable logistic regression analysis was conducted to identify factors associated with dilation. RESULTS: There was no difference in clinical success rate between the dilation (+) and dilation (-) groups (91% vs. 95%, P = 0.545). The AE rate (P = 0.013) and long procedure time (P = 0.017) were significantly higher in the dilation (+) group than in the dilation (-) group before and after propensity score matching. Factors associated with dilation were plastic stent placement (odds ratio [OR], 6.96; 95% confidence interval [CI], 1.68-28.7; P = 0.007) and puncture angle of ≤ 90° (OR, 44.6; 95% CI, 5.1-390; P < 0.001). CONCLUSIONS: A dilation procedure in EUS-HGS may not always be necessary. However, patients with an angle of ≤ 90° between the needle and intrahepatic biliary tract or plastic stent deployment require dilation procedures.

A clinical analysis on functioning pancreatic neuroendocrine tumors (focusing on VIPomas): a single-center experience.

VIPomas are generally rare functioning pancreatic neuroendocrine tumors (PanNETs) that cause watery diarrhea, hypokalemia, and achlorhydria. Due to their extreme rarity, the clinicopathological features and outcomes of VIPomas have not been well reported. This study aimed to determine the diagnostic and therapeutic characteristics and prognosis of VIPomas and to compare them with other PanNETs at a Japanese reference hospital. Medical records of 293 patients with PanNETs were collected. Patient and tumor characteristics and outcomes were retrospectively reviewed. This cohort had only 1.4% (four patients) of patients with VIPomas, and three of these patients changed from non-functioning (NF-) PanNETs during their disease course. Recurrences of hormonal symptoms were observed in all patients despite the initial controls, and all of them died from their disease, more specifically mainly from hormonal symptoms. Compared to the other PanNETs, VIPomas were all located at the pancreatic tail, were larger, and had a higher Ki-67 index and more metastasis. The median survival time was significantly shorter for patients with VIPoma than for those with NF-PanNET (5.9 vs. 26.7 years, p < 0.0001), insulinoma (21.8 years, p < 0.0001), and gastrinoma (12.3 years, p = 0.0325). This study presents the possibility of shifting from non-symptomatic to symptomatic VIPomas as they grow or of transforming from NF-PanNETs to VIPomas. VIPomas should be considered in patients with relatively large NF-PanNETs, especially those located in the pancreatic tail, when diarrhea is continuously observed. As hormonal symptoms are an important cause of death in VIPomas, long-term symptomatic control, which is relatively difficult, is of great significance.

Bazedoxifene Ameliorates Homocysteine-Induced Apoptosis via NADPH Oxidase-Interleukin 1ß and 6 Pathway in Osteocyte-like Cells.

Homocysteine (Hcy) increases oxidation and inflammation; however, the mechanism of Hcy-induced bone fragility remains unclear. Because selective estrogen modulators (SERMs) have an anti-oxidative effect, SERMs may rescue the Hcy-induced bone fragility. We aimed to examine whether oxidative stress and pro-inflammatory cytokines such as interleukin (IL)-1ß and IL-6 are involved in the Hcy-induced apoptosis of osteocytes and whether bazedoxifene (BZA) inhibits the detrimental effects of Hcy. We used mouse osteocyte-like cell lines MLO-Y4-A2 and Ocy454. Apoptosis was examined by DNA fragmentation ELISA and TUNEL staining, and gene expression was evaluated by real-time PCR. Hcy 5 mM significantly increased expressions of NADPH oxidase (Nox)1, Nox2, IL-1ß, and IL-6 as well as apoptosis in MLO-Y4-A2 cells. Nox inhibitors, diphenyleneiodonium chloride and apocynin, significantly suppressed Hcy-induced IL-1ß and IL-6 expressions. In contrast, an IL-1ß receptor antagonist and an IL-6 receptor monoclonal antibody had no effects on Hcy-induced Nox1 and Nox2 expressions, but significantly rescued Hcy-induced apoptosis. BZA (1 nM-1 µM) and 17ß estradiol 100 nM significantly rescued Hcy-induced apoptosis, while an estrogen receptor blocker ICI 182,780 reversed the effects of BZA and 17ß estradiol. BZA also rescued Hcy-induced apoptosis of Ocy454 cell, and ICI canceled the effect of BZD. Moreover, BZA significantly ameliorated Hcy-induced expressions of Nox1, Nox2, IL-1ß, and IL-6, and ICI canceled the effects of BZA on their expressions. Hcy increases apoptosis through stimulating Nox 1 and Nox 2-IL-1ß and IL-6 expressions in osteocyte-like cells. BZA inhibits the detrimental effects of Hcy on osteocytes via estrogen receptor.

Insulin-Like Growth Factor-I Protects Against the Detrimental Effects of Advanced Glycation End Products and High Glucose in Myoblastic C2C12 Cells.

Previous studies suggested that advanced glycation end products (AGEs) and insulin-like growth factor-I (IGF-I) are involved in the mechanism of diabetes-induced sarcopenia. In this study, we examined effects of treatments with AGEs and/or IGF-I for 24 h on myogenic differentiation and apoptosis in mouse myoblastic C2C12 cells. Real-time PCR and Western blot were performed to investigate mRNA and protein expressions, and apoptosis was examined by using a DNA fragment detection ELISA kit. AGE3 significantly decreased mRNA and protein expressions of MyoD and Myogenin, whereas IGF-I significantly increased them and attenuated the effects of AGE3. AGEs significantly decreased endogenous IGF-I mRNA expression and suppressed IGF-I-induced Akt activation. High glucose (22 mM) significantly increased mRNA expression of Rage, a receptor for AGEs, while IGF-I significantly decreased it. DNA fragment ELISA showed that AGE2 and AGE3 significantly increased apoptosis of C2C12 cells, whereas IGF-I significantly suppressed the AGE2- and AGE3-induced apoptosis. In contrast, high glucose enhanced AGE3-induced apoptosis. IGF-I significantly attenuated the effects of high glucose plus AGE3 on the mRNA and protein expressions of MyoD and Myogenin as well as the apoptosis. These findings indicate that AGEs inhibit myogenic differentiation and increase apoptosis in C2C12 cells, and that high glucose increases RAGE and enhances the AGE3-induced apoptosis, suggesting that AGEs and high glucose might contribute to the reduction of muscle mass and function. Moreover, IGF-I attenuated the detrimental effects of AGEs and high glucose in myoblastic cells; thus, IGF-I-Akt signal could be a therapeutic target of DM-induced sarcopenia.

Overweight and underweight are risk factors for vertebral fractures in patients with type 2 diabetes mellitus.

The aim of this cross-sectional study was to examine the association between body mass index (BMI) and the prevalence of vertebral fracture (VF) in Japanese patients with type 2 diabetes (T2DM). A total of 798 patients with T2DM were enrolled. VF was determined semi-quantitatively using lateral X-ray films. The association between BMI quartiles (Q1: ≤ 21.2 kg/m2, Q2: 21.3-23.4 kg/m2, Q3: 23.5-25.8 kg/m2, Q4: 25.9≤ kg/m2) and the presence of VF was examined. Multiple logistic regression analyses adjusted for age, sex, diabetes duration, hemoglobin A1c (HbA1c), estimated glomerular filtration rate, and albumin showed that Q1, Q3, and Q4 were significantly associated with an increased VF risk compared to Q2, which served as a reference [Q1; odds ratio (OR) = 1.91, 95% confidence interval (CI) 1.24-2.95, p = 0.004, Q3; OR = 1.65, 95% CI 1.07-2.55, p = 0.023, and Q4; OR = 2.18, 95% CI 1.39-3.41, p < 0.001]. Moreover, these associations remained significant after additional adjustment for femoral neck T-score, a bone resorption marker, urinary N-terminal cross-linked telopeptide of type-I collagen, and use of insulin and thiazolidinedione. Our study shows for the first time that both overweight and underweight were associated with the bone mineral density (BMD)-independent risk of VF in patients with T2DM. Therefore, body weight control should be considered as a protective measure against diabetes-related bone fragility.

Osteoporosis and vertebral fracture are associated with deterioration of activities of daily living and quality of life in patients with type 2 diabetes mellitus.

Patients with type 2 diabetes mellitus (T2DM) have an increased risk of fragility fracture. However, whether diabetes-related osteoporosis independently contributes to the deterioration of activities of daily living (ADLs) and quality of life (QOL) is unclear. This cross-sectional study investigated the association between osteoporosis, ADLs, and QOL in 309 patients with T2DM. ADLs and QOL were assessed using Barthel Index (BI) and a SF-36 questionnaire. Multiple logistic regression analyses adjusted for age, gender, T2DM duration, body mass index, hemoglobin A1c, estimated GFR, diabetic neuropathy, retinopathy, nephropathy, cardiovascular disease, cerebrovascular disease, peripheral artery disease, and anti-diabetic treatments were conducted. The number of patients with osteoporosis or vertebral fracture was 166 (53.7%) and 118 (38.2%), respectively. Osteoporosis was significantly associated with lower general health (GH), social functioning (SF), and role emotional (RE) (OR 2.56, 1.79, and 1.92, respectively; all p values < 0.05 at least) and marginally associated with lower BI (OR 2.39, p = 0.068). Moreover, the presence of vertebral fracture grade 2 or 3 was significantly associated with lower BI, bodily pain (BP), GH, vitality, SF, and RE (OR 2.58, 2.01, 3.64, 1.99, 2.18, and 1.97, respectively; all p values < 0.05 at least). Osteoporosis and severe vertebral fracture were associated with the deterioration of ADLs and QOL independently of other diabetic complications. Therefore, the management of diabetes-related osteoporosis is an important strategy to avoid the deterioration of ADLs and QOL in T2DM.

Late-onset isolated adrenocorticotropic hormone deficiency caused by nivolumab: a case report.

BACKGROUND: Immune checkpoint inhibitors including nivolumab, an anti-programmed cell death protein 1 antibody, are recently developed cancer immunotherapy agents. Immune checkpoint inhibitors are known to cause autoimmune-related side effects including endocrine dysfunctions. However, there are few reports on late-onset isolated adrenocorticotropic hormone (ACTH) deficiency caused by nivolumab. CASE PRESENTATION: The patient was a 72-year-old female. When she was 64 years old, she was diagnosed with malignant melanoma of the left thigh accompanied by left inguinal lymph node metastases, and she received several courses of chemotherapy for malignant melanoma followed by the resection of these lesions. At 71 years of age, multiple metastases were found and treatment with nivolumab 2 mg/kg every 3 weeks was initiated. Six months later, replacement with levothyroxine was started because of hypothyroidism following mild transient thyrotoxicosis. Eleven months after the beginning of nivolumab, the treatment was discontinued because of tumor expansion. Four months after the discontinuation of nivolumab, general malaise and appetite loss worsened, and 2 months later, hyponatremia (Na; 120-127 mEq/L) and hypoglycemia (fasting plasma glucose; 62 mg/dL) appeared. Her ACTH and cortisol levels were extremely low (ACTH; 9.6 pg/mL, cortisol; undetectable). Challenge tests for anterior pituitary hormones showed that responses of ACTH and cortisol secretion to corticotropin-releasing hormone were disappeared, although responses of other anterior pituitary hormones were preserved. Thus, she was diagnosed with isolated ACTH deficiency. Her symptoms were improved after treatment with hydrocortisone. CONCLUSIONS: The present report showed a case of late-onset isolated ACTH deficiency accompanied by hyponatremia, which was diagnosed 6 months after the discontinuation of nivolumab. The effects of nivolumab last for a long time and the side effects of nivolumab can also appear several months after discontinuation of the drug. Repeated monitoring of serum sodium levels may be a beneficial strategy to find the unexpected development of adrenal insufficiency even after discontinuation of nivolumab.

Successful reduction of ACTH secretion in a case of intractable Cushing's disease with pituitary Crooke's cell adenoma by combined modality therapy including temozolomide.

Crooke's cell adenoma (CCA) is an aggressive subtype of corticotroph adenoma; however, CCA is associated with a high incidence of low expression of methyl guanine methyl transferase (MGMT), suggesting that temozolomide (TMZ) treatment might be effective for this tumor type. The case of a 56-year-old woman with Cushing's disease caused by a pituitary CCA is presented. At the age of 38 years, the patient presented to our hospital with polyuria and a visual field defect. MRI and laboratory studies showed a 4.5-cm-diameter pituitary tumor with plasma adrenocorticotropic hormone (ACTH) and serum cortisol levels of more than 500 pg/mL and 40 µg/dL, respectively. At 39 years of age, the patient underwent a craniotomy, and her plasma ACTH and cortisol levels decreased to less than 200 pg/mL and 10 µg/dL, respectively; however, these hormone levels increased gradually to 3,940 pg/mL and 70 µg/dL, respectively, by the time the patient was 56 years old. Histopathological re-examination of the previously resected specimen showed that the pituitary tumor was MGMT-negative CCA. TMZ treatment after the second operation decreased the plasma ACTH levels from 600-800 pg/mL to 70-300 pg/mL. No signs of recurrence were observed in the seven years following these treatments with added prophylactic radiation therapy. These clinical findings suggest that TMZ treatment to patients with CCA accompanied with elevated ACTH may be good indication to induce lowering ACTH levels and tumor shrinkage.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway.

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.

Glucose uptake inhibition decreases expressions of receptor activator of nuclear factor-kappa B ligand (RANKL) and osteocalcin in osteocytic MLO-Y4-A2 cells.

Bone and glucose metabolism are closely associated with each other. Both osteoblast and osteoclast functions are important for the action of osteocalcin, which plays pivotal roles as an endocrine hormone regulating glucose metabolism. However, it is unknown whether osteocytes are involved in the interaction between bone and glucose metabolism. We used MLO-Y4-A2, a murine long bone-derived osteocytic cell line, to investigate effects of glucose uptake inhibition on expressions of osteocalcin and bone-remodeling modulators in osteocytes. We found that glucose transporter 1 (GLUT1) is expressed in MLO-Y4-A2 cells and that treatment with phloretin, a GLUT inhibitor, significantly inhibited glucose uptake. Real-time PCR and Western blot showed that phloretin significantly and dose-dependently decreased the expressions of RANKL and osteocalcin, whereas osteoprotegerin or sclerostin was not affected. Moreover, phloretin activated AMP-activated protein kinase (AMPK), an intracellular energy sensor. Coincubation of ara-A, an AMPK inhibitor, with phloretin canceled the phloretin-induced decrease in osteocalcin expression, but not RANKL. In contrast, phloretin suppressed phosphorylation of ERK1/2, JNK, and p38 MAPK, and treatments with the p38 inhibitor SB203580 and the MEK inhibitor PD98059, but not the JNK inhibitor SP600125, significantly decreased expressions of RANKL and osteocalcin. These results indicate that glucose uptake by GLUT1 is required for RANKL and osteocalcin expressions in osteocytes, and that inhibition of glucose uptake decreases their expressions through AMPK, ERK1/2, and p38 MAPK pathways. These findings suggest that lowering glucose uptake into osteocytes may contribute to maintain blood glucose levels by decreasing osteocalcin expression and RANKL-induced bone resorption.

Osteoblast AMP-activated protein kinase regulates glucose metabolism and bone mass in adult mice.

Previous studies have shown that AMP-activated protein kinase (AMPK), a crucial regulator of energy homeostasis, plays important roles in osteoblast differentiation and mineralization. However, little is known about in vivo roles of osteoblastic AMPK in glucose metabolism and bone mass regulation in adult mice. Here, we used the inducible Cre system to control the onset of Ampk disruption after birth by removing doxycycline supplementation. We conditionally inactivated Ampk in osterix (Osx)-expressing cells in 3-week-old Ampk-/- mice. After 6 months of Ampk inactivation, the Ampk-/- mice displayed lower serum osteocalcin levels as well as glucose intolerance and insulin resistance, as indicated by glucose tolerance and insulin tolerance tests, respectively, when compared with wild-type mice. After 18 months of Ampk inactivation, micro computed tomography showed significant reductions in trabecular bone volume and cortical bone thickness in the femur of Ampk-/- mice when compared with wild-type mice. Moreover, bone stiffness was significantly lower in Ampk-/- mice than in wild-type mice. This is the first study to show that osteoblast AMPK plays an important roles in glucose metabolism and in maintaining trabecular bone volume, cortical thickness, and bone strength in adult mice.

Inhibition of adenosine monophosphate-activated protein kinase suppresses bone morphogenetic protein-2-induced mineralization of osteoblasts via Smad-independent mechanisms.

Previous studies showed that adenosine monophosphate-activated protein kinase (AMPK), which plays as an intracellular energy sensor, promotes the differentiation and mineralization of osteoblasts via enhancing expression of bone morphogenetic protein (BMP)-2, which is a potent inducer of osteoblastogenesis. Thus, the aim of this study was to examine the roles of AMPK in BMP-2-induced osteoblastogenesis. We used a murine osteoblastic cell line MC3T3-E1 and a murine marrow stromal cell line ST2. BMP-2 (50 and 100 ng/mL) stimulated alkaline phosphatase (ALP) activity and enhanced mineralization of MC3T3-E1 cells, while the effects of BMP-2 were partly abolished by an inhibitor of AMPK, ara-A (0.1 mM). Real-time PCR showed that BMP-2 significantly increased the mRNA expressions of Alp, osteocalcin (Ocn), Runx2, Osterix and Dlx-5 in MC3T3-E1 cells, while co-incubation of ara-A significantly decreased the BMP-2-stimulated expression of Alp, Ocn, and Runx2. Moreover, co-incubation of ara-A suppressed the BMP-2-induced upregulation of Alp and Ocn in ST2 cells. Western blot analysis showed that BMP-2 phosphorylated Smad1/5 although it did not affect AMPK phosphorylation in MC3T3-E1 cells. Furthermore, a BMP receptor inhibitor LDN-193189 inhibited the phosphorylation of Smad1/5, but did not affect AMPK. In addition, co-incubation of ara-A did not affect BMP-2-induced phosphorylation of Smad1/5. These findings suggest that the inhibition of AMPK activation reduces the osteo-inductive effects of BMP-2 by decreasing the expression of Alp, Ocn, and Runx2 through Smad-independent mechanisms in osteoblastic cells.

A case report of fulminant type 1 diabetes mellitus associated with drug-induced hypersensitivity syndrome in an elderly patient with coxsackie B4 virus infection and human leukocyte antigen-A24 haplotype.

Drug-induced hypersensitivity syndrome (DIHS) is a severe systemic adverse drug reaction. Previous studies showed that DIHS is associated with the onset of fulminant type 1 diabetes mellitus (FT1D). Although genetic background and abnormalities in immune response or viral infection are considered to be associated with pathogenesis of FT1D, it remains unclear whether virus infection and specific human leukocyte antigen (HLA) typing are involved in DIHS-associated FT1D. Here, we report a case of a 78-year-old female patient with FT1D after DIHS treatment. She was diagnosed as DIHS caused by carbamazepine, and treatment with predonisolone was initiated. After 46 days from the occurrence of DIHS, she was admitted to our hospital because of type 1 diabetes mellitus and diabetic ketoacidosis. Although her Hemoglobin A1c (HbA1c) was elevated by predonisolone treatment (HbA1c: 9.2%), we diagnosed her as fulminant type 1 diabetes mellitus considering the abrupt onset of the ketoacidosis. Her general condition was improved by treatment with fluid infusion and insulin administration. During her clinical course, the infection of coxsackie B4 virus was observed. In addition, the examination of HLA typing showed HLA-A24 haplotype. These findings suggest that the coxsackie B4 virus infection may be involved in the pathogenesis of DIHS-induced FT1D, and that HLA-A24 haplotype might relate to DIHS-associated FT1D.

Phloretin Promotes Adipogenesis via Mitogen-Activated Protein Kinase Pathways in Mouse Marrow Stromal ST2 Cells.

Phloretin, a glucose transporter (GLUT) inhibitor, has pleiotropic effects. The present study examined the effects of phloretin on the commitment of marrow stromal cells to adipocytes, using the mouse marrow stromal cell line ST2. Oil red O staining showed that treatment with phloretin 10⁻100 µM promoted lipid accumulation. Real-time PCR showed that phloretin significantly increased the expression of adipogenic markers, including PPARγ, C/EBPα, fatty acid synthase, fatty acid-binding protein 4, and adiponectin. Western blotting showed that phloretin inhibited ERK1/2 and JNK but activated p38 MAPK. Treatment with a MAPK/ERK kinase inhibitor and a JNK inhibitor enhanced adipogenesis, similar to phloretin. In contrast, a p38 MAPK inhibitor suppressed phloretin-induced adipogenesis. Although phloretin phosphorylated AMP-activated protein kinase (AMPK), co-incubation with an AMPK inhibitor did not block phloretin-induced adipogenesis. The 2-deoxyglucose colorimetric assay showed that phloretin and siRNA silencing of GLUT1 decreased glucose uptake. However, unlike phloretin treatment, GLUT1 silencing inhibited adipogenesis. In addition, phloretin enhanced adipogenesis in GLUT1 knocked-down cells. Taken together, phloretin induced adipogenesis of marrow stromal cells by inhibiting ERK1/2 and JNK and by activating p38 MAPK. The adipogenic effects of phloretin were independent of glucose uptake inhibition. Phloretin may affect energy metabolism by influencing adipogenesis and adiponectin expression.

Advanced Glycation End Product 3 (AGE3) Increases Apoptosis and the Expression of Sclerostin by Stimulating TGF-ß Expression and Secretion in Osteocyte-Like MLO-Y4-A2 Cells.

Advanced glycation end products (AGEs) cause bone fragility due to deterioration in bone quality. We previously reported that AGE3 induced apoptosis and inhibited differentiation via increased transforming growth factor (TGF)-ß signaling in osteoblastic cells. Additionally, we demonstrated that AGE3 increased apoptosis and sclerostin expression and decreased receptor activator of nuclear factor-κB ligand (RANKL) expression in osteocyte-like cells. However, it remains unclear whether TGF-ß signaling is involved in the effects of AGEs on apoptosis and the expression of sclerostin and RANKL in osteocytes. Effects of AGE3 on apoptosis of mouse osteocyte-like MLO-Y4-A2 cells were examined by DNA fragmentation ELISA. Expression of TGF-ß, sclerostin, and RANKL was evaluated using real-time PCR, Western blotting, and ELISA kits. To block TGF-ß signaling, we used SD208, a TGF-ß type I receptor kinase inhibitor. AGE3 (200 µg/mL) significantly increased apoptosis and mRNA expression of Sost, the gene encoding sclerostin, and decreased Rankl mRNA expression in MLO-Y4-A2 cells. AGE3 significantly increased the expression of TGF-ß. Co-incubation of SD208 with AGE3 significantly rescued AGE3-induced apoptosis in a dose-dependent manner. Moreover, SD208 restored AGE3-increased mRNA and protein expression of sclerostin. In contrast, SD208 did not affect AGE3-decreased mRNA and protein expression of RANKL. These findings suggest that AGE3 increases apoptosis and sclerostin expression through increasing TGF-ß expression in osteocytes, and that AGE3 decreases RANKL expression independent of TGF-ß signaling.

Activation of AMP-activated protein kinase decreases receptor activator of NF-κB ligand expression and increases sclerostin expression by inhibiting the mevalonate pathway in osteocytic MLO-Y4 cells.

BACKGROUND: AMP-activated protein kinase (AMPK) plays important roles in bone metabolism; however, little is known about its role in osteocytes. This study investigated the effects of AMPK activation on the expression of receptor activator of NF-κB ligand (RANKL) and sclerostin in osteocytes. RESULTS: Real-time PCR showed that AMPK activation by 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) significantly decreased the expression of Rankl in a dose- and time-dependent manner and significantly increased the expression of Sost, the gene encoding sclerostin, in osteocytic MLO-Y4 cells. Western blotting confirmed that AICAR decreased RANKL protein levels and increased sclerostin levels. In addition, suppression of AMPKα1 by siRNA significantly increased the expression of Rankl on 4 days after the transfection of siRNA, while Sost expression was not changed. Simvastatin, an inhibitor of HMG-CoA reductase, significantly decreased Rankl expression and increased Sost expression in MLO-Y4 cells. Supplementation with mevalonate or geranylgeranyl pyrophosphate, which are downstream metabolites of HMG-CoA reductase, significantly reversed the effects of AICAR. CONCLUSION: These findings indicated that AMPK regulated RANKL and sclerostin expression through the mevalonate pathway in osteocytes.

Administration of anti-receptor activator of nuclear factor-kappa B ligand (RANKL) antibody for the treatment of osteoporosis was associated with amelioration of hepatitis in a female patient with growth hormone deficiency: a case report.

BACKGROUND: Growth hormone deficiency (GHD) is associated with non-alcoholic fatty liver disease (NAFLD). A recent animal study showed that hepatocyte-specific receptor activator of nuclear factor-kappa B (RANK) knockout mice had significantly lower liver fat content compared with control mice concomitant with a decrease in production of inflammatory cytokines such as tumor necrosis factor-α (TNF-α) from hepatocytes and kupffer cells. The role of anti-RANK ligand (RANKL) antibody for osteoporosis on hepatitis in patients with aGHD is still unknown. CASE PRESENTATION: A forty-seven-year-old female patient was referred to our hospital to investigate chronic hepatitis caused by unknown etiology. She had past history of craniopharyngioma treated with craniotomy and post-surgical radiotherapy. She was for the first time diagnosed as panhypopituitarism including growth hormone deficiency and osteoporosis by endocrine examinations and bone mineral densitometry, respectively. In addition, non-alcoholic steatohepatitis (NASH) was histologically confirmed by liver biopsy in this time. Sixty mg anti-RANKL antibody, which was subcutaneously injected to treat the osteoporosis every six months after replacement of 5 mg hydrocortisone and 30 µg oral desmopressin, rapidly decreased the levels of her liver enzymes (ALT and γGTP were 133 to 72 U/L and 284 to 99 U/L at 16 months after the beginning of the treatment, respectively). Additional amelioration of liver dysfunction was not observed after growth hormone replacement. CONCLUSIONS: The clinical course of the present case suggested that RANKL-RANK signaling may be a key pathological mechanism in establishment or development of NAFLD or NASH in patients with panhypopituitarism including GHD.

Simvastatin rescues homocysteine-induced apoptosis of osteocytic MLO-Y4 cells by decreasing the expressions of NADPH oxidase 1 and 2.

Clinical studies have shown that hyperhomocysteinemia is associated with bone fragility. Homocysteine (Hcy) induces apoptosis of osteoblastic cell lineage by increasing oxidative stress, which may contribute to Hcy-induced bone fragility. Statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, ameliorate oxidative stress by regulating oxidant and anti-oxidant enzymes. However, the effects of statins on Hcy-induced apoptosis of osteocytes are unknown. This study was thus aimed to investigate whether or not statins prevent Hcy-induced apoptosis of osteocytic MLO-Y4 cells and regulate NADPH oxidase (Nox) expression. TUNEL staining showed that 5 mM Hcy induced apoptosis of MLO-Y4 cells, and that co-incubation of 10(-9) or 10(-8) M simvastatin significantly suppressed the apoptotic effect. Moreover, we confirmed the beneficial effect of simvastatin against Hcy's apoptotic effect by using a DNA fragment ELISA assay. However, TUNEL staining showed no significant effects of pravastatin, a hydrophilic statin, on the Hcy-induced apoptosis. Real-time PCR showed that Hcy increased the mRNA expressions of Nox1 and Nox2, whereas simvastatin inhibited the stimulation of Nox1 and Nox2 expressions by Hcy. In contrast, neither Hcy nor simvastatin had any effect on Nox4 expression. These findings indicate that simvastatin prevents the detrimental effects of Hcy on the apoptosis of osteocytes by regulating the expressions of Nox1 and Nox2, suggesting that statins may be beneficial for preventing Hcy-induced osteocyte apoptosis and the resulting bone fragility.