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The following handle holds various files of this Leiden University dissertation:
http://hdl.handle.net/1887/61829
Author: Hoeke, G.
Title: A fatty battle: towards identification of novel genetic targets to comBAT cardiometabolic diseases
Issue Date: 2018-05-03
Chapter
Short-term cooling increases serum triglycerides and small HDL levels in humans
Geerte Hoeke*, Kimberly J. Nahon*, Leontine E.H. Bakker, Sabine S. C. Norkauer, Donna L.M. Dinnes, Maaike Kockx Laeticia Lichtenstein, Diana Drettwan, Anne Reifel-Miller,
Tamer Coskun, Philipp Pagel, Fred P.H.T.M. Romijn, Christa M. Cobbaert, Ingrid M. Jazet, Laurent O. Martinez Leonard Kritharides, Jimmy F.P. Berbée, Mariëtte R. Boon, Patrick C.N. Rensen
*Contributed equally
J Clin Lipidol 2017; 11: 920-928
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ABSTRACT
Background
Cold exposure and β3-adrenergic receptor agonism, which both activate brown adipose tissue, markedly influences lipoprotein metabolism by enhancing LPL-mediated catabolism of triglyceride-rich lipoproteins and increasing plasma HDL levels and functionality in mice. However, the effect of short-term cooling on human lipid and lipoprotein metabolism remained largely elusive.
Objective
To assess the effect of short-term cooling on the serum lipoprotein profile and HDL functionality in men.
Methods
BMI-matched young, lean men were exposed to a personalized cooling protocol for 2 h. Before and after cooling, serum samples were collected for analysis of lipids and lipoprotein composition by 1H-nuclear magnetic resonance. ABCA1-mediated cholesterol efflux capacity of HDL was measured using [3H]cholesterol-loaded ABCA1- transfected CHO cells.
Results
Short-term cooling increased serum levels of free fatty acids, triglycerides and cholesterol.
Cooling increased the concentration of large VLDL particles accompanied by increased mean size of VLDL particles. In addition, cooling enhanced the concentration of small LDL and small HDL particles as well as the cholesterol levels within these particles. The increase in small HDL was accompanied by increased ABCA1-dependent cholesterol efflux in vitro.
Conclusions
Our data show that short-term cooling increases the concentration of large VLDL particles and increases the generation of small LDL and HDL particles. We interpret that cooling increases VLDL production and turnover, which results in formation of surface remnants that form small HDL particles that attract cellular cholesterol.
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INTRODUCTION
Cold exposure has been instrumental to show that brown adipose tissue (BAT) is present in adult humans (1-3), and is metabolically active to take up large amounts of glucose and fatty acids (FA) (4, 5). Especially FA are combusted and used for the generation of heat (6).
BAT activity, as determined by the uptake of [18F]fluorodeoxyglucose ([18F]FDG), is inversely correlated with BMI and fat mass (1, 2, 7, 8), and prolonged cold exposure reduces body fat mass (9). Taken together, these data underscore the potential of BAT activation to combat obesity and associated disorders.
Cold exposure enhances sympathetic outflow to activate brown adipocytes through binding of noradrenalin to the β3-adrenergic receptor. As a consequence, triglycerides (TG) stored within numerous intracellular lipid droplets are hydrolysed. FA are subsequently directed towards the mitochondria where they activate uncoupling protein 1 (UCP-1) and are oxidized, resulting in production of heat instead of ATP synthesis (10). To replenish intracellular lipid stores, BAT takes up TG-derived FA from the blood, which are liberated by the high local concentration of lipoprotein lipase (LPL) (11). Cold exposure also increases sympathetic outflow to other organs including WAT, resulting in increased intracellular lipolysis and release of free FA (FFA) (12), and possibly also to the liver, which increases hepatic VLDL-TG production (13, 14).
Recently we showed in mice that rapid LPL-mediated lipolysis of TG-rich lipoproteins as a consequence of BAT activation results in the formation of cholesterol-enriched remnants that are taken up by the liver via the apolipoprotein E (apoE) - low-density lipoprotein receptor (LDLr) pathway (15). Via this mechanism, exposure of hyperlipidemic mice to cold or treatment with a β3-adrenergic receptor agonist markedly reduces hypertriglyceridemia (15, 16) as well as hypercholesterolemia (15). Furthermore, BAT activation increases high- density lipoprotein (HDL) levels and enhances reverse cholesterol transport (RCT) in mice (17). Collectively, this resulted in reduced atherosclerosis development in mice (15).
In humans, there is little evidence that cold exposure contributes to lipid metabolism. Short- term mild cooling increases the uptake of the FFA-tracer [18F]fluoro-6-thia-heptadecanoic acid ([18F]FTHA) by BAT (7), but data on the effects of cooling on lipoprotein metabolism are scarce. Therefore, the aim of the current study was to determine the effect of short- term mild cooling on the serum lipid and lipoprotein profile in young healthy lean men.
MATERIALS AND METHODS
Participants
Blood samples were collected as part of a clinical study, registered with the Netherlands Trial Register (2473), which aimed to investigate the volume and activity of BAT in Dutch white Caucasian and Dutch South Asian individuals (18). The study has been approved by the Medical Ethical Committee of the Leiden University Medical Center (LUMC) and was undertaken in accordance with the principles of the revised Declaration of Helsinki.
All volunteers provided written informed consent before participation. Twenty-four Dutch healthy, lean (BMI < 25 kg/m2) males of white Caucasian (n=12) and South Asian (n=12) origin between 18 and 28 years of age were included. Subjects were matched for age and BMI. For this study, cold-induced measurements of two white Caucasians were excluded due to the development of hyperventilation after [18F]FDG administration and difficulties with blood withdrawal respectively. One South Asian participant suffered from familiar hypercholesterolemia and was excluded as well. Subjects underwent a medical screening including their medical history, a physical examination, blood chemistry tests and an oral glucose tolerance test to exclude individuals with type 2 diabetes according to the American Diabetes Association 2010 criteria. Other exclusion criteria were rigorous exercise, smoking and recent body weight change up to 3 months prior to the start of the study.
Study design
The study was conducted in Alrijne Hospital, Leiderdorp (The Netherlands). Subjects were studied in the morning after a 10-hour overnight fast and were not allowed to exercise 24 hours prior to the study. At the start of the study day, a cannula was inserted in the left antecubital vein for blood withdrawal and [18F]FDG injection. Subjects were exposed to an individualized cooling protocol using two water perfused cooling mattresses (Blanketrol® III, Cincinatti Sub-Zero (CSZ) Products, Cincinnati, OH, USA) that covered the anterior and posterior sides of the body of the subject, as previously described (19).
In short, the protocol started with a baseline period of one hour in thermoneutrality (water temperature cooling mattresses 32ºC), after which blood samples were taken.
Then, water temperature was gradually decreased until shivering occurred. When the shivering temperature had been reached, the subject was warmed for 3 min so that shivering stopped and the water temperature was raised by 3°C. From that moment, the cooling period of two hours was started (tcold=0min). In case of shivering, temperature
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was raised by steps of 1°C until shivering just stopped. In this manner, non-shivering thermogenesis was maximized for each individual. At the end of the first hour of cooling (tcold=60 min), [18F]FDG was injected intravenously (2 MBq/kg). Both in thermoneutral and cold-induced condition (tcold=110 min) indirect calorimetry was performed with a ventilated hood (Oxycon Pro™, CareFusion, Germany) (tcold=80-110 min). After the second hour of cooling (tcold=120 min) blood samples were taken again and [18F]FDG-PET-CT (PET-CT from Gemini TF PET-CT, Philips, The Netherlands) imaging was performed to quantify BAT.
Lipoprotein profiling
Lipoproteins were analyzed by 1H-nuclear magnetic resonance (NMR) spectroscopy at numares AG (Regensburg, Germany), using the AXINON® lipoFIT®-S100 test system.
Serum (630 μL) was gently mixed with 70 μL of an additives solution containing reference substances, NaN3 and D2O, and 600 μL of the mixture were transferred into 5 mm NMR tubes with barcode-labeled caps. Briefly, 1H-NMR spectra were recorded at a temperature of 310 K on a shielded 600 MHz Avance III HD NMR spectrometer (Bruker Biospin, Rheinstetten, Germany) with a 5 mm triple resonance TXI probe head including deuterium lock channel, a z-gradient coil and automatic frequency tuning and matching. Prior to each analytical run, calibration was performed using a calibration sample comprising an aqueous solution of various calibration substances with different molecular masses, 0.01% (w/v) NaN3, 10% (v/v) D2O as a locking substance and 1%
glycerol to adjust viscosity. Two identical control samples were measured directly after calibration and at the end of each run. Each spectrum was referenced, normalized and subjected to a set of quality checks including checks of baseline properties, noise level, shift, width, and symmetry properties of quality control signals. Lipoprotein analysis was conducted via deconvolution of the broad methyl group signal at about 0.9-0.8 ppm.
In this process, lipoprotein subclasses are reflected by a fixed number of predefined bell-shaped (e.g. Gaussian or Lorentzian) base functions, each of which has a constant position and defined width. The concentrations of lipoprotein particles and cholesterol in lipoprotein (sub)classes as well as the average particle size (23 lipoprotein-related parameters in total) were calculated based on the integrals attributable to specific base functions. Fit quality was checked by calculating the residual deviation between fit and spectrum intensity.
Protein and FFA measurement
Total protein was determined in serum samples after 100x dilution in sodium hydroxide using a Pierce BCA protein assay Kit (Fisher Scientific, Landsmeer, The Netherlands). Serum FFA levels were measured using the NEFA C kit (Wako Diagnostics; Instruchemie, The Netherlands). Both assays were performed according to the manufacturers’ instructions.
Cholesterol efflux
ApoB-depleted serum was prepared as described previously (20). In short, 400 μL of serum was transferred to a fresh tube, mixed with 160 μL of PEG solution (20% PEG, 200 mM glycine, pH 7.4) and incubated at room temperature for 30 min, then centrifuged at 2,000 x g for 20 min. The supernatant, representing apoB-depleted serum, was transferred to a clean tube and stored at 4˚C until use (within 24 h). Chinese hamster ovary (CHO) cells stably expressing inducible ABCA1,(21, 22) were used to determine ABCA1-dependent cholesterol efflux. Cells were seeded into 24-well plates at 40,000 cells per well in 500 μL Ham’s F-12/10% heat-inactivated FCS. The following day the medium was changed to labelling medium containing Ham’s F-12, heat-inactivated FCS (10%), [3H]cholesterol (2 μCi/mL) without and with tetracycline (1 μg/mL) for 24 hours. The cells were washed and incubated for 1 hour in serum-free Ham’s F-12 containing 0.1%
BSA without and with tetracycline. The cells were then used to measure [3H]cholesterol efflux during 4 hours to 1.4% apoB-depleted serum (representing 1% original serum) in Ham’s F-12 without and with tetracycline. At the end of the efflux period of 4 hours, the media were removed, centrifuged at 2,000 x g for 5 min to remove any detached cells and an aliquot was counted for radioactivity. Cells were lysed in 500 μL of 0.1% Triton X-100 and an aliquot was counted for radioactivity. Efflux counts in the medium for each sample was expressed as a percentage of the total radioactivity in the sample. ABCA1- dependent efflux was calculated as the difference in efflux rates between cells incubated with (ABCA1-expressing) and without (control) tetracycline.
Quantitative proteomics
Principle: We used bottom up proteomics and a semi-automated approach to quantify apolipoproteins apoAI, B, CI, CII, CIII and E. Serum samples were prepared on a fully- covered (i.e. darkened) Bravo liquid handling platform equipped with a 96LT disposable tip head (Agilent Technologies, Amstelveen, The Netherlands). Eight μL of 20-fold diluted serum was added to a 40 μL mixture of DOC (0.40 % w/v), TCEP (2.3 mmol/L), and ISmix (14.3% v/v) in 100 mmol/L TRIS in a 96-well plate. The samples were denatured and
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reduced for 30 min at 56oC under gentle shaking (700 rpm) before addition of 20 μL 4.6 mmol/L iodoacetamide for alkylation (30 min at room temperature). Trypsin digestion was performed at 37oC at a 1:35 w/w trypsin-to-protein ratio in 92 μL total volume and gentle shaking (700 rpm). During all heated incubations, the sample plate was sealed and covered with a temperature-controlled heated lid (Inheco, Martinsried, Germany).
After 3 h, the digestion was quenched by addition of 106 μL 0.6% (v/v) formic acid in 5%
(v/v) methanol in water and the samples were centrifuged for 10 min at 2,000 x g. Finally, 150 μL supernatant was transferred to a clean 96-well plate. We performed LC-MS/MS analysis on an Agilent 1290 UHPLC system coupled to an Agilent 6490 triple-quadrupole mass spectrometer operated in positive-ion multiple reaction monitoring (MRM) mode.
Signature peptide selection, internal standards and external calibrators: We selected 2 signature peptides per apolipoprotein on the basis of previous work (23) and signal intensities during LC-MS/MS analyses of tryptic peptide candidates. The peptide with the most reproducible results was selected as the quantification peptide, whereas the other peptide was used for confirmation. We used stable isotope–labeled (SIL) peptides as internal standards, whereas external calibrators were used for standardization of the test.
The serum calibrators were value-assigned for apoAI and apoB and are traceable to WHO international reference materials SP1–01 and SP3–07, respectively. Serum calibrators for apoCII, apoCIII, and apoE were value-assigned in-house by immunoturbidimetric assays (ITA) with a Modular P800 analyzer (Roche Diagnostics, Almere, The Netherlands) using product calibrators with Conformité Européenne marking (Randox Laboratories, Wülfrath, Germany). The serum calibrator used for apoCI quantification was value- assigned using an ELISA (Assaypro, St Charles, MO, USA).
Statistics
The effects of mild cooling on lipoproteins (constituents) were assessed using a paired Student t-test with the SPSS 20 software package for Windows (SPSS Inc, Chicago, IL, USA). Correlations between metabolic parameters and serum lipoproteins were analysed using linear regression analysis. No adjustments (for e.g. BMI or age) were made, since these subjects were already matched for these parameters. Differences at probability values less than 0.05 were considered statistically significant. Data are presented as mean ± SEM.
RESULTS
Clinical characteristics
We included healthy lean male Dutch subjects of white Caucasian (n=10) and South Asian (n=11) descent. Clinical characteristics of the participants were described previously (18). Subjects were matched for age (white Caucasians: 24.7 ± 2.7 years; South Asians:
23.3 ± 2.7 years) and BMI (white Caucasians: 22.3 ± 1.4 kg/m2; South Asians: 21.4 ± 2.1 kg/m2) and exposed to cold for approximately 2 hours (18). Cooling activated BAT, as evidenced by the uptake of [18F]FDG. BAT volume was not significantly different between the white Caucasians and South Asian subjects (299 ± 54 mL vs 199± 22 mL) that were included in this study. Since all the baseline characteristics did not differ between the white Caucasian and South Asian subjects, we pooled the data of the two ethnicities to evaluate the effect of cooling on lipid and lipoprotein parameters.
Figure 1: Short-term cooling increases serum free fatty acids, triglyceride and cholesterol levels. Serum was collected before cooling (thermoneutral, TN) and after cooling (COLD). Serum concentrations of free fatty acids (FFA) (A) were measured with an enzymatic kit. 1H-NMR was used to measure serum concentrations of triglycerides (B), total cholesterol (C), VLDL-cholesterol (-C) (D), LDL-C (E) and HDL-C (F). Data are presented as mean ± SEM and a paired Student t-test was used for statistical comparison. **p< 0.01; ***p< 0.001 TN vs COLD.
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Short-term cooling increases large VLDL in addition to small and medium LDL Since cooling increased VLDL-C (Fig. 1D) and LDL-C (Fig. 1E), we determined the effect of cooling on other (V)LDL parameters. Cooling increased the average VLDL size (+3%;
p<0.05; Fig. 2A) as reflected by a markedly increased concentration of large VLDL particles (+62%; p<0.001; Fig. 2B). Cooling decreased the average LDL size (-1%; p<0.001; Fig. 2C) and increased the concentration of total LDL particles (+12%; p<0.001;
Figure 2: Short-term cooling increases large VLDL in addition to small and medium LDL. Serum was collected before cooling (thermoneutral, TN) and after cooling (COLD). 1H-NMR was used to measure (V)LDL parameters.
The average VLDL size (A), large VLDL particle (-p) concentration (B) were determined as well as the average LDL size (C) and the total LDL-p concentration (D). LDL particle concentration was subdivided into particle concentrations of small LDL (E) and large LDL (F). Levels of small LDL-cholesterol (-C) (G), medium LDL-C (H) and large LDL-C (I) were also measured. Data are presented as mean ± SEM and a paired Student t-test was used for statistical comparison. *p < 0.05; **p< 0.01; ***p< 0.001 TN vs COLD.
Fig. 2D). Consistent with these findings, cooling markedly increased the concentration of small LDL particles (+30%; p<0.001; Fig. 2E) but not large LDL particles (Fig. 2F). The increase in LDL-C was caused by increased cholesterol in small (+22%; p<0.01; Fig. 2G) and medium LDL (+37%; p<0.001; Fig. 2H), but not in large LDL (Fig. 2I). The increased concentration of (V)LDL particles after cooling was accompanied by an increase in their apolipoproteins. Cooling markedly increased the concentration of all apolipoproteins measured, i.e. apoB, apoE, apoCI, apoCII and apoCIII, with approx. 20% (all p<0.001;
Suppl. Fig. 1A-E).
Figure 3: Short-term cooling increases small HDL and ABCA1-dependent cholesterol efflux Serum was collected before cooling (thermoneutral, TN) and after cooling (COLD). 1H-NMR was used to measure the average HDL size (A), and the total HDL particle (-p) concentration (B), which was subdivided into particle concentrations of small HDL (C) and large HDL (D). Levels of small HDL-cholesterol (-C) (E), medium HDL-C (F) and large HDL-C (G) were measured. ABCA1-dependent [3H]cholesterol efflux from ABCA1-transfected CHO cells to ApoB-depleted serum obtained before and after cooling was determined (H). Correlation between ABCA1-dependent cholesterol efflux and small HDL-p concentration (A). Data are presented as mean ± SEM and a paired Student t-test was used for statistical comparison. *p< 0.05; **p< 0.01; ***p< 0.001 TN vs COLD.
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DISCUSSION
In the present study, we provide evidence that short-term cooling, the most important physiological activator of BAT, increases serum concentration of TG and apoB due to the an increase in large VLDL particles and small LDL particles, accompanied by increased cholesterol levels within small and medium LDL. Moreover, cooling increased the concentration and cholesterol content of small HDL particles, accompanied by increased ABCA1-dependent cholesterol efflux to HDL in vitro. Overall, these results point to cooling as a major modulator of plasma lipoprotein metabolism.
Cooling increased the serum concentration of large VLDL particles, which is unlikely to be caused by reduced lipid catabolism, e.g. due to vasoconstriction. In mice, cooling accelerates the LPL-dependent clearance of VLDL-TG by BAT, to provide BAT with FA for combustion to generate heat, a process that increases whole-body energy expenditure (15, 24). Since we (18) and others (7) have previously shown that cooling also increases FA oxidation and energy expenditure in humans, it is conceivable that human cooling also accelerates the LPL-dependent clearance of VLDL-TG-derived FA by BAT. The increase in large VLDL is likely explained by concomitantly increased hepatic VLDL production related to enhanced systemic sympathetic outflow. Although we were unable to measure serum catecholamines in the present study, we recently observed a 3-fold increase in noradrenalin levels upon a similar cooling protocol in overweight middle-aged men (Nahon & Rensen, unpublished).
Indeed, hepatic VLDL-TG secretion is increased in rats after exposure to cold (25) or specific activation of the sympathetic nervous system (13). Taken together, the increase in VLDL is likely due to increased hepatic VLDL particle secretion, which outweighs the accelerated VLDL particle clearance upon BAT activation.
In addition, we observed that cooling increased the serum concentration of small LDL particles, a phenomenon that is often accompanied by elevated TG levels (26-28). We can only speculate on the underlying mechanism(s). Possibly, cooling leads to the hepatic production of VLDL particles that are enriched in apoCIII relative to apoE, resulting in slow lipolysis into small dense LDL before being cleared (28). Alternatively, the higher serum VLDL-TG levels may cause increased cholesteryl ester transfer protein (CETP)-mediated transfer of VLDL-TG to LDL, followed by subsequent hydrolysis by lipases including LPL, hepatic lipase (HL) and endothelial lipase (EL), yielding more dense LDL particles (29). Finally, we cannot exclude that cooling reduces LDL receptor expression that could have contributed to this metabolic shift.
Finally, cooling increased the serum concentration of small HDL particles. This corroborates our recent observation that cooling of these subjects also increases subclasses of phosphatidylcholine and sphingomyelin in serum (30). Similar to the increase in small dense LDL, an increase in small HDL may be caused by CETP-mediated transfer of TG to HDL with subsequent lipolytic processing by HL and EL (29). Alternatively, increased lipolytic conversion of VLDL as a consequence of BAT activation will generate phospholipid-rich surface remnants will that can bind lipid-poor apoAI (31-34) and cause conversion of discoidal HDL into small HDL (35), as reviewed by Lewis and Rader (36) and Barter (32). In fact, incubation of purified lipid-poor apoAI with VLDL and LPL leads to the formation pre-β1 particles (33). In addition, we observed a relative enrichment of the small HDL particles with cholesterol, which likely reflects increased attraction of cellular cholesterol. Indeed, cooling increased the capacity of HDL to induce ABCA1- dependent cholesterol efflux from cholesterol-loaded ABCA1-transfected CHO cells in vitro. ABCA1-specific cholesterol efflux correlated with the concentration of small HDL particles rather than with large particles, consistent with the reported role of ABCA1 in cholesterol efflux to small HDL (21). In fact, we recently observed that cooling and more specific BAT activation by β3-adrenergic receptor agonism increased RCT in mice (17).
Taken together, cooling enhances the functionality of HDL with respect to stimulating cholesterol mobilisation and possibly RCT.
In conclusion, we show that short-term cooling markedly modulates lipid and lipoprotein metabolism in healthy lean men. Our data point to enhanced hepatic VLDL secretion, which was accompanied by increased concentrations of small cholesterol-enriched LDL.
Moreover, cooling increased the formation of small HDL particles that had increased ABCA1-dependent cholesterol uptake capacity. Future studies should investigate the dynamics of changes in lipid levels lipoprotein composition during cold exposure, and the potential contribution involvement of BAT. In addition, studies are warranted to investigate whether these data translate to other study populations, including women and dyslipidemic subjects, and to conditions of chronic cold exposure.
SOURCES OF FUNDING
We acknowledge the support from the Netherlands Cardiovascular Research Initiative: an initiative with support of the Dutch Heart Foundation (CVON2011-9 GENIUS). This work was also supported by Eli Lilly and Company through the Lilly Research Program. M.R.
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Boon is supported by an NWO-Rubicon grant (grant 825.13.021) and by a Dutch Diabetes Research Foundation Fellowship (grant 2015.81.1808). P.C.N. Rensen is an Established Investigator of the Netherlands Heart Foundation (grant 2009T038). L Kritharides is supported by the National Health and Medical Research Council of Australia (Program Grant 1037903). The Blanketrol III cooling device was kindly provided by FMH Medical (Veenendaal, Netherlands).
CONFLICTS OF INTEREST
P.C.N. Rensen receives research funding from a Lilly Research Award Program (LRAP) grant. A. Miller-Reifel and T. Coskun are employees of Lilly. Others have nothing to disclose.
AUTHOR CONTRIBUTIONS
Dr Hoeke was involved in the concept of the work, collection of data, data analysis and interpretation of the results. She was responsible for writing and submitting the manuscript. Dr Nahon was involved in the concept of the work, collection of data, data analysis and interpretation of the results. She was responsible for writing the manuscript.
Dr Bakker was involved in the concept of the work and interpretation of results. She approved final version of the manuscript. Dr Norkauer was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Dinnes was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Kockx was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Lichtenstein was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Drettwan was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Reifel-Miller was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Coskun was involved in data collection and interpretation of the results.
He approved the final version of the manuscript. Dr Pagel was involved in data collection and interpretation of the results. He approved the final version of the manuscript. Dr Romijn was involved in data collection and interpretation of the results. He approved the final version of the manuscript. Dr Cobbaert was involved in data collection and interpretation of the results. She approved the final version of the manuscript. Dr Jazet
was involved in the concept of the work and interpretation of the results. She approved the final version of the manuscript. Dr Martinez was involved in data collection and interpretation of the results. He approved final version of the manuscript. Dr Kritharides was involved in data collection and interpretation of the results. He approved final version of the manuscript. Dr Berbée was involved in the concept of the work and interpretation of the results. He critically reviewed first draft of the work and approved the final version of the manuscript. Dr Boon was involved in the concept of the work and interpretation of the results. She critically reviewed first draft of the work and approved the final version of the manuscript. Dr Rensen was involved in the concept of the work and interpretation of the results. He critically reviewed first draft of the work and approved the final version of the manuscript.
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SUPPLEMENTAL APPENDIX
Supplemental Figure 1
Supplemental Figure 1: Short-term cooling increases apolipoprotein concentrations. Serum was collected before cooling (thermoneutral, TN) and after cooling (COLD). LC-MS/MS was used as to measure concentrations of apoB (A), apoE (B), apoCI (C), apoCII (D) and apoCIII (E). Data are presented as mean ± SEM and a paired Student t-test was used for statistical comparison. ***p< 0.001 TN vs COLD.
5
Supplemental Figure 2
Supplemental Figure 2: Short-term cooling increases apolipoprotein AI concentration. Serum was collected before cooling (thermoneutral, TN) and after cooling (COLD). LC-MS/MS was used to measure concentrations of apoAI (A). Scatterplot of the correlation between ABCA1-dependent cholesterol efflux and concentrations of total HDL particles (-p) (B) and large HDL-p (C) for which the correlation was analysed using linear regression analysis. Data are presented as mean ± SEM and a paired Student t-test was used for statistical comparison. ***p< 0.001 TN vs COLD.
