Changes in quantity plant-based protein intake on type 2 diabetes remission in coronary heart disease patients: from the CORDIOPREV study

The current work was conducted within the framework of the CORDIOPREV study (Clinicaltrials.gov NTC00924937), a randomized, single-blinded, controlled dietary intervention trial including 1002 patients with CHD, who had their last coronary event more than six months before their enrolment in two different dietary models for seven years, in addition to following the conventional treatment for CHD [13]. The participants were randomized to receive two diets: a Mediterranean diet or a low-fat diet. Rationale, methodology and baseline characteristics of study participants have been published elsewhere [13]. The protocol was written in accordance with the principles of the Declaration of Helsinki. The respective Institutional Review Board (IRB) by the Human Investigation Review Committee of the Reina Sofia University Hospital (Córdoba, Spain) approved the study protocol. Recruitment took place from July 2009 to February 2012. All subjects provided written informed consent. The ethics approval and study protocol can be found on the IMIBIC website. At baseline and during every year of follow-up study participants passed medical and dietary interviews and underwent blood and oral glucose tolerance test (OGTT). The biochemical and laboratory measurements were performed as previously described [14, 15].

Patients who had diabetes diagnosed at the beginning of the study and were not receiving glucose-lowering treatment were included in the CORDIOPREV-DIRECT study (190 out of 1002 patients). The diabetes diagnosis was carried out according to the diagnosis criteria of the American Diabetes Association (ADA) (fasting glucose ≥ 126 mg/dL, 2 h glucose during OGTT ≥ 200 mg/dL, or HbA1c ≥ 6.5%) [16]. Of these, seven patients could not be included due to the inability to perform the diagnostic test used in this work. Since the aim of this sub-study were to evaluate changes in food and nutrients consumption from baseline to first year of follow-up, six more patients were excluded for not been disposable the food-frequency questionnaires (FFQ) at first year visit due to these patients denied continuing with dietary follow-up. Thus, for this sub-study, the final number of patients analyzed was 177.

T2DM remission was defined as HbA1c < 6.5%, fasting plasma glucose < 126 mg/dL and 2 h plasma glucose after 75 gr OGTT < 200 mg/dL and maintaining these levels for at least 2 consecutive years without the use of diabetes medication to lower blood glucose levels [17, 18]. Patients were tested yearly from the first year of follow-up and classified as remission or maintaining diabetes at fifth year of the study. From the finally 177 patients included in the present work (those with biochemical tests and completed FFQ disposable), 72 patients reverted from T2DM after five years of dietary intervention, while 105 participants had not achieved remission by the end of the follow-up period.

The participants were randomized to receive two diets: a Mediterranean diet or a low-fat diet. The low-fat diet consisted of < 30% total fat (< 10% saturated fat, 12–14% monounsaturated fat (MUFA), and 6–8% polyunsaturated fat (PUFA)), 15% protein, and a minimum of 55% carbohydrates. The Mediterranean diet comprised a minimum of 35% of calories as fat (22% MUFA fat, 6% PUFA fat, and < 10% saturated fat), 15% proteins, and a maximum of 50% carbohydrates [13]. In both diets, the cholesterol content was adjusted to < 300 mg/d.

The dietary assessment has been described recently [19]. Participants in both intervention groups received the same intensive dietary counseling. Nutritionists carried out individual interviews at baseline and every six months, and quarterly group education sessions were held with up to 20 participants per session and separate sessions for each group. Information on habitual dietary intake was gathered by registered dietitians at baseline and at each year of follow-up using a validated 137-item semi-quantitative FFQ [20, 21]. To complete the FFQ, patients reported their average intake of different foods and beverages over the previous 12 months. Consumption frequencies were registered in nine categories ranging from “never or less than one time per month” to “six or more times per day”. Energy and nutrient intake were calculated using the Spanish Food Composition Tables [22, 23]. Percentage of contribution of foods and nutrients to the mean daily energy intake was also calculated. A more detailed description of the dietary intervention is shown in Quintana-Navarro et al. (2019) [19]. To investigate the association between changes in the consumption of plant-based proteins at the expense of proteins from animal source and T2DM remission, study participants were classified into two groups according to the median of change (Δ) in plant protein intake (median = 0.13%), expressed as percentage of energy (% E) (changes produced between post- and pre-intervention, calculated as the value of percentage of energy from plant protein after one year of dietary intervention minus the value at baseline), regardless of the type of consumed diet. Thus, patients whose Δ in plant-based protein consumption was below the median were sorted into the group which decreased plant protein intake (n = 89), and patients whose Δ in plant-based protein consumption was above the median were sorted into the group which increased plant protein intake (n = 88). With the former classification, it was possible to select study participants whose dietary habits were more noticeably modified towards a shift in plant protein intake after one year of follow-up thanks to dietary intervention.

Following a 12 h fasting period, patients were admitted to the laboratory for anthropometric and biochemical test [body mass index (BMI), waist circumference, systolic blood pressure, diastolic blood pressure, HDL-cholesterol (c-HDL), LDL-cholesterol (c-LDL), triglycerides (TG), total cholesterol, highly sensitive C-reactive protein (hs-CRP), Alanine Aminotransferase (ALT), glucose, and HbA1c]. Smoking status, alcohol intake, and drug therapy were also registered for each participant.

Anthropometric parameters were measured by trained dietitians using calibrated scales (BF511 body composition analyzer/scale, OMROM, Japan) and a wall-mounted stadiometer (Seca 242, HealthCheck Systems, Brooklyn, NY). Waist circumference was measured midway between the lowest rib and the iliac crest. BMI was calculated as weight per square meter (kg/m2). Blood pressure was measured with a validated digital automated blood pressure monitor, and hypertension was defined as a systolic blood pressure ≥ 130 mmHg, diastolic blood pressure ≥ 85 mmHg, and/or current use of antihypertensive agents.

Venous blood samples were collected from the antecubital vein in Vacutainer ™ tubes containing EDTA or no anticoagulant. Serum parameters were measured by spectrophotometry using an Architect c-16000 analyser (Abbott^®, Chicago, IL, USA): hexokinase method for glucose and oxidation-peroxidation for c-HDL, total cholesterol, and TG. C-LDL was calculated using the Friedewald formula provided serum TG levels were < 400 mg/dL. Plasma levels of insulin were measured by chemiluminescent microparticle immunoassay using an i-2000 Abbott Architect^® analyser. The plasma concentrations of hs-CRP were determined by high-sensitivity ELISA (BioCheck, Inc., Foster City, CA, USA).

SPSS statistical software (IBM SPSS Statistics version 21.0) and R version 4.0.3 software (R Foundation for Statistical Computing, Vienna, Austria) [24] on the RStudio platform, and corrplot R package version 0.92 were used for statistical analysis of data. The normal distribution of variables was assessed using the Kolmogorov–Smirnov test. Data are represented as the mean ± SEM for continuous variables and as frequencies for categorical variables. P values ≤ 0.05 were considered statistically significant. The statistical differences in the metabolic variables between groups were evaluated by one-way ANOVA. Qualitative variables were compared using the Chi-square test. A repeated-measures ANOVA test was used to determine the statistical differences between variables at baseline and during the follow-up period. The post hoc statistical analysis was completed using Bonferroni's multiple comparison tests. Correlations between the Δ (1-year post-intervention minus baseline values) in energy, nutrients and food intake were calculated by means of a Spearman’s rank correlation.

Probability of T2DM remission was calculated using the Kaplan–Meier method of estimating the cumulative probability of an event in the groups of patients who had a Δ in plant protein (% E) intake above or below the median of the population after receiving dietary counselling (median = 0.13%). Time-dependent Cox regression models were used to identify significant factors associated with the time of incidence (full model was implemented with the following variables: sex, age, intervention group, baseline levels of c-HDL, TG, BMI, glucose, Hb1Ac, hs-CRP, insulin, and waist circumference, statin use, smoking status (never, former, or current smoker), alcohol intake, and familial history of diabetes).

Characteristics of the subjects classified according to the median of Δ in plant protein consumption (expressed as % energy intake) at baseline are shown in Table 1. No statistically significant differences were observed in any of the anthropometric or clinical parameters. The proportion of men and women were differentially distributed among the two groups of Δ in plant protein intake with greater percentage of men in those who increased the plant protein intake (p = 0.034). Regarding the differences in energy, nutrients and food consumed at the beginning of the study (before initiating the dietary intervention), we found that those who increased the plant protein intake during the first years of follow-up, consumed more fat, MUFA, and PUFA, and less vegetal protein, carbohydrates, fiber, vegetables, cereals and derivates and whole grains at the baseline of the study (all p < 0.05) (Table supplementary 1).

The probability of T2DM remission depending on the Δ of plant protein intake during dietary intervention was estimated using a Kaplan–Meier survival curve (Fig. 1). To that end, patients were classified according to median in Δ plant protein intake (first year of follow-up vs baseline). We observed in the table adjacent to survival curve that majority of T2DM remission occurred at first and second year of follow-up, and that the number of patients that achieved remission in the third year onwards diminished compared to previous years. We also found that patients who increased plant protein intake had higher probability of T2DM remission than those who decreased plant protein intake, after adjustment with a hazard ratio (HR) of 1.71 (1.05–2.77) (p = 0.031). Hazard ratios for T2DM remission according to the groups studied are presented in Table 2.

Participants belonging to the group of patients who increased plant protein intake significantly modified the amount of plant protein in their diet compared to baseline and maintained the changes for at least 3 years of follow-up (Fig. 2). Patients who decreased the plant protein intake modified the amount of plant protein in its diet compared to baseline only until the first year of follow-up, after that they maintained the amount of plant protein they consumed at baseline. However, the amount of plant protein intake by the group who increased their consumption was maintained higher than those consumed by the other group during the follow-up until the third year (Fig. 2).

Table 3 summarizes mean changes in energy, nutrients, and food intake, according to the data collected in the FFQs, as the values at first year of follow-up minus the baseline. In the two studied groups, modification of baseline dietary habits led to a significant reduction in total energy, cholesterol, and SFA (expressed as % of total fat), and increment in PUFA (%total fat), and fruit consumption. On the other hand, those who increased the plant protein intake had reduction in fat, mainly SFA (%E) and MUFA (%E) but without changes as expressed in %total fat, and animal protein consumption, and significant increment in plant protein, carbohydrates, fiber, vegetables, legumes, tree nuts, cereals, and whole grains intake. Furthermore, those patients who decreased the plant protein intake reduced the plant protein, carbohydrates, tree nuts and cereals intake with increment in animal protein consumption. A more detailed description of the changes in nutrients and food intake along the follow-up can be found in Quintana-Navarro et al. [19].

We evaluated changes in biochemical and anthropometrics parameters after the first year of follow-up in both groups of patients studied. We found that those patients who increased the plant protein consumption reduced the waist to height ratio (WHTR, a biomarker of ectopic fat deposit), and HbA1c, whilst no changes were found in those patients who decreased the plant protein intake. On the other hand, both groups of patients decreased the levels of ALT and TG, but with greater reduction in the group of patients who decreased plant protein consumption (Table 4).

To assess the interrelation between changes in dietary variables adjusted by energy, a Spearman correlation matrix was used (Fig. 3). Changes in plant protein intake were positively correlated with changes in the intake of carbohydrates, fiber, legumes, tree nuts, cereals and derivatives, and whole grains, and negatively correlated with changes in the intake of SFA, MUFA, and total fat, cholesterol, and animal protein.