| Objective:Numerous studies showed that adipose tissue is a highly active metabolic and endocrine organ. The function of adipose tissue plays a crucial role in obesity-related complications. Adipose tissue dysfunction in obesity would lead to insulin resistance, increaed monocyte/macrophage infiltration in adipose tissue, and is associated with cardiovascular disease and type2diabetes. Recent animal studies suggest that adipose tissue hypoxia could induce low-grade inflammation. Current human studies on ATH are mainly focused on subcutaneous adipose tissue using invasive methods; however, visceral adipose tissue exerts more adverse metabolic effects than subcutaneous fat on insulin sensitivity, which could represent a more appropriate target for oxygenation measurement. Blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) offers a non-invasive and reliable tool for assessing organ/tissue hypoxia under different disease conditions. To our knowledge, BOLD-MRI has not been used in assessing human ATH. Recent evidence have shown that inflammatory cells, especially macrophage, have a great contribution to ATH. Human peripheral circulation monocytes (as the precursors for local macrophages) are divided into three subsets with distinct physiological functions, including the CD14++CD16-(classical), the CD14++CD16+(intermediate), and CD14+CD16++(non-classical). Among them, the CD14++CD16+monocytes were considered as "pro-inflammatory" monocytes, and are critical for a variety of inflammatory diseases. The role of monocyte/macrophage infiltration in adipose tissue in initiating insulin resistance has been well acknowledged. Which subset contributes to monocyte/macrophage accumulation in adipose tissue, and whether high salt loading play an important role in adipose hypoxia remains unclear. Therefore, the purposes of present study are (1) to investigate the feasibility of BOLD-MRI in evaluating adipose hypoxia under altered lipid and glucose homeostasis induced by dietary salt loading/depletion in human abdominal visceral adipose tissue; and (2) to investigate the associations between the three monocyte subsets and adipose hypoxia during high-salt loading.Methods:A total of23healthy non-smoking volunteers were recruited in our dietary intervention trial. We conducted a three-phase dietary intervention study including usual-salt phase, high-salt phase and low-salt phase. According to our recent investigation in aged inhabitants in rural northern China, we thus chose15g salt/day as high-salt loading. The low salt criterion is in accordance with World Health Organization’s recommendations(5g salt/day). During the investigation, all beverages and foods were prepared and provided by the investigators. Blood pressure and heart rate were measured at baseline,7th day after high-salt diet (HS7d), and7th day after low-salt diet (LS7d), as well as24-hour urine collection for urinary electrolytes and creatinine measurements. Overnight fasting blood samples were drawn at the ending of baseline, HS7d and LS7d. Circulating monocyte subsets were analyzed using a three-color flow cytometric protocol, based on CD86, CD14and CD16. The abdominal visceral adipose tissue (VAT) BOLD-MRI scanning were performed on a3.0T MR imager to determine the VAT R2*value at three stages in the same period. BOLD-MRI T2*-weighted images were recorded during a single breath holding and with a respiratory-triggered FFE sequence. Sixteen slices with a thickness of5mm and a gap of0mm were obtained in the coronal plane through the abdomen. Ficoll density gradient centrifugation was used to isolate peripheral blood mononuclear cells (PBMCs). PBMCs were further purified by positive selection with anti-CD14magnetic microbeads to purify CD14+monocytes at baseline, HS7D and LS7D. Total RNA of purified monocytes was isolated using Trizol. Real-time PCR was used to analyze the mRNA expression of related pro-inflammatory molecules.Results:All23volunteers finished the dietary intervention study.(1) Compared to the baseline, the24-hour urinary volume(1898±180mL vs.2588±169mL, P<0.001),24-hour urinary sodium (161±10.8mmol vs.360±13.9mmol, P<0.001) and24-hour urinary potassium(37.3±2.93mmol vs.46.8±3.2mmol, P<0.001) of HS7d were significantly higher, and significantly decrease at LS7d [24-hour urinary volume (1898±180mL vs.1558±110mL, P<0.05);24-hour urinary sodium (161±10.8mmol vs.89.3±8.12mmol P<0.001);24-hour urinary potassium (37.3±2.93mmol vs.28.7±1.9mmol, P<0.001)]. Compared to the high salt period, the24-hour urinary volume, urinary sodium and urinary potassium of LS7d were significantly lower (P<0.001, respectively). The24-hour urinary creatinine was significant high in HS7d (P<0.05), and declined at LS7d (P<0.001).(2) There was a dramatic increase of the CD14+CD16++monocyte count and percent at HS7d (P<0.001), and returned to the baseline level at LS7d (P<0.001). However, similar dynamic changes were not observed in CD14++CD16-and CD14+CD16++monocytes. The change of total monocyte count were not have statistical difference at any intervention stage(baseline:218±13.1cells/μL; HS7D:230±9.5cells/μL; LS7D:227±12.2cells/μL, P=0.6124).(3) Compared with baseline, the R2*value of VAT were significantly increased after taken7days high salt (25.2±0.90s-1vs.21.5±0.71s-1, P<0.001), and at the end of low-salt diet, the R2*value of VAT were descent to baseline levels (25.2±0.90s-1vs.21.3±0.70s-1, P<0.001). Compared with high-salt stage, the R2*values of VAT were significantly lower after taken7days low-salt diet (P<0.001).(4) At HS7d, there was a dramatic change in lipid and glucose metabolism, as shown by elevated fasting blood glucose levels (P<0.01, P<0.01, respectively), decreased insulin and C peptide,(P<0.01, P<0.01, respectively). HOMA2model also confirmed reduced insulin sensitivity (HOMA2-%IR decreased:P<0.05) and compromised pancreatic (3cell function (HOMA2-%B decreased:P<0.05) during high-salt feeding. These alterations regressed at the end of the low-salt phase.(5) During dietary intervention, there were no significant changes in blood pressure. Compared with BS and LS7d, heart rate was increased at HS7d (P<0.05).Conclusions:This work provides proof-of-principle evidence supporting the feasibility of BOLD-MRI in monitoring visceral ATH in humans with different statuses of insulin sensitivity. Moreover, for the first time, we demonstrate a close relationship between the CD14++CD16+monocytes and ATH during high salt intake, suggesting a plausible mechanistic link between monocyte phenotype and adipose tissue macrophage accumulation. |
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