Type 2 diabetes mellitus (T2DM) has become a major global health problem in recent decades due to its rising incidence, prevalence, and its tight causal association with diverse comorbidities, including cardiovascular disease (CVD) [1
]. In the context of CVD prevention, lifestyle and behavioral interventions have been shown as advantageous approaches with less associated costs and side effects compared to available medical treatments [3
]. In particular, several healthy dietary patterns have been shown to be useful tools for the management of T2DM and the concomitant reduction of cardiovascular risk. This is the case of the Mediterranean diet (Med diet), Dietary Approaches to Stop Hypertension (DASH), vegetarian diet, and the low-fat (LF) high carbohydrates diet recommended by the National Cholesterol Education Program and the American Diabetes Association (ADA) [2
]. In line with this, our group has shown that within the Coronary Diet Intervention with Olive Oil and Cardiovascular Prevention (CORDIOPREV) study, a prospective, randomized, controlled trial that includes CVD patients at high-risk of T2DM development [6
], long-term consumption of a Med diet rich in olive oil and a LF diet have beneficial effects on the patients, improving insulin sensitivity and beta-cell function [7
]. In this context, early identification of patients at higher risk of T2DM development is critical for prevention of new cardiovascular events [8
]. Interestingly, in searching for novel associated molecular mechanisms and predictive markers, we recently discovered that the expression pattern of certain splicing machinery elements in the peripheral blood mononuclear cells (PBMCs) of patients is tightly associated with the risk of T2DM and could accurately predict T2DM development in those individuals from the CORDIOPREV study, outperforming the capacity of classical predictors of T2DM development, such as glycated hemoglobin (HbA1c) or predictive scores (FINDRISK) [10
], two established strategies that have limitations and cannot precisely predict an individual’s risk of developing T2DM [11
The splicing machinery comprises the spliceosome, a sophisticated macromolecular complex with a functional core comprising several small nuclear ribonucleoprotein (snRNP) subunits, which interact dynamically to control the splicing process. The activity of the spliceosome is precisely modulated by more than 300 auxiliary proteins, the so-called splicing factors, that recognize specific sequences in exons and introns [13
]. An emerging body of evidence indicates that, under adverse health conditions, there is a profound dysregulation of certain spliceosomal components and splicing factors that results in altered and even aberrant splicing processes which, in turn, substantially contribute to the development of severe pathologies, including cancer, neurodegeneration, liver disease and diabetes [13
]. Indeed, the correct function of the splicing machinery is essential to maintain cell homeostasis [17
]. In this scenario, it has been proposed that nutrients can influence processes essential for cell homeostasis by altering gene expression and, in particular, modulating the splicing of pre-mRNAs encoding key regulatory proteins (e.g., leptin receptor, insulin receptor) [24
]. Moreover, several studies have shown that the gene expression pattern of PBMCs is severely influenced by the diet [25
] and might reflect metabolic and immune responses of adipocytes or hepatocytes [29
], thus providing valuable information to advance in the study of diseases such as T2DM and CVDs, using less invasive sampling methods [10
Based on all the above, the aim of this work was to evaluate the influence of the dietary intervention in the expression pattern of the components of the splicing machinery in PBMCs from patients included in the CORDIOPREV study. Specifically, we sought to ascertain if the consumption of two healthy diets (Med diet and LF diet) during three years modified the expression profile of the splicing machinery in PBMCs from CVD patients.
This study represents, to the best of our knowledge, the first comprehensive analysis of the regulatory role of healthy dietary interventions on the expression of the components of the splicing machinery, including spliceosome elements and splicing factors. This study was implemented using PBMCs from CVD patients at high risk of T2DM development included in the CORDIOPREV trial, in that we have previously demonstrated that the expression pattern of certain splicing machinery components is associated with the risk of T2DM development and could accurately predict this development in individuals with coronary heart disease [10
Our present study provides primary evidence that a dietary intervention can distinctly alter the expression pattern of the splicing machinery, both spliceosome components and splicing factors, in CVD patients at high risk of T2DM. In particular, these results demonstrate that the consumption of two healthy diets (Med diet and LF diet) during three years can modulate the expression pattern of key spliceosome components and splicing factors in PBMCs from the patients enrolled in the CORDIOPREV study, including the overexpression of some molecular components, like SPFQ, RBM45, RNU6, etc. and the downregulation of others, including RNU2 and SRSF6. Interestingly, some of the changes observed in the expression levels of certain splicing machinery components were closely associated with relevant biochemical parameters and clinical features, as is the case of the increase in the expression levels of the splicing factor SPFQ, which was inversely correlated with the decrease in HOMA-IR and HIRI indexes observed in the population.
The finding of a diet-related long-term modulation of the expression of the splicing machinery components could represent a novel valuable piece of information for two reasons. First, because it unveils that the splicing process may represent an adaptive mechanism in response to different nutritional conditions, and that this mechanism could be in place not only in circulating PBMCs but may also operate in cell types from other tissues and organs tightly coupled to nutrient-dependent metabolic homeostasis (e.g., liver, pancreas, adipose tissue), an avenue that is indeed worth exploring. Actually, we and others have already discovered the delicate and important role that the regulation of the splicing machinery can play in those organs [10
]. Secondly, inasmuch as PBMCs can be an accessible and suitable sentinel to detect relevant changes related to nutrient- and diet-dependent metabolic homeostasis, our current results support the idea that changes in the expression of key splicing machinery components could provide a fine screening marker for the development or progression of T2DM and their diet-related dynamics. Indeed, within the CORDIOPREV study, the long-term intake of a Med diet, rich in olive oil, or a LF diet similarly improved insulin sensitivity and beta-cell function [7
] and, therefore, the increase in the expression of specific splicing factors found herein under both diets, and their inverse correlation with insulin resistance indexes, strongly suggest that the molecular changes might be related to the beneficial consequence of consumption of a healthy diet. Furthermore, the dynamic, concomitant changes in expression observed suggest that splicing factors and, hence, the alternative splicing process, could represent novel elements within the complex mechanisms linking healthy dietary intervention and the improvement of patients’ metabolic status and the consequent protection from cardiovascular complications. Given the very scarce information available on the functional roles and implications of many of the molecules identified in the present study to be altered in PBMCs (e.g., SPFQ
, etc.), the present findings open novel, unexplored avenues in this field of research.
One of the findings from this study that we consider most noteworthy is that the diet-induced alterations in the splicing machinery of PBMCs was independent of the type of healthy diet in which CORDIOPREV participants were enrolled (Med diet or LF diet), except for three splicing factors (SNW1
) that showed a more pronounced modulation in patients under the LF diet. To date, and to the best of our knowledge, no data were reported regarding the influence of diet intervention in the modulation of the expression of SNW1
. However, some of these factors have been described to contribute to the alternative splicing of key genes whose splicing processing changes in response to a fatty diet [34
]. In particular, NOVA1
expression has been shown to be modulated under high-fat diet-induced obesity and to be responsible for the regulation of the splicing process of key genes under these conditions [34
]. Of note, it has been proposed that NOVA1
is a master regulator of alternative splicing in pancreatic beta cells, where it controls the expression of key genes involved in insulin transcription and secretion [36
]. Conversely, to date, the regulation and possible role of this splicing factors in PBMCs have not been explored in detail.
Previously, several studies have shown that PBMCs’ gene expression pattern is influenced by the diet [25
] and that this might reflect changes related to both metabolic and immune responses [29
]. In addition, it has been demonstrated that the splicing process of key regulatory proteins for metabolic homeostasis, like the receptors for insulin or leptin, can be markedly influenced by nutrient metabolism, directly or indirectly [24
]. Thus, it seems reasonable to think that those splicing-related changes would rely on upstream changes in the function of the machinery responsible for generating the splice variants. However, little or nothing is known in this regard in PBMCs, for there are no reports on how diet can influence the expression of the components of the spliceosome and the splicing factors, which altogether are responsible of the modulation of the splicing process. Nevertheless, in this context, some studies have highlighted that nutritional status can induce changes in the activity of serine-arginine (SR) proteins [38
], an important family of splicing factors, further supporting the contention that different nutrients may be able to modulate the expression of metabolic genes at the level of its splicing processing. Specifically, it has been shown that insulin signaling can up-regulate the expression of the splicing factor SRSF1 in pancreatic beta cells, inducing the splicing of the insulin receptor to generate the INSR-B isoform [39
]. The same study also found a regulation of the protein levels of the splicing factor MBNL1 by high glucose levels. In addition, other splicing factors belonging to the SR proteins family, SRSF2, are decreased under a vitamin E-deficient diet in the liver [40
]. Thus, although still limited, the evidence is growing, and by inclusion of the results from this study, pointing to a link between diet and nutrient and regulation of the splicing process, including its underlying operating machinery.
Another intriguing implication of our present results relates to the predictive capacity of studying changes in the splicing machinery in at-risk patients. To be more specific, nutrient-induced changes in specific splicing machinery components may provide hints on the predictive potential and possible functional correlation of key molecules, which had not been explored hitherto in this regard. Thus, within this study, regardless of the type of dietary intervention, the expression of some of the splicing factors studied was differentially altered in patients that develop T2DM after the 5 years of the study compared to non-T2DM subjects. For example, RNU12
, a component of the minor spliceosome, showed a significant increase after the 3 years of dietary intervention in non-T2DM controls. Interestingly, we have previously described how the expression of this small nuclear RNA (snRNA), which is essential to form U12 snRNP and carry out the appropriate splicing of type 12 introns [41
], was lower, at baseline (inclusion of the study), in Incident-T2DM compared to non-T2DM subjects and that this was associated with the risk of T2DM development [10
]. Therefore, since lower expression levels of RNU12
were associated with a higher risk of T2DM, it is reasonable to infer that the dietary-induced increase in the expression of this component in non-T2DM might be related to the protective effects of the healthy dietary consumption. In support to this notion, in that same study, a 4 h-incubation with baseline postprandial serum from Incident-T2DM patients induced a significant reduction of RNU12
expression compared to non-T2DM treated PBMCs from healthy patients [10
]. Therefore, from a more general standpoint, it can be proposed that modulation of the expression of specific spliceosomal components may represent a link between dietary intervention and beneficial effects on the patient metabolic status. Remarkably, the difference in RNU12
expression, at year 3, between incident patients and non-T2DM subjects, was more pronounced under the LF diet. Thus, although the possible mechanisms linking nutrient-induced changes in the splicing machinery, their functional consequences and the regulatory implications are still to be fully elucidated, our present study provides supportive evidence to further explore both the mechanistic/functional and the predictive components of this plausible link, for it may provide original, valuable biological knowledge as well as practical information for the patients.
This study has strengths and limitations. Among the latter, it should be mentioned that (1) the analyses of the expression patterns of key splicing machinery elements was carried out in the heterogeneous PBMC population, but not in specific cell types; (2) the sample sizes of T2DM patients and non-T2DM controls were relatively small; and (3) the non-T2DM subjects analyzed were CVD patients within the CORDIOPREV study and, therefore, were not non-T2DM healthy controls. On the other hand, one of the strengths of our work derives from its nested case-control study nature, which enabled both the T2DM patients and non-T2DM subjects to be sampled from the longitudinal CORDIOPREV study. Also, expression patterns of a wide array of spliceosome components and splicing factors were studied.
In conclusion, this study reveals that expression of the splicing machinery components in PBMCs from CVD patients at risk of T2DM can be notably and selectively influenced by long-term dietary intervention; also, that the two dietary interventions tested herein, Med diet and LF diet, induced remarkably similar changes on the expression of spliceosome components; and, finally, that there are distinct, diet type-induced changes in PBMCs from both non-T2DM and incident-T2DM patients, that may have an as yet unknown functional significance. Therefore, we propose that the machinery that controls and performs the alternative splicing process, which is consequently responsible for changes in the pattern of functionally and pathologically relevant splice variants involved in the regulation of metabolic homeostasis, is a plausible target to be operated by dietary intervention. As such, our results pave the way to explore in experimental models the possible mechanistic role and relevance of the splicing machinery and its components in diet-related metabolic regulation, and to investigate the value of screening changes in specific splicing machinery components to monitor and predict early the relevant diet-related changes in CVD patients at risk of T2DM.