Sechium edule var. nigrum spinosum (chayote) Increases the Expression of mRNA of Genes Encoding Sirtuins in Older Adults with Type 2 Diabetes Mellitus

1. Introduction

Type 2 diabetes mellitus (T2DM) is a metabolic disorder that occurs frequently in older adults. Its pathophysiology has been linked to processes such as oxidative stress (OS) and alterations in markers associated with the hallmarks of aging, including chronic inflammation, telomere shortening, mitochondrial dysfunction, and genomic instability. Its high frequency and the debilitating nature of its complications pose challenges for healthcare systems worldwide. [1,2,3,4,5,6,7,8,9].

Hence, the need to explore new therapeutic targets that focus on the pathophysiological mechanisms underlying T2DM, such as the activation of the family of enzymes structurally related to the silent mating-type regulatory protein 2 (Sir2), better known as sirtuins (SIRTs) or longevity proteins. These enzymes are involved in DNA damage repair, stabilization of telomere shortening, regulation of the inflammatory process and glycemic metabolism, control of mitochondrial dysfunction, and protection against OS. Among the nutritional interventions that improve the glycemic response and also exert direct effects on sirtuins are those focused on the use of polyphenols, bioactive compounds abundant in various fruits, such as berries, blueberries, raspberries, and grapefruit, and, as recently demonstrated, in Sechium edule var. nigrum spinosum, whose effects have been determined by our research group [10,11,12].

Previous studies have shown that consuming Sechium edule powder capsules has hypoglycemic, anti-inflammatory, hypotensive, and antioxidant effects in older adults with metabolic syndrome (MetS), impacting even at the molecular level by modulating the expression of mRNA of genes encoding enzymes with antioxidant functions and nuclear factor erythroid 2-related factor 2 (Nrf2) [13,14,15,16,17,18].

In this regard, sirtuins have been shown to deacetylate this factor, thereby promoting its activation and, consequently, the activation of antioxidant protection mechanisms, thereby reducing reactive oxygen species (ROS) levels [19]. Therefore, the objective of this study was to evaluate whether Sechium edule supplementation acts as a potential activator of mRNA expression of genes encoding sirtuins in older adults with T2DM.

2. Results

Table 1 presents the values of the anthropometric and clinical parameters, as well as the participants’ ages. No statistically significant differences were found in the measurements taken.

Glucose, cholesterol, triglycerides, HDL, uric acid, urea, albumin, and %HbA1c levels did not show statistically significant changes in either group at 3 and 6 months post-treatment (Table 2).

Regarding total oxidative capacity (Table 3), a statistically significant decrease was observed at six months post-treatment in the EG group (baseline, 6.4 ± 2.9 vs. post., 3.2 ± 2.1), as well as in the OSI group (baseline, 6.6 ± 3.3 vs. post., 2.0 ± 1.4). Conversely, total antioxidant capacity increased (baseline, 0.9 ± 0.3 vs. post., 1.3 ± 0.2) in the same group.

Figure 1 shows the relative expression of mRNA from genes encoding proteins that regulate the responses to metabolic or energy stress. SIRT1 (Figure 1A), SIRT3 (Figure 1C), SIRT5 (Figure 1E), and SIRT6 (Figure 1F) showed a statistically significant increase at 6 months post-treatment in the EG group compared to the PG group, at 52%, 69%, 62%, and 69%, respectively.

3. Discussion

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by chronic hyperglycemia secondary to defects in insulin secretion and/or action. Individuals with T2DM have a greater predisposition to developing pathologies such as cerebrovascular and cardiac diseases, infections, among others [20,21]. It is estimated that by 2050, approximately 1.31 billion people will suffer from T2DM [22]; therefore, this pathology is and will continue to be a serious public health problem worldwide. Hence, there is a need to prevent its complications.

In this regard, Sechium edule is a fruit that has been attributed with hypotensive, anti-inflammatory, hypoglycemic, antioxidant, and even geroprotective properties in older adults with metabolic syndrome (MetS) [17,21,23,24,25]. Therefore, it is possible to assume that Sechium edule could also be an adjunct in the treatment of T2DM in older adults [26,27].

However, this investigation found no significant effect on clinical and biochemical markers in patients with T2DM. These findings can be explained by considering the specific differences between our population and those previously studied. It is important to note that the previous studies, although conducted in older adults, were performed on patients with MetS, who could present with hyperglycemia or glucose intolerance, but not diabetes. Therefore, given the pathophysiology of T2DM, it is reasonable to assume that the effects of Sechium edule were insufficient to induce significant changes in this population. This proposal can be supported by findings from a systematic review and meta-analysis showing that the hypoglycemic effect of Sechium edule is modest (approximately a 1% reduction in HbA1c), which may be useful in pre-diabetic patients [28]. In this regard, we assume that, given the pathophysiology of T2DM, its natural history, and the development of metabolic memory, achieving significant changes in the identified markers is more complex. This explains why only some non-significant trends were observed in SBP, DBP, and HbA1c%.

On the other hand, it is interesting to observe the effect of Sechium edule on sirtuins and OS markers. Sirtuins are enzymes belonging to a family of seven members (SIRT1-SIRT7) with deacetylase (SIRT1-SIRT3, SIRT5-SIRT7) or ADP-ribosyltransferase (SIRT4 and SIRT6) activity, dependent on nicotinamide adenine dinucleotide (NAD+), and respond to various stressors, such as genotoxic and oxidative stressors. Accumulating evidence indicates that certain nutraceuticals present in a wide variety of fruits enhance sirtuin activity, leading to beneficial clinical outcomes in the treatment of cardiovascular diseases, arthritis, osteoporosis, dementia, and T2DM [29,30,31,32].

SIRT1 is a histone deacetylase that acts as a nutrient sensor. Its expression increases with caloric restriction and decreases with overfeeding. A decrease in SIRT1 expression leads to the recruitment or infiltration of macrophages into adipose tissue, resulting in histone hyperacetylation and, consequently, ectopic inflammatory expression. Conversely, its overexpression prevents this [33]. In human monocytes from patients with MetS, decreased SIRT1 expression levels have been associated with insulin resistance and atherosclerosis. Therefore, both glucotoxicity and lipotoxicity affect its expression [34]. In contrast, caloric restriction increases SIRT1 expression, which is associated with reduced inflammation and histological renal lesions in diabetic models. Consequently, its use has been proposed as a promising therapy for preventing diabetic nephropathy with an anti-aging focus [35,36].

Along these same lines, it has been observed that certain nutraceuticals in Sechium edule enhance the expression of this sirtuin, thereby significantly improving mitochondrial function by mitigating OS and inflammation. In the case of myricetin administration, it has been reported to promote mitochondrial biogenesis via SIRT1 by deacetylating Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha (PGC-1α) in experimental models [37].

It has also been observed that naringenin mitigates OS and inflammation and regulates mitochondrial function in human granulosa cells (KGN) by promoting SIRT1 expression [38]. Likewise, it has been reported that resveratrol consumption for six months increased SIRT1 levels by 50% in individuals with T2DM [29]. In this regard, in the present study, we observed a 52% increase in SIRT1 mRNA expression in the EG group compared to the PG group, which is likely associated with the effects observed in oxidation markers [39].

Regarding this, it has been noted that SIRT1 activation increases the post-translational activity of Nrf2, a transcription factor that regulates antioxidant protection mechanisms [40]. Furthermore, Sechium edule has been shown to increase mRNA levels of genes encoding Nrf2 and antioxidant enzymes, such as superoxide dismutase (SOD) and catalase (CAT), in patients with MetS [18]. This may be related to the results of the present study, where we found a 44% increase in TAS in the EG group six months post-treatment. This could imply that Sechium edule consumption improves antioxidant capacity by increasing SIRT1 expression.

On the other hand, our results showed that Sechium edule consumption had no effect on SIRT2 and SIRT4 expression, probably because these enzymes maintain a delicate balance to maintain cellular homeostasis, so both extremes (overexpression or downregulation) can be dangerous. In this regard, SIRT2 is inhibited in people with T2DM. This effect results in the restoration of pancreatic β-cell ability to enter the cell cycle and counteract the decrease in these cells [41]. For its part, SIRT4 overexpression leads to dyslipidemia, lipogenesis, and insulin secretion inhibition [42]. At the same time, its absence increases insulin levels, leading to accelerated development of insulin resistance and OS in in vivo models [43]. As can be seen, the effects can vary, suggesting that its regulation is a controlled process, in which the components of Sechium edule failed to exert any effect.

Regarding SIRT3, as with SIRT1, it regulates mitochondrial acetylation levels, protecting mitochondria from a wide range of damage, including oxidative injury [44,45]. Therefore, it is reasonable to assume that both sirtuins participate in similar protective mechanisms. In this regard, it has been proposed that naringenin promotes mitochondrial biogenesis by reducing oxidative damage, thereby attenuating ischemia-reperfusion injury and cardiac damage via AMPK/SIRT3 signaling [46]. In cardiomyocytes from diabetic models, SIRT3 overexpression has been observed to attenuate hypertrophy and fibrosis and reduce ROS formation [47].

In this respect, in the present study, we observed a 44% increase in TAS in the EG group, along with decreases in TOS and OSI (50% and 70%, respectively). These changes can be attributed to increased SIRT3 mRNA expression. This may indicate that Sechium edule supplementation promotes a balance between TOS/TAS levels, suggesting maintenance of cellular homeostasis in these individuals and potentially leading to effective ROS elimination [48,49]. SIRT5, like SIRT3, promotes antioxidant defense mechanisms; its inactivation negatively impacts NADPH and reduced glutathione (GSH) production. Therefore, the 62% increase in its expression in the EG group may be associated with the antioxidant effect observed in this group.

This behavior may be attributable to the presence of bioactive compounds in Sechium edule. One of them is quercetin, a flavonoid that promotes the desuccinylation of isocitrate dehydrogenase 2 (IDH2), a major source of NADPH, thereby supporting mitochondrial homeostasis, protecting against inflammation, and reducing oxidative damage [50]. In this regard, it is worth noting that supplementation with Sechium edule has been reported to reduce oxidative damage to lipids, proteins, and DNA [17].

Similarly, quercetin has been shown to activate not only SIRT5 but also SIRT6, which protects against age-related metabolic diseases and regulates chromatin homeostasis for telomere maintenance [51,52]. Its decrease leads to telomere dysfunction, premature cellular senescence, and chromosome end fusions [53]. In this regard, our research group has shown that Sechium edule consumption prevents telomere shortening [54]. Furthermore, it is important to note that, like SIRT1, SIRT6 is essential for Nrf2 transcriptional activation under OS conditions. This reinforces the findings of the present study, which show an increase in TAS and decreases in TOS and OSI in the EG [55]. Therefore, the 69% increase in SIRT6 gene expression at six months post-treatment in the EG may also be related to greater antioxidant protection [56,57].

On the other hand, it is well established that NAD+ levels decrease during the aging process, leading to impaired nuclear and mitochondrial function [58]. Therefore, based on our results, we suggest that the antioxidant effect of Sechium edule supplementation may be associated with the restoration of NAD+ levels, as evidenced by the evident increase in sirtuin transcriptional levels. This points to an increase in deacetylation [59,60], favoring the maintenance of redox homeostasis, which is mediated by Nrf2. Similarly, sirtuins can directly deacetylate Nrf2 at lysine residues, promoting its nuclear translocation, its ability to bind the antioxidant response element (ARE) in DNA, and/or inhibiting its ubiquitination via Keap1 [61]. Furthermore, it has been noted that the flavonoids present in Sechium edule mimic the effect of caloric restriction (CRM) by modulating metabolic pathways such as AMPK (which detects cellular energy levels and increases NAD levels) [62]. Hence, a possible synergistic interaction exists between the bioactive components of Sechium edule, involving the SIRT-AMPK-Nrf2 axis, which simultaneously integrates energy sensing and defense against oxidative stress [61]. This coincides with the observed results, both in increased expression of SIRT1, 3, 5, and 6 mRNA and in total antioxidant/oxidative capacity.

4. Materials and Methods

4.1. Experimental Design

This study was approved by the Research and Biosafety Bioethics Committee of the Faculty of Higher Studies Zaragoza, UNAM (FESZ/DEPI/CE/023/22/; October 21, 2022) with trial registration number (ISRCTN: 43215432).

All procedures were conducted in accordance with the ethical principles of the Declaration of Helsinki of the World Medical Association. Informed consent was obtained from each participant. The fruits of Sechium edule, varietal group nigrum spinosum, were donated by the Interdisciplinary Research Group on Sechium edule A.C. (GISeM) of the RED-Chayote, of the Agricultural Genetic Resources Subcommittee of the National Seed Inspection and Certification Service (NSICS), focused on the conservation, improvement, characterization, and enrichment of the genus Sechium in Mexico in the Municipality of Huatusco in the State of Veracruz, where the fruits used to make the capsules used in the study were harvested [63].

The fruit characterization was carried out according to the guidelines proposed by the International Union for the Protection of New Varieties of Plants (UPOV) and validated, including morphological, phenotypic, and chromosomal characterization, as carried out by GISeM [16,63].

The biological material was collected at horticultural maturity (the condition in which the fruit is suitable for consumption), selected, washed, disinfected, and sectioned into slices, which were then dried at 40°C and pulverized (epidermis, seeds, and spines). Previously, our research group identified the secondary metabolites present in each chayote capsule, determined using HPLC. These metabolites are found in ascending order of concentration as follows: 0.71 μg of cucurbitacin I, 6.11 μg of cucurbitacin D, 89.9 μg of cucurbitacin B and 154.8 μg of cucurbitacin E; flavonoids: 0.014 μg of apigenin, 1.3 μg of quercetin, 2.38 μg of myricetin, 14.2 μg of pholirizin, 45.5 μg of rutin and 48.8 μg of naringenin and phenolic acids: 0.11 μg of p-hydroxybenzoic, 1.4 μg of chlorogenic, 1.7 μg of p-coumaric, 3.3 μg of protocatechuic, 7.0 μg of ferulic, 8.7 μg of syringic, 9.3 μg of caffeic and 38.8 μg of gallic [17] (Suppl. S1).

4.2. Intervention

The capsule formulation was designed in the pharmaceutical development laboratory of FES Zaragoza. The placebo was prepared using pharmaceutical-grade lactose monohydrate and talc (United States Pharmacopeia, USP) (Sigma, St. Louis, MO, USA). The optimal particle size of the Sechium powder was determined, and rheological studies were conducted to ensure capsule filling, weight, homogeneity, and stability. In accordance with the design, the treatments were manufactured and packaged by a pharmaceutical company specializing in nutraceutical products. The intervention consisted of consuming three capsules (placebo or active) per day (500 mg of powdered Sechium edule, one before each meal) for six months. Dose selection based on the safety profile has been previously described by our research group [17,54]. A convenience sampling method was used to recruit 43 older adults with type 2 diabetes mellitus (T2DM), with a mean age of 66 years. Participants were randomly assigned to the experimental group (EG; n=22) or the placebo group (PG; n=21). Of these patients, only some participants chose to donate venous blood for gene expression measurements throughout the study (PG, n=14; EG, n=12). (Figure 2) In both groups, all measurements were taken at baseline (before treatment) and at 3 and 6 months (post-treatment).

4.3. Anthropometric and Blood Pressure Measurements

Body weight (kg) and waist circumference (cm) were recorded. Body weight was determined using a calibrated medical scale (SECA, Hamburg, Germany), while waist circumference was measured at the level of the umbilicus with a medical measuring tape (SECA, Hamburg, Germany). These measurements were performed by trained nursing staff [64]. Systolic (SBP) and diastolic (DBP) blood pressure were measured using a calibrated mercury sphygmomanometer. Patients were asked to rest for at least five minutes before the measurement, seated in a chair with a backrest, with their back straight, legs uncrossed, and feet flat on the floor. Finally, the Osler technique was used to identify pseudohypertension [65].

4.4. Biochemical Analysis

For blood sampling, participants were asked to fast for at least 8 hours. Samples were obtained by venipuncture and collected in vacuum tubes without anticoagulant. For clinical chemistry determinations (glucose, cholesterol, triglycerides, high-density lipoproteins (HDL-c), uric acid, urea, and albumin), colorimetric techniques were used with a Selectra Junior automated clinical chemistry analyzer (Vital Scientific, Dieren, The Netherlands). An immunoturbidimetric assay with the same clinical chemistry analyzer determined the percentage of glycated hemoglobin. Total antioxidant and oxidant status (TOS/TAS) were determined from heparinized plasma, and, finally, samples were collected in tubes containing the anticoagulant EDTA for lymphocyte isolation.

4.5. Total Oxidation Status (TOS)

The TOS was determined using a commercial kit (Rel Assay Diagnostics, Gaziantep, TR). In this kit, the oxidants present in the sample can oxidize the ferrous ion-chelating complex to ferric ions. In an acidic medium, this ion forms a colored complex with the chromogen, which can be measured spectrophotometrically. Therefore, the color intensity is directly associated with the amount of oxidants present in the sample. This test uses hydrogen peroxide (H₂O₂) as a calibrator.

4.6. Total Antioxidant Status (TAS)

The TAS was quantified using a kit (Randox Laboratories Ltd., Antrim, UK) that uses metmyoglobin and H₂O₂, along with 2,2-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), to produce a blue-green stain from the ABTS+ cationic radical. The ABTS+ cation is relatively stable and was measured at 600 nm. Color intensity is inversely proportional to the amount of antioxidants present in the sample.

4.7. Oxidative Stress Index (OSI)

The OSI was determined by the ratio of TOS to TAS concentrations (TOS/TAS) [66].

4.8. Lymphocyte Isolation and RNA Extraction

Lymphocyte separation was performed using 5 mL of venous blood diluted in equal parts sterile phosphate-buffered saline (PBS) (Sigma, St. Louis, MO, USA) and 2% fetal bovine serum (FBS) (ThermoFisher Scientific, Waltham, MA, USA). Four mL of Ficoll-Cold Pack (Gibco ThermoFisher Scientific, Waltham, MA, USA) were added. The mixture was centrifuged at 200 x g, and the opaque interface was transferred to a sterile tube. RNA extraction from 2 × 10⁶ lymphocytes was performed using the RNeasy Mini isolation kit (Qiagen, Hilden, Düsseldorf, Germany) according to the manufacturer´s recommendations, and the samples were stored at 70°C until use. RNA was quantified and its integrity determined from 5 µg on a 1% agarose gel with ethidium bromide and Tris-acetate-EDTA buffer, visualized using Kodak Molecular Imaging software (v.4.5.1). Simultaneously, its purity was calculated using the A260/A280 ratio. All RNA samples used were considered intact and of optimal purity.

4.9. Gene Expression Analysis

All reactions were performed using 10 ng of RNA and forward and reverse primers at a final concentration of 100 nM (IDT, Coralville, IA, USA) (Table 4). The primers were generated using a primer design tool (NCBI Primer-BLAST tool from NIH) [67].

The QuantiFast SYBR Green RT-PCR kit (one-step RT-PCR) (Qiagen, Hilden, Düsseldorf, Germany) was also used for gene expression analysis, which allows simultaneous execution of reverse transcription and PCR reactions. The reaction conditions are shown in Figure 3. The mean crossing threshold (Ct) of each gene was normalized to the mean Ct of the housekeeping gene β-actin.

Statistical Analysis

Results are presented as mean ± standard deviation and were analyzed usingrepeated measures ANOVA. Associations between sirtuin gene expression and the parameters analyzed were determined using Pearson´s correlation coefficient in IBM SPSS V 20 statistical software (Armonk, NY, USA). Results were considered statistically significant when p < 0.05. All determinations were performed duplicate.

5. Conclusions

Our findings suggest that consuming Sechium edule for six months increases transcriptional levels of the genes encoding SIRT 1, 3, 5, and 6, which promotes antioxidant protection mechanisms, supporting the proposal that consuming this fruit as an adjunct treatment to prevent complications of diabetes mellitus