Effects of a short‐term cold exposure on circulating microRNAs and metabolic parameters in healthy adult subjects

Abstract This discovery study investigated in healthy subjects whether a short‐term cold exposure may alter circulating microRNAs and metabolic parameters and if co‐expression networks between these factors could be identified. This open randomized crossover (cold vs no cold exposure) study with blind end‐ point evaluation was conducted at 1 center with 10 healthy adult male volunteers. Wearing a cooling vest perfused at 14°C for 2 h reduced the local skin temperature without triggering shivering, increased norepinephrine and blood pressure while decreasing copeptin, C‐peptide and heart rate. Circulating microRNAs measured before and after wearing the cooling vest twice (4 time points) identified 196 mature microRNAs with excellent reproducibility over 72 h. Significant correlations of microRNA expression with copeptin, norepinephrine and C‐peptide were found. A co‐expression‐based microRNA‐microRNA network, as well as microRNA pairs displaying differential correlation as a function of temperature were also detected. This study demonstrates that circulating miRNAs are differentially expressed and coregulated upon cold exposure in humans, supporting their use as predictive and dynamic biomarkers of cardio‐metabolic disorders.


| INTRODUC TI ON
MicroRNAs (miRNAs) play important roles in the pathogenesis of several metabolic disorders including obesity, type 2 diabetes, dyslipidaemia and metabolic dysfunction-associated fatty liver disease (MAFLD). [1][2][3][4][5][6] Circulating miRNAs are associated with vesicles (exosomes, microparticles and apoptotic bodies), protein complexes (Ago2, NPM1) and lipoprotein complexes (HDL, LDL complexes). 7,8 Circulating miRNAs are regarded as promising noninvasive biomarkers for risk stratification, diagnosis and prognosis of various cardiometabolic diseases. [9][10][11][12] In addition, circulating miRNAs are mediators of intercellular signalling. 13 It has been proposed that circulating miRNAs behave like hormones in intercellular communications. 14 For instance, the adipose tissue is a major source of circulating miRNAs that can regulate gene expression in distant metabolic tissues and organs. 15 miRNAs have also been identified as extensive regulators of adipocyte development, differentiation and biologic functions, 16 often through their effects on glucose and lipid metabolism. 6 This has raised the exciting prospect of using miRNAs as therapeutic targets in obesity and obesity-associated metabolic dysfunction. 6,[17][18][19] We recently provided evidence for this prospect by demonstrating that inhibition of miRNA-22-3p could lead to a potent treatment of fat accumulation, insulin resistance and related complex metabolic disorders like obesity, type 2 diabetes mellitus and nonalcoholic fatty liver disease. 20,21 Short-term cold exposure activates oxidative metabolism in adipose tissues and increases total energy expenditure. [22][23][24] While many aspects of cold exposure, especially those pertaining to brown fat activation, have been widely studied in animal models, there is less known about short-term cold exposures in humans. Even less information exists on the molecular consequences of such exposures, and the association between molecular mediators and circulating metabolic effectors. In this study, we have investigated the effects of short-term cold exposure of adult human subjects on changes in circulating miR-NAs and their association with circulating metabolic effectors also regulated by such activation. Current advances in network biology indicate that molecular regulators such as mRNAs and miRNAs seldom function alone but rather make joint contributions through functional associations such as gene co-expression networks. It is also becoming increasingly clear that differential correlation among molecular mediators (in addition to differential expression) is an equally important mediator of disease processes, such that changes in co-expression, rather than differential expression, among molecular regulators become important. 25 Based on these premises, we have characterized circulating 'miRNA-miRNA' and 'miRNA-metabolite' co-expression networks among robustly expressed miRNAs, and also examined the extent of differential correlation among miRNAs as a function of cold exposure. To our knowledge, such a network-based analysis of circulating miRNAs and their association with metabolic mediators in response to brief cold exposures in humans have not been reported.

| Study design
The primary objective of the study was to characterize the effect of short-term cold exposure on the profile of miRNAs, metabolic and hormonal parameters released in the peripheral circulation of healthy adults. The secondary objective was to look for correlations between such miRNAs and metabolic or hormonal parameters.
Safety measurements included adverse event assessment and physical examination, blood pressure (BP), heart rate (HR), electrocardiogram (ECG) and laboratory parameters (haematology, biochemistry, urinalysis, serologies and drugs screening), skin and core temperatures and visual analogue scale grading discomfort symptoms.
This was an open randomized crossover (cold vs. the absence of cold exposure) study with blind end-point evaluation conducted at 1 centre in healthy adult male volunteers (Appendix 1). To alter body temperature, the participants put on an appropriately sized surgeon's cooling vest (Cool Flow Fitted Vest System ® , POLAR Products, Figure S1). The use of the cooling vests provided for the participants involved in the study was strictly restricted to those participants and disinfected before each use. Following a predefined randomization code, the temperature of the water circulating inside the vest was set at 14°C (cold test) or kept at room temperature (RT, sham test) and monitored by a digital thermometer.
The protocol was approved before study initiation by the The execution of the study is detailed in Appendix 1. A sufficient number (20) of subjects were included, so that a total of 10 subjects will complete the study.
To be included in the study, the subjects had to meet the follow- drinks per week, alcohol consumption within 2 days prior to inclusion into the study, illicit drug use within the last 12 months, therapies resulting in potential dependence (e.g., sedatives and hypnotics), positive urine drug screen, blood donation during the prior 3 months or the intention to give blood during the next 3 months, blood transfusion within the last 12 months.

| Laboratory parameters
The metabolic and hormonal parameters measured during the study are listed in Appendix 1. The assays were performed locally following manufacturers' recommendations. The circulating miRNAs were extracted using the miRNeasy serum/plasma kit from Qiagen (miRNeasy Serum/Plasma Advanced Kit, Cat No./ID: 217204). miRNA sequencing was performed at the Genome Sequencing and Analysis Facility at the University of Texas at Austin on an Illumina HiSeq 2500 (Run SR50) system following the manufacturer's protocol. The results are expressed in reads per million mapped reads (rpm, linked to depth of sequencing) and not in reads per kilobase per million mapped reads (rpkm, linked to gene length) as the analysis was centred on miRNAs.

| Statistical analyses
Based on our own experience and prior reports involving human subjects, we considered 10 completed subjects to provide adequate information for descriptive statistical analyses. Results in the text and data points in the figures are shown as the mean ± SEM.
Statistical analysis used ANOVA and Student's t-test, unless nonparametric tests were selected, based on data distribution (GraphPad Prism 8). The type I error of the statistical analyses was set at 5%.
As the study is exploratory, no adjustment to control the type I error was used. Descriptive statistics are provided by period (cold vs sham exposure). The effect was investigated using an analysis of variance appropriate for crossover design. The study baseline is the last observation before the first period wearing the surgeon's cooling vest and the period baseline is the last observation before wearing the surgeon's cooling vest at each period.

| Bioinformatic analysis of miRNA expression data
Data obtained from miRNA sequencing were analysed to generate normalized read counts for each sample. 26

| Participants
Appendix 1 summarizes the baseline characteristics of the 10 subjects who completed the study. As outdoor temperature can rapidly affect nonshivering thermogenesis, we performed both tests (vest worn at RT and at 14°C) in each volunteer only 3 days apart. The study of the 10 volunteers was completed over the span of 4 weeks.
Female were not included in the study as breast tissue may have affected the cooling effect of the vest and to avoid hormonal variations related to menstrual cycle.

| Effects of cold exposure on various haemodynamic, metabolic and hormonal parameters
Based on the visual analogue scale of discomfort ( Figure S2), wearing the cooling vest at RT produced no discomfort (score: 0 on a scale from 0 to 10) in 7 subjects and mild discomfort (score: 1) in 3 subjects. Wearing the cooling vest at 14°C for 2 h produced no discomfort in 4 subjects and mild discomfort (score 1 to 3) in 6 subjects.
No shivering was reported nor observed.
Wearing the cooling vest at 14°C produced a significant decrease of local skin temperature from an average of 35.
Three distinct patterns of circulating hormonal and metabolic variations were observed during cold exposure ( Figure 3 and Table 1). First, there were metabolites that varied significantly only during cold exposure: increased norepinephrine as well as decreased copeptin and C-peptide upon cold exposure. The second pattern involved metabolites (ACTH, aldosterone and cortisol) that trended downwards with time both at RT and during cold exposure, reflecting the hypothalamic-pituitary-adrenal (HPA) axis circadian rhythm. The third class included metabolites that did not show significant temporal variation either at RT or cold exposure, under the given conditions of the study (epinephrine, dopamine, renin, insulin, glucose, T3, T4, adiponectin, FGF21, lactic acid and pyruvic acid).
We next investigated the relation among the expressed circulating miRNAs to infer a co-expression network based on pairwise partial correlations among miRNAs. For this analysis, 118 miRNAs with nonzero expression values in all measurements were considered. Additionally, correlation of miRNA expression to copeptin, norepinephrine and C-peptide were also considered during network generation. Figure 5 shows the miRNA-miRNA and miRNA-analyte networks based on nodes with a probability cut-off for nonzero correlation >0.8. In the network, miRNAs are sized by their number of connections to other miRNAs/analytes. Some of the highly connected miRNAs include mir-30a-5p, mir-148a-3p, mir-378a-3p and mir-143-3p. The full output correlation analysis is provided in Table S3.
An additional analysis was conducted to identify miRNA pairs that are differentially correlated with each other at 14°C compared to RT. The same set of 118 miRNAs with nonzero expression values at all measurements were used for this analysis. A total of 73 miRNA pairs were found to be significantly differentially correlated (p < 0.01) (full results provided in Table S4). The correlogram in Figure 6a shows a hierarchically clustered view of the differences in correlation values between miRNA pairs. Some specific examples of differential correlation are depicted in Figure 6b and include cases where the correlation signs are reversed between 14°C and RT (mir-182-5p/mir-103a-1-3p, mir-340-5p/148a-3p) or is weaker in one condition relative to the other (mir-423-3p/mir-27b-3p).

| DISCUSS ION
The communication between circulating miRNAs and target cells may lead to a series of effects on both physiological and pathological conditions. 27 Cell-secreted miRNAs facilitate the exchange of genetic information between cells and play an important role in intercellular communication. 28 They are also implicated in physiological processes such as the regulation of immunity and angiogenesis or cellular migration, while they are also involved in various pathological conditions. Importantly, miRNA released from cells can be detected in various human body fluids including saliva, urine, blood, serum, plasma, seminal fluid and pleural effusion. 29,30 The expression profile of extracellular miRNAs in different bio-fluids under different pathophysiological conditions displays specific patterns suggesting that such miRNAs are not passively released from the necrotic or injured cells, but rather selectively released from specific cells. 29,30 Circulating miRNAs exist in two distinct populations as vesicle-associated and nonvesicle-associated (e.g., as a ribonucleoprotein complex), respectively, with the majority existing in the nonvesicle-associated form. In this study, we assayed total circulating miRNAs which reflects the abundances in the predominant vesicle-free form. Short-term cold exposure protocols have been used to explore the activation of thermogenesis in humans without triggering shivering and related metabolic responses due to increased muscular activity. [40][41][42][43] Our crossover protocol produced a maximum 5°C reduction in the thoracic skin temperature when the cooling vest was perfused at 14°C. The impact on core body temperature was a maximum decrease of −0.3°C without shivering. These findings are similar to those (−4.5 ± 0.3°C for skin temperature and −0.4 ± 0.1°C for core temperature) reported by Blondin et al. during subjects' exposure to a suit cooled at 18°C for 180 min. 41 The BP increase and HR decrease observed in our study were also similar to that reported by Cypess et al. who exposed healthy subjects to a surgeon's cooling vest cooled at 14°C for 120 min, 43 presumably related to peripheral vasoconstriction and increase of central blood volume. Under similar conditions, we observed upon cold exposure a significant increase of norepinephrine reflecting the activation of the beta-adrenergic signalling and peripheral sympathetic nervous system in responsive tissues such as brown fat. 44 Our findings are in line with other studies that observed increased plasma norepinephrine, and increased norepinephrine turnover due to sympathetic activation of BAT following cold exposure. 45,46 The other circulating peptide significantly altered by cold exposure was copeptin, a surrogate biomarker of arginine-vasopressin (AVP) secretion. AVP levels are most notably regulated through changes in plasma osmolality, as well as adaptations to physiological stress. 47 In the absence of osmotic changes in the current study, the decrease in copeptin during cold exposure may suggest an alternative haemodynamic adjustment such as an increase in the central volume sensed by central baroreceptors. Notably, a decline of plasma vasopressin has also been reported during cold exposure in the context of normal hydration. 48 As copeptin levels have also been associated with additional metabolic effects including lipid oxidation hyperinsulinaemia, metabolic syndrome and future type 2 diabetes, the observed changes in copeptin in our study might encompass additional physiologic processes besides haemodynamic control. 49,50 Finally, the observed changes in circulating insulin C-peptide levels upon cold exposure is in line with existing literature linking insulin's requirement for cold-induced thermogenesis, presumably due to alterations in insulin sensitivity following cold exposure. 51 However, in a study involving human subjects exposed to thermoneutrality (22°C) or moderate cold (18°C) for 100 min, plasma C-peptide was found to be unaltered, in contrast to our findings. 40 The shorter exposure to a milder temperature reduction in that study may explain these  54 To put these findings in context, both miR-122-5p and miR-92a-3p were highly expressed in our study, and miR-122-5p expression was further reduced during cold exposure with nominal significance (p < 0.05) (miR-92a-3p was not significantly altered). However, cold exposure-induced circulating miRNAs are expected to also associate with other metabolic endpoints in addition to BAT activation. This possibility has been addressed in our study where the correlation of circulating miRNAs to cold-induced changes in circulating hormones were compared. We further expanded the scope of examination beyond single miRNAmetabolite correlations and investigated the co-expression structure of circulating miRNAs and differences in such co-expression networks as a function of cold exposure. These are the novel contributions from this study.

F I G U R E 4
Correlation between circulating miRNAs and temperature-sensitive blood metabolites. Correlation analysis was performed on miRNAs with nonzero expression values in all samples. Hormone/peptide levels are plotted on the x-axis (pmol/L) and miRNA expression on the y-axis (log2 scale). Open and closed circles represent data for RT and 14°C respectively. The correlation value and its significance are listed for each plot. Only correlations with p < 0.05 are shown. (A) Correlations between circulating miRNAs and copeptin levels, (B) correlations between circulating miRNAs and norepinephrine levels, (C) correlations between circulating miRNAs and C-peptide levels The number of circulating miRNAs found in our study is within the range reported by others in 12 body fluids. 55,56 The correlations between circulating miRNAs and the metabolic/hormonal parameters that were significantly altered during the cold exposure (copeptin, norepinephrine and C-peptide) reveal that miR-30d-5p was correlated with all 3 parameters. Both positive and negative correlation values were observed, suggesting a complex interplay between circulating miRNA and analyte levels.
Expanding on the individual miRNA-metabolite correlations, we generated a co-expression network based on miRNA-miRNA and miRNA-metabolite partial correlation estimates. The network allowed us to further characterize the miRNAs based on their relationships with other miRNAs. For example, several miRNAs, such as mir-30a-5p, mir-148a-3p, mir-378a-3p, mir-143-3p, were found to be highly connected to multiple other miRNAs in the network (high degree), whereas other miRNAs, such as mir-199a-1-3p and mir-199a-2-3p, were only associated with each other and with no other miRNAs. Interestingly, the largest node belonged to miR-30a-5p, which has been reported to promote browning of adipocytes and insulin sensitivity. [57][58] Also, mir-378-3p, a regulator of energy and glucose homeostasis, was found to be one of the highly connected elements in the miRNA co-expression network. 59 While the co-expression network provided a general view of miRNA-miRNA associations, we also investigated if a subset of these associations is differentially regulated as a function of temperature.
Of the significantly differentially correlated miRNAs included are mir-148b-3p and mir-151a-5p which are also some of the highly connected nodes in the miRNA co-expression network. Although the functional relevance of these observations is currently unknown, they nevertheless point to a complex and dynamic interplay among F I G U R E 5 miRNA-miRNA and miRNA-metabolite correlation networks. Partial correlation based co-expression networks were constructed from 118 miRNAs and 3 metabolite (copeptin, norepinephrine, C-peptide) expression data. Network is restricted to nodes with a probability cut-off of nonzero correlation >0.8. Nodes are shaped based on molecule type (miRNA, green circle; metabolite, orange diamond). Node size is directly proportional to node degree (density of connectivity to other nodes). Edges are colour coded based on the sign of partial correlation (red, positive; blue, negative). Network was generated in Cytoscape using the yFiles organic layout circulating miRNA levels, possibly as a consequence or adaptation to a changing thermal stimulus.
Some limitations of the current study are now discussed. The small sample size limits the power to detect small differences in miRNA expression between the different time points, due to which we have not focused on differential miRNA expression in the study. Also, the

ACK N OWLED G EM ENTS
We thank the following individuals for helpful discussions and sug- for overseeing the approval and execution of the study.

CO N FLI C T O F I NTE R E S T
There is no conflict of interest to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data generated or analysed during this study are included in this published article or in the data repositories listed in References.

Study Execution
All subjects reported to the CIC for 1 screening visit, 2 one-day inpatient stays, and 1 follow-up visit. All study assessments and procedures were timed related to the first CIC in-patient stay. Subjects were permitted a window of ±2 days for each scheduled visit.
a. During the initial screening visit (Day-28 to day-7) in the CIC: • The study protocol was fully explained to the subjects and their questions answered.
• Signed and dated written informed consent forms were obtained.
• A full history and physical examination were performed.
• The subjects were asked to try on an appropriately sized Cooling Vest to verify that: 1. The vest can be well adjusted to their morphology and 2. The subjects feel comfortable wearing the vest • A licensed phlebotomist drew blood (amount of up to 30 ml) from an arm vein for routine and safety lab work.
• A calendar of the actions to be taken during the next weeks as part of the study was given to the study participants.
• For the upcoming weeks, the participants were asked to follow the same healthy lifestyle and maintain their weight.
Involvement in strenuous physical activity was prohibited.
Proper hydration was maintained.
• The participants were asked to refrain from caffeine and alcohol intake for 48 h prior to reporting to the CIC the evening before Study Day 1 and Study Day 3.
b. On Study Day 1: • The participants were admitted to the CIC in the evening (20h00).
• A normocaloric meal was served at around 20h30 • RT was maintained at 23°C throughout the stay in the CIC.
• Fasting was initiated at midnight onwards.
c. On Study Day 1 in the CIC: • Upon waking up at 07h00, the participants wore a standard hospital scrub suit.
• A physical examination was performed.
• Vital signs (blood pressure (BP), heart rate (HR), skin (thorax level) and core temperatures were recorded every 15 min from 08h00 to 12h00 in supine position.
• Eighty (80) ml of venous blood was drawn before 09h00 for baseline miRNAs and metabolic/hormonal parameters measurements • Cutaneous and central temperature will be measured every 15 min from 08h00 to 12h00 with skin thermistors placed under the vest and electronic thermometers placed in the ear.
• At 09h00, the participants put on a surgeon's cooling vest (Cool Flow Fitted Vest System ® , POLAR Products, Figure S1).
• Following a predefined randomization code (RANDI2, http:// www.randi2.org/), the temperature of the water circulating inside the vest was set at 14°C (cold test) or kept at RT (sham cold) and monitored by a digital thermometer.
• The participants rested comfortably in bed in a supine position and were exposed to a total of 120 min of cold (or sham cold) from 09h00 to 11h00.
• At the end of the cold (or sham cold) exposure period (11h00), 80 ml of venous blood was drawn for cold phase (or sham cold) miRNAs and metabolic/hormonal parameter measurements.
• By the end of the morning, the subjects were fed and discharged from the CIC. They were given the date and time of the second stay in the CIC and were reminded to continue the same healthy lifestyle and to avoid caffeine and alcohol intake.
d. On Study Day 2: • The participants were admitted to the CIC in the evening (20h00).
• A normocaloric meal was served at around 20h30 • They were fasting from midnight onwards.
• RT was maintained at 23ºC throughout the stay in the CIC.
e. On Study Day 3 in the CIC: • The protocol applied on Study Day 1 in the CIC was repeated, but each subject was wearing the vest set at the temperature, which was not applied on Study Day 1.
• By the end of the morning, which was the final visit: 1. A full physical examination was completed.
2. Assessment of adverse events was performed.
3. The subjects were discharged from the CIC after being fed lunch.
Five venous blood draws totalling 350 ml of blood were collected over the duration of the study.