*2.7. Barley*

Barley (Hordeum vulgare) is a cereal grain that is grown in temperate climates worldwide. It is one of the oldest cultivated cereals, originally in the Fertile Crescent area of the Middle East and Egypt. Barley is used commonly as animal fodder. Concerning human nutrition, two types of barley are commonly found: hulled barley, which requires a long cooking time and preventive soaking, and pearl barley, which undergoes a refining process (similar to whitening rice) to remove the outermost part. This can be used without prior soaking and cooking time is shorter. Barley is used for the preparation of soups and stews and also for cooking barley bread. From the coarse ground, coarse semolina is obtained, suitable for typical North African dishes similar to couscous. Roasted in the oven at temperatures around 170–180 ◦C, and very finely ground until obtaining a powder similar to flour, and freeze-dried, it is used to quickly prepare drinks by adding hot water or milk or used as a substitute for coffee. Roasted fine flours are also obtained from the roasting of barley and are used in the preparation of sweets or pastries. Barley grains are commonly made into malt as a source of fermentable material for beer and distilled beverages, like whisky.

## 2.7.1. Nutrients

A 100 g of barley provides 352 calories. Barley is made up of about 28% carbohydrates, 57% dietary fiber, 2% fat, and 20% protein. Barley is also a good source of B vitamins and minerals including copper, iron, magnesium, manganese, phosphorus, selenium, and zinc [21].

#### 2.7.2. Barley β-Glucan

B-glucan constitutes approximatively 75% of dry weight of endosperm cell walls, and arabinoxylan constitutes 25% [75]. The percentages of beta-glucan content in the barley grain vary according to the different polymorphisms of the genes that encode the corresponding synthase and endohydrolase enzymes [76]. Barley β-glucans also have cholesterol-lowering properties [77,78], however, lower than oats [79]. In addition, it is known that β-glucans from barley reduce post-prandial glycemic response with lowering blood glucose. This effect is due not because of the high viscosity of the β-glucans, but rather to the direct inhibition of the activities of glucose transporters and intestinal brush border enzymes [80,81]. More evidence has shown that β-glucans exert their beneficial effects on lipid and glucose metabolism and reduce CVD risk by the increase in colonic microbial population and activity, particularly favoring the increase in Lactobacillus over Bacteroidetes spp, yielding short-chain fatty acids as end products [48,82]. In animal models, these health benefits, on microbial gu<sup>t</sup> flora, are also associated with an increase in lifespan and better locomotor activity, muscle coordination, and balancing activity [83].

#### **3. Diet Pattern and Risk of Frailty and Mortality**

It is now known that fruits and vegetables, in addition to the intake of whole grains, monounsaturated and omega-3 fatty acids, and moderate amounts of alcohol are fundamental elements of a cardioprotective diet [84]. Furthermore, it is known that a prevalent consumption of fruit, vegetables, and WG, even in a dietary model different from traditional MD, is protective (i.e., associated with a reduced risk of frailty) [85].

Lo et al. [86] analyzed data from the Nutrition and Health Survey in Taiwan. They showed that elderly subjects in the higher tertile of the dietary pattern score (i.e., with a high consumption of fruit, nuts and seeds, tea, vegetables, WG, omega-3-rich deep-sea fish, and shellfish and milk as protein-rich foods) had a reduced risk (Odds Ratio, OR) of frailty (OR = 0.12, 95% CI 0.02–0.76, *p* = 0.019) or pre-frailty (OR = 0.40, 95% CI 0.19–0.83, *p* = 0.015).

Following the studies by Ancel Keys, MD has been proposed as a model of healthy eating, associated with a reduced risk of developing cardiovascular and metabolic diseases [5]. Subsequently, Trichopoulou et al. showed that high adhesion to MD was associated with a reduction in the risk of total mortality [6,87].

Subsequently, the PREDIMED study [8,9,88] demonstrated that high-risk CVD subjects who followed an MD pattern, in which monounsaturated and antioxidant fatty acids came from taking EVOO, or alternatively taking omega-3 fatty acids from nut consumption, had a reduced risk of acute myocardial infarction, stroke, or death from CVD (MD with EVOO: HR = 0.70, 95% CI: 0.53–0.91, *p* = 0.009; MD with nuts: HR = 0.70, 95% CI: 0.53–0.94, *p* = 0.02), but not of total mortality (MD with EVOO: HR = 0.81, 95% CI: 0.63–1.05, *p* = 0.11; MD with nuts: HR = 0.95, 95% CI: 0.73–1.23, *p* = 0.68).

A meta-analysis [10] that involved 1,574,299 subjects followed for a time period of 3–18 years found a significant direct association between higher adherence to MD, improved health status, and reduced mortality risk (Rate Risk, RR) (RR = 0.91, 95% CI 0.89–0.94; *p* < 0.0001), particularly in mortality due to CHD (RR = 0.91, 95% CI: 0.87–0.95, *p* < 0.0001) and cancer (RR = 0.94; 95% CI: 0.92–0.96; *p* < 0.0001).

Another meta-analysis by the same authors [11] also showed a significant association between higher MD adherence, improved health and quality of life, and reduced overall mortality (RR = 0.92, CI 95%: 0.90–0.94, *p* < 0.00001). In particular, the authors showed a significant reduction in mortality from CHD (RR = 0.90; 95% CI: 0.87–0.93; *p* < 0.00001) or from cancer (RR = 0.94; 95% CI: 0.92–0.96; *p* < 0.00001).

Kromhout et al. [89] confirmed the association between a higher adherence to a dietary model with the characteristics of MD with a reduction in CHD mortality (r = −0.91). The authors further highlighted the protective role in the diet of cereals (r = −0.52), vegetables (r = −0.52), and legumes (r = −0.62) as well as the intake of a moderate amount of alcohol (r = −0.54).

Subsequently, Zaslavsky et al. [90] analyzed a sample of 10,431 women aged 65– 84 years from the Women's Health Initiative Observational Study [91,92] with complete frailty according to Fried's criteria [93]. MD pattern adherence was assessed using the alternative MD (aMed) index [6,94], which considered the intake of fruit, vegetables, nuts, legumes, WG, fish, ratio of monounsaturated to saturated fat, red and processed meats, and alcohol. The authors further showed the association between a higher intake of vegetables, nuts, and WG with a significant reduction in mortality risk (HR = 0.91, 95% CI: 0.84–0.99, *p* = 0.02; HR = 0.87, 95% CI: 0.80–0.94, *p* < 0.001; HR = 0.83, 95% CI: 0.77–0.90, *p* < 0.001, respectively). The relative contribution of these components to the reduction of mortality risk, obtained by subtracting each component from the aMed Index, was respectively 21% (vegetables), 42% (nuts) and 57% (WG).

More recently, Campanella et al. [95] performed a survival analysis involving 4896 subjects from Castellana Grotte and Putignano (Apulia, Italy) included in the MICOL study [96] and in the NUTRIHEP study [97], respectively. The relative Mediterranean scoring system (rMED) [98] was used to measure adherence to MD. The rMED considers the intake of fruit (excluding fruit juices), vegetables (excluding potatoes), legumes, cereals, fresh fish, olive oil, meat and dairy products, and alcohol. The authors noted that higher MD adherence was directly correlated with longer lifespan. In particular, among subjects with greater adherence to MD at the baseline, the mean time to death was estimated to be postponed from 6.21 to 8.28 years compared to subjects with lower MD adherence.

The protective effect of the MD [99] are certainly due to the lipid-lowering effect, protection against oxidative stress, inflammation, and platelet aggregation, modification of hormones and growth factors involved in the pathogenesis of cancer, inhibition of nutrient sensing pathways by specific amino acid restriction, and gu<sup>t</sup> microbiota-mediated production of metabolites influencing metabolic health. Specifically, the moderate energy restriction provided by the high consumption of fiber-rich energy-poor plant foods and the specific restriction of sulfur compounds, branch-chain amino acids, and saturated fatty acids, characteristics of the MD, play a prominent role in mediating the beneficial effects on the health and longevity of this dietary model. In addition, the intestinal microbiome, which is actively involved in the processing of many plant foods rich in fiber as well as several vitamins and phytochemicals, plays a vital role in maintaining both metabolic and molecular health.

Hernaez et al. [100] reported the results of a study conducted on a subsample of 296 subjects at high cardiovascular risk, extracted from the cohort of the PREDIMED study [8,9]. The authors confirmed the beneficial effects of the intake of EVOO, nuts, legumes, WG, and fish. Mostly, they showed that increase for one year in the intake of these cardioprotective foods was linked to an improvement in HDL biological functions. In particular, increase in daily intake of 10 g of EVOO and 25 g of whole grain was associated with increment in cholesterol efflux capacity, in other words, the capacity of HDL to pick up cholesterol (+0.7%, *p* = 0.026; +0.6%, *p* = 0.017, respectively). Increase in daily intake of 30 g of nuts and 25 g of legumes and 25 g of fresh fish was linked to increment in the activity of paraoxonase-1, a key HDL-bound antioxidant enzyme (+12.2%, *p* = 0.049; +11.7%, *p* = 0.043; +3.9%, *p* = 0.030, respectively). Increase in legumes and fish consumption was also related to decreases in the activity of cholesteryl ester transfer protein, pro-atherogenic when excessively active (–4.8%, *p* = 0.028; –1.6%, *p* = 0.021, respectively).

The above evidence reaffirms a fundamental concept, namely, that it is not the single nutrient or the single antioxidant that is effective in reducing mortality, nor the risk of frailty, but the set of nutrients in the diet. Another key point is that diet is not intended as an effective therapy or as something to be taken for a defined time. In contrast, the diet should be understood as a diet to be practiced for life and in the context of a healthy lifestyle, as observed, for example, in populations following the traditional MD or the Okinawa diet.

All the above-mentioned studies are shown in Table 3.


**Table 3.** Diet pattern and risk of frailty, cardiovascular risk, and mortality.


#### **Table 3.** *Cont.*

#### **4. Whole Grains Intake, Cardiovascular Risk Factors, and Body Weight**

According to the HEALTHGRAIN Consortium definition [101], whole grain (WG) means "the intact, ground, cracked or flaked kernel after the removal of inedible parts such as the hull and husk. The principal anatomical components, as the starchy endosperm, germ, and bran, are present in the same relative proportions, as they exist in the intact kernel. Small losses of components, that is, less than 2% of the grain/10% of the bran, that occur through processing methods consistent with safety and quality are allowed".

intake of 25 g of fish.

Maras et al. [102] analyzed data from the Baltimore Longitudinal Study on Aging [103] and identified the main sources of WG as breakfast cereals (57.5%), multi-grain and whole wheat bread (16.5%), corn chips snack type (4.2%), popcorn (3.8%), and rye bread (3.6%).

Sette et al. [104] calculated whole grain intakes in an Italian sample of 2830 adults and older adults and of 440 children and adolescents from the INRAN-SCAI 2005–06 Study. The main source of total WG intake among adults and older adults were bread (46%), biscuits (20%), savory fine bakery products (15%), breakfast cereals (7%), and wheat and other cereals (6%).

Subsequently, Ruggiero et al. [105] calculated WG intakes in a different Italian sample of 2830 adults and older adults and of 440 children and adolescents from the Italian Nutrition & Health Survey (INHES) Study. In this study, the major food sources of WG among adults and older adults were bread (53.3%), biscuits (27.4%), pasta (13.1%), breakfast cereals (4.8%), and soups (1.3%). Figure 4 summarizes the different intake of whole grain among U.S. and Italian populations.

**Figure 4.** Intake of whole grain: U.S. vs. Italian population. Adapted from Ruggiero et al. [105].

There is now growing epidemiological evidence that WG exerts beneficial effects on human health, especially concerning the metabolic profile [106]. In particular, the consumption of WG has been associated with a reduction in cardiovascular risk factors such as postprandial insulin, blood lipid profile, and finally, the intestinal microbiome [107–109], as summarized in Figure 5.

Kelly et al. [110] conducted a systematic review to evaluate the effect of WG diets on total cardiovascular mortality, cardiovascular events, and cardiovascular risk factors (blood lipids, blood pressure). Nine randomized clinical trials (RCTs) published from 2008 to 2014 were included involving 1414 subjects. All included studies reported the effect of WG on major CVD risk factors such as body weight, blood lipids, and blood pressure. The authors did not find any study that clearly reported any effect of WG diets on total cardiovascular mortality or on cardiovascular events (i.e., total myocardial infarction, unstable angina, coronary artery bypass graft surgery, percutaneous transluminal coronary angioplasty, total stroke). Furthermore, the authors specified that all studies involved primary prevention populations and had an unclear or high risk of bias, and no studies had a duration of intervention greater than 16 weeks.

**Figure 5.** WG(whole grain)'s main mechanisms in reducing mortality and slowing aging.

Kirwan et al. [111] reported the results of a double-blind randomized case-control study conducted in a sample of 40 men and women aged <50 years, with no known history of CVD but who were overweight or frankly obese to compare the effects on body composition and metabolism of a diet containing WG versus an energy diet with refined grains. Each group followed the two diets for eight weeks; a washout period of 10 weeks was interposed between the two diets. The authors described an improvement in diastolic blood pressure (DBP) among overweight and obese adults that was >3 times greater at the end of the feeding period with the WG diet compared to the period of consumption of refined grains (−5.8 mm Hg, 95% CI: −7.7–−4.0 mm Hg; −1.6 mm Hg, 95% CI: −4.4– 1.3 mm Hg; *p* = 0.01, respectively). Regarding systolic blood pressure (SBP), the authors did not observe any significant differences in the magnitude of reduction between WG diet and refined-grain diet group (*p* = 0.80). In addition, the authors observed a lower decrease in plasma adiponectin levels after the whole-grain diet compared with the control diet (−0.1 μg/mL, 95% CI: −0.9–0.7; −1.4 μg/mL, 95% CI: −2.6–−0.3, *p* = 0.05, respectively). The preserved total circulating adiponectin concentrations were related to the concentration of circulating adiponectin (r = 0.35, *p* = 0.04).

Subsequently, Marventano et al. [112] performed a meta-analysis including 41 RCTs to evaluate the effect of WG-containing foods on glycemic control and insulin sensitivity in healthy individuals in the short-, medium-, and long-term by analyzing changes from baseline fasting blood glucose and insulin levels and insulin levels by measuring the area under the curve (iAUC). The authors showed that WG foods induced a significant reduction in the post-prandial values of the glucose iAUC and of insulin iAUC at 120 min by −29.71 mmol min/L and by −2.01 nmol min/L, respectively. They concluded by stating that in healthy subjects, the consumption of WG foods improved postprandial glycemia, insulin response as well as insulin and glucose homeostasis compared to the consumption of refined grain derivatives. The authors suggested, as a possible mechanism that could explain these effects of WG, both the slower digestion rate and the action produced by the microbiome in the large intestine through the fermentation of resistant fibers and starches, with the consequent production of short-chain fatty acids (SCFAs). These short-chain fatty acids, once in the liver, would improve glucose homeostasis and insulin sensitivity by increasing glucose oxidation, the reduction of fatty acid release, and by augmenting insulin clearance [113].

In addition, Musa-Veloso et al. [114] conducted a meta-analysis on 20 full-text articles with the aim of evaluating the effects induced by the consumption of WG wheat, WG rice, or WG rye on postprandial glycemia by comparing the glycemic values after the consumption of the same refined grains. They reported that a significant reduction in blood glucose AUC was observed only after consumption of WG rice compared to white rice ( −40.5 mmol/L × min; 95% C = −59.6– −21, 3; *p* < 0.001). In contrast, no significant change in blood glucose AUC was reported, either after the consumption of whole wheat, compared to white wheat, or after the consumption of whole-meal rye compared to refined rye ( −6.7 mmol/L × min, 95% CI = −25.1–11.7, *p* = 0.477; −5.5 mmol/L × min; 95% CI = −24.8–13.8; *p* = 0.576, respectively).

Kirø et al. [115] analyzed data from the Diet, Cancer, and Health cohort study [116]. They reported a reduction in the risk of type 2 diabetes of 11% for men and 7% for women, for each increase in consumption of WG (mainly rye) of 16 g/day (HR = 0.89, CI 95% = 0.87, 0.91; HR = 0.93, 95% CI = 0.91. 0.96, respectively). The highest quartile group of WG consumption had a reduction in the risk of type 2 diabetes of 34% for men and 22% for women (HR = 0.66, 95% CI: 0.60–0.72, *p* < 0.0001; HR = 0.78, 95% CI: 0.70–0.86, *p* < 0.0001, respectively). The authors also observed a reduced risk of 12% type 2 diabetes mellitus for men and 7% for women for every increase in the consumption of WG products (mainly rye bread) 50 g/day (HR = 0.88, 95% CI = 0.86–0.90; HR = 0.93, 95% CI = 0.90–0.96, respectively). In addition, a 37% reduction in the risk of type 2 diabetes mellitus for men and 20% for women was observed in the highest quartile group of consumption of WG products (HR = 0.63, CI 95%: 0.58–0.69, *p* < 0.0001; HR = 0.80, 95% CI: 0.72–0.88, *p* < 0.0001, respectively).

Maki et al. [117] performed a meta-regression analysis of cross-sectional data from 12 observational studies involving 136,834 subjects, and a meta-analysis of nine RCTs (WG versus controls) that involved 973 subjects to examine the relationship of WG intake with body weight; they also qualitatively reviewed six prospective cohort publications. The meta-regression analysis from cross-sectional studies indicated a significant inverse correlation between WG intake and body mass index (BMI) (r = −0.526, *p* = 0.0001). The review of the results of the qualitative analysis from the prospective cohort studies, with a follow-up period from five to 20 years, showed an inverse correlation between WG consumption and body weight change. Meta-analysis of RCTs, with a length from 12 to 16 weeks, did not show any significant difference in weight change (standardized mean difference = −0.049 Kg; 95% CI = −0.388–0.199; *p* = 0.698).

The discordant results are easily explained by the short duration of some of the studies. Twelve or even 16 weeks is too short a period to observe a significant reduction in cardiovascular events and mortality. The most significant results were observed in long-term prospective studies. This further reinforces the key concept that the intake of WGs should not be compared to taking a drug therapy, which in any case has effects in the short- and medium-term. The intake of WG in the diet must be contextualized, and the above evidence confirms it as part of a healthy diet.

All studies are summarized in Table 4.

**Table 4.** Whole grains(WG) intake, cardiovascular risk factors and body weight.



#### **Table 4.** *Cont.*

#### **5. Whole Grains Intake and Reduction of Mortality**

Accumulating evidence indicates that high intake of WG decreases the risks of mortality from all causes, CVD, and cancer in the general population.

Ma et al. [118] conducted a meta-analysis of prospective cohort studies involving 843,749 subjects and 101,282 deaths to quantify the association between WG intake and all-cause mortality. They showed that high WG intake was associated with a reduction of 18% for all-cause mortality risk (RR = 0.82, 95% CI = 0.78–0.87). In addition, the authors reported a 7% reduction in the risk of mortality from all causes for each increment of 16 g/day of WG consumption (RR = 0.93, 95% CI = 0.89 to 0.97).

A subsequent interesting meta-analysis by Zong et al. [119] involving 786,076 subjects with 97,867 total deaths confirmed the association between WG intake and reduction in mortality. The authors showed that a high WG intake was associated with a significative reduction in total mortality, CVD mortality, and cancer mortality (RR = 0.84, 95% CI = 0.80– 0.88, *p* < 0.001; RR = 0.82, 95% CI = 0.79–0.85, *p* < 0.001; RR = 0.88, 95% CI = 0.83–0.94, *p* < 0.001, respectively). The authors also estimated that each serving/day increase in WG intake was associated to a reduction of 7% for total mortality (RR = 0.93, 95% CI = 0.92– 0.94), 9% for CVD mortality (RR = 0.91, 95% CI = 0.90–0.93), and 5% for cancer mortality (RR = 0.95, 95% CI = 0.94–0.96).

The meta-analysis by Wei et al. [120] involving 816,599 subjects with 89,251 all-cause deaths, 23,280 CVD deaths, and 35,189 cancer deaths obtained similar results. The authors found that a high WG intake was associated with a signification reduction in risk (summary relative risk, SRR) for total mortality, CVD mortality, and cancer mortality (SRR = 0.87, 95% CI = 0.84–0.90; SRR = 0.81, 95% CI = 0.75–0.89; SRR = 0.89, 95% CI = 0.82–0.96, respectively). The dose-response analysis showed a reduction in overall mortality risk of 19% as well as a reduction of CVD mortality risk and cancer mortality risk of 26% and 9%, respectively (SRR = 0.81, 95% CI = 0.76–0.85; SRR = 0.74, 95% CI = 0.66–0.83; SRR = 0.91, 95% CI = 0.84–0.98), for every three servings/day increase in WG consumption.

Furthermore, a meta-analysis conducted by Aune et al. [36] confirmed the association between WG intake and reduction of mortality. The meta-analysis was performed on 45 prospective studies involving 245,012 to 705,253 participants with 7068 cases of coronary heart disease, 2337 cases of stroke, 26,243 cases of cardiovascular disease, 34,346 deaths from cancer, and 100,726 all cause deaths. In their study, the authors found that a high WG intake was associated with a signification reduction in CHD (RR = 0.79, 95% CI = 0.73–0.86), stroke (RR = 0.87, 95% CI = 0.72–1.05), and CVD (RR = 0.84, 95% CI = 0.80–0.87). The authors also reported a reduction in the risk of CHD, stroke, and CVD respectively of 19% (RR = 0.81, 95% CI = 0.75–0.87), 12% (RR = 0.88, 95% CI = 0.75–1.03), and 22% (RR = 0.78, 95% CI = 0.73–0.85) for each increase of 90 g/day (three servings/day) of the consumption of WG. In addition, the authors estimated that a high WG intake was associated with a signification reduction in mortality risk for CHD (RR = 0.65, 95% CI = 0.52–0.83), stroke (RR = 0.85, 95% CI = 0.64–1.13), CVD (RR = 0.81, 95% CI = 0.75–0.87), cancer (RR = 0.89, 95% CI = 0.82–0.96), and mortality for all-cause (RR = 0.82, 95% CI = 0.77–0.88). In particular, a reduced risk of coronary heart disease, stroke, CVD, cancer and overall mortality was observed by 19% (RR = 0.81, 95% CI = 0.74–0.89), 14% (RR = 0.86, 95% CI = 0.74–0.99), 29% (RR = 0.71, 95% CI = 0.61–0.82), 15% (RR = 0, 85, 95% CI = 0.80–0.91), and 17% (RR = 0.83, 95% CI = 0.77–0.90), respectively, for each increase in consumption of 90 g/day (three portions/day) of WG.

Another meta-analysis performed by Benisi-Kohansal et al. [121] involving 2,282,603 participants from 20 prospective cohort studies further confirmed the association between WG intake and reduction in mortality. Authors found that higher consumption of WG was associated with a reduction in overall mortality (RR = 0.87; 95% CI = 0.84–0.91), CVD mortality (RR = 0.84; 95% CI = 0.78–0.89), and cancer mortality (RR = 0.94; 95% CI = 0.91, 0.98). The authors also estimated a reduction of overall mortality, CVD, and cancer mortality of 17% (SRR = 0.83; 95% CI = 0.79–0.88), 25% (SRR = 0, 75; 95% CI = 0.68– 0.83) and 10% (SRR = 0.90; 95% CI = 0.83–0.98), respectively, for each additional three servings/day (90 g/day) of the consumption of WG.

A further confirmation of the beneficial effects of WG consumption on the reduction of overall mortality risk as well as CVD and cancer mortality risk was provided by Zhang et al. [122], which conducted a meta-analysis on 19 prospective cohort studies involving 1,041,692 subjects. The authors confirmed the relationship between a high intake of WG and the risk reduction of all-cause mortality (RR = 0.84; 95% CI = 0.81–0.88). The authors also confirmed that higher WG consumption was related with a reduction in mortality risk for both CVD (RR = 0.83; 95% CI = 0.79–0.86) and for cancer (RR = 0.94; 95% CI = 0.87–1.01). After performing the dose-response analysis, the author estimated that each serving/day intake of whole grain could reduce the overall mortality by 9% (RR = 0.91; 95% CI = 0.90– 0.93), CVD mortality by 14% (RR = 0.86; 95% CI = 0.83–0.89), and cancer mortality by 3% (RR = 0.97; 95% CI = 0.95–0.99).

This latest evidence on the reduction in total cardiovascular mortality and even of mortality from neoplastic disease obtained from long-term perspective studies, further confirms the key concept that the benefits of taking WG cannot be interpreted as the beneficial effect of an isolated nutrient, but must be contextualized as part of a healthy diet in a healthy lifestyle. All studies are reported in Table 5.



It is therefore evident that a high intake of WG, vegetables, fruits, nuts, and coffee is associated with a reduced risk of mortality whereas a high intake of red and processed meat is related to a higher mortality risk. High-quality diets such as MD are associated with a reduced risk of all-cause mortality [123].

Few studies relate the effects of a diet high in fibers on gastrointestinal function, glycemic or lipid metabolism, or on body weight [124]. Gopinath et al. [125] examined the relationship between total dietary carbohydrate intake, glycemic index (GI), glycemic load (GL), and fiber intake, with the state of successful aging [3,4] and with mortality risk, for a follow-up period of 10 years in a cohort of 1609 adults from The Blue Mountains Eye Study [126]. The authors showed that higher intake of total fiber, and particularly vegetable fibers and fruit fibers, was associated with greater odds of successful aging (OR = 1.79, 95% CI = 1.13–2.84; OR = 1.26, 95% CI = 0.83–1.91; OR = 1.81, 95% CI = 1.15–2.83, respectively).

Nevertheless, there have been a small number of studies examining the effect of WG on outcomes other than cardio-metabolic function or gastro-enteric function, or glycemic or lipid metabolism, or body weight. For example, there are very few studies analyzing the effect of WG in the diet on aging.

In this regard, Foscolou et al. [127] conducted an interesting study on a sample of 3349 elderly subjects from the ATTICA study and from the MEDIS study [36,128], both aimed to assess the association between WG intake with the diet and successful aging, and evaluated with the successful aging index (SAI) [129]. By applying the linear regression models, the authors observed a significant association between low vs. high intake of WG and SAI (b ± SE = −0.278 ± 0.091, *p* = 0.002). They did not observe any significant association between low vs. moderate WG intake and SAI (b ± SE = 0.010 ± 0.083, *p* = 0.901), and between moderate vs. high WG intake and SAI (b ± SE = −0.178 ± 0.095, *p* = 0.062).

#### **6. Reduction of Protein to Carbohydrates Ratio Influence Aging and Lifespan**

There is a consensus among gerontology researchers that dietary interventions can slow down aging, that is, prevent or delay the onset of numerous age-related chronic diseases [130–132]. The study of the relationship between nutrition and healthy aging has increasingly become a subject of grea<sup>t</sup> interest. Caloric restriction (CR), avoiding malnutrition, is the most studied dietary intervention known to extend life in many organisms [133–135]. To date, CR has been the focus of most non-genetic nutritional interventions. CR has been shown to improve several markers of health [136,137]. Fasting is the most extreme of the CR interventions, which requires the complete elimination of nutrients. Indeed, one of the more evaluated forms of fasting in both rodent and human studies is intermittent fasting [IF]. IF reduces body weight, body fat, and particularly abdominal fat, plasma insulin concentrations in both men and women, and reduces blood pressure, improving insulin sensitivity and lipid profile [138–140].

As reported by de Cabo and Mattson [141], cells exposed to fasting produce an adaptive stress response that leads to an increased expression of the antioxidant defenses, DNA repair, and control of protein quality, mitochondrial biogenesis, and autophagy, and downregulation of inflammation. In particular, the cell in intermittent fasting regimen showed a better and stronger resistance to a wide range of potentially harmful insults that involve metabolic, oxidative, ionic, traumatic, and proteotoxic stress. The protective effects of intermittent fasting are mediated by the stimulation of autophagy and mitophagy and by the inhibition of the mTOR (mammal target of rapamycin) protein synthesis pathway [142]. These responses allow cells to remove damaged proteins and mitochondria from oxidation and to recycle the molecular constituents not damaged by temporarily reducing the overall protein synthesis to conserve energy and molecular resources. In humans, intermittent fasting interventions induce health benefits than can largely be attributed to simply reducing caloric intake. These benefits are due to the loss of fat mass, and consequently to the decrease in fasting insulin levels and to the increase in insulin sensitivity, resulting in a decrease of insulin resistance, dyslipidemia, hypertension, and a pro-inflammatory state typical of advanced age. Nevertheless, the IF needs medical supervision as it may cause

serious adverse effects in patients with extremely low BMI or between the frail and elderly patients [132].

Further evidence has suggested that macronutrient balance, rather than simple calorie restriction, plays a more important role in extending lifespan [143–145]. That is, modulating protein and carbohydrate intake, rather than simplistically reducing the entire energy intake, may offer a more feasible nutritional intervention in humans [146]. That is, it has become clear that specific nutrients and nutritional balance (i.e., the result of interactions between nutrients) play an important role in the biology of aging.

The "Geometric Framework for Nutrition" (GNF) [147,148] is a method developed in nutritional ecology with the purpose of understanding the nutritional interactions of animals with their environments by explicitly distinguishing the roles of calories, individual nutrients, and nutrient balance. In this model, the nutritional requirements of an animal can be schematized in a two-dimensional or three-dimensional Cartesian space, called the nutrient space. The axes that define this space each represent a functionally important food component, for example, proteins, carbohydrates, and fats. The intake target (IT) represents the balance and quantity of functional nutrients for regulatory mechanisms (e.g., proteins and carbohydrates). The animal can reach the IT if appropriate foods are available. As shown in Figure 6, foods are represented by radials or nutritional tracks (T), which are projected into the space between nutrients according to angles determined by the ratio of the nutrients they contain. The animal can reach its target state by selecting Food 1, which is nutritionally balanced with respect to its target, or by mixing its intake with nutritionally complementary foods (Food 2 and Food 3). Therefore, when on the T1, the animal is unbalanced toward Food 2, that is, off course with respect to its target; however, it can ge<sup>t</sup> closer to the target by approaching Food 3, then passing into T2, and a further passage to Food 2 brings it closer to its nutrition target.

**Figure 6.** Dietary imbalance in nutritional geometry. Adapted from Raubenheimer et al. [148].

In this regard, basic research studies conducted on Drosophila have shown that longevity was maximal when the diet included a 1:16 ratio of proteins to carbohydrates, while reproductive capacity, measured in insects through egg production, was maximum with a ratio of proteins and carbohydrates between 1:2 and 1:4. This increased lifespan observed with a low-protein diet was attributed to a reduction in initial mortality and a delayed acceleration of age-dependent mortality [149,150]. Further studies conducted on male decorated crickets have confirmed that a low protein and high carbohydrate diet could induce higher immune functions and consequently lower mortality [151]. Similar results were observed in studies conducted on Gasterosteus aculeatus, or stickleback fish, where a significant increase in lifespan was observed in fish with a diet with a lower protein content than carbohydrates, as opposed to an increased reproductive capacity observed in a diet higher in protein than carbohydrates [152].

Evidence showed that these models of a low protein, high carbohydrate diet would induce a reduced TOR signaling [145,153]. In this regard, Senior et al. [154] examined data from the Solon-Biet study [145] to evaluate how the macronutrient content in the diet could influence life expectancy and mortality in a sample of mice. They showed that the mouse's self-selected diet, which was composed of 22% protein, 47% carbohydrate, and 31% fat, with a protein–carbohydrate ratio lower than one, was associated with a long-life expectancy, low mortality in early and middle age, and high mortality in old age. In contrast, a diet rich in proteins or fats relative to carbohydrates produces low life expectancy with high mortality rates across all age classes.

There are no studies on humans that have applied nutritional geometry. However, the grea<sup>t</sup> longevity of populations from Sardinia in Italy or from Okinawa in Japan is well known as is the generally low mortality of the populations of the Mediterranean basin who follow a traditional Mediterranean diet [6,7,16]. The explanation for this high life expectancy lies in traditional eating habits, both in the Mediterranean diet and in the Okinawan diet, which are rich in carbohydrates taken with cereals (wheat or rice) or its derivatives and with low protein content, with a protein/carbohydrate ratio for both diets of about 1:10 [155].

Regarding protein intake, Pedersen et al. [156] conducted an interesting review that aimed to assess the health effects of protein intake in healthy adults. The 64 papers that were included in the study were classified according to the grade of evidence as "convincing", "probable", "suggestive", or "inconclusive". The authors assessed as "suggestive" the evidence regarding the increased risk of all-cause mortality in relation to a low carbohydrates high protein (LCHP) diet, where total protein intake of at least 20–23% of total energy; they also assessed as "suggestive" the evidence concerning relations between vegetable protein intake and low risk of cardiovascular mortality.

With regard to the carbohydrates, it is known that molecules are essential for many cellular processes, mainly for the production of energy, after having been converted into glucose by the cells. Furthermore, high blood glucose levels are known to be responsible for the progression of chronic diseases such as diabetes mellitus. More importantly, glucose is one of the most studied nutrient molecules, influencing lifespan in various model organisms. For example, diets enriched with glucose reduce lifespan in Caenorhabditis elegans by inhibiting the insulin/IGF-1 (IIS) signaling pathway. Diets enriched with glucose act by inhibiting DAF-16/FOXO, HSF-1, and SKN-1/nuclear factor erythroid-related factor (NRF), which regulate the expression of several target genes in the IIS pathway. Treatments with high glucose content also produce negative effects of aging on human endothelial progenitor cells (EPCs) and fibroblasts. In these human cells, elevated glucose treatment accelerates various aging-related phenotypes through the activation of the p38 mitogenactivated protein kinase (MAPK). High-glucose treatments induce downregulation of sirtuins; this leads to a reduction in FOXO activity and accelerates cellular senescence. In these glucose-rich conditions, we observed in the EPC some cellular aging phenotypes such as increased levels of b-gal staining SA, reduced cell proliferation, irregular morphology, and increased levels of ROS [150].

Regarding the relation between dietary carbohydrate intake and mortality, Seidelmann [157] conducted a prospective cohort study aimed at investigating the association of carbohydrate intake with mortality and residual life span in a large cohort of adults from the risk of community atherosclerosis (ARIC), which involved 15,428 subjects with a 25-year follow-up. The authors also investigated whether the replacement of carbohydrates with animal or vegetable sources of fats and proteins changed the observed associations. As a benefit of the study, they combined their findings with data from seven studies from North America, Europe, Asia, and multinationals involving 432,179 participants to contextualize all findings in a meta-analysis. The author showed that an increased risk of mortality was

related to low carbohydrate consumption (low versus moderate carbohydrate consumption: HR = 1.20; 95% CI = 1.09–1.32; *p* < 0.0001) and high carbohydrate consumption (high versus moderate carbohydrate consumption: HR = 1.23; 95% CI = 1.11–1.36; *p* < 0.0001). After exploring the association between mortality and alternative source of fat and protein to carbohydrate intake, the authors found that increasing the protein and animal fat intake instead of carbohydrates was associated with a significantly increased mortality risk (HR = 1.18; 95% CI = 1.08–1.29; *p* < 0.0001). Alternatively, an increased intake of protein and vegetable fats instead of carbohydrates has been related to a significant reduction in mortality risk (HR = 0.82; 95% CI = 1.78–1.87; *p* < 0.0001). In conclusion, the authors stated that a diet with a low carbohydrate content, in which carbohydrates are replaced with fat and protein mainly of plant origin, might be associated with a higher life expectancy and a long-term approach could be considered to promote healthy aging. It would be simplistic, in fact, if we wanted to evaluate the effects of nutrition on aging and longevity, consider separately the intake of proteins or carbohydrates, or extrapolate from the context caloric intake total daily, for example, by simply reducing energy intake.

## **7. Discussion**

The application of the GNF method to study the effects of macronutrients and caloric intake on aging and lifespan has allowed us to understand that not the single nutrient but the interaction between macronutrients affects age-related health and lifespan. Animal model studies, which applied the GNF method, have shown that a low-protein, highcarbohydrate diet increases lifespan in many species. These studies were performed using ad libitum feeding regimes, which considered the eating behavior in a not strictly organized environment (i.e., less artificial), which produces more reliable results, if we want to translate them into human populations. Food intake is recorded so that diet content and calorie and macronutrient intake can be assessed [158]. All these studies have shown that longer lifespans are generated by diets low in protein and high in carbohydrates (low protein high carbohydrates, LPHC diet) where the optimal ratio of protein and carbohydrates is about 1:10, with the protein content of diet about 10% or less. All these studies have also shown that simple reduced caloric intake has no effect or negative effect on lifespan, which is clearly in contrast to many previous studies on CR.

Although CR overall has shown established benefits for health and aging, this model is not readily feasible in practice in both humans and animals that have free access to food. In contrast, alternative dietary models such as the LPHC diet, which allow ad libitum access to food, are more likely to be feasible as health interventions. Comparing the results of LPHC diets with those of caloric restriction, we can find similarities such as the reduction of insulin and inactivation of mTOR, and differences, the most interesting of those concerning mitochondrial biogenesis between cellular mechanisms concerning aging and life span [159]. Interestingly, LPHC diets are associated with reduced mitochondrial number and reduced expression of the master regulator of mitochondrial biogenesis, peroxisome proliferator-activated receptor gamma coactivator 1-alpha [PGC-1 α], unlike CR, where there is an increase in the number of mitochondria associated with a greater expression of PGC-1 α.

The concept of "mythormesis" could explain the paradox that both LPHC and CR diets increase lifespan but induce opposite effects on mitochondria. After postulating that low levels of oxidative stress induce the activation of systemic defense mechanisms beneficial for aging such as the activation of endogenous antioxidant enzymes, LPHC diets can increase the production of hydrogen peroxide sufficiently to generate hormetic benefits without producing mitochondrial damage.

It is now an irrefutable fact that a healthy lifestyle during a younger age, which includes consuming healthy foods in an amount appropriate to both health and physical activity, smoking cessation, and even taking moderate amounts of alcohol, prepares for successful aging. It is also an irrefutable fact that one of the main factors in increasing the average life span in the last two centuries has been the improvement in the nutritional status of the population. In contrast, a poor-quality diet is still the main risk factor of mortality, but above all, of disability in older age, even in developed and wealthy nations [160].

The most recent evidence has shown that diets that are rich in low glycemic index carbohydrates, combined with low amounts of proteins, are optimal to determine a longer and healthier life expectancy. In addition, diets that combine high amounts of refined, starch rich, high glycemic index carbohydrates with high contents of animal-derived proteins and fats determine a higher mortality rate, especially from CVD [161–164].

Dietary patterns that demonstrated greater adherence to diets that emphasized fruit and vegetables, WG rather than refined grains, low-fat dairy, lean meats, legumes, and nuts were inversely associated with mortality [165,166]. We already know that adherence to a diet pattern such as the traditional MD [6] was associated with a reduction in overall mortality, coronary heart disease, and cardiovascular disease. An overall high-quality diet that emphasizes a high consumption of polyunsaturated and monounsaturated fatty acids, raw vegetables, dairy, legumes, low-fat lean meats fat, fresh fish, bread (especially whole-grain bread), and wine in moderation, has been inversely associated with overall mortality, especially in the elderly [167,168]. In addition, a healthy diet of high quality can increase the number of years without disease and without disabilities [169]. Evidence also suggests that higher adherence to a dietary pattern that includes mainly legumes, fruit, vegetables, cereals, bread, olive oil, and dairy products, more occasionally meat, fish, and seafood, is associated with lower risk of becoming frail in old age [170]. In contrast, an eating style characterized by a high consumption of refined cereals has been associated with a greater risk of total mortality, especially mortality from major CVD [171].

Concerning protein intake, an established dietary bias is that older people need to obtain more protein with their diet, even though they are not malnourished. The main objective of this advice is to first prevent sarcopenia, then to maintain a good state of health, which allows for the prevention of malnutrition, improving wound healing and faster recovery from acute illness [167]. Instead, these recommendations are in contrast with the results of basic research on animal models and with the results of observational studies on population cohorts, which on the contrary have shown that a low-protein, high-carbohydrate diet (LPHC) can delay aging and extend lifespan [123,133,142,159,172].

Residents of the Japanese island of Okinawa and people living in the central-eastern mountainous area of the Italian island of Sardinia [173,174], although so distant, share a unique characteristic: both populations show one of the highest concentrations of centenarians in the world, whose ages have been carefully validated. Several factors contribute to the exceptional longevity of these populations. Among these are a moderate caloric intake that is never excessive, a high quality of food, constant physical activity, and genetic predisposition.

Regarding centenarians in Okinawa, their dietary energy intake comes from 85% carbohydrates and only 9% from proteins. [175]. Furthermore, the ratio of proteins to carbohydrates is extremely low [1:10], like what has been discovered to optimize lifespan in aging studies in animal models [159].

Concerning Sardinian centenarians [174], the consumption of sourdough bread, which is prepared from WG with a microbial yeas<sup>t</sup> containing lactobacilli called "mother yeast", with chemical and physical characteristics quite different from bread bought from the ovens, and a plant soup called "minestrone" containing fresh vegetables (onions, fennel, carrots, celery) and legumes (beans, broad beans, peas) is very widespread. Honey was generally used as a sweetener. Meat consumption does not exceed 2–4 portions per month. As for the consumption of dairy products, these people make extensive use of ricotta (whey cheese and dry curd), both goa<sup>t</sup> and sheep, rather than mature cheese, and a local fresh sour cheese called "casu axedu" in the local dialect, which is rich in lactobacilli.

Indeed, the consumption of sourdough bread is very widespread in the traditional MD in southern Italy. This type of bread can reduce blood glucose and postprandial insulin levels by 25%, thus being able to preserve the function of pancreatic insulin-secreting cells and prevent obesity and diabetes [176].

Other predisposing health promoting factors between both people from Sardinia and from Okinawa are physical activity, low stress levels, and strong community support. We have already stated [177] that the benefits of MD should not be simply attributed to the high content of fiber, antioxidants, and proteins of vegetable origin. Nevertheless, it should be reiterated that the benefits of MD should be considered as part of a cultural context where food, together with the convivial aspect, is part of a "Mediterranean" lifestyle [178,179].
