Taurine and Nitrate Supplementation on Thermoregulatory in T2DM

WBSR will be measured by taking a nude body mass (kg). Participants will be shown to a private changing facility in which the scales (Industrial Electronic Weight Indicator, Model 10, Ohaus Corporation, Parsippany, NJ, US) are located, with the screen for the value reading located outside to ensure privacy of the participant. The pre-mass measure will occur during every experimental visit, before the instrumentation section of the visit. Towel dried nude body mass will then be recorded once de-instrumentation has occurred. The equation below will be used to calculate WBSR.

WBSR (L/h) equation:

(Pre body mass (kg) - Post body mass (kg)) + Fluid intake (L) - Urine output (L) Duration of test (hrs)

The Q-sweat (Qsweat Quantitative Sweat Measurement System, WR Medical Electronics Co., Minnesota, USA) will be used to understand LSR during the heat event. Participants will be instrumented with the ventilated sweat capsule and placed on the upper back (mid scapula) and onto the mid anterior forearm. Additionally, measuring the forearm may provide insight into regional differences and any autonomic dysfunction in sudomotor response. This will be measured continuously throughout the investigation through the software TestWorks (TestWorks software, WR Medical Electronics Co., Maplewood, MN, USA). Live data will be gathered during the trials and subsequent data will be processed into one-minute averages across each trial.

Trec will be measured continuously (1s intervals) throughout each trial. This will be measured via a rectal thermistor (PROACT Temperature Probes, Northamptonshire, United Kingdom).

Tsk will be assessed using four skin thermistors (Grant Instruments, Cambridge, UK), at the sites on the right side of the body at the locations, pectoralis major, biceps brachii, mid-thigh and the gastrocnemius. Thermistors will be placed during the instrumentation part at the start of the protocol and will remain on location until the session has concluded. Data will be recorded throughout the whole duration of the experimental protocol. These will be held in place by a Tegaderm™ dressing (Tegaderm™ Dressings, 3M, St. Paul, Minnesota, USA) with additional tape anchoring them to the skin to avoid movement. Minute averages will be recorded for the duration of the protocol and plotted to understand the change in temperature throughout each trial, to provide information on the specific temperatures that have been identified.

Mean skin temperature = (0.3*Tpectorlis) + (0.3*Tbicep) + (0.2*Tthigh) + (0.2*Tcalf)

*expressed as an indication of multiplication.

Mean body temperature will be measured to provide an overall measure of heat gain for the individual through the combination of Trec and Tsk. The weighting of the different measures of temperature is determined by the ambient temperature of the surrounding, as the protocol will be of a hot environment the below equation was chosen over other weightings, which is as below in Equation two (Burton et al., 2025). This will be measured at all time points that Trec and Tsk are taken in conjunction with each other. This will be measured as absolute values in degrees Celsius, for all time points and also be expressed as a delta change from the baseline temperature or between other specific time points chosen, to provide an understanding of the rate of change.

Mean body temperature = Tˉsk=0.3(Tchest+Tarm)+0.2(Tthigh+Tcalf)

Using thoracic bio-reactance technology (Physioflow), which will derive cardiac output at rest. Four electrodes are placed on the participant's chest between which a small, painless current is passed.

Brachial artery flow mediated dilation (FMD) will be used as a measure of endothelial function. Endothelial-dependent function is assessed by measuring FMD in response to a 5 minute ischaemic stimulus, induced by forearm cuff inflation. Measurements will be performed in the supine position on the right arm with the cuff placed distal to the olecranon process. High-resolution duplex ultrasound (SonoSite Edge II, Bothell, Washington, USA) with a 12-MHz multifrequency linear array probe will be used to image the brachial artery at the distal third of the upper arm and simultaneously record the longitudinal B-mode image and Doppler blood velocity trace. The angle of Doppler insonation will be 60°. Images will be optimised, and settings (depth, focus position, and gain) will be maintained between FMD assessments within each individual visit, and the location of the transducer will be recorded and marked on the skin using an indelible marker.

Briefly, following cleaning of the skin surface with water for injection, two perspex rings will be attached to the skin with one acting as an anode, and the other as the cathode. These electrodes will be connected to the iontophoresis controller (MIC 2, Moor Instruments, UK). Both chambers have an 8 mm inner diameter. The anode chamber will be filled with ~ 0.5 mL of ACh (Braun, Melsungen, Germany), with a 1% concentration dissolved in water for injection. The cathode chamber will be filled with ~ 0.5 mL of SNP (Sigma-aldirch, Missouri, USA) with a 0.01% concentration dissolved in water for injection. The protocol for electrical pulses included: four at 25 μA, followed by a single pulse of 50 μA, 100 μA, 150 µA and 200 µA. These pulses will last for 20 seconds with 120 seconds intervals between each pulse where no current will be applied. An interval of five minutes will be given between testing each site (forearm, finger and foot).

During the experimental visits participants will wear a Heart Rate monitor (Polar M430, Kempele, Finland) which will continually record data throughout the session. It will be fitted during the instrumentation section of the protocol. This will provide real time data on participants current HR and allow assessment of the task demands placed on the individual. HR will be averaged to one-minute intervals upon analysis of the data.

blood samples will be collected through venepuncture of a vein in the cubital fossa. The collection of the plasma during the investigation session will be taken pre and post simulated heat events. Sample will be drawn at each specified visit/time point into ethylenediaminetetraacetic acid (EDTA) tube (K2, BD, USA) for analysis of plasma [Taurine] and [cysteine], (inflammation markers) [IL-6], [IL-10], [TNF-α], (intestinal permeability), [LBP] and [iFABP] and into lithium heparin (K2, BD, USA) tubes for [nitrate] and [nitrite].

Near-infrared spectroscopy (NIRS) will be used to measure oxygenation at the periphery of the medial gastrocnemius. Instrumentation and recording of the measure will be done in accordance with previous published methodology in consuming ketone drink for people with T2DM which is as followed. The NIRS device (NIRS; Portamon Artinis Medical Systems, The Netherlands) will be placed 10 cm inferior to the tibial tubercle and 10 cm lateral to the anterior tibial crest. Upon measurement completion, cleaning and shaving (if required) of the area will be undertaken following marking of the area to ensure correct placement of the device. These measures and markings will take place during trial visits. Participants will be shown the device and where it will be placed during the familiarisation visit. Supporting tape will hold the device in place with a darker colour dressing placed over the device to minimise the light affecting the data.

Participants will be required to provide a urine sample on entry to the laboratory on experimental visits. This will be measured via an osmolality device [Osmocheck, Vitech Scientific, West Sussex, UK] to ensure hydration levels are below >600 mOsm kg-1, for euhydration (Oppliger et al., 2005). Upon obtaining the sample, a pipette will be used to aliquot the urine onto the prism surface of the sensor well. Should hydration levels be greater than this, participants will be required to consume 500 ml of water and wait 30 minutes (Page et al., 2019) before being able to enter the simulated heat event. Participants will be asked to consume 500 ml of water before entering the laboratory on experimental days (Kirby et al., 2024; Notley et al., 2021). After the experimental sessions, participants will provide a further urine sample (if possible), to assess dehydration pre and post simulated heat event.

Thermal sensation will be assessed during the investigation to provide a subjective rating of how the participants are feeling within the environment. For thermal comfort participants will be asked to rate their perception of satisfaction of the environment from their state of mind. For thermal sensation, participants will be asked to rate their perception of the sensory feeling of the environment around them. Both questions will be measured on a six-point and six-point scale respectively. This will provide a more in detail understanding of the sensory and state of mind of thermal perception. Participants will be provided with a pen and will be asked to draw a line onto the scale regarding their answer. This will then be measured on a visual analogue scale, and be recorded in cm (Stevens et al., 2025). The marker location will be taken as the uppermost and leftmost extent of the ink marking drawn by participants.

The heat illness symptom index (HISI) will be a perceptual based measure used during the experimental protocol. This measure will be used to assess the risk from the heat exposure that may be being experienced by the participant. The measure will allow a greater informed decision making in the safety of the participant during the simulated heat event. Participants will be encouraged throughout to express any feelings of discomfort or illness at any moment and not waiting for the index measure to take place. This will be measured at baseline (room temperature) upon entry and exit of the environmental chamber, every ten minutes.

Higher HISI = worse (more severe heat illness symptoms) Lower HISI = better (fewer or no symptoms)

Each symptom is score on a scale of, 0, 1, 2 and 3.

The scale that will be used is the Borg (1982) rate of perceived exertion (RPE) scale ranging from 6 (no exertion at all) to 20 (maximal exertion). This measure is widely established scale and will be used to help understand how physically demanding the participants are experiencing the protocol. This will be measured in accordance with the timings of the perceptual measures of heat stress. Participants will be familiarised with this scale during the baseline visit. During the experiment, participants will be asked to verbally tell the research team which number on the scale that is currently being experienced.

Using thoracic bio-reactance technology (Physioflow), which will derive stroke volume at rest. Four electrodes are placed on the participant's chest between which a small, painless current is passed.

Using thoracic bio-reactance technology (Physioflow), which will total peripheral resistance, at rest. Four electrodes are placed on the participant's chest between which a small, painless current is passed.

Using thoracic bio-reactance technology (Physioflow), which will derive heart rate at rest. Four electrodes are placed on the participant's chest between which a small, painless current is passed.

Thermal comfort will be used during the investigation to provide a subjective rating of how the participants are feeling within the environment. For thermal comfort participants will be asked to rate their perception of satisfaction of the environment from their state of mind. For thermal sensation, participants will be asked to rate their perception of the sensory feeling of the environment around them. Both questions will be measured on a six-point and six-point scale respectively. This will provide a more in detail understanding of the sensory and state of mind of thermal perception. Participants will be provided with a pen and will be asked to draw a line onto the scale regarding their answer. This will then be measured on a visual analogue scale, and be recorded in cm. The marker location will be taken as the uppermost and leftmost extent of the ink marking drawn by participants.