SREBP2 regulates the endothelial response to cytokines via direct transcriptional activation of KLF6

The transcription factor SREBP2 is the main regulator of cholesterol homeostasis and is central to the mechanism of action of lipid-lowering drugs, such as statins, which are responsible for the largest overall reduction in cardiovascular risk and mortality in humans with atherosclerotic disease. Recently, SREBP2 has been implicated in leukocyte innate and adaptive immune responses by upregulation of cholesterol flux or direct transcriptional activation of pro-inflammatory genes. Here, we investigate the role of SREBP2 in endothelial cells (ECs), since ECs are at the interface of circulating lipids with tissues and crucial to the pathogenesis of cardiovascular disease. Loss of SREBF2 inhibits the production of pro-inflammatory chemokines but amplifies type I interferon response genes in response to inflammatory stimulus. Furthermore, SREBP2 regulates chemokine expression not through enhancement of endogenous cholesterol synthesis or lipoprotein uptake but partially through direct transcriptional activation. Chromatin immunoprecipitation sequencing of endogenous SREBP2 reveals that SREBP2 bound to the promoter regions of two nonclassical sterol responsive genes involved in immune modulation, BHLHE40 and KLF6. SREBP2 upregulation of KLF6 was responsible for the downstream amplification of chemokine expression, highlighting a novel relationship between cholesterol homeostasis and inflammatory phenotypes in ECs

Skeletal muscle autophagy and mitophagy in endurance-trained runners before and after a high-fat meal

Objective: We tested the hypothesis that skeletal muscle of endurance-trained male runners would exhibit elevated autophagy and mitophagy markers, which would be associated with greater metabolic flexibility following a high-fat meal (HFM). Methods: Muscle biopsies were collected to determine differences in autophagy and mitophagy protein markers and metabolic flexibility under fasting conditions and 4 h following a HFM between endurance-trained male runners (n = 10) and sedentary, non-obese controls (n = 9). Results: Maximal oxygen consumption (ml·kg·min−1) was approximately 50% higher (p  0.05), but increased in response to the HFM in endurance-trained athletes only (p < 0.005). Key mitophagy markers, phospho-Pink1Thr257 and phospho-ParkinS65 (r = 0.64, p < 0.005), and phospo-ParkinSer65 and phospho-Drp1Ser616 (r = 0.70, p < 0.05) were correlated only within the endurance-trained group. Autophagy and mitophagy markers were not correlated with metabolic flexibility. Conclusion: In summary, mitophagy may be enhanced in endurance-trained runners based on elevated markers of mitophagy and mitochondrial dynamics. The HFM did not alter autophagy or mitophagy in either group. The absence of a relationship between mitophagy markers and metabolic flexibility suggests that mitophagy is not a key determinant of metabolic flexibility in a healthy population, but further investigation is warranted. Author Video: Author Video Watch what authors say about their articles Keywords: Metabolic flexibility, Autophagy, Mitophagy, Endurance training, Skeletal muscl

Overfeeding and Substrate Availability, But Not Age or BMI, Alter Human Satellite Cell Function

Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18&ndash;30 years) and sedentary older (60&ndash;80 years) men with varying BMIs (18&ndash;32 kg/m2), as well as young sedentary men before and after four weeks of overfeeding (OVF) (55% fat/ + 1000 kcal, n = 4). The isolated SCs were then treated in vitro with a control (5 mM glucose, 10% fetal bovine serum (FBS)) or a high substrate growth media (HSM) (10% FBS, 25 mM glucose, and 400 &mu;M 2:1 oleate&ndash;palmitate). Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (&ndash;19% p &lt; 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor&rsquo;s body mass index (BMI) (r2 = 0.6121, p &lt; 0.01). When exposed to HSM, SCs from normal weight (BMI 18&ndash;25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p &lt; 0.05), while SCs from participants with higher BMIs (BMI 25&ndash;32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p &lt; 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet

Optimized and Automated Radiosynthesis of [18F]DHMT for Translational Imaging of Reactive Oxygen Species with Positron Emission Tomography

Reactive oxygen species (ROS) play important roles in cell signaling and homeostasis. However, an abnormally high level of ROS is toxic, and is implicated in a number of diseases. Positron emission tomography (PET) imaging of ROS can assist in the detection of these diseases. For the purpose of clinical translation of [18F]6-(4-((1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)methoxy)phenyl)-5-methyl-5,6-dihydrophenanthridine-3,8-diamine ([18F]DHMT), a promising ROS PET radiotracer, we first manually optimized the large-scale radiosynthesis conditions and then implemented them in an automated synthesis module. Our manual synthesis procedure afforded [18F]DHMT in 120 min with overall radiochemical yield (RCY) of 31.6% ± 9.3% (n = 2, decay-uncorrected) and specific activity of 426 ± 272 GBq/µmol (n = 2). Fully automated radiosynthesis of [18F]DHMT was achieved within 77 min with overall isolated RCY of 6.9% ± 2.8% (n = 7, decay-uncorrected) and specific activity of 155 ± 153 GBq/µmol (n = 7) at the end of synthesis. This study is the first demonstration of producing 2-[18F]fluoroethyl azide by an automated module, which can be used for a variety of PET tracers through click chemistry. It is also the first time that [18F]DHMT was successfully tested for PET imaging in a healthy beagle dog

Serum endotoxin, gut permeability and skeletal muscle metabolic adaptations following a short term high fat diet in humans

Background: Our previous work demonstrated that a short-term high fat diet (HFD) increased fasting serum endotoxin, altered postprandial excursions of serum endotoxin, and led to metabolic and transcriptional responses in skeletal muscle in young, healthy male humans. Purpose: The purpose of the present study was to determine if a short-term high fat diet: 1) increases intestinal permeability and, in turn, fasting endotoxin concentrations and 2) decreases postprandial skeletal muscle fat oxidation. Methods: Thirteen normal weight young adult males (BMI 23.1 +/- 0.8 kg/m(2), age 22.2 +/- 0.4 years) were fed a control diet (55% carbohydrate, 30% fat, 9% of which was saturated, 15% protein) for two weeks, followed by 5 days of an isocaloric HFD (30% carbohydrate, 55% fat, 25% of which was saturated, 15% protein, isocaloric to the control diet). Intestinal permeability (via four sugar probe test) was assessed in the fasting state. Both before and after the HFD, a high fat meal challenge (HFM, 820 kcal, 25% carbohydrate, 63% fat, 26% of which was saturated, and 12% protein) was administered. After an overnight fast, blood samples were collected before and every hour for 4 h after the HFM to assess endotoxin, and other serum blood measures. Muscle biopsies were obtained from the vastus lateralis before and 4 h after the HFM in order to assess substrate oxidation (glucose, fatty acid and pyruvate) using radiolabeled techniques. Insulin sensitivity was assessed via intravenous glucose tolerance test. Intestinal permeability, blood samples and muscle biopsies were assessed in the same manner before and following the HFD. Main findings: Intestinal permeability was not affected by HFD (p > 0.05), but fasting endotoxin increased two fold following the HFD (p = 0.04). Glucose oxidation and fatty acid oxidation in skeletal muscle homogenates significantly increased after the HFM before the HFD (+97%, and +106% respectively) but declined after the HFM following 5 days of the HFD (-24% and +16% respectively). Fatty acid suppressibility of pyruvate oxidation increased significantly after the HFM (+32%) but this physiological effect was abolished following 5 days of the HFD (+7%). Insulin sensitivity did not change following the HFD. Conclusion: These findings demonstrate that in healthy young men, consuming an isocaloric HFD for 5 days increases fasting endotoxin, independent of changes in gut permeability. These changes in endotoxin are accompanied by a broad effect on skeletal muscle substrate metabolism including increases in postprandial fat oxidation. Importantly, the latter occurs independent of changes in body weight and whole-body insulin sensitivity. (C) 2019 The Authors. Published by Elsevier Inc