矢作 直也

With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Type 2 diabetes (T2D) is a progressive disorder denoted by hyperglycemia and impaired insulin secretion. Although a decrease in β-cell function and mass is a well-known trigger for diabetes, the comprehensive mechanism is still unidentified. Here we carried out single-cell RNA sequencing (scRNA-seq) of pancreatic islets from prediabetic and diabetic db/db mice, an animal model of T2D. We discovered a diabetes-specific transcriptome landscape of endocrine and nonendocrine cell types with subpopulations of β and α cells. We recognized a new prediabetic gene, Anxa10, that was induced by, and regulated Ca2+ influx from metabolic stresses. Anxa10-overexpressed β cells displayed suppression of glucose-stimulated intracellular Ca2+ elevation and potassiuminduced insulin secretion. Pseudotime analysis of β cells predicted this Ca2+-surge responder-cluster proceeded to mitochondria dysfunction and also endoplasmic reticulum stress. Other trajectories comprised dedifferentiation and transdifferentiation, emphasizing acinar-like cells in diabetic islets. Altogether, our data provides a new insight into Ca2+ allostasis and β cell failure processes.

During periods of fasting, the body undergoes a metabolic shift from carbohydrate utilization to the use of fats and ketones as an energy source, as well as the inhibition of de novo lipogenesis and the initiation of gluconeogenesis in the liver. The transcription factor sterol regulatory element-binding protein-1 (SREBP-1), which plays a critical role in the regulation of lipogenesis, is suppressed during fasting, resulting in the suppression of hepatic lipogenesis. We previously demonstrated that the interaction of fasting-induced Kruppel-like factor 15 (KLF15) with liver X receptor serves as the essential mechanism for the nutritional regulation of SREBP-1 expression. However, the underlying mechanisms of KLF15 induction during fasting remain unclear. In this study, we show that the glucocorticoid receptor (GR) regulates the hepatic expression of KLF15 and, subsequently, lipogenesis through the KLF15-SREBP-1 pathway during fasting. KLF15 is necessary for the suppression of SREBP-1 by GR, as demonstrated through experiments using KLF15 knockout mice. Additionally, we show that GR is involved in the fasting response, with heightened binding to the KLF15 enhancer. It has been widely known that the hypothalamic-pituitary-adrenal (HPA) axis regulates the secretion of glucocorticoids and plays a significant role in the metabolic response to undernutrition. These findings demonstrate the importance of the HPA-axis-regulated GR-KLF15 pathway in the regulation of lipid metabolism in the liver during fasting.

Nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes are interacting comorbidities of obesity, and increased hepatic de novo lipogenesis (DNL), driven by hyperinsulinemia and carbohydrate overload, contributes to their pathogenesis. Fatty acid synthase (FASN), a key enzyme of hepatic DNL, is upregulated in association with insulin resistance. However, the therapeutic potential of targeting FASN in hepatocytes for obesity-associated metabolic diseases is unknown. Here, we show that hepatic FASN deficiency differentially affects NAFLD and diabetes depending on the etiology of obesity. Hepatocyte-specific ablation of FASN ameliorated NAFLD and diabetes in melanocortin 4 receptor-deficient mice but not in mice with diet-induced obesity. In leptin-deficient mice, FASN ablation alleviated hepatic steatosis and improved glucose tolerance but exacerbated fed hyperglycemia and liver dysfunction. The beneficial effects of hepatic FASN deficiency on NAFLD and glucose metabolism were associated with suppression of DNL and attenuation of gluconeogenesis and fatty acid oxidation, respectively. The exacerbation of fed hyperglycemia by FASN ablation in leptin-deficient mice appeared attributable to impairment of hepatic glucose uptake triggered by glycogen accumulation and citrate-mediated inhibition of glycolysis. Further investigation of the therapeutic potential of hepatic FASN inhibition for NAFLD and diabetes in humans should thus consider the etiology of obesity.

Abstract Background Mild cognitive impairment (MCI) is not just a prodrome to dementia, but a very important intervention point to prevent dementia caused by Alzheimer's disease (AD). It has long been known that people with AD have a higher frequency of falls with some gait instability. Recent evidence suggests that vestibular impairment is disproportionately prevalent among individuals with MCI and dementia due to AD. Therefore, we hypothesized that the measurement of balance capability is helpful to identify individuals with MCI. Methods First, we developed a useful method to evaluate balance capability as well as vestibular function using Nintendo Wii balance board as a stabilometer and foam rubber on it. Then, 49 healthy volunteers aged from 56 to 75 with no clinically apparent cognitive impairment were recruited and the association between their balance capability and cognitive function was examined. Cognitive functions were assessed by MoCA, MMSE, CDR, and TMT-A and -B tests. Results The new balance capability indicator, termed visual dependency index of postural stability (VPS), was highly associated with cognitive impairment assessed by MoCA, and the area under the receiver operating characteristic (ROC) curve was more than 0.8, demonstrating high sensitivity and specificity (app. 80% and 60%, respectively). Conclusions Early evidence suggests that VPS measured using Nintendo Wii balance board as a stabilometer helps identify individuals with MCI at an early and preclinical stage with high sensitivity, establishing a useful method to screen MCI.

ELOVL fatty acid elongase 6 (ELOVL6) controls cellular fatty acid (FA) composition by catalyzing the elongation of palmitate (C16:0) to stearate (C18:0) and palmitoleate (C16:1n-7) to vaccinate (C18:1n-7). Although the transcriptional regulation of ELOVL6 has been well studied, the post-transcriptional regulation of ELOVL6 is not fully understood. Therefore, this study aims to evaluate the role of microRNAs (miRNAs) in regulating human ELOVL6. Bioinformatic analysis identified five putative miRNAs: miR-135b-5p, miR-135a-5p, miR-125a-5p, miR-125b-5p, and miR-22-3p, which potentially bind ELOVL6 3'-untranslated region (UTR). Results from dual-luciferase assays revealed that these miRNAs downregulate ELOVL6 by directly interacting with the 3'-UTR of ELOVL6 mRNA. Moreover, miR-135b-5p and miR-135a-5p suppress cell proliferation and migration in glioblastoma multiforme cells by inhibiting ELOVL6 at the mRNA and protein levels. Taken together, our results provide novel regulatory mechanisms for ELOVL6 at the post-transcriptional level and identify potential candidates for the treatment of patients with glioblastoma multiforme.

Although branched-chain amino acids (BCAA) are known to stimulate myofibrillar protein synthesis and affect insulin signaling and kynurenine metabolism (the latter being a metabolite of tryptophan associated with depression and dementia), the effects of BCAA supplementation on type 2 diabetes (T2D) are not clear. Therefore, a 24-week, prospective randomized open blinded-endpoint trial was conducted to evaluate the effects of supplementation of 8 g of BCAA or 7.5 g of soy protein on skeletal muscle and glycemic control as well as adverse events in elderly individuals with T2D. Thirty-six participants were randomly assigned to the BCAA group (n = 21) and the soy protein group (n = 15). Skeletal muscle mass and HbA1c, which were primary endpoints, did not change over time or differ between groups. However, knee extension muscle strength was significantly increased in the soy protein group and showed a tendency to increase in the BCAA group. Homeostasis model assessment for insulin resistance did not significantly change during the trial. Depressive symptoms were significantly improved in the BCAA group but the difference between groups was not significant. Results suggested that BCAA supplementation may not affect skeletal muscle mass and glycemic control and may improve depressive symptoms in elderly individuals with T2D.

We aimed to investigate the status of falls and to identify important risk factors for falls in persons with type 2 diabetes (T2D) including the non-elderly. Participants were 316 persons with T2D who were assessed for medical history, laboratory data and physical capabilities during hospitalization and given a questionnaire on falls one year after discharge. Two different statistical models, logistic regression and random forest classifier, were used to identify the important predictors of falls. The response rate to the survey was 72%; of the 226 respondents, there were 129 males and 97 females (median age 62 years). The fall rate during the first year after discharge was 19%. Logistic regression revealed that knee extension strength, fasting C-peptide (F-CPR) level and dorsiflexion strength were independent predictors of falls. The random forest classifier placed grip strength, F-CPR, knee extension strength, dorsiflexion strength and proliferative diabetic retinopathy among the 5 most important variables for falls. Lower extremity muscle weakness, elevated F-CPR levels and reduced grip strength were shown to be important risk factors for falls in T2D. Analysis by random forest can identify new risk factors for falls in addition to logistic regression.

The pancreatic islet vasculature is of fundamental importance to the β-cell response to obesity-associated insulin resistance. To explore islet vascular alterations in the pathogenesis of type 2 diabetes, we evaluated two insulin resistance models: ob/ob mice, which sustain large β-cell mass and hyperinsulinemia, and db/db mice, which progress to diabetes due to secondary β-cell compensation failure for insulin secretion. Time-dependent changes in islet vasculature and blood flow were investigated using tomato lectin staining and in vivo live imaging. Marked islet capillary dilation was observed in ob/ob mice, but this adaptive change was blunted in db/db mice. Islet blood flow volume was augmented in ob/ob mice, whereas it was reduced in db/db mice. The protein concentrations of total and phosphorylated endothelial nitric oxide synthase (eNOS) at Ser1177 were increased in ob/ob islets, while they were diminished in db/db mice, indicating decreased eNOS activity. This was accompanied by an increased retention of advanced glycation end-products in db/db blood vessels. Amelioration of diabetes by Elovl6 deficiency involved a restoration of capillary dilation, blood flow, and eNOS phosphorylation in db/db islets. Our findings suggest that the disability of islet capillary dilation due to endothelial dysfunction impairs local islet blood flow, which may play a role in the loss of β-cell function and further exacerbate type 2 diabetes.

High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.

KLF15 is a transcription factor that plays an important role in the activation of gluconeogenesis from amino acids as well as the suppression of lipogenesis from glucose. Here we identified the transcription start site of liver-specific KLF15 transcript and showed that FoxO1/3 transcriptionally regulates Klf15 gene expression by directly binding to the liver-specific Klf15 promoter. To achieve this, we performed a precise in vivo promoter analysis combined with the genome-wide transcription-factor-screening method "TFEL scan", using our original Transcription Factor Expression Library (TFEL), which covers nearly all the transcription factors in the mouse genome. Hepatic Klf15 expression is significantly increased via FoxOs by attenuating insulin signaling. Furthermore, FoxOs elevate the expression levels of amino acid catabolic enzymes and suppress SREBP-1c via KLF15, resulting in accelerated amino acid breakdown and suppressed lipogenesis during fasting. Thus, the FoxO-KLF15 pathway contributes to switching the macronutrient flow in the liver under the control of insulin.

While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically.

Engineered synthetic biomolecular devices that integrate elaborate information processing and precisely regulate living cell behavior have potential in various applications. Although devices that directly regulate key biomolecules constituting inherent biological systems exist, no devices have been developed to control intracellular membrane architecture, contributing to the spatiotemporal functions of these biomolecules. This study developed a synthetic biomolecular device, termed inducible counter mitochondrial morphology (iCMM), to manipulate mitochondrial morphology, an emerging informative property for understanding physiopathological cellular behaviors, on a minute timescale by using a chemically inducible dimerization system. Using iCMM, we determined cellular changes by altering mitochondrial morphology in an unprecedented manner. This approach serves as a platform for developing more sophisticated synthetic biomolecular devices to regulate biological systems by extending manipulation targets from conventional biomolecules to mitochondria. Furthermore, iCMM might serve as a tool for uncovering the biological significance of mitochondrial morphology in various physiopathological cellular processes.

In Japan, a new opportunistic community-based walk-in HbA1c testing program at pharmacies was enabled in 2014. An economic evaluation of this program from societal perspective has previously been published. This study examines the effect of a subsidy program for walk-in HbA1c-testing at community pharmacies in Japan on public health care expenditure by conducting a budget impact analysis from payer's perspective. The study focused on Adachi Ward in Tokyo, where a pioneering subsidy program was implemented. It examined the budget impact of the subsidy program over a 15 years. The total subsidy paid by the local authority during the first year was 2909 USD. Public expenditure remained positive for the first five years before becoming negative in the sixth year, eventually resulting in savings of 221,000 USD in the 15th year. The cost of treating type 2 diabetes that is detected early is offset by a reduction in expensive treatments for complicated cases. Subsidizing walk-in fingertip HbA1c testing in community pharmacies is likely to result in a significant reduction in public expenditure over the mid-to long term. Our result suggests that a similar strategy in other areas could also have a potentially favorable budget impact.

The identification of upstream transcription factors regulating the expression of a gene is generally not an easy process.  To facilitate this task, we constructed an expression cDNA library named Transcription Factor Expression Library (TFEL), which is composed of nearly all the transcription factors in the mouse genome. Genome-wide screening using this library (TFEL scan method) enables us to easily identify transcription factors controlling any given promoter or enhancer of interest in a chromosomal context-dependent manner. Thus, TFEL scan method is a powerful approach to explore transcriptional regulatory networks.

Aim: We investigated the relationship between cognitive function and olfactory and physical functions in middle-aged persons with and without type 2 diabetes (T2D) to examine the potential of olfactory and physical functions as biomarkers for early cognitive impairment. Methods: Enrolled were 70 T2D patients (age 40 to <65 y) and 81 age-matched control participants without diabetes. Cognitive function was assessed by the Montreal Cognitive Assessment (MoCA), Trail Making Test parts A and B (TMT-A/-B), Wisconsin Card Sorting Test (WCST), Quick Inventory of Depressive Symptomatology Self-Report (QIDS), and Starkstein Apathy Scale (SAS). Multiple linear regression analyses were performed. Results: Odor identification was an independent determinant shown in the results of the TMT-A in the entire participant group and was independently associated with the MoCA and TMT-B in the T2D group. Balance capability assessed with a stabilometer was independently associated with all cognitive function tests except for QISD and SAS in the entire participant group and the T2D group and was independently associated with TMT-A in the control group. Knee extension strength was independently associated with the SAS in the entire participant group and the T2D group. Conclusions: Odor identification, balance capability, and knee extension strength were potential markers for cognitive decline in middle-aged persons with T2D.

The World Anti-Doping Agency has prohibited gene doping in the context of progress in gene therapy. There is a risk that the artificial regulation of genes using plasmids could be applied for gene doping. However, no gold standard method to detect this has been established. Here, we aimed to develop a method to detect multiple transgene fragments as proof of gene doping. First, gene delivery model mice as a mimic of gene doping were created by injecting firefly luciferase plasmid with polyethylenimine (PEI) into the abdominal cavity. The results confirmed successful establishment of the model, with sufficient luminescence upon in vivo imaging. Next, multiple transgene fragments in the model were detected in plasma cell-free (cf)DNA, blood-cell-fraction DNA, and stool DNA using the TaqMan-qPCR assay, with the highest levels in plasma cfDNA. Using just a single drop of whole blood from the model, we also attempted long-term detection. The results showed that multiple transgene fragments were detected until 11 days. These findings indicate that the combination of plasma cfDNA or just one drop of whole blood with TaqMan-qPCR assay is feasible to detect plasmid-PEI-based gene doping. Our findings could accelerate the development of methods for detecting gene doping in humans.

BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic β-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 μmol/l (226-416 μmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 μg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic β-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic β-cell functions that deserve further scrutiny.

The aim of this study was to clarify degradation characteristics in each tissue of the knee complex of a medial meniscectomy (MMx)-induced knee osteoarthritis (KOA) animal model using classical methods and an alternative comprehensive evaluation method called contrast-enhanced X-ray micro-computed tomography (CEX-μCT), which was developed in the study. Surgical MMx was performed in the right knee joints of five male Wistar rats to induce KOA. At four weeks post-surgery, the synovitis was evaluated using quantitative polymerase chain reaction (qPCR). Degradations of the articular cartilage of the tibial plateau were evaluated using classical methods and CEX-μCT. Evaluation of the synovitis demonstrated significantly increased expression levels of inflammation-associated marker genes in MMx-treated knees compared with those in sham-treated knees. Evaluation of the articular cartilage using classical methods showed that MMx fully induced degradation of the cartilage. Evaluation using CEX-μCT showed that local areas of the medial cartilage of the tibial plateau were significantly reduced in MMx-treated knees compared with those in sham-treated knees. On the other hand, total cartilage volumes were significantly increased in MMx-treated knees. On the basis of the findings of this study, the method could be relevant to study new treatments in KOA research.

The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-β (TGF-β) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.

Background: With the rapid progress of genetic engineering and gene therapy methods, the World Anti-Doping Agency has raised concerns regarding gene doping, which is prohibited in sports. However, there is no standard method available for detecting transgenes delivered by injection of naked plasmids. Here, we developed a detection method for detecting transgenes delivered by injection of naked plasmids in a mouse model that mimics gene doping. Methods: Whole blood from the tail tip and one piece of stool were used as pre-samples of injection. Next, a plasmid vector containing the human erythropoietin (hEPO) gene was injected into mice through intravenous (IV), intraperitoneal (IP), or local muscular (IM) injection. At 1, 2, 3, 6, 12, 24, and 48 h after injection, approximately 50 µL whole blood was collected from the tail tip. One piece of stool was collected at 6, 12, 24, and 48 h. From each sample, total DNA was extracted and transgene fragments were analyzed by Taqman quantitative PCR (qPCR) and SYBR green qPCR. Results: In whole blood DNA samples evaluated by Taqman qPCR, the transgene fragments were detected at all time points in the IP sample and at 1, 2, 3, 6, and 12 h in the IV and IM samples. In the stool-DNA samples, the transgene fragments were detected at 6, 12, 24, and 48 h in the IV and IM samples by Taqman qPCR. In the analysis by SYBR green qPCR, the transgene fragments were detected at some time point in both specimens; however, many non-specific amplicons were detected. Conclusions: These results indicate that transgene fragments evaluated after each injection method of naked plasmids were detected in whole-blood and stool DNA samples. These findings may facilitate the development of methods for detecting gene doping.

With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.

Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injury. However, its molecular mechanisms are unknown. To clarify these mechanisms, in this study, we applied one to three 15-min cold stimulations at 4 &amp;deg;C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cAMP response element-binding protein 1 (CREB1) was markedly phosphorylated (as pCREB1) and CREB-binding protein (CBP) was recruited to pCREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 &amp;deg;C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes such as Pgc-1&amp;alpha; involved in mitochondrial biogenesis. The foregoing results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes in a manner dependent on cold stimulation frequency and duration. This information may serve as an impetus for further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.

Background and aims: Oxidized low-density lipoprotein (oxLDL) causes the development of atherosclerosis and kidney injury. Although circulating oxLDL levels were reportedly increased in type 2 diabetic patients with macroalbuminuria, it remains unclear whether albuminuria or the reduced glomerular filtration rate (GFR) is independently associated with the circulating oxLDL level. This study aimed to elucidate the association between the stage of diabetic nephropathy and serum malondialdehyde-modified LDL (MDA-LDL) and the ratio of MDA-LDL to LDL-cholesterol (MDA-LDL/LDL). Methods and results: This retroactive cross-sectional study used data from 402 patients with type 2 diabetes. Patients undergoing hemodialysis were excluded. Serum MDA-LDL levels were significantly increased with increases in severity of albuminuria (103 ± 44 U/L, 109 ± 54 U/L, and 135 ± 72 U/L for normoalbuminuria, microalbuminuria, and macroalbuminuria, respectively P for trend = 0.020) but not according to the estimated GFR (eGFR). An increased MDA-LDL/LDL ratio was significantly associated with both increased albuminuria (35 ± 13, 37 ± 14, and 40 ± 15 for normoalbuminuria, microalbuminuria, and macroalbuminuria, respectively P for trend = 0.003) and reduced eGFR (34 ± 13, 36 ± 13, 38 ± 12, and 51 ± 28 for grade 1, 2, 3 and 4, respectively P for trend = 0.002). Multiple linear regression analysis showed that neither the albumin excretion rate nor eGFR but ln-transformed triglycerides and LDL-C levels were independent determinants of both serum MDA-LDL levels and MDA-LDL/LDL ratios. Conclusion: Serum MDA-LDL levels and MDA-LDL/LDL ratios were increased in those with dyslipidemia associated with diabetic kidney disease.

Sterol regulatory element-binding protein (SREBP)-1a is a key transcription factor that activates the expression of genes involved in the synthesis of fatty acids, triglycerides (TGs), and cholesterol. Transgenic mice that overexpress the nuclear form of SREBP-1a under the control of the phosphoenolpyruvate carboxykinase promoter (Tg-1a) were previously shown to display a lipodystrophic phenotype characterized by enlarged and fatty livers, diminished peripheral white adipose tissue (WAT), and insulin resistance. In the current study, we crossed these Tg-1a mice with genetically obese (ob/ob) mice (Tg-1a ob/ob) and examined change in fat distribution between liver and adipose tissues in severe obesity and mechanism underlying the lipodystrophic phenotype in mice with Tg-1a. Tg-1a ob/ob mice developed more severe steatohepatitis but had reducedWAT mass and body weight compared with ob/obmice. The reduction ofWATmass in Tg-1a and Tg-1a ob/obmice was accompanied by enhanced lipogenesis and lipid uptake in the liver, reduced plasma lipid levels, impaired adipocyte differentiation, reduced food intake, enhanced energy expenditure, and extended macrophage infiltration and fibrosis in WAT. Despite the improved glucose tolerance, Tg-1a ob/ob mice showed severe peripheral insulin resistance. Adenoviral hepatic expression of SREBP-1a mimicked these phenotypes. The 'fat steal'-like lipodystrophy phenotype of the Tg-1a ob/ob model demonstrates that hepatic SREBP-1a activation has a strong impact on the partition of TG accumulation, resulting in adipose-tissue remodeling by inflammation and fibrosis and insulin resistance.

OBJECTIVE A new opportunistic community-based strategy was launched in Japan in April 2014 to detect lifestyle-related diseases, including diabetes, by creating Specimen Measurement Offices (SMOs). SMOs offer walk-in fingertip HbA1c testing. This article aimed to assess the value-for-money of HbA1c testing services at SMOs by conducting a cost-effectiveness analysis. RESEARCH DESIGN AND METHODS We compared two scenarios: 1) status quo, defined as HbA1c testing that is available only through conventional screening, and 2) HbA1c testing available at SMOs as a complement to the status quo scenario. The model consisted of a screening module with a decision tree and a disease progression module with a Markov model. We calculated incremental cost-effectiveness ratios (i.e., cost per quality-adjusted lifeyears [QALYs]) over the lifetime analytic horizon as the primary end point of the cost-effectiveness analysis. In this model, we assumed the participant cohort to be people 40-74 years of age who sought walk-in fingertip HbA1c testing at SMOs on the premises of community pharmacies. Costs and outcomes were discounted at a rate of 3%. The cost-effectiveness was analyzed from a societal perspective. RESULTS The incremental cost per individual for those 40-74 years of age was estimated to be 2527 U.S. dollars (USD) (252,722 Japanese yen [JPY]) for HbA1c testing at SMOs compared with the status quo. Incremental effectiveness was estimated to be 0.0203 QALYs for HbA1c testing at SMOs compared with the status quo. Therefore, this cost-effectiveness analysis showed that compared with the status quo, HbA1c testing at SMOs was more effective and had lower cost for the population studied. CONCLUSIONS We consider our results to be robust because most simulations were under the threshold of USD 50,000 (JPY 5,000,000) per QALYs gained, by sensitivity analysis. These results will be useful to managers of pharmacies or other health institutions and/or policy makers in local government.

The SNP rs7903146 at the transcription factor 7-like 2 (TCF7L2) locus is established as the strongest known genetic marker for type 2 diabetes via genome-wide association studies. However, the functional SNPs regulating TCF7L2 expression remain unclear. Here, we show that the SNP rs7074440 is a candidate functional SNP highly linked with rs7903146. A reporter plasmid with rs7074440 normal allele sequence exhibited 15-fold higher luciferase activity compared with risk allele sequence in hepatocytes, demonstrating a strong enhancer activity at rs7074440. Additionally, we identified C-FOS as an activator binding to the rs7074440 enhancer using a TFEL genome-wide screen method. Consistently, knockdown of C-FOS significantly reduced TCF7L2 expression in hepatocytes. Collectively, a novel enhancer regulating TCF7L2 expression was revealed through searching for functional SNPs.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFU), a highly selective SGLT2 inhibitor, for 3 weeks. TOFU-treated mice showed a decrease in fat mass and the effect of TOFU was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFU treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PICA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PICA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry. (C) 2017 Elsevier Inc. All rights reserved.

Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around −100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.

Although the human liver comprises approximately 2.8% of the body weight, it plays a central role in the control of energy metabolism. While the biochemistry of energy substrates such as glucose, fatty acids, and ketone bodies in the liver is well understood, many aspects of the overall control system for hepatic metabolism remain largely unknown. These include mechanisms underlying the ascertainment of its energy metabolism status by the liver, and the way in which this information is used to communicate and function together with adipose tissues and other organs involved in energy metabolism. This review article summarizes hepatic control of energy metabolism via the autonomic nervous system.

Aim: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin’s action. Methods: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. Results: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. Conclusions: We clarify for the first time the role of HSL in leptin’s effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.

Objective: The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by Creb3l3) is highly expressed in the liver and small intestine. Hepatic CREBH contributes to glucose and triglyceride metabolism by regulating fibroblast growth factor 21 (Fgf21) expression. However, the intestinal CREBH function remains unknown. Methods: To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions. Results: Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of NiemannePick C1-like 1 (Npc1l1), a rate-limiting transporter mediating intestinal cholesterol absorption, was reduced in the small intestine of CREBH Tg mice. Adenosine triphosphate-binding cassette transporter A1 (Abca1), Abcg5/8, and scavenger receptor class B, member 1 (Srb1) expression levels were also reduced in CREBH Tg mice. Promoter assays revealed that CREBH directly regulates Npc1l1 expression. Conversely, CREBH null mice exhibited higher intestinal Npc1l1 expression, elevated plasma and hepatic cholesterol, and lower fecal output. Conclusion: Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia. (C) 2016 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).