Chromogenic in situ hybridization reveals specific expression pattern of long non-coding RNA DRAIC in formalin-fixed paraffin-embedded specimen.

Long non-coding RNA (lncRNA) plays an important role in the regulation of gene expression in normal and cancer cells. We previously discovered a novel tumor-suppressive lncRNA, DRAIC, in prostate cancer cells. Subsequent studies have demonstrated that DRAIC is dysregulated in various malignancies and exhibits a tumor-suppressive or pro-oncogenic function. However, details regarding its expression pattern in normal and cancerous tissues remain largely unknown. In this study, we performed chromogenic in situ hybridization (CISH) using RNAscope technology to assess DRAIC expression in formalin-fixed paraffin-embedded (FFPE) specimens. In the neuroendocrine-differentiated cancer cell line VMRC-LCD, CISH revealed a diffuse localization of DRAIC in the cytoplasm as well as specific accumulation in the nuclear compartment. DRAIC expression was comprehensively analyzed using tissue microarrays containing 89 normal and 155 tumor tissue samples. DRAIC was weakly expressed in normal epithelial cells of the colon, bronchiole, kidney, prostate, and testis. Conversely, DRAIC was moderately to highly expressed in some cancer tissues, including prostate adenocarcinoma, invasive ductal carcinoma of the breast, neuroendocrine carcinoma of the esophagus, lung adenocarcinoma, and small cell lung carcinoma. While DRAIC knockdown did not affect VMRC-LCD cellular viability and invasive ability, gene expression related to the neuroendocrine and cancer-related pathways was altered. Our expression analysis revealed the specific expression pattern of DRAIC in normal and cancerous FFPE tissues. The results presented here may lead to the elucidation of additional novel functions of DRAIC.

MBNL1-Associated Mitochondrial Dysfunction and Apoptosis in C2C12 Myotubes and Mouse Skeletal Muscle.

We explored the interrelationship between a tissue-specific alternative splicing factor muscleblind-like 1 (MBNL1) and peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), B-cell lymphoma 2 (Bcl-2) or Bcl-2-associated X protein (Bax) in C2C12 myotubes and mouse skeletal muscle to investigate a possible physiological role of MBNL1 in mitochondrial-associated apoptosis of skeletal muscle. Expression level of PGC-1α and mitochondrial membrane potential evaluated by the fluorescence ratio of JC-1 aggregate to monomer in C2C12 myotubes were suppressed by knockdown of MBNL1. Conversely, the ratio of Bax to Bcl-2 as well as the apoptotic index in C2C12 myotubes was increased by MBNL1 knockdown. In plantaris muscle, on the other hand, not only the minimum muscle fiber diameter but also the expression level of MBNL1 and PGC-1α in of 100-week-old mice were significantly lower than that of 10-week-old mice. Furthermore, the ratio of Bax to Bcl-2 in mouse plantaris muscle was increased by aging. These results suggest that MBNL1 may play a key role in aging-associated muscle atrophy accompanied with mitochondrial dysfunction and apoptosis via mediating PGC-1α expression in skeletal muscle.

Activation of adiponectin receptors has negative impact on muscle mass in C2C12 myotubes and fast-type mouse skeletal muscle.

This study investigated the effects of AdipoRon, which is an agonist for adiponectin receptor 1 (AdipoR1) and AdipoR2, on the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells and skeletal muscle mass in C57BL/6J mice. AdipoRon suppressed the protein content, myotube diameter, and number of nuclei per myotube of C2C12 cells of C2C12 myotubes in a dose-dependent manner. Adiponectin-associated decline of protein content, diameter, and number of nuclei per myotube in C2C12 myotubes was partially rescued by knockdown of AdipoR1 and/or AdipoR2. Phosphorylation level of AMPK showed a trend to be increased by AdipoRon. A significant increase in phosphorylation level of AMPK was observed at 20 µM AdipoRon. Knockdown of AdipoR1 and/or AdipoR2 rescued AdipoRon-associated decrease in protein content of C2C12 myotubes. AdipoRon-associated increase in phosphorylation level of AMPK in C2C12 myotubes was suppressed by knockdown of AdipoR1 and/or AdipoR2. Successive intravenous injections of AdipoRon into mice caused a decrease in the wet weight of plantaris muscle (PLA), but not in soleus muscle (SOL). Mean fiber cross-sectional area of PLA, but not of SOL, was significantly decreased by AdipoRon administration. On the one hand, the expression level of phosphorylated AMPK and ubiquitinated protein in SOL and PLA muscles was upregulated by AdipoRon administration. On the other hand, AdipoRon administration induced no changes in the expression level of puromycin-labeled proteins in both SOL and PLA muscles. Expression level of adiponectin in extensor digitorum longus (EDL) muscle was increased by aging, but not in SOL muscle. Aging had no effect on the expression level of AdipoR1 and AdipoR2 in both muscles. Phosphorylation level of AMPK in EDL was increased by aging, but not SOL muscle. Results from this study suggest that high level of circulating adiponectin may induce skeletal muscle atrophy, especially fast-type muscle.

Nuclear Accumulation of HSP70 in Mouse Skeletal Muscles in Response to Heat Stress, Aging, and Unloading With or Without Reloading.

The purpose of this study was to investigate the nuclear accumulation of heat shock protein 70 (HSP70), a molecular chaperonin in mouse skeletal muscle in response to aging, heat stress, and hindlimb unloading with or without reloading. Profiles of HSP70-specific nuclear transporter Hikeshi in skeletal muscles were also evaluated. Heat stress-associated nuclear accumulation of HSP70 was observed in slow soleus (SOL) and fast plantaris (PLA) muscles of young (10-week-old) mice. Mean nuclear expression level of HSP70 in slow medial gastrocnemius (MGAS) and PLA muscles of aged (100-week-old) mice increased ~4.8 and ~1.7 times, compared to that of young (10-week-old) mice. Reloading following 2-week hindlimb unloading caused accumulation of HSP70 in myonuclei in MGAS and PLA of young mice ( p < 0.05). However, reloading-associated nuclear accumulation of HSP70 was not observed in both types of muscles of aged mice. On the other hand, 2-week hindlimb unloading had no impact on the nuclear accumulation of HSP70 in both muscles of young and aged mice. Nuclear expression level of Hikeshi in both MGAS and PLA in mice was suppressed by aging. No significant changes in the nuclear Hikeshi in both muscles were induced by unloading with or without reloading. Results of this study indicate that the nuclear accumulation of HSP70 might show a protective response against cellular stresses in skeletal muscle and that the protective response may be suppressed by aging. Protective response to aging might depend on muscle fiber types.

Teneligliptin, a Dipeptidyl Peptidase-4 Inhibitor, Improves Early-Phase Insulin Secretion in Drug-Naïve Patients with Type 2 Diabetes.

INTRODUCTION: It remains unknown whether dipeptidyl peptidase-4 (DPP-4) inhibitors improve early-phase insulin secretion in Japanese patients with type 2 diabetes (T2D), a disease characterized by impaired insulin secretion. We investigated the changes in insulin secretion before and after treatment with the DPP-4 inhibitor teneligliptin in patients with T2D with a low insulinogenic index (IGI) determined by the oral glucose tolerance test (OGTT). METHODS: An open-label, prospective clinical study was conducted. Thirteen drug-naïve patients (mean age 55.5 ± 3.9 years) with T2D underwent OGTT before and after teneligliptin 20 mg/day monotherapy. Plasma levels of glucose (PG), insulin, and C-peptide were measured at 0, 30, 60, 90, and 120 min after glucose loading in the OGTT. Homeostasis model assessment (HOMA)-ß, IGI, and the total or incremental area under the curve (AUC) for PG and insulin were measured. AUC120min for the secretory units of islets in transplantation (SUIT) index was also measured. RESULTS: HbA1c significantly decreased from 8.3 ± 0.4% at baseline to 6.3 ± 0.2% after 12 weeks of teneligliptin treatment (p < 0.05). Incremental AUC120min PG also significantly decreased, and ß-cell function assessed by IGI30min, AUC120min insulin, and the AUC120min SUIT index significantly increased (0.16 ± 0.05 vs. 0.28 ± 0.06, 2692 ± 333 µU·2h/mL vs. 3537 ± 361 µU·2h/mL, and 4261 ± 442 vs. 8290 ± 1147, respectively; all p < 0.05). HOMA-ß was unchanged. The reduction in incremental AUC120min PG was significantly associated with the augmentation of IGI30min and the AUC120min SUIT index. No severe adverse events were observed. CONCLUSIONS: Twelve weeks of teneligliptin treatment improved IGI30min, AUC120min, and the SUIT index in drug-naïve Japanese patients with T2D.