The effects of a ketogenic diet on endurance capacity and post-exercise recovery

早稲田大学博士(スポーツ科学)早大学位記番号:新8247doctoral thesi

Glucose Ingestion Inhibits Endurance Exercise-Induced IL-6 Producing Macrophage Infiltration in Mice Muscle

BACKGROUND: Carbohydrate (CHO) supplementation during exercise attenuates exercise-induced increases in plasma Interleukin (IL)-6 concentration. However, the effects of CHO supplementation on muscle IL-6 production during endurance exercise is controversial. The purpose of this study was to investigate the effects of CHO supplementation on muscle IL-6 production during endurance exercise with a special focus on the IL-6 producing cells. METHODS: C57BL/6J mice were divided into three groups-sedentary with water ingestion group as the control (Con; n = 10), exercise with water ingestion group (Ex; n = 10), and exercise with 6% glucose ingestion group (Ex + glucose; n = 10). The Ex and Ex + glucose groups completed 3 h of treadmill running (24 m/min, 7% incline) and were sacrificed immediately after exercise. RESULTS: The exercise-induced increases of plasma IL-6 concentration and gastrocnemius IL-6 gene expression were attenuated by glucose ingestion. However, the increases of soleus IL-6 gene expression and gastrocnemius and soleus IL-6 protein expression were not attenuated by glucose ingestion. Furthermore, we observed that macrophages that infiltrated muscle produce IL-6 and glucose ingestion attenuated the infiltration of IL-6-producing macrophages. CONCLUSION: This study revealed that infiltrating macrophages may be one type of IL-6-producing cells during endurance exercise, and the infiltration of these cells in muscle was attenuated by glucose ingestion. However, the effects of glucose ingestion on muscle IL-6 production were limited

Postnatal nutrition environment reprograms renal DNA methylation patterns in offspring of maternal protein-restricted stroke-prone spontaneously hypertensive rats

Maternal malnutrition hampers the offspring health by manipulating the epigenome. Recent studies indicate that the changes in DNA methylation could be reversed by afterbirth nutrition supplementation. In this study, we used DNA methylation arrays to comprehensively investigate the DNA methylation status of the renal promoter regions and the effects of postnatal protein intake on DNA methylation. We fed stroke-prone spontaneously hypertensive (SHRSP) rat dams a normal diet or a low-protein diet during pregnancy, and their 4-week-old male offspring were fed a normal diet or a high−/low-protein diet for 2 weeks. We found that the methylation status of 2,395 differentially methylated DNA regions was reprogrammed, and 34 genes were reset by different levels of postnatal protein intake in the offspring. Among these genes, Adora2b, Trpc5, Ar, Xrcc2, and Atp1b1 are involved in renal disease and blood pressure regulation. Our findings indicate that postnatal nutritional interventions can potentially reprogram epigenetic changes, providing novel therapeutic and preventive epigenetic targets for salt-sensitive hypertension

PO-305 An 8-week, low carbohydrate, high fat, ketogenic diet enhanced exercise capacity through improved ketolysis and lipolysis in mice

Objective Carbohydrates, lipids and proteins are utilized both for energy production and structure of body. Among them, protein is the most important component of our body, carbohydrates and lipids are more flexible for energy supply system. Due to carbohydrate pitfall and lipid reserve abundance, coaches and elite athletes aspirate for an effective way to enhance fat utilization. Meanwhile, intramuscular triacylglycerol (IMTG) is a special way for skeletal muscle to store lipids. During exercise, IMTG may contribute up to 20% of total energy turnover, thus contribute significantly for ATP synthesis during exercise. However, abnormal or excessive fat deposition in skeletal muscle may induce insulin resistance as well. Intramuscular lipolysis regulation is crucial for energy supply system during exercise. It is reported by Amati and colleagues that well-trained athletes exhibit higher levels of IMTG and diacylglycerol (DAG) as well as well-preserved sensitivity to insulin, indicating lipolysis ability may be enhanced during exercise. In our previous study, we reported that an 8-week, a low carbohydrate, ketogenic diet increased running time till exhausted in male C57BL6/J mice, presuming the mechanism to be enhanced fat utilization. In the present study, we observed the alternation pattern of messenger RNAs related to lipid mobilization, fatty acid utilization and ketone body oxidation in muscle and adipose tissue immediately after exercise in both Type 1 and Type 2 muscle fibers. Materials and Methods Male C57BL/6J mice (n = 35) were purchased from Takasugi Experimental Animals Supply (Kasukabe, Japan) at 7 weeks of age. All mice were randomly divided into four groups: chow diet (control: Con), including chow diet, sedentary (n = 8) and chow diet plus exercise (Con + Ex, n = 9), ketogenic diet (KD), including KD, sedentary, n = 9, and KD plus exercise (KD + Ex, n = 9) groups. A KD diet TP-201450 (consisting of 76.1% fat, 8.9% protein and 3.5% carbohydrate, 7.342 kcal/g) and a chow diet AIN93G (consisting of 7% fat, 17.8% protein and 64.3% carbohydrate, 3.601 kcal/g) wt/wt were obtained from Trophic (TROPHIC Animal Feed High-tech Co., Ltd., Nantong, Jiangsu, China). Mice were maintained on ad libitum chow diet or KD. Total RNA was extracted from the gastrocnemius muscle, soleus muscle and epididymal adipose tissue using the RNeasy Mini Kit or RNeasy Lipid Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. PCR was performed with the Fast 7500 real-time PCR system (Applied Biosystems) using the Fast SYBR® Green PCR Master Mix (Applied Biosystems). Plasma IL-6 was measured using a R&D Mouse ELISA Duo set (R&D Systems, Minneapolis, USA) according to the manufacture’s instructions. Plasma glycerol was measured using Glycerol Colorimetric Assay Kit (Cayman Chemical Co., Ann Arbor, MI, USA). Results and Discussion 3.1 IL-6 concentration and exercise-induced myokine IL-6 mRNA alternation in both muscle fiber IL-6 plays essential roles in immune responses. However, exercise induced IL-6 is reported to be able to stimulate lipolysis both in IMTG pool intramuscularly and adipocytes. Defined as exercise factor, or so-called myokines, muscle-derived IL-6 exhibits regulating function in various experiment circumstances. Recombinant human IL-6 infusion showed an enhanced lipolysis and fat oxidation capacity in human subjects. Genetically IL-6 deficient mice presented a reduced ability on lipolysis and fatty acid oxidation. During KD administration, fat oxidation is no doubt the predominant, if not the only origin of energy, this make us to suspect IL-6 may be altered by acute exercise. As shown in Figure 1, IL-6 mRNA increased rapidly, with a nearly 100-fold change in slow-twitch muscle fiber, and KD helped to this up-regulation. Transcription IL-6 level increased significantly in KD, compared to Con group subject, in soleus muscle, under the context of exhaustive exercise. This makes us to suspect that IL-6 may contribute to enhanced lipolysis and fatty acid mobilization. However, the effect is not observed in fast-twitch muscle. The result indicated that IL-6 mRNA expression exhibited a muscle fiber specification. Slow-twitch muscle fiber contributes more to endurance exercise, as fast-twitch muscle fiber mainly contributes to explosive strength and acceleration. The difference of fiber function leads to a different secretion mode of IL-6. The error bar is high in both exercise group. We observed an interesting phenomenon: the mice who quit at around 200 minutes has the highest IL-6 mRNA expression (gastrocnemius muscle) and plasma IL-6 concentration in both groups. One reason may be that, as the exercise taking on and gradually reach final fatigue, the call for fatty acid decreases with time. As shown in Figure 2, both muscular IL-6 protein and plasma IL-6 were increased by exhaustive exercise. However, plasma IL-6 is significantly lower in the KD plus exercise group, though IL-6 rose nearly 5-fold in control feed group, it only rose to 2.5-fold in the KD group after exercise.  For this phenomenon, the best explanation is that the well-adaption of lipid-centered metabolism, including metabolic flexibility and increased IMTG reservoir weakened the need to pull the trigger; this may also be the answer why KD mice had lower weight. 3.2 Fatty acid mobilation related RNA alternation after exhaustive exercise under endogenous ketosis in epididymal adipose tissue Adipose triglyceride lipase (ATGL) is also known as desnutrin in the first place, is a kind of lipase whose substance is patatin-like phospholipase domain-containing protein. Hormone-sensitive lipase (HSL) is also known as cholesteryl ester hydrolase (CEH), is another intracellular neutral lipase. ATGL and HSL cooperated to break apart fatty acids from TG, after which IMTG-origin fatty acid will be directly used for beta-oxidation, or lipid drop-origin fatty acids will be transported though lipoprotein shipping in the form of VLDL from adipose tissue into muscle fibers during exercise. As shown in Figure 3, mRNA expression levels of lipase were significantly enhanced by KD or exercise, indicating the up-regulated lipid mobilization and utilization ability is enhanced by exercise in adipocytes. However, KD plus exercise reversed this increase. One plausible explain for this phenomenon is the lack of plasma IL-6, thus the ability to mobilize fatty acid from adipose tissue is reduced. Adrenergic blocking agents are reported to harm fatty acid mobilization during fasting, and IL-6 is reported to function as adrenergic hormone. Adipocyte-specific HSL deficiency mice present lowered submaximal exercise capacity. Our experiment design, the protocol for treadmill running is similar to a submaximal exercise. Under this circumstance, fat mobilization seems to be critical. Loss of this mobilizing ability, while exercise capacity is yet enhanced, makes us to suspect whether IMTG plays dominant role in this process. 3.3 Ketolytic RNA alternation after exhaustive exercise under conditions of endogenous ketosis in Type 1 and Type 2 muscle fiber Ketolysis is a complete oxidation of ketone bodies. Ketone bodies are utilized by mitochondria of extrahepatic tissues via a series of enzymatic reactions. Ketolysis is regulated by a rate-limiting enzyme 3-oxoacid CoA-transferase 1 (OXCT)-1 and hydroxybutyrate dehydrogenase (HBDH). Thus, we measured the transcriptional alternation of these enzymes in different muscle tissues. In our previous study, plasma ketone body (KB) increased rapidly in the sedentary KD group. However, after exhaustive exercise, blood KB of those KD mice dropped dramatically, while situation of blood KD in the Con mice showed a different figure. These results indicated that 1n 8-week KD administration has improved ketolysis, the ability for subjects to utilize KB. To investigate the mechanism of this enhancement, we assessed key enzymes in ketolysis in both fiber types. As shown in Figure 4, gene expressions of these enzymes also present a fiber-specification. Since fast-twitch muscle fiber plays a second role in endurance exercise, exercise did not alter ketolytic enzymes in the transcriptional level, in gastrocnemius tissue. However, in the slow-twitch muscle fiber, it was changed. HBDH is up-regulated significantly in the case of KD plus exercise. Results here indicated that HBDH plays the key role in the improvement of exercise capacity by an 8-week KD. 3.4 Lipolysis- and fatty acid oxidation related RNA alternation after exhaustive exercise during endogenous ketosis in Type 1 and Type 2muscle fiber After reaching working site, muscle lipoprotein lipase (LPL) hydrolyzes VLDL and harvests fatty acids at last, which will be finally utilized as primary fuel. Carnitine palmitoyl transferase (CPT)-1A, acyl-CoA oxidase (ACO), hydroxyacyl-coenzyme A dehydrogenase (HADH), medium chain acyl-CoA dehydrogenase (MCAD) and malonyl-CoA decarboxylase (MCD) are key regulating enzymes during fatty acid beta-oxidation. And for IMTG, the fatty acid is harvest by intramuscular lipase, ATGL and HSL. In an article published several years ago, the authors called adipose ATGL and HSL, “the mover and shaker of muscle lipolysis”. As shown in Figure 5, both ATGL and HSL mRNA expression are up-regulated by KD, but not by exhaustive exercise. Combined with results in the above part, enhanced mobilization of fatty acid intramuscularly is the main factor, but not the mobilization from adipocyte. LPL mRNA expression in gastrocnemius exhibited the same pattern of lipase mRNA synthesized by adipose tissue. Combined together, the reduced fatty mobilization from adipocyte, partly being the results of higher blood NEFA and TG, accompanied with enhanced fatty acid gain from IMTG pool, need for LPL was reduced.  As shown in Figure 6, in fast-twitch muscle fiber, CPT-1a, ACO and HADH mRNA expressions are enhanced by KD during exhaustive exercise. In slow-twitch muscle fiber, feed played as a main factor regulating fatty acid oxidation. CPT1a, MCAD and MCD mRNA expressions are enhanced. In summary, though tissue specific specificity were observed, overall ability of intramuscular fatty acid mobilization and fatty acid oxidation were enhanced by an 8-week KD feeding, thus contributed to exercise capacity. Compared to a glucose-centered metabolic system, a long-term KD feeding leads to establishment of a fatty acid oxidation-centered metabolic system. Metabolic flexibility is used as a term for the ability to adapt to conditional change in metabolic demand, and an 8-week KD helped established lipid-focused metabolic system through keto-adaption, thus increasing the metabolic flexibility. This is not a denial for the conception “glycogen loading” before competitions, while adequate KD meal may help our body to be more flexible during fuel choosing. Moderate training may enhance the ability to utilize ketone bodies as well as fatty acid, or to increase fatty acid mobilization from adipose tissue. Further investigation is urged to be carried out.   Conclusions In the present study, we investigated how an 8-week KD remodeled adipose and muscle metabolic adaptation towards ketolysis, lipolysis and fatty acid oxidation under the circumstance of exhaustive exercise. Along with enhanced fatty acid oxidation capacity, KD also enhanced fatty acid mobilization capacity, ketolysis and lipolysis. These results revealed that an 8-week KD administration enhance exercise performance by up-regulated ketolytic and free fatty acid oxidation ability, indicating KD being a promising diet approach in athletes

The Optimal Axial Anatomical Site for a Single-Slice Area to Quantify the Total Volume of Visceral Adipose Tissue in Quantitative CT

PurposeDetermine the association between cross-sectional visceral adipose tissue (VAT) area of different anatomic locations and total abdominopelvic VAT volume; identify the optimal measurement site in a single-slice to quantify the total VAT volume.MethodParticipants who underwent non-contrast abdominal scan by quantitative CT (QCT) were enrolled from May 2021 to October 2021. The VAT area (cm2) at different anatomic sites as upper-pole, lower-pole, and hilum of the kidney, intervertebral disc of L2/L3 and L5/S1, and umbilical level were measured on QCT PRO BMD workstation (Mindways QCT PRO workstation). The total VAT volume (cm3) from the upper pole of kidney to the L5/S1 intervertebral disc of the pelvis (abdominopelvic region) was obtained by using Siemens Healthineers Syngo via Frontier cardiac risk assessment. Regression models were used to identify the optimal single-slice in different gender for estimating VAT volume. Statistical significance was established at P < 0.05.ResultsTotal of 311 Chinese participants including 179 men [age, 55.1 ± 14.9 years; body mass index (BMI), 24.2 ± 3.2 kg/m2; total VAT volume, 2482.6 ± 1276.5 mL] and 132 women [age, 54.3 ± 14.9; BMI, 23.5 ± 2.9; total VAT volume, 1761.5 ± 876.4]. Pearson’s correlation analysis revealed a strong association between the VAT area and total abdominopelvic VAT volume at the hilum of the kidney in both men (r=0.938, P<0.001) and women (r=0.916, P<0.001). Adjust for covariates including age, BMI, and waist circumference make a relatively small effect on predicting the total VAT volume.ConclusionsMeasurement of cross-sectional areas at the hilum of the kidney in both genders showed a strongest relation to TVAT volume. Our results may provide an identifiable and valuable axial landmark for measuring visceral adipose tissue in clinical practice

Immunostimulatory Activity of Protein Hydrolysate from Oviductus Ranae on Macrophage In Vitro

Oviductus Ranae is the dry oviduct of Rana chensinensis, which is also called R. chensinensis oil. Oviductus Ranae is a valuable Chinese crude drug and is recorded in the Pharmacopoeia of the People’s Republic of China. The aim of this study was to investigate the immunostimulatory activity of protein hydrolysate of Oviductus Ranae (ORPH) and to assess its possible mechanism. Immunomodulatory activity of ORPH was examined in murine macrophage RAW 264.7 cells. The effect of ORPH on the phagocytic activity of macrophages was determined by the neutral red uptake assay. After treatment with ORPH, NO production levels in the culture supernatant were investigated by Griess assay. The mRNA and protein expressions of inducible nitric oxide synthase (iNOS) were detected by RT-PCR and Western blotting. The production of TNF-α, IL-1β, and IL-6 after treatment with ORPH was measured using ELISA assay. In addition, NF-κB levels were also investigated by Western blot. The results showed that ORPH enhanced the phagocytosis of macrophage, increased productions of TNF-α, IL-1β, IL-6, and NO in RAW 264.7 cells, and upregulated the mRNA and protein expression of iNOS. Besides, NF-κB, levels in RAW 264.7 cells were elevated after ORPH treatment. These findings suggested that ORPH might stimulate macrophage activities by activating the NF-κB pathway

Prolonged administration of total glucosides of paeony improves intestinal immune imbalance and epithelial barrier damage in collagen-induced arthritis rats based on metabolomics-network pharmacology integrated analysis

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and joint damage with complex pathological mechanisms. In recent years, many studies have shown that the dysregulation of intestinal mucosal immunity and the damage of the epithelial barrier are closely related to the occurrence of RA. Total glucosides of paeony (TGP) have been used clinically for the treatment of RA in China for decades, while the pharmacological mechanism is still uncertain. The purpose of this study was to investigate the regulatory effect and mechanism of TGP on intestinal immunity and epithelial barrier in RA model rats. The results showed that TGP alleviated immune hyperfunction by regulating the ratio of CD3+, CD4+ and CD8+ in different lymphocyte synthesis sites of the small intestine, including Peyer’s patches (PPs), intraepithelial lymphocytes (IELs), and lamina propria lymphocytes (LPLs). Specially, TGP first exhibited immunomodulatory effects on sites close to the intestinal lumen (IELs and LPLs), and then on PPs far away from the intestinal lumen as the administration time prolonged. Meanwhile, TGP restores the intestinal epithelial barrier by upregulating the ratio of villi height (V)/crypt depth (C) and expression of tight junction proteins (ZO-1, occludin). Finally, the integrated analysis of metabolomics-network pharmacology was also used to explore the possible regulation mechanism of TGP on the intestinal tract. Metabolomics analysis revealed that TGP reversed the intestinal metabolic profile disturbance in CIA rats, and identified 32 biomarkers and 163 corresponding targets; network pharmacology analysis identified 111 potential targets for TGP to treat RA. By intersecting the results of the two, three key targets such as ADA, PNP and TYR were determined. Pharmacological verification experiments showed that the levels of ADA and PNP in the small intestine of CIA rats were significantly increased, while TGP significantly decreased their ADA and PNP levels. In conclusion, purine metabolism may play an important role in the process of TGP improving RA-induced intestinal immune imbalance and impaired epithelial barrier

Analysis of care-seeking and diagnosis delay among pulmonary tuberculosis patients in Beijing, China

BackgroundTuberculosis (TB) remains a significant public health challenge in China. Early detection and diagnosis of TB cases are crucial to interrupt disease transmission and prevent its progression. This study aims to describe the delay in seeking care and diagnosis among patients with pulmonary tuberculosis (PTB) and identify the influencing factors in two counties in Beijing.MethodsA retrospective analysis was carried out to investigate care-seeking and diagnosis delay in two counties in Beijing. Basic information of PTB patients from January 1 to December 31, 2021, was extracted from the Tuberculosis Information Management System of China (TBIMS), and all enrolled patients were interviewed via telephone using a standard questionnaire. Statistical description was performed using the median and interquartile range (IQR). Chi-square test and multivariate logistic regression model were used to analyze the influencing factors.Results537 patients were enrolled. The median duration of care-seeking and diagnosis delay was 11 (IQR: 5–26) days and 8 (IQR: 0–18) days, with 41.71 and 35.20% of patients experiencing delays (>14 days). The study found that being asymptomatic (OR = 2.791, 95%CI: 1.710–4.555) before seeking medical care and not attending work during treatment (OR = 2.990, 95%CI: 1.419–6.298) were identified as risk factors for care-seeking delay. Patients who were tracked (OR = 2.632, 95%CI: 1.062–6.521) and diagnosed at tuberculosis control and prevention institutions (OR = 1.843, 95%CI: 1.061–3.202) had higher odds of diagnostic delays. 44.69% of patients presented a total delay (>28 days), with a median duration of 25 (IQR: 13–39) days. A multivariate logistic regression analysis showed that healthy examination (OR = 0.136, 95%CI: 0.043–0.425) was a protective factor for total delay.ConclusionPublic interventions are necessary to improve the efficiency of PTB patients detection and treatment in Beijing. Medical services should focus on the target population and improve access to medical care to further reduce delays for PTB patients