Insomnia´s Impact on DNA Stability, Cardio-metabolic Health and Lifestyle Behaviors

Condition(s) studied

Investigational drug(s) / intervention(s)

Exercise: A cardiopulmonary exercise test in a treadmill will be performed after an overnight fasting. Gas exchange, heart rate, and muscle oxygen saturation levels will be assessed continuously during the entire test. Blood pressure and rate of perceived exertion will be recorded prior the end of each stage whereas blood glucose concentration will be assessed before and after the test. All the hemodynamic parameters will be assessed at rest to standardize the cardio-metabolic condition of the participants prior to exercise testing. Maximal exertion will be requested from all participants except elders, in whom a submaximal test will be applied, following the guidelines provided by the American College of Sports Medicine. From this trial, maximal oxygen uptake, ventilatory thresholds/dynamics, chronotropic and inotropic response, and metabolic flexibility will be examined. A trained exercise physiologist will supervise all the tests and validate the obtained data.

Bioelectrical Impedance: Bioelectrical impedance analysis will be used to estimate body compartments through a mechanism of resistance and reactance (InBody 770, KOR) . The compartments measured include body cell mass, fat mass, extracellular tissue, lean mass, muscle mass, visceral adipose tissue area, phase angle, among others.

Micronucleus Assay: The frequency of micronuclei, nuclear buds, basal cells, binucleated cells, condensed chromatin cells, karyorrhexis, karyolysis, and pyknosis will be determined through microscopic examination of exfoliated oral mucosal cells from each participant.

Physical activity and sedentary time: Physical activity and sedentary time will be assessed through the Spanish version of the International Questionnaire of Physical activity. In half of the analyzed subjects a wearable device (Fitbit luxe, Fit bit Inc) will be also used to record daily steps, energy expenditure and heart rate patterns.

Sleep: Insomnia severity index, sleep quality, somnolence and chronotype will be subjectively assessed in all participants, using validated scales such as the Pittsburgh, Epworth and ISI questionnaires. In half of the participants, sleep quality will be tracked by wearing a Fit band luxe device in the wrist.

blood sampling: The concentrations of glucose, glycated hemoglobin, and lipid profile will be determined through serum analysis using a blood chemistry analyzer (Spin120, Spinreact, ESP). Serum samples will be obtained by centrifuging peripheral venous blood samples (4-6 mL) collected in Vacutainer tubes at 2500 rpm for 10 minutes. From the same serum samples, circulating levels of insulin, ovarian hormones (estradiol and progesterone), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) will be assessed using enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA), following the specifications provided by each kit manufacturer (Biotechne, USA; AccuBind, GUA). Glucose, insulin and triglyceride values obtained from the blood chemistry analyzer and ELISA assays will be used to calculate the Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) and the triglyceride-glucose index.

psychometrics: Perceived stress will be obtained from the Perceived Stress Scale adapted for Mexican population. Additionally, depression and anxiety scores will be obtained from the Becks Depression Inventory, validated in Mexican adults. Ruminative thinking will be assessed through the short version of the Ruminative Responses Scale and substance abuse will be examined through the 10-items Drug Abuse Screening Test. Social media disorder and screen time will be also investigated from the Social Media Disorder Test and a cell-phone screen capture.

Nutritional Supplement: A semi quantitative food frequency questionnaire will be applied to collect data about macronutrient intake and supplements consumption. Hierarchical consumption of each food groups will be also assessed. Last meal consumed prior to exercise testing will be also recorded in conjunction with fasting time.

Genotyping: The P10L polymorphism of the OPN4 gene, previously associated with chronic insomnia and severe daytime sleepiness in the Mexican population will be determined using RFLP-PCR assays. DNA will be extracted from previously collected blood samples (4 ml) using the Master Pure DNA purification kit from Epicentre (Illumina Inc., US).

Study summary

Insomnia is a highly prevalent sleep disorder worldwide, with an increasing incidence in athletes, older adults, university students, and individuals with metabolic syndrome. This disorder has been associated with metabolic dysfunction, cardiovascular diseases, and genetic instability. Recent evidence suggests that insomnia negatively impacts cardiorespiratory fitness, insulin sensitivity, and overall health through mechanisms involving neuroendocrine dysregulation, oxidative stress, and alterations in energy metabolism. However, the specific biological and environmental factors that contribute to its prevalence and health consequences remain poorly understood, particularly across different populations. Understanding these associations is crucial for developing effective prevention and intervention strategies to mitigate the long-term impact of insomnia. Therefore, this study aims to analyze the impact of insomnia on body composition, cardiorespiratory fitness, metabolic flexibility, and DNA integrity, across populations with high prevalence of insomnia, including athletes, older adults, university students, and individuals with metabolic syndrome. This study will also investigate which biological and lifestyle behaviors contribute to insomnia and its health consequences. By doing so, this work will provide critical insights into the physiological and molecular mechanisms that link insomnia with metabolic and cardiovascular dysfunction, contributing to the development of targeted interventions for at-risk populations

Eligibility

Primary outcome measure(s)

• Insomnia severity index — Day 1
  A Spanish version of the insomnia severity index scale will be applied to examine the absence or presence of clinical insomnia. This five-item scale provides a range score between 0-28 that is interpreted as follows: * 0-7= No clinically significant insomnia * 8-14 subthreshold insomnia * 15-21 Clinical insomnia (moderate severity) * 22-28 Clinical insomnia (severe)
• Daytime sleepiness — Day 1
  A Spanish version of the Epworth Sleepiness Scale will be applied to examine day-time sleepiness. This is an 8-items self-administered questionnaire that provides a range score between 0-24, interpreted as follows: * 0-5 Lower Normal Daytime Sleepiness * 6-10 Higher Normal Daytime Sleepiness * 11-12 Mild Excessive Daytime Sleepiness * 13-15 Moderate Excessive Daytime Sleepiness * 16-24 Severe Excessive Daytime Sleepiness
• Sleep quality Index — Day 1
  A Spanish version of the Pittsburgh Sleep Quality Index scale will be applied to examine sleep quality. This 9-item scale provides a range score between 0-21 that is interpreted as follows: \*\<5 good sleep quality \*\>5 poor sleep quality
• Body composition — Day 1
  Bioelectrical impedance analysis (BIA) will be used to measure the components of the body, including fat mass (kg, %), lean mass (kg, %), muscle mass (kg), visceral adipose tissue area (cm2), phase angle (°), and total body water (L), through a mechanism of resistance and reactance. A multi-frequency device (1-1000 kHz), with 8-point Tactile Electrode System will be used for this measurements (InBody 770, KOR). This system also provides a segmental analysis of fat mass (kg, %), and lean mass (kg, %) (right/left arms or legs, and trunk). A body composition score, ranging from 0-100, will be retrieved from a predetermined algorithm provided by the fabricant.
• DNA integrity — Day 1
  The frequency of micronuclei, nuclear buds, basal cells, binucleated cells, condensed chromatin cells, karyorrhexis, karyolysis, and pyknosis will be determined through microscopic examination of exfoliated oral mucosal cells from each participant.
• OPN4 Single nucleotide polymorphism — Day 1
  The P10L polymorphism of the OPN4 gene, previously associated with chronic insomnia and severe daytime sleepiness in the Mexican population will be determined using RFLP-PCR assays. DNA will be extracted from previously collected blood samples (4 ml) using the Master Pure DNA purification kit from Epicentre (Illumina Inc., US).
• Cardiorespiratory fitness — Day 1
  A cardiopulmonary exercise test in a treadmill will be performed after an overnight fasting. Gas exchange, heart rate, and muscle oxygen saturation levels will be assessed continuously during the entire test. Blood pressure and rate of perceived exertion will be recorded prior the end of each stage whereas blood glucose concentration will be assessed before and after the test. All the hemodynamic parameters will be assessed at rest to standardize the cardio-metabolic condition of the participants prior to exercise testing. Maximal exertion will be requested from all participants except elders, in whom a submaximal test will be applied, following the guidelines provided by the American College of Sports Medicine. From this trial, maximal oxygen uptake, ventilatory thresholds/dynamics, chronotropic and inotropic response, and will be examined. A trained exercise physiologist will supervise all the tests and validate the obtained data.
• Metabolic flexibility — Day 1
  A cardiopulmonary exercise test in a treadmill will be performed after an overnight fasting. Gas exchange will be assessed continuously during the entire test to determine fat and carbohydrate oxidation. The maximal rate of fat oxidation, its shape and area under the curve will be defined in conjunction with the workload at which carbohydrates become the predominant energy source (cross over point). The increase in fat oxidation from rest to exercise will be also relativized to the change in energy expenditure to characterize metabolic flexibility. Resting metabolic rate and respiratory quotient will be also assessed at rest to standardize the metabolic condition of the participants prior to exercise testing. A trained exercise physiologist will supervise all the tests and validate the obtained data.
• Homeostatic Model Assessment of Insulin Resistance — Day 1
  The serum concentrations of glucose and insulin will be determined under fasting conditions, using a blood chemistry analyzer (Spin120, Spinreact, ESP), and enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA) . Serum samples will be obtained by centrifuging peripheral venous blood samples collected in vacutainer tubes under fasting conditions (4-6 mL; 2500 rpm for 10 minutes). The Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) will be then calculated with the following equation: HOMA-IR = glucose (mg/dL) x insulin (uU/mL) / 405
• Cholesterol — Day 1
  Total cholesterol (mg/dL) will be will be assessed in a blood chemistry analyzer (Spin120, Spinreact, ESP). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes under fasting conditions (4-6 mL; 2500 rpm for 10 minutes).
• Triglycerides — Day 1
  Triglycerides levels (mg/dL) will be will be assessed with a blood chemistry analyzer (Spin120, Spinreact, ESP). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes under fasting conditions (4-6 mL; 2500 rpm for 10 minutes).
• High-density lipoproteins — Day 1
  High-density lipoproteins concentration (mg/dL) will be will be assessed with a blood chemistry analyzer (Spin120, Spinreact, ESP). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes under fasting conditions (4-6 mL; 2500 rpm for 10 minutes).
• Insulin-like growth factor 1 — Day 1
  The concentration of Insulin-like growth factor 1 (0.1 - 30 ng/mL) will be will be assessed through an enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes (4-6 mL; 2500 rpm for 10 minutes).
• Vascular endothelial growth factor — Day 1
  The concentration of vascular endothelial growth factor (31.3 - 2,000 pg/mL) will be will be assessed through an enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes (4-6 mL; 2500 rpm for 10 minutes).
• Estradiol — Day 1
  The concentration of Estradiol (10 pg/ml - 4300 pg/mL) in young women will be will be assessed through an enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes (4-6 mL; 2500 rpm for 10 minutes). Current stage of the menstrual cycle and contraceptive use will be recorded for a proper interpretation of Estradiol levels.
• Progesterone — Day 1
  The concentration of Progesterone (0.15 ng/mL - 128 ng/mL) in young women will be will be assessed through an enzyme-linked immunosorbent assays (ELISA) on a microplate reader (iMark, BIORAD, USA). For this, serum samples will be obtained by centrifuging peripheral venous blood samples collected in Vacutainer tubes (4-6 mL; 2500 rpm for 10 minutes). Current stage of the menstrual cycle and contraceptive use will be recorded for a proper interpretation of Estradiol levels.

Trial sites (1)

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