Brain and muscle arnt-like protein 1 (BMAL1), is a transcription factor known to regulate circadian rhythm. BMAL1 was originally characterized by its high expression in the skeletal muscle. Since the skeletal muscle is the dominant organ system in energy metabolism, the possible functions of BMAL1 in the skeletal muscle include the control of metabolism. Here, we established that its involvement in the regulation of oxidative capacity in the skeletal muscle. Muscle-specific Bmal1 KO mice (MKO mice) displayed several physiological hallmarks for the increase of oxidative capacity. This included increased energy expenditure and oxygen consumption, high running endurance and resistance to obesity with improved metabolic profiles. Also, the phosphorylation status of AMP-activated protein kinase and its downstream signaling substrate acetyl-CoA carboxylase in the MKO mice were substantially higher than those in the Bmal1^flox/flox mice. In addition, biochemical and histological studies confirmed the substantial activation of oxidative fibers in the skeletal muscle of the MKO mice. The mechanism includes the regulation of Cacna1s expression, followed by the activation of calcium—nuclear factor of activated T cells (NFAT) axis. We thus conclude that BMAL1 is a critical regulator of the muscular fatty acid level under nutrition overloading and that the mechanism involves the control of oxidative capacity.

Prostaglandin E2 (PGE2) exerts various biological effects by binding to E-prostanoid receptors (EP1-4). Although recent studies have shown that PGE2 induces cell differentiation in some neuronal cells such as mouse DRG neurons and sensory neuron-like ND7/23 cells, it is unclear whether PGE2 plays a role in differentiation of motor neurons. In the present study, we investigated the mechanism of PGE2-induced differentiation of motor neurons using NSC-34, a mouse motor neuron-like cell line. Exposure of undifferentiated NSC-34 cells to PGE2 and butaprost, an EP2-selective agonist, resulted in a reduction of MTT reduction activity without increase the number of propidium iodide-positive cells and in an increase in the number of neurite-bearing cells. Sulprostone, an EP1/3 agonist, also significantly lowered MTT reduction activity by 20%; however, no increase in the number of neurite-bearing cells was observed within the concentration range tested. PGE2-induced neurite outgrowth was attenuated significantly in the presence of PF-0441848, an EP2-selective antagonist. Treatment of these cells with dibutyryl-cAMP increased the number of neurite-bearing cells with no effect on cell proliferation. These results suggest that PGE2 promotes neurite outgrowth and suppresses cell proliferation by activating the EP2 subtype, and that the cAMP-signaling pathway is involved in PGE2-induced differentiation of NSC-34 cells.

Several reports have shown that some dopamine receptor ligands modulate the ischemia–reperfusion injury in animal models; however, its underling mechanisms are still unclear. In this study, we sought to establish an in vitro experimental model of hypoxia/reoxygenation (H/R) using HT22 cells that originated from mouse hippocampal neurons and to examine protective the effect of dopamine-receptor ligands against H/R-induced cell injury. The treatment with hypoxia for 18 h followed by reoxygenation for 6 h induced the elevation of intracellular reactive oxygen species (ROS) and reduction of mitochondrial membrane potential; however, lactate dehydrogenase (LDH) release was not changed at this time point. LDH release was increased after reoxygenation for 18 h and longer, and this increase in LDH release was suppressed by dopamine receptor agonists such as apomorphine and apocodeine. The suppressive effects of these agonists were reversibly inhibited by L750667, a D4-receptor antagonist but not by D2- or D3-receptor antagonists. In addition, PD168077, a selective dopamine D4–receptor agonist, also protected against H/R-induced cell death. These results suggest that H/R causes oxidative stress–induced cell death and that the activation of dopamine D4 receptors protects against H/R-induced cell death in HT22 cells.

Motor neuron diseases are a group of progressive neurological disorders that degenerate motor neurons. The neuroblastoma × spinal cord hybrid cell line NSC-34 is widely used as an experimental model in studies of motor neuron diseases. However, the differentiation efficiency of NSC-34 cells to neurons is not always sufficient. We have found that prostaglandin E₂ (PGE₂) induces morphological differentiation in NSC-34 cells. The present study investigated the functional properties of PGE₂-differentiated NSC-34 cells. Retinoic acid (RA), a widely-used agent inducing cell differentiation, facilitated neuritogenesis, which peaked on day 7, whereas PGE₂-induced neuritogenesis took only 2 days to reach the same level. Whole-cell patch-clamp recordings showed that the current threshold of PGE₂-treated cell action potentials was lower than that of RA-treated cells. PGE₂ and RA increased the protein expression levels of neuronal differentiation markers, microtubule-associated protein 2c and synaptophysin, and to the same extent, motor neuron-specific markers HB9 and Islet-1. On the other hand, protein levels of choline acetyltransferase and basal release of acetylcholine in PGE₂-treated cells were higher than in RA-treated cells. These results suggest that PGE₂ is a rapid and efficient differentiation-inducing factor for the preparation of functionally mature motor neurons from NSC-34 cells.

Prostaglandins are a group of physiologically active lipid compounds derived from arachidonic acid. Our previous study has found that prostaglandin E₂ promotes neurite outgrowth in NSC-34 cells, which are a model for motor neuron development. However, the effects of other prostaglandins on neuronal differentiation are poorly understood. The present study investigated the effect of prostaglandin D₂ (PGD₂) on neuritogenesis in NSC-34 cells. Exposure to PGD₂ resulted in increased percentages of neurite-bearing cells and neurite length. Although D-prostanoid receptor (DP) 1 and DP2 were dominantly expressed in the cells, BW245C (a DP1 agonist) and 15(R)-15-methyl PGD₂ (a DP2 agonist) had no effect on neurite outgrowth. Enzyme-linked immunosorbent assay demonstrated that PGD₂ was converted to 15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) under cell-free conditions. Exogenously applied 15d-PGJ₂ mimicked the effect of PGD₂ on neurite outgrowth. GW9662, a peroxisome proliferator-activated receptor–gamma (PPARγ) antagonist, suppressed PGD₂-induced neurite outgrowth. Moreover, PGD₂ and 15d-PGJ₂ increased the protein expression of Islet-1 (the earliest marker of developing motor neurons), and these increases were suppressed by co-treatment with GW9662. These results suggest that PGD₂ induces neuritogenesis in NSC-34 cells and that PGD₂-induced neurite outgrowth was mediated by the activation of PPARγ through the metabolite 15d-PGJ₂.