Crucial role of iron in epigenetic rewriting during adipocyte differentiation mediated by JMJD1A and TET2 activity.

Iron metabolism is closely associated with the pathogenesis of obesity. However, the mechanism of the iron-dependent regulation of adipocyte differentiation remains unclear. Here, we show that iron is essential for rewriting of epigenetic marks during adipocyte differentiation. Iron supply through lysosome-mediated ferritinophagy was found to be crucial during the early stage of adipocyte differentiation, and iron deficiency during this period suppressed subsequent terminal differentiation. This was associated with demethylation of both repressive histone marks and DNA in the genomic regions of adipocyte differentiation-associated genes, 　including Pparg, which encodes PPARγ, the master regulator of adipocyte differentiation. In addition, we identified several epigenetic demethylases to be responsible for iron-dependent adipocyte differentiation, with the histone demethylase jumonji domain-containing 1A and the DNA demethylase ten-eleven translocation 2 as the major enzymes. The interrelationship between repressive histone marks and DNA methylation was indicated by an integrated genome-wide association analysis, and was also supported by the findings that both histone and DNA demethylation were suppressed by either the inhibition of lysosomal ferritin flux or the knockdown of iron chaperone poly(rC)-binding protein 2. In summary, epigenetic regulations through iron-dependent control of epigenetic enzyme activities play an important role in the organized gene expression mechanisms of adipogenesis.

Actin-binding protein filamin B regulates the cell-surface retention of endothelial sphingosine 1-phosphate receptor 1.

Sphingosine 1-phosphate receptor 1 (S1PR1) is a G protein-coupled receptor essential for vascular development and postnatal vascular homeostasis. When exposed to sphingosine 1-phosphate (S1P) in the blood of ∼1 µM, S1PR1 in endothelial cells retains cell-surface localization, while lymphocyte S1PR1 shows almost complete internalization, suggesting the cell-surface retention of S1PR1 is endothelial cell specific. To identify regulating factors that function to retain S1PR1 on the endothelial cell surface, here we utilized an enzyme-catalyzed proximity labeling technique followed by proteomic analyses. We identified Filamin B (FLNB), an actin-binding protein involved in F-actin cross-linking, as a candidate regulating protein. We show FLNB knockdown by RNA interference induced massive internalization of S1PR1 into early endosomes, which was partially ligand dependent and required receptor phosphorylation. Further investigation showed FLNB was also important for the recycling of internalized S1PR1 back to the cell surface. FLNB knockdown did not affect the localization of S1PR3, another S1P receptor subtype expressed in endothelial cells, nor did it affect localization of ectopically expressed ß2-adrenergic receptor. Functionally, we show FLNB knockdown in endothelial cells impaired S1P-induced intracellular phosphorylation events and directed cell migration and enhancement of the vascular barrier. Taken together, our results demonstrate that FLNB is a novel regulator critical for S1PR1 cell-surface localization and thereby proper endothelial cell function.

Impact of GAD65 and/or GAD67 deficiency on perinatal development in rats.

GABA is a major neurotransmitter in the mammalian central nervous system. Glutamate decarboxylase (GAD) synthesizes GABA from glutamate, and two isoforms of GAD, GAD65, and GAD67, are separately encoded by the Gad2 and Gad1 genes, respectively. The phenotypes differ in severity between GAD single isoform-deficient mice and rats. For example, GAD67 deficiency causes cleft palate and/or omphalocele in mice but not in rats. In this study, to further investigate the functional roles of GAD65 and/or GAD67 and to determine the contribution of these isoforms to GABA synthesis during development, we generated various kinds of GAD isoform(s)-deficient rats and characterized their phenotypes. The age of death was different among Gad mutant rat genotypes. In particular, all Gad1-/- ; Gad2-/- rats died at postnatal day 0 and showed little alveolar space in their lungs, suggesting that the cause of their death was respiratory failure. All Gad1-/- ; Gad2-/- rats and 18% of Gad1-/- ; Gad2+/- rats showed cleft palate. In contrast, none of the Gad mutant rats including Gad1-/- ; Gad2-/- rats, showed omphalocele. These results suggest that both rat GAD65 and GAD67 are involved in palate formation, while neither isoform is critical for abdominal wall formation. The GABA content in Gad1-/- ; Gad2-/- rat forebrains and retinas at embryonic day 20 was extremely low, indicating that almost all GABA was synthesized from glutamate by GADs in the perinatal period. The present study shows that Gad mutant rats are a good model for further defining the role of GABA during development.

CD36 Expression Is Associated with Cancer Aggressiveness and Energy Source in Esophageal Squamous Cell Carcinoma.

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is an important cause of cancer-related death worldwide. CD36, a long-chain fatty acid (FA) receptor, can initiate metastasis in human oral squamous cell carcinoma (SCC), and its expression is associated with poor prognosis in several cancers. The clinical significance of CD36 expression and its function in ESCC remain unknown. METHODS: We examined the clinical significance of CD36 expression in 160 ESCC samples using immunohistochemical staining. Functional analysis was performed to determine the association between CD36 and ESCC characteristics (proliferative ability, invasive ability, and energy source dependency). RESULTS: Thirty (18.8%) ESCC cases showed high CD36 expression, indicating a significant association with progression. CD36 suppression inhibited proliferation and invasiveness in ESCC cells. ESCC cells with CD36 suppression used specific essential amino acids (EAAs) as energy sources. Cell viability depended on FAs under CD36 expression. The viability of ESCC cells with CD36 suppression depended on EAAs but not FAs. CONCLUSIONS: CD36 may be a good biomarker and therapeutic target in ESCC. Our data provide new insights into the basic mechanism of CD36-dependent energy utilization for ESCC survival. CD36 might be a key regulator of the dependency of FAs as energy source in ESCC cells.

Sphingosine 1-phosphate and inflammation.

AbstractSphingosine 1-phosphate (S1P), a sphingolipid mediator, regulates various cellular functions via high-affinity G protein-coupled receptors, S1P1-5. The S1P-S1P receptor signaling system plays important roles in lymphocyte trafficking and maintenance of vascular integrity, thus contributing to the regulation of complex inflammatory processes. S1P is enriched in blood and lymph while maintained low in intracellular or interstitial fluids, creating a steep S1P gradient that is utilized to facilitate efficient egress of lymphocytes from lymphoid organs. Blockage of the S1P-S1P receptor signaling system results in a marked decrease in circulating lymphocytes because of a failure of lymphocyte egress from lymphoid organs. This provides a basis of immunomodulatory drugs targeting S1P1 receptor such as FTY720, an immunosuppressive drug approved in 2010 as the first oral treatment for relapsing-remitting multiple sclerosis. The S1P-S1P receptor signaling system also plays important roles in maintenance of vascular integrity since it suppresses sprouting angiogenesis and regulates vascular permeability. Dysfunction of the S1P-S1P receptor signaling system results in various vascular defects, such as exaggerated angiogenesis in developing retina and augmented inflammation due to increased permeability. Endothelial-specific deletion of S1P1 receptor in mice fed high-fat diet leads to increased formation of atherosclerotic lesions. This review highlights the importance of the S1P-S1P receptor signaling system in inflammatory processes. We also describe our recent findings regarding a specific S1P chaperone, apolipoprotein M, that anchors to high-density lipoprotein and contributes to shaping the endothelial-protective and anti-inflammatory properties of high-density lipoprotein.

Identification of ApoA4 as a sphingosine 1-phosphate chaperone in ApoM- and albumin-deficient mice.

HDL-bound ApoM and albumin are protein chaperones for the circulating bioactive lipid, sphingosine 1-phosphate (S1P); in this role, they support essential extracellular S1P signaling functions in the vascular and immune systems. We previously showed that ApoM- and albumin-bound S1P exhibit differences in receptor activation and biological functions. Whether the physiological functions of S1P require chaperones is not clear. We examined ApoM-deficient, albumin-deficient, and double-KO (DKO) mice for circulatory S1P and its biological functions. In albumin-deficient mice, ApoM was upregulated, thus enabling S1P functions in embryonic development and postnatal adult life. The Apom:Alb DKO mice reproduced, were viable, and exhibited largely normal vascular and immune functions, which suggested sufficient extracellular S1P signaling. However, Apom:Alb DKO mice had reduced levels (∼25%) of plasma S1P, suggesting that novel S1P chaperones exist to mediate S1P functions. In this study, we report the identification of ApoA4 as a novel S1P binding protein. Recombinant ApoA4 bound to S1P, activated multiple S1P receptors, and promoted vascular endothelial barrier function, all reflective of its function as a S1P chaperone in the absence of ApoM and albumin. We suggest that multiple S1P chaperones evolved to support complex and essential extracellular signaling functions of this lysolipid mediator in a redundant manner.

Activation of the Sphingosine 1 Phosphate-Rho Pathway in Pterygium and in Ultraviolet-Irradiated Normal Conjunctiva.

Sphingosine 1 phosphate (S1P) is a bioactive lipid that regulates cellular activity, including proliferation, cytoskeletal organization, migration, and fibrosis. In this study, the potential relevance of S1P-Rho signaling in pterygium formation and the effects of ultraviolet (UV) irradiation on activation of the S1P/S1P receptor axis and fibrotic responses were investigated in vitro. Expressions of the S1P2, S1P4, and S1P5 receptors were significantly higher in pterygium tissue than in normal conjunctiva, and the concentration of S1P was significantly elevated in the lysate of normal conjunctival fibroblast cell (NCFC) irradiated with UV (UV-NCFCs). RhoA activity was significantly upregulated in pterygium fibroblast cells (PFCs) and UV-NCFCs, and myosin phosphatase-Rho interacting protein (MRIP) was upregulated, and myosin phosphatase target subunit 1 (MYPT1) was downregulated in PFCs. Fibrogenic changes were significantly upregulated in both PFCs and UV-NCFCs compared to NCFCs. We found that the activation of the S1P receptor-Rho cascade was observed in pterygium tissue. Additionally, in vitro examination showed S1P-rho activation and fibrogenic changes in PFCs and UV-NCFCs. S1P elevation and the resulting upregulation of the downstream Rho signaling pathway may be important in pterygium formation; this pathway offers a potential therapeutic target for suppressing pterygium generation.

Light Stress-Induced Increase of Sphingosine 1-Phosphate in Photoreceptors and Its Relevance to Retinal Degeneration.

Sphingosine 1-phosphate (S1P) is a potent lipid mediator that modulates inflammation and angiogenesis. In this study, we investigated the possible involvement of S1P in the pathology of light-induced retinal degeneration in vivo and in vitro. The intracellular S1P and sphingosine kinase (SphK) activity in a photoreceptor cell line (661W cells) was significantly increased by exposure to light. The enhancement of SphK1 expression was dependent on illumination, and all-trans-retinal significantly promoted SphK1 expression. S1P treatment reduced protein kinase B (Akt) phosphorylation and increased the protein expression of cleaved caspase-3, and induced photoreceptor cell apoptosis. In vivo, light exposure enhanced the expression of SphK1 in the outer segments of photoreceptors. Intravitreal injection of a SphK inhibitor significantly suppressed the thinning of the outer nuclear layer and ameliorated the attenuation of the amplitudes of a-waves and b-waves of electroretinograms during light-induced retinal degeneration. These findings imply that light exposure induces the synthesis of S1P in photoreceptors by upregulating SphK1, which is facilitated by all-trans-retinal, causing retinal degeneration. Inhibition of this enhancement may be a therapeutic target of outer retinal degeneration, including age-related macular degeneration.

Metabolic activity by 18F-FDG-PET/CT is predictive of early response after nivolumab in previously treated NSCLC.

BACKGROUND: Nivolumab, an anti-programmed death-1 (PD-1) antibody, is administered in patients with previously treated non-small cell lung cancer. However, little is known about the established biomarker predicting the efficacy of nivolumab. Here, we conducted a preliminary study to investigate whether 18F-FDG-PET/CT could predict the therapeutic response of nivolumab at the early phase. METHODS: Twenty-four patients were enrolled in this study. 18F-FDG-PET/CT was carried out before and 1 month after nivolumab therapy. SUVmax, metabolic tumour volume (MTV), and total lesion glycolysis (TLG) were calculated. Immunohistochemical analysis of PD-L1 expression and tumour-infiltrating lymphocytes was conducted. RESULTS: Among all patients, a partial metabolic response to nivolumab was observed in 29% on SUVmax, 25% on MTV, and 33% on TLG, whereas seven (29%) patients achieved a partial response (PR) based on RECIST v1.1. The predictive probability of PR (100% vs. 29%, p = 0.021) and progressive disease (100% vs. 22.2%, p = 0.002) at 1 month after nivolumab initiation was significantly higher in 18F-FDG on PET/CT than in CT scans. Multivariate analysis confirmed that 18F-FDG uptake after administration of nivolumab was an independent prognostic factor. PD-L1 expression and nivolumab plasma concentration could not precisely predict the early therapeutic efficacy of nivolumab. CONCLUSION: Metabolic response by 18F-FDG was effective in predicting efficacy and survival at 1 month after nivolumab treatment.

Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1.

Apolipoprotein M (ApoM) transports sphingosine-1-phosphate (S1P) in plasma, and ApoM-deficient mice (Apom(-/-)) have ∼50% reduced plasma S1P levels. There are 5 known S1P receptors, and S1P induces adherens junction formation between endothelial cells through the S1P1 receptor, which in turn suppresses vascular leak. Increased vascular permeability is a hallmark of inflammation. The purpose of this study was to explore the relationships between vascular leakage in ApoM deficiency and S1P1 function in normal physiology and in inflammation. Vascular permeability in the lungs was assessed by accumulation of dextran molecules (70 kDa) and was increased ∼40% in Apom(-/-) mice compared to WT (C57Bl6/j) mice. Reconstitution of plasma ApoM/S1P or treatment with an S1P1 receptor agonist (SEW2871) rapidly reversed the vascular leakage to a level similar to that in WT mice, suggesting that it is caused by decreased plasma levels of S1P and reduced S1P1 stimulation. In a carrageenan-induced model of inflammation, Apom(-/-) mice had increased vascular leakage compared with that in WT mice. Adenoviral overexpression of ApoM in Apom(-/-) mice decreased the vascular leakage compared to adenoviral overexpression of green fluorescent protein. The study suggests that vascular leakage of albumin-sized particles in ApoM deficiency is S1P- and S1P1-dependent and this dependency exacerbates the response to inflammatory stimuli.-Christensen, P. M., Liu, C. H., Swendeman, S. L., Obinata, H., Qvortrup, K., Nielsen, L B., Hla, T., Di Lorenzo, A., Christoffersen, C. Impaired endothelial barrier function in apolipoprotein M-deficient mice is dependent on sphingosine-1-phosphate receptor 1.

Individual variation of human S1P1 coding sequence leads to heterogeneity in receptor function and drug interactions.

Sphingosine 1-phosphate receptor 1 (S1P1), an abundantly-expressed G protein-coupled receptor which regulates key vascular and immune responses, is a therapeutic target in autoimmune diseases. Fingolimod/Gilenya (FTY720), an oral medication for relapsing-remitting multiple sclerosis, targets S1P1 receptors on immune and neural cells to suppress neuroinflammation. However, suppression of endothelial S1P1 receptors is associated with cardiac and vascular adverse effects. Here we report the genetic variations of the S1P1 coding region from exon sequencing of >12,000 individuals and their functional consequences. We conducted functional analyses of 14 nonsynonymous single nucleotide polymorphisms (SNPs) of the S1PR1 gene. One SNP mutant (Arg¹²° to Pro) failed to transmit sphingosine 1-phosphate (S1P)-induced intracellular signals such as calcium increase and activation of p44/42 MAPK and Akt. Two other mutants (Ile45 to Thr and Gly³°5 to Cys) showed normal intracellular signals but impaired S1P-induced endocytosis, which made the receptor resistant to FTY720-induced degradation. Another SNP mutant (Arg¹³ to Gly) demonstrated protection from coronary artery disease in a high cardiovascular risk population. Individuals with this mutation showed a significantly lower percentage of multi-vessel coronary obstruction in a risk factor-matched case-control study. This study suggests that individual genetic variations of S1P1 can influence receptor function and, therefore, infer differential disease risks and interaction with S1P1-targeted therapeutics.

Endothelium-protective sphingosine-1-phosphate provided by HDL-associated apolipoprotein M.

Protection of the endothelium is provided by circulating sphingosine-1-phosphate (S1P), which maintains vascular integrity. We show that HDL-associated S1P is bound specifically to both human and murine apolipoprotein M (apoM). Thus, isolated human ApoM(+) HDL contained S1P, whereas ApoM(-) HDL did not. Moreover, HDL in Apom(-/-) mice contains no S1P, whereas HDL in transgenic mice overexpressing human apoM has an increased S1P content. The 1.7-Å structure of the S1P-human apoM complex reveals that S1P interacts specifically with an amphiphilic pocket in the lipocalin fold of apoM. Human ApoM(+) HDL induced S1P(1) receptor internalization, downstream MAPK and Akt activation, endothelial cell migration, and formation of endothelial adherens junctions, whereas apoM(-) HDL did not. Importantly, lack of S1P in the HDL fraction of Apom(-/-) mice decreased basal endothelial barrier function in lung tissue. Our results demonstrate that apoM, by delivering S1P to the S1P(1) receptor on endothelial cells, is a vasculoprotective constituent of HDL.

Aquaporin-5 Protein Is Selectively Reduced in Rat Parotid Glands under Conditions of Fasting or a Liquid Diet.

Aquaporin-5 (AQP5) water channel, transmembrane protein 16A (TMEM16A) Ca2+-activated Cl- channel, and Na+-K+-2Cl- cotransporter (NKCC1) are membrane proteins on salivary gland acinar cells that function in watery saliva secretion. We examined their expression changes in rat parotid glands under reduced mastication. Rats were either fed regular chow as a control group, fasted for 48 hr or fed a liquid diet for 48 hr or 1 week to reduce mastication. The parotid glands were then resected to analyze the protein and mRNA levels by immunofluorescence, immunoblotting, and reverse-transcription quantitative PCR (RT-qPCR). AQP5 protein was significantly decreased in both liquid diet groups and the fasting group but its mRNA levels showed no apparent changes compared with the control group. The protein and mRNA levels of TMEM16A and NKCC1 showed no significant changes between any of the groups other than an increase in NKCC1 mRNA in the 1-week liquid diet group. These results suggest that reduced mastication may increase the AQP5 protein degradation, but not that of other membrane proteins necessary for saliva secretion.

Apolipoprotein M supports S1P production and conservation and mediates prolonged Akt activation via S1PR1 and S1PR3.

Sphingosine 1-phosphate (S1P) is one of the lipid mediators involved in diverse physiological functions. S1P circulates in blood and lymph bound to carrier proteins. Three S1P carrier proteins have been reported, albumin, apolipoprotein M (ApoM) and apolipoprotein A4 (ApoA4). The carrier-bound S1P exerts its functions via specific S1P receptors (S1PR1-5) on target cells. Previous studies showed several differences in physiological functions between albumin-bound S1P and ApoM-bound S1P. However, molecular mechanisms underlying the carrier-dependent differences have not been clarified. In addition, ApoA4 is a recently identified S1P carrier protein, and its functional differences from albumin and ApoM have not been addressed. Here, we compared the three carrier proteins in the processes of S1P degradation, release from S1P-producing cells and receptor activation. ApoM retained S1P more stable than albumin and ApoA4 in the cell culture medium when compared in the equimolar amounts. ApoM facilitated theS1P release from endothelial cells most efficiently. Furthermore, ApoM-bound S1P showed a tendency to induce prolonged activation of Akt via S1PR1 and S1PR3. These results suggest that the carrier-dependent functional differences of S1P are partly ascribed to the differences in the S1P stability, S1P-releasing efficiency and signaling duration.

Pilot study of plasma creatine riboside as a potential biomarker for cervical cancer.

This pilot study aimed primarily to evaluate plasma levels of a novel metabolite, creatine riboside, in patients with cervical cancer (discovery and validation cohorts, n = 11 for each) compared with non-cancer subjects (controls, n = 30). We found that the pre-treatment plasma creatine riboside level was significantly higher in the discovery cohort than in controls. The cut-off value determined from the discovery cohort distinguished 90.9% of the patients in the validation cohort from controls. Unbiased principal component analysis of plasma metabolites in high-creatine riboside samples demonstrated enrichment of pathways involved in arginine and creatine metabolism. These data indicate the potential utility of plasma creatine riboside as a biomarker of cervical cancer.

2,2,6,6-Tetramethylpiperidine-1-oxyl acts as a volatile inhibitor of ferroptosis and neurological injury.

Ferroptosis, a type of oxidative stress cell death, has been implicated in cell injury in several diseases, and treatments with specific inhibitors have been shown to protect cells and tissues. Here we demonstrated that a treatment with the nitroxide radical, 2,2,6,6-tetramethylpiperidine-N-oxyl (TEMPO), prevented the ferroptotic cell death in an airborne manner. Other TEMPO derivatives and lipophilic antioxidants, such as Trolox and ferrostatin-1, also prevented cell death induced by erastin and RSL3; however, only TEMPO exhibited inhibitory activity from a physically distant location. TEMPO vaporized without decomposing and then dissolved again into a nearby water solution. Volatilized TEMPO inhibited glutamate-induced cell death in mouse hippocampal cell lines and also reduced neuronal cell death in a mouse ischemia model. These results suggest that TEMPO is a unique cell protective agent that acts in a volatility-mediated manner.

Global knockdown of glutamate decarboxylase 67 elicits emotional abnormality in mice.

Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1tTA/STOP-tetO biallelic knock-in mice using Gad1STOP-tetO and Gad1tTA knock-in mice, and compared them with Gad1+/+ mice. The expression level of GAD67 protein in brains of Gad1tTA/STOP-tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1tTA/STOP-tetO mice. In the open-field test, Dox-treated Gad1tTA/STOP-tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1tTA/STOP-tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.

Reduced Fatty Acid Use from CD36 Deficiency Deteriorates Streptozotocin-Induced Diabetic Cardiomyopathy in Mice.

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.

Disordered branched chain amino acid catabolism in pancreatic islets is associated with postprandial hypersecretion of glucagon in diabetic mice.

Dysregulation of glucagon is associated with the pathophysiology of type 2 diabetes. We previously reported that postprandial hyperglucagonemia is more obvious than fasting hyperglucagonemia in type 2 diabetes patients. However, which nutrient stimulates glucagon secretion in the diabetic state and the underlying mechanism after nutrient intake are unclear. To answer these questions, we measured plasma glucagon levels in diabetic mice after oral administration of various nutrients. The effects of nutrients on glucagon secretion were assessed using islets isolated from diabetic mice and palmitate-treated islets. In addition, we analyzed the expression levels of branched chain amino acid (BCAA) catabolism-related enzymes and their metabolites in diabetic islets. We found that protein, but not carbohydrate or lipid, increased plasma glucagon levels in diabetic mice. Among amino acids, BCAAs, but not the other essential or nonessential amino acids, increased plasma glucagon levels. BCAAs also directly increased the intracellular calcium concentration in α cells. When BCAAs transport was suppressed by an inhibitor of system L-amino acid transporters, glucagon secretion was reduced even in the presence of BCAAs. We also found that the expression levels of BCAA catabolism-related enzymes and their metabolite contents were altered in diabetic islets and palmitate-treated islets compared to control islets, indicating disordered BCAA catabolism in diabetic islets. Furthermore, BCKDK inhibitor BT2 suppressed BCAA-induced hypersecretion of glucagon in diabetic islets and palmitate-treated islets. Taken together, postprandial hypersecretion of glucagon in the diabetic state is attributable to disordered BCAA catabolism in pancreatic islet cells.

64Cu-ATSM Predicts Efficacy of Carbon Ion Radiotherapy Associated with Cellular Antioxidant Capacity.

Carbon ion radiotherapy is an emerging cancer treatment modality that has a greater therapeutic window than conventional photon radiotherapy. To maximize the efficacy of this extremely scarce medical resource, it is important to identify predictive biomarkers of higher carbon ion relative biological effectiveness (RBE) over photons. We addressed this issue by focusing on cellular antioxidant capacity and investigated 64Cu(II)-diacetyl-bis(N4-methylthiosemicarbazone) (64Cu-ATSM), a potential radioligand that reflects an over-reduced intracellular environment. We found that the carbon ion RBE correlated with 64Cu-ATSM uptake both in vitro and in vivo. High RBE/64Cu-ATSM cells showed greater steady-state levels of antioxidant proteins and increased capacity to scavenge reactive oxygen species in response to X-rays than low RBE/64Cu-ATSM counterparts; this upregulation of antioxidant systems was associated with downregulation of TCA cycle intermediates. Furthermore, inhibition of nuclear factor erythroid 2-related factor 2 (Nrf2) sensitized high RBE/64Cu-ATSM cells to X-rays, thereby reducing RBE values to levels comparable to those in low RBE/64Cu-ATSM cells. These data suggest that the cellular activity of Nrf2-driven antioxidant systems is a possible determinant of carbon ion RBE predictable by 64Cu-ATSM uptake. These new findings highlight the potential clinical utility of 64Cu-ATSM imaging to identify high RBE tumors that will benefit from carbon ion radiotherapy.