A total of 20 male T2DM subjects with the mean age of 51.75±7.71 years based on convenient sampling were recruited form a residential holistic health center, South India. Sample size was not calculated based on any previous study, which is one of the limitations of the study. Male subject with the history of T2DM with more than one year and on regular medication and the ones who were willing to participate in the study were included in the study. Subject with the history of Type 1 diabetes mellitus or uncontrolled T2DM with complications including cardiovascular complications, sensitive to cold application, and subject who underwent CAP for the past one week were excluded from the study. Study was approved by the Institutional Ethics Committee and a written/informed consent was taken from each subject included in the study.

A single group pre-test and post-test design was adopted. All the subjects underwent a single session of CAP for the duration of 20 minutes. Assessments were taken before and after the intervention.

Random Blood Glucose (RBG) level: It was assessed before and after the intervention using a glucometer (hemocue 201+, Angelholm, Swedan).

Blood Pressure (BP) and Pulse Rate (PR): Assessment of Systolic Blood Pressure (SBP), Diastolic Blood Pressure (DBP) and PR were measured before and after the intervention using a non-invasive arm type automatic BP monitor (Omron HEM-7120, Kyoyto, Japan). Minimum of two measurements with the rest period of one minute between the measurements were taken and averaged to get a final value. In case of difference between the two measurements by >10 mmHg, a three measurement was taken after one minute of rest period followed by the second measurement and the average of the two measurements which did not differ >10 mmHg was averaged to get a final value. By using the following formula, assessments like pulse pressure (PP), Mean Arterial Pressure (MAP), Rate Pressure Product (RPP), and Double Product (Do-P) were calculated. PP was calculated as (SBP-DBP); MAP as (DBP+1/3 PP); RPP as (PR×SBP/100); and Do-P as (HR×MAP/100) [9].

All the subjects underwent CAP that consists of a cotton cloth dipped in water (15-16^oC) and wrung out and then wrapped around the abdomen from lower part of the ribs to the groin. A dry cloth and the flannel were wrapped over the wet cloth in order to reduce evaporation. The duration of the intervention was kept 20 minutes since a previous study reported that the physiological effects of cold therapy appear when it continues for 20 minutes [7,10].

Data were checked for the normality using Kolmogorov-Smirnov test. Statistical analysis of was performed using Student’s paired samples t-test with the use of SPSS for Windows, version 16.0. (Chicago, SPSS Inc.). A p-value <0.05 was considered as significant.

Recruited 20 subject’s demographic details are given in [Table/Fig-1]. Results of this study showed a significant reduction in RBG levels, PR, SBP, MAP, RPP and Do-P compared to its respective pre-test and no such significant changes were observed in DBP and PP [Table/Fig-2].

Increased blood glucose level (hyperglycaemia) is the most important characteristics of T2DM [1]. Since, the aetiology, pathophysiology, severity, age of the onset, and the management of the Type 1 and T2DM is quite different, the response to the treatment may also be different. Moreover, prevalence of the Type 2 diabetes is quite more than that of Type 1 diabetes [8]. Hence, we thought of having the Type 2 diabetes patients as present subjects. Moreover, to get the homogeneity in the study group we have excluded the subjects with Type 1 diabetes. Result of this study showed a significant reduction in RBG level. It suggests that the application of 20 minutes of CAP might be effective in reducing the blood glucose level in patient with T2DM. The possible mechanisms for the reduction of blood glucose level are as follows: 1) Utilisation of glucose by increased metabolism; 2) Increased insulin release through activation of (closure) of Adenosine Triphosphate (ATP) sensitive potassium (KATP) channels and Calcium ion (Ca²⁺) permeability; 3) Increased insulin sensitivity; and 4) Activation of Transient Receptor Potential Melastatin-like 8 (TRPM 8) ion channel and brown adipose tissue.

Cold induced thermogenesis is the ability to generate heat by increasing metabolism in response to cold, to maintain a stable core body temperature which is the basic property of endothermic thermoregulation [11]. When we expose a small surface area to cold as like CAP application, cold fibers evoke transient afferent discharges, inducing cold sensation and heat-gain responses while the temperature of skin decreases cold exposure is associated with hypothalamic signals to constrict the peripheral blood vessels [8,12,13]. This in turn produces a compensatory vasodilatation in deeper vascular system resulting increased blood flow to the tissues underlying the site of exposure and that increase the metabolic rate in order to maintain constant deep tissue temperature. For example, according to a previous study, immersion in cold water of 20°C almost doubles metabolic rate, while at 14°C it is more than quadrupled [12]. Hence, application of CAP might have produced an increased metabolic rate by the utilisation of blood glucose.

The KATP channels play a key physiological roles in many tissues. In pancreatic β cells, these channels become the target for sulfonylurea drugs that is used to treat T2DM and regulate glucose dependent insulin secretion. By regulating membrane K⁺ fluxes, KATP channels couple cell metabolism to electrical activity of the plasma membrane. Opening K⁺ channels followed by K⁺ efflux, membrane hyperpolarisation, and suppression of electrical activity occurs when metabolism reduces. Conversely, closure of KATP channels followed membrane depolarisation and stimulation of electrical activity occurs when metabolism increases [14]. A previous study reported that a brief cold exposure produces increased blood flow by compensatory vasodilatation in deeper vascular system to the underlying site of exposure and that increase the metabolic rate [12]. Similarly, the application of CAP might have produced an increase in plasma glucose through increased blood flow to internal organs including pancreas which stimulates glucose uptake and metabolism by the β cell, producing changes in cytosolic nucleotide concentrations that result in closure of KATP channels. This leads to insulin secretion by producing a membrane depolarisation that opens voltage gated Ca²⁺ channels, initiating β cell electrical activity, Ca²⁺ influx, a rise in intracellular calcium concentration [14]. In a study, cooling of a particular part was reported to induce an inward cold current which is a non selective cation current with high Ca²⁺ permeability [13]. This implies that receptor potential increases from resting potential toward 0 mV, when whole cell conductance is activated fully by cooling. Hence, application of CAP might produce high Ca²⁺ permeability in pancreatic beta cells that leads to the secretion of insulin [13].

Hyperinsulinemia is known to be present in patients with T2DM due to reduced insulin sensitivity while, cold exposure has been suggested to improve glycaemic control for T2DM through improving insulin sensitivity [8]. In a study, 10 days of cold acclimation (14-15^oC) has shown to induce a 43% increase in insulin sensitivity which was explained by a 60% increase in GLUT-4 translocation (i.e., the membrane channel that allows glucose to enter a muscle cell or adipocyte) in patients with T2DM. Hence, application of CAP might have produced increase in insulin sensitivity and thereby reduced the blood glucose level [8,15].

Transient Receptor Potential (TRP) ion channels like TRPM 8, TRP Ankyrin rich 1 (TRPA1) and TRP Canonical 5 (TRPC5) have been found to be cold sensitive ion channels. Among all TRPM8 plays a crucial role in detecting cool to cold temperature in vivo [16]. In humans, TRPM8 activation has been associated with ‘browning’ of white adipose tissue there by possibly promoting energy utilisation [16]. Cold exposure was reported to be a potential therapy for diabetes by increasing brown adipose tissue mass and activity [15]. Cold exposure is associated with increase in metabolic heat production (i.e., shivering and non shivering thermogenesis) to prevent dangerous drops in core temperature. Although, shivering is the major contributor to increases in heat production, during mild cold exposure, non shivering thermogenesis play a vital role to produce physiological response. Non shivering thermogenesis is primarily mediated by metabolically active brown adipose tissue. Hence, application of CAP is also believed to reduce the blood glucose level by producing energy utilisation and non shivering thermogenesis through activation of TRPM8 ion channel and brown adipose tissue activity respectively [8].

Results of this study also showed a significant reduction in PR, SBP and MAP compared to its respective pre-test. However, no such significant changes were observed in DBP and PP. It suggests that 20 minutes of CAP were effective in improving various cardiovascular variables except DBP and PP. Significant reductions in SBP followed by 20 minutes of CAP might attribute to the effects on either baroreceptor reflex or significant reduction of HR/PR. Because SBP=cardiac output (CO)×peripheral resistance, wherein CO=HR × stroke volume and thus HR forms one of the determinants of SBP [9]. Significant reduction in MAP might attribute to the reduction in SBP (a component of PP, i.e., PP=SBP-DBP).

Autonomic dysfunction is one of the major complications of T2DM. The cardiac autonomic neuropathy is one of the leading causes of morbidity and mortality in diabetics [17]. Results of this study also showed a significant reduction in RPP and Do-P. This reduction might attribute to the reduction in PR and BP. RPP and Do-P are the important indirect indicators of myocardial oxygen consumption and load on the heart. Reduction of these variables in this study suggests a strain lowering effects on the heart. When HR Variability (HRV) analysis is not available, the RPP can be used as a simple measure of overall HRV [9]. Hence, a significant reduction in both RPP and Do-P after 20 minutes of CAP indicates its strain lowering effect and better autonomic regulation of the heart. Significant reduction in both RBG level and other cardiovascular functions, suggests that the 20 minutes of CAP was effective not only in improving blood glucose level but also effective in improving cardiovascular functions in patients with T2DM. Hence, CAP might be considered as one of the therapeutic tools for the prevention and the management of T2DM and its cardiovascular complications.

It is a well known fact that the patients with T2DM have a lowered immunity than the people with non diabetes and that is one of the complications of diabetes [2]. Whereas, acute cold exposure has reported to have a immune stimulating effects. Because, the major effectors of adaptive and innate tumour immunity such as Natural Killer (NK) cells and peripheral cytotoxic T-lymphocytes respectively were reported to increase both in numbers and its activity after brief cold stress. Hence, we believe that, a brief cold stress like CAP might help in improving the immunity in patients with T2DM [12].

None of the subjects reported any side effects during the intervention. Use of drugs has its own drawbacks, such as drug dependency, drug resistance and adverse effects, if used for a long time [2,4]. However, CAP is a safe, simple, feasible and cost effective treatment modality that can be used without any technical assistant for the management of T2DM. And, based on the present study findings studies on large sample size and covering all the limitations should be conducted.

The study was conducted in male patients with T2DM that limits the scope of this study in its application to the female patients with T2DM. Small sample size and it was not calculated based on the previous study. Assessments such as fasting blood glucose level and/or post prandial blood glucose levels, insulin sensitivity, continuous BP monitoring, peripheral resistance and baroreceptor sensitivity would have given better understanding. Current study assessed only the immediate effects of CAP on blood glucose level and cardiovascular functions but did not assess its long-term effects and its underlying mechanisms. Hence, further studies are required with large sample size, longer duration and advanced techniques for the better understanding of its effects and its underlying mechanisms.

Results of this study suggest that 20 minutes of CAP might be effective in improving blood glucose level and cardiovascular functions of patients with T2DM. Based on the present study findings the use of CAP in T2DM patients and patients with cardiovascular diseases is recommended.