Evaluating the Therapeutic Potential of Exercise in Hypoxia and Low-Carbohydrate High-Fat Diet in Managing Hypertension in Elderly Type 2 Diabetes Patients: A Novel Intervention Approach

1. Introduction

Diabetes is a metabolic disease that shows itself clinically as chronic hyperglycemia. Poorly or uncontrolled diabetes could be associated with many physiological processes that may affect lipid metabolism, regulation of inflammation, vasodilatation, vascular, immunological, and hematological parameters [1]. In light of this, hypertension (HTN), defined by systolic blood pressure ≥ 140mmHg or diastolic blood pressure ≥90mmHg [2], is prevalent in over 50% of individuals with type 2 diabetes (T2DM) and significantly escalates the risk of cardiovascular diseases (CVD) by fourfold compared to normotensive non-diabetic individuals [3]. Notably, individuals with T2DM who also have HTN at the time of diagnosis exhibit elevated rates of mortality and cardiovascular events, particularly pronounced in older populations [4], suggesting that much of this excess risk is attributable to coexistent HTN [5], underscoring the critical need for effective therapeutic strategies [6].

Most T2DM patients are not active [7] and a sedentary lifestyle, together with poor nutrition, are considered as the major risk factors for T2DM and its complications [8]. Current international guidelines recommend aerobic and resistance exercise training for T2DM patients for improving blood lipids, inflammation, glycemic and blood pressure control [9]. Exercise and Sports Science Australia guidelines [10] recommends supervised exercise as an effective modality for improving weight loss and both blood pressure and glycemic in T2DM patients.

Hyperglycemia in T2DM was negatively correlated with some hematological indices, as red blood cells (RBC), hemoglobin (Hb) and mean corpuscular hemoglobin (MCH), due the augmented oxidative stress [11]. Improvements in these biomarkers, mainly by the production of erythropoietin (EPO) and its consequent erythropoiesis, can be a helpful physiological mechanism, but do not occur as a result of an exercise training in normoxia [12]. Conversely, adding hypoxia to exercise promotes relevant hematological adaptations [12,13]. Recent investigations have shown promising outcomes in elderly patients with CVD [14] and in individuals with T2DM [15] performing exercise in hypoxia (EH) at simulated altitudes, suggesting EH may offer additional benefits compared to exercise in normoxia.

Physiological adaptations occur in hypoxic environments [16], mainly driven by hypoxia inducible factor 1α (HIF-1α) activation [17] and increased expression of hypoxia-responsive genes [18]. EH-induced erythropoiesis and angiogenesis may contribute to improved blood pressure control and enhanced tissue oxygenation. Not least, HIF-1α promotes lowering of blood glucose levels through the stimulating on glycolysis and as a result of the increased expression of GLUT4 glucose transporters in muscle tissue independently of muscle work [19]. Collectively, these adaptations could potentially mitigate vascular, hematological and metabolic complications associated with T2DM and HTN.

The optimal dietary approach remains a subject of debate among experts [20,21]. The low-carbohydrate, high-fat (LCHF) diet has emerged as a promising therapeutic option for individuals with T2DM and HTN, endorsed by the American Diabetes Association for glycemic control and weight management [22]. Notably, LCHF diets have demonstrated efficacy in reducing blood pressure [23], even surpassing the Dietary Approaches to Stop Hypertension (DASH) diet [24], the standard recommendation for blood pressure management by the American Heart Association [21].

Furthermore, LCHF diets have also been associated with hematological adaptations, including reductions in MCH and in mean corpuscular hemoglobin concentration (MCHC)[25]. Decreases in MCH and MCHC were inversely linked with insulin resistance and high blood pressure [26]. It was previously shown that hematological changes could occur as an effect of oxidative stress promoted by T2DM [11], and that these biomarkers may also serve as predictors of the disease's evolution [27], due to their role as determinants of blood viscosity [28]. Increased blood viscosity could contribute to the development of T2DM [29] and HTN [26,30].

Thus, this study aimed to evaluate the effects of an eight-week normobaric EH intervention at 3000m of simulated altitude combined with an LCHF diet on the hematological and lipid profiles, inflammation and blood pressure in older patients with T2DM and coexistent HTN. We hypothesize that the combination of chronic EH and LCHF dietary modifications will lead to improvements in blood pressure, with associated changes in hemoglobin levels, offering novel insights into therapeutic interventions for this high-risk population.

2. Materials and Methods

2.1. Ethical Considerations

The study was approved by the Ethics Committee of the Faculty of Nutrition and Food Sciences, University of Porto (Approval Number 45/2021/CEFCNAUP/2021 in July 2021. It was conducted following the declaration of Helsinki for studies in humans [31]. It has been registered in the Clinical Trial database (NCT05094505).

2.2. Study Design

This is a controlled, single-blind, three-arm, parallel, randomized controlled trial (RCT). Participants were randomly assigned into three independent groups (n=14, in each): control group (control diet + exercise in normoxia), EH group (control diet + EH) and EH+LCHF group [32]. The experimental design consisted of (1) pre-intervention tests, (2) a familiarization period, (3) an experimental intervention, and (4) post-intervention tests [33].

2.3. Participants

To be included, participants met the following criteria: (1) old individuals (>65 years) of either sex diagnosed with T2DM and HTN by their medical doctor for at least one year; (2) hemoglobin A1c (HbA1c) between 6.5% and 10%; (3) systolic blood pressure ≥ 140mmHg or diastolic blood pressure ≥ 90mmHg (4) pharmacological regimen stabilized for at least three months; (5) previous participation in supervised exercise programs in the last six months, and (6) absence of smoking in the last six months. Participants were excluded if: (a) they were insulin-dependent; (b) had uncontrolled microvascular or macrovascular complications related to diabetes, such as retinopathy, nephropathy, diabetic foot and atherosclerosis, diabetic cardiomyopathy or acute myocardial infarction; (c) presented other uncontrolled metabolic or vascular comorbidities; (d) were sedentary or (e) had a physical limitation that prevented them from exercising.

2.4. Dietary Plan

To each participant an individualized dietary plan was prescribed using the Dietbox® software, version 7.0. The energy content of the dietary plan met 100% of the estimated energy requirement (EER) for each participant. EER was calculated by multiplying the resting metabolic rate obtained by the Harris-Benedict equation, the most accurate at the individual level for older adults [34], by a physical activity level, assessed using the International Physical Activity Questionnaire (IPAQ short form, last seven days, elderly, self-administered format). Participants were categorically rated into one of three levels of physical activity: low, moderate or high [35].

The energy distribution by macronutrients was 60% from carbohydrates, 20% from protein and 20% from fat for the control diet, and 40% of energy from carbohydrates, 20% from protein, and 40% from fat for the LCHF diet. Both diets emphasized low–glycemic index foods to convey conventional dietary guidelines [36]. According to the classification of diets as (1) very low-carbohydrate: less than 26% of the energy intake; (2) low-carbohydrate: 26-45% of the energy intake; and (3) high carbohydrate ≥ 45% of the energy intake [37], we compared a low-carbohydrate diet (EH+LCHF group) to a high-carbohydrate diet (EH group). Compliance with the dietary plan was assessed using weekly 24-hour recalls. Participants met individually in appointments with a nutritionist twice over the eight weeks to encourage adherence to the dietary plan.

2.5. Exercise Protocol

Exercise sessions, either in normoxia or hypoxia (at 3000m of simulated altitude, via nitrogen dilution), took place in a hypoxic chamber at CMEP – Exercise Medical Center, 3 times per week during a period intervention of eight weeks. The chamber allows the control of O₂ (11% to 20.97%), temperature (up to 50ºC), relative humidity (up to 80%), and altitude above sea level (up to 8000m). Altitude rating can be defined as (1) high altitude: 1500 to 3500m; (2) very-high altitude: 3500 to 5500m; and (3) extreme altitude: above 5500m [38].

Before starting the intervention period, six familiarization sessions were held for two weeks so the participants could learn the exercise techniques and acclimate to the simulated altitude, with an increment of 500m at each visit until reaching 3000m of altitude. Exercise intensity was set at 75% of the heart rate reserve, quantified in the pre-intervention cardiopulmonary test (CPET). Heart rate and oxygen saturation were constantly monitored using a finger pulse oximeter (Globus YM201, Milan, Italy), and the Borg Rating of Perceived Exertion (RPE) was recorded after each exercise session [39]. All exercise sessions occurred at the same time of day (± 1 hour), and visits were separated by at least 48 hours of recovery.

The total duration of each exercise session was approximately 60min, which included a 5min warm-up with body mobilization and dynamic stretching, followed by 40min of moderate aerobic exercise, alternated every 9min on a cycle ergometer (Life Fitness, Illinois, United States) and a treadmill (Life Fitness, Illinois, United States), with a 1min rest between them. At the end of each session, and alternately between weeks, three strength exercises were performed (pectoral, shoulders, back, arms, thighs, legs and abdominals) using a structure of 3 series of 12 to 15 repetitions per exercise, with a 1min rest between sets, totaling ~ 15min of strength exercises.

2.6. Measurements

In all groups, hematological parameters, blood lipids, inflammation marker, and blood pressure were evaluated at baseline and 48h after the last exercise session (eighth week), after fasting for 12 hours and without any strenuous exercise in the last 24 hours and no alcohol consumption in the previous 72 hours.

2.7. Blood Samples Analyses

Hematological markers were determined, including erythrocytes (L), hemoglobin (g/dL), globular volume, (MCV, %), mean corpuscular hemoglobin (MCH, pg), mean corpuscular hemoglobin concentration (MCH, g/dL), red cell distribution width (RDW, %), leukocytes (L), neutrophils (%), eosinophils (%), basophils (%), lymphocytes (%), monocytes (%) and platelets (L). Lipid parameters included total cholesterol (mmol/L), high-density lipoprotein cholesterol (HDL-c, mmol/L), low-density lipoprotein cholesterol (LDL-c, mmol/L), triglycerides (TAG, mmol/L); and inflammatory marker, as CRP (mg/dL) were collected and analyzed.

2.8. Blood Pressure

Systolic, diastolic, and mean blood pressure (SBP, DBP and MAP) were measured using an automated sphygmomanometer (Dinamap Pro; Florida, United States) in the left arm, in a sitting position and after ten minutes of rest in the same day of blood sample collection. The measurements were performed in triplicate, and the average was expressed in mmHg.

2.9. Statistical Analysis

For sample and power calculations, this study was powered based on changes in hemoglobin A1c in the RCTs included in the meta-analysis by Zuuren et al. [40]. To detect an effect size of Cohen’s d=1.14 with 80% power (alpha=0.05, two-tailed), G*Power software [41] suggested we would need 28 participants in a paired samples t-test, totaling 42 individuals to the three experimental groups. Assuming a 20% dropout rate, it was recruited 48 participants. All data was reported as mean (standard deviation SD). Normality was assessed using Shapiro-Wilk’s test. Continuous variables that did not follow a normal distribution were transformed using the logarithm function: y=log(x-L) with L < minimum of x, if skewness was positive; or y=log(H-x) with H > maximum of x, if the skewness was negative; except for the diastolic blood pressures, that were transformed using the Box-Cox transformation with parameter λ=0.75. A two-way repeated measures ANOVA was used to examine changes in hematological markers, CRP, lipid profile and blood pressure over the chronic exercise period (zero vs. eighth weeks) and whether the magnitude of the chronic exercise-mediated adaptations differ in time and differ among groups. A Tukey post hoc test for multiple pairwise comparisons was performed to identify differences between groups when a significant main- or interaction effect was found. Statistical analysis was performed using SPSS Statistics software version 28.0, 2021 (IBM Company, Chicago, United States), and statistical significance was assumed at p≤0.05.

3. Results

3.1. Baseline Characteristics

The baseline characteristics of the participants are shown in Table 1. None of the participants got injured or had adverse responses to the EH or LCHF diet. The groups did not differ significantly at this moment, neither in terms of gender or age.

3.2. Dietary and Exercise Interventions

The detailed data regarding exercise and diet interventions were previously published [33]. Briefly, the EH+LCHF group had a lower carbohydrate intake (p<0.001), a higher total fat intake (p<0.001), while no significant differences were found between groups about energy (p=0.69) and fiber (p=0.49) intakes. Physiological parameters obtained during eight weeks of exercise sessions, showed that the groups that exercised in hypoxia (EH and EH+LCHF) presented lower mean values of oxygen saturation when compared to the CTRL group (p<0.001), but the average heart rate was similar among groups (p=0.63). Also, subjective effort perception was collected and the EH and EH+LCHF groups achieved the highest score of RPE (p=<0.001) used to measure the level of subjective intensity of physical exercise.

3.3. Cardiovascular Risk Factors

Cardiovascular risk factors were evaluated by lipid profile and CRP. No effect from baseline to post-interventions was observed on total cholesterol (p=0.08), HDL-c (p=0.987), LDL-c (p=0.501) and TAG (p=0.435), and among the three groups (p=0.135; p00.511; p=0.119; p=0.518), respectively. CRP did not show differences from baseline to the eighth week of intervention (p=0.090), and did not differ among groups (p=0.66, Table 2).

3.4. Hematological Parameters

The hemogram of the study participants is shown in Table 3. There were no significative differences from pre to post interventions regarding erythrocytes (p=0.585), Hb (p=0.355), globular volume (MCV, p=0.460), RDW (p=0.059), leukocytes (p=0.999), neutrophils (p=0.192), eosinophils (p=0.863), basophils (p=0.691), lymphocytes (p=0.279%), monocytes (p=0.303) and platelets (p=0.105). There were no differences between the moments for MCH (p=0.733) and MCHC (p=0.669), but MCH increased only in the EH group (p=0.027), and MCHC was reduced only in the EH+LCHF group (p=0.046).

3.5. Blood Pressure

SBP, DBP and MAP significantly reduced (p<0.001) from pre- to post-intervention (Table 4). While SBP, DBP, and MAP values decreased after interventions, post-hoc analysis revealed that these changes did not reach statistical significance between groups (p=0.151; p=0.124; p=0.18, respectively). No differences were found in resting heart rate regarding moments (p=0.090) and groups (p=0.660).

4. Discussion

This is the first RCT examining the responses of chronic EH with and without a LCHF diet on hematological and lipid profile, inflammation and blood pressure in patients with T2DM and coexistent HTN. The study’s main findings revealed an increased in MCH in patients who exercised in hypoxia, while a reduction in MCH concentration was observed in patients following an LCHF diet. Additionally, the study identified a significant reduction in HTN following eight weeks of exercise in normoxia and hypoxia, with no substantial disparity in the efficacy of EH compared to normoxia, irrespective of dietary carbohydrate content, which contradicts our hypothesis. These findings align with prior research indicating comparable blood pressure outcomes between EH and normoxia-based exercise interventions in older individuals over an eight-week period with same oxygen levels (~15% of FIO₂) [42].

However, contrasting results from existing studies suggest superior effects of regular EH on blood pressure regulation. For instance, moderate exercise at a natural altitude of 1700m exhibited notable reductions in SBP and DBP in individuals with metabolic syndrome over a three-week period [43]. A reduction of 10 mmHg and 7 mmHg in SBP was observed after four weeks of chronic EH at 16.4 and 14.5% of FIO₂, respectively [44,45]. In contrast, other researchers observed a reduction only in DBP after a 13-week of aerobic and strength exercise in normobaric hypoxia simulating 2000–3350m altitude [46]. It is noteworthy that most of these studies reported an improved in body composition after EH intervention [43,44,46], a crucial factor in decreasing blood pressure by exercise [47].

Moreover, there is evidence suggesting that older individuals may exhibit resistance to the reduction of exercise-induced blood pressure [47], and this resistance appears to be closely tied to changes in body composition [44,47]. Published data from the current RCT showed reductions in weight loss, body mass index (BMI) and body fat after eight weeks of interventions [33], and there were no significant additional benefits observed in the groups exercising in hypoxia compared to those exercising in normoxia. Similarly, our investigation into blood pressure failed to reveal any notable advantages of exercising in hypoxia over normoxia, irrespective of dietary carbohydrate content. In agreement, it was demonstrated that an eight-month training program led to improvements in weight, BMI, waist and hip circumference over time, with no discernible differences between the normoxia and hypoxia exercise groups [48].

Despite observing similar improvements in blood pressure with both normoxic and hypoxic exercise interventions, along with comparable levels of Hb mass, MCH, and MCHC before and after chronic EH and LCHF diet interventions, our study revealed higher MCH levels in the EH group. It is widely acknowledged that achieving a substantial increase in hemoglobin content requires an adequate hypoxic dose of >12 hours per day at a sufficient altitude for >21 days (approximately 300 hours) [49,50]. This suggests that the similarity in hemogram biomarkers from pre- to post-eight weeks in the current study may be attributed to the short exposure to simulated altitude, specifically 3 hours per week and 24 hours during the total intervention period.

Compensatory elevation in MCH within a specific range is recognized as a fundamental physiological response to high-altitude hypoxia [51], enhancing blood´s oxygen-carrying capacity and improving tissue oxygenation without increasing cardiac output [52]. This adjustment translates to an increase of approximately 0.30–0.47 g/dL per 1000 meters of altitude [50]. However, when Hb production significantly exceeds the reference range, an increase in cardiac output is required to sustain oxygen transport [52], potentially contributing to blood pressure elevation. Given that Hb production remains within the normal range proposed for sea-level [53], and when corrected by high-altitudes [54], it appears that the increase in MCH production within the minimum cut-off values in patients who exercised in hypoxia was not sufficient to induce significant changes in blood pressure compared to exercise in normoxia, at least with 24h of hypoxic exposure at 3000m simulated-altitude.

Contrary to our findings, previous studies have demonstrated improvements in SPB and DBP following long-term exposure (approximately 6 and 12 months) to natural high-altitude, accompanied by increased Hb levels [55]. Other authors found similar improvements after 15 sessions of hypoxia exposure (14-10% FiO₂) compared to normoxia [56]. This discrepancy may be attributed to altered hydration status in the EH group, increasing Hb content when dehydrated [53]. However, the MCV values in the current study do not support this hypothesis.

Additionally, existing evidence has demonstrated that incorporating hypoxia into exercise, even during short periods and at moderate high altitudes, yields greater benefits in blood pressure, independent of hematological parameters. This suggests the involvement of alternative mechanisms beyond MCH in this response [44,45,46]. Chronic EH has been linked to reduced arterial stiffness [57], and improvements in metabolic risk factors such as body fat and insulin resistance [58], all of which play a role in regulating blood pressure. EH also induces vasodilation and lowers blood pressure in patients with T2DM [59]. These effects are, in part, mediated by increased HIF-1α protein expression, which is inversely associated with systemic blood pressure [16]. HIF-1α also triggers VEGF activation [60], impacting blood pressure regulation via nitric oxide synthase expression and nitric oxide activity [59]. Unfortunately, we did not evaluate HIF-1α and VEGF levels in our study.

It is well known that elevated levels of Hb, MCH and MCHC are associated with increased blood viscosity [61], which induces decreased blood flow to skeletal muscles and fat tissues, contributing to peripheral vascular resistance and elevating the risk for T2DM development, interfering with insulin-mediated glucose uptake [29], and potentially elevating blood pressure [62] contributing to the development of HTN [26,30]. While the EH group exhibited increased MCH levels within normal ranges, restricting carbohydrate intake appeared to decrease MCH concentration, albeit within appropriate levels [53,54]. Consistent with this, prior research reported that a LCHF diet over twelve weeks reduced iron intake and lowered both MCH and MCHC in male endurance athletes [25]. This reduction could be attributed to increased inflammation associated with the LCHF diet, affecting the iron regulatory hormone hepcidin [63]. Hepcidin production is stimulated by iron and inflammation but inhibited by hypoxia [64]. Considering that Hb comprises nearly 70% of iron in the body, the LCHF diet's reduction in iron consumption, coupled with hypoxia-induced decrease in hepcidin production, could explain the reduced MCHC values observed in our study.

Moreover, MCHC has been linked to metabolic disorders, with levels increased in obesity and decreased by medications improving cellular insulin sensitivity [64]. Prediabetic patients with higher Hb content have been shown to exhibit impaired blood pressure, HDL-c levels, and waist circumference [65], contributing to a proinflammatory state and worsening metabolic dysfunction and CVD development [66]. Although our study found no differences in lipid profile and CRP levels among time and groups, evidence suggests a positive association between reduced MCHC and carbohydrate-restricted diets, with consequent improvement on insulin resistance and blood pressure, highlighting a crucial mechanism for further exploration in patients with T2DM and coexistent HTN.

In summary, while EH interventions did not demonstrate superior benefits in reducing blood pressure compared to normoxia-based exercise, our findings underscore the intricate interplay between exercise, diet, and hematological parameters in managing metabolic disorders and hypertension in patients with T2DM. Further research is needed to elucidate underlying mechanisms and optimize therapeutic strategies for this population.

Strengths and Limitations

The study had a 100% adherence rate, with no dropouts throughout the testing and intervention phases, surpassing the general cutoff point for sufficient adherence in older adults by 30pp [67]. To uphold participation levels, the research team maintained constant contact with participants throughout the eight-week intervention period, motivating them to complete their participation regularly during the eight weeks of intervention. This proactive approach helped motivate participants, ensuring their consistent involvement in the study. Recognizing potential barriers to attendance, such as transportation challenges, the study provided chauffeured transportation for participants to attend exercise sessions at the clinic, removing logistical hurdles. Regular meetings were convened to assess food consumption and address any queries, fostering correct adherence to the prescribed dietary plan among all participants. This personalized support contributed to the overall adherence success of the intervention.

Some limitations should also be mentioned. Due to logistical and budgetary constraints, the study was limited to an eight-week duration, resulting in a total of 24h of hypoxia exposure. While sufficient for certain outcomes, this timeframe may have been insufficient to induce substantial changes in hematological parameters [50], such as Hb, MCH and MCHC, and their influence on blood pressure regulation. The reasons mentioned above precluded the determination of key biomarkers, including HIF-1α, VEGF, iron deficiency markers, and hepcidin levels. These markers are vital for understanding the mechanistic underpinnings of the intervention and its effects on blood pressure regulation. Future research should delve into these biomarkers, as well as increase the time of exposure to hypoxia, to elucidate their role in the treatment of HTN in type 2 diabetic patients undergoing EH and LCHF dietary interventions.

5. Conclusions

In conclusion, diets and exercise lowered HTN, with no additional benefits from added hypoxia and restrict carbohydrate. Future research is needed to provide a deeper understanding of the precise mechanisms underlying hematological adaptations and their subsequent impact on blood pressure regulation. Additional elucidation of these mechanisms is imperative for a comprehensive understanding of the therapeutic potential and optimization of EH and LCHF dietary interventions in managing related HTN complications in individuals with T2DM.