➢Caloric restriction
Another possible anti-ageing strategy, able to reduce the biological effects of NF-κB signaling pathway in ageing, is the notable caloric restriction (CR) [
75]. Restricting the intake of calories has been practiced as a method for increasing both the length and quality of life for over 500 years. Experimental work confirming the success of this approach in animals has accumulated over the last 100 years. CR may extend life by up to 50% in rodents, with progressively less impact the later in life it is started. This effect is matched by profound impacts on age-related diseases, including reduced risk of cancer, neurodegenerative disorders, autoimmune disease, CVD and T2DM [
75]. The disposable soma theory of ageing suggests that CR evolved as a somatic protection response to enable animals to survive periods of food shortage [
4]. The shutdown of reproductive function during CR is consistent with this suggestion, but other features of the phenomenon are less consistent with this theory. Some researchers have, indeed, proposed that in rodents it may be mostly an artifact of domestication. CR induces profound effects on animals at all levels from the transcriptome to whole animal physiology and behavior. Animals under CR lose weight which is disproportionately contributed to by white adipose tissue. Generally animals on CR change their activity patterns. Thus, they are more active prior to food delivery each day, but total activity may be unchanged or reduced [
75]. Considerable debate has occurred over the effects of CR on resting metabolic rate (RMR). Total RMR declines, but as body mass and body composition also change it is unclear whether metabolism at the tissue level also declines, is unchanged or even increases. Body temperature universally decreases. Hunger is increased and does not seem to decline even with very long term restriction. Circulating adipokines are reduced reflecting the reduction in white adipose tissue mass under CR [
75]. There is also a large reduction in circulating insulin and glucose levels. There are profound tissue level changes in metabolism with a generalized shift from carbohydrate to fat metabolism.
Four pathways have been implicated in mediating the CR effects. They are the insulin/IGF-1signaling pathway, the Sirtuin pathway, the adenosine monophosphate (AMP) activated protein kinase (AMPK) pathway and mTOR pathway [
75]. These different pathways may interact and all play important roles mediating different aspects of CR response. Exactly how they generate the health benefits remains open for debate. However, one of the major impact of CR is the reduction of oxidative stress [
76]. As described above, the major cellular source of ROS are the mitochondria. Isolated mitochondria from animals under CR show a reduced ROS production. In particular, CR results in an increase in the level and activation of adenine nucleotide translocase and uncoupling proteins able to reduce the mitochondrial membrane potential. This results in a decline in superoxide radical (O2) production and a less damage to the lipids in the mitochondrial membrane reduced ulteriorly by increases in the membrane lipid saturation [
76]. Increases in superoxide dismutase convert superoxide into hydrogen peroxide and increased levels of Se-dependent glutathione peroxidase and catalase convert this to water reducing the production of the toxic hydroxyl radical (OH). Lowered levels of OH diminish the oxidative damage to proteins and DNA, which is further ameliorated by enhanced levels of degradation and base excision repair respectively [
76]. Furthermore, CR induces mitochondrial biogenesis, as evidenced by changes in mtDNA levels, and protein levels [
77]. Such effects on mitochondrial biogenesis are consistent with the idea that there may be a tissue level increase in oxygen consumption under CR which is accommodated in the reduced overall energy budget by the reduced amount of metabolizing tissue.
In addition, CR increases the levels of a member of Sirtuin family (SIRT1 to SIRT 7), NAD + dependent deactylases involved in the regulation of the activity of many proteins, energy metabolism, cell survival and longevity [
78,
79]. In particular, CR increases the expression of SIRT1 in multiple tissues, even if this effect does not appear to be uniform in all tissues or across different studies [
80]. It has been demonstrated that SIRT1 interacts with p65/RelA protein and specifically cleaves the acetyl group form, the lysine-310 of p65 protein, involved in enhancing the trans-activation efficiency of NF-κB system [
81,
82]. Thus, SIRT1 is a potent inhibitor of NF-κB system.
An enhanced autophagy is also induced by CR via the inhibition of mTOR or the activation of AMPK pathway. This last is an evolutionary conserved sensor for disturbances in cellular energy balance and a major inductor of autophagy. Thus, CR acts directly or indirectly as inhibitor of NF-κB system [
75,
81,
82].
Based on these observations, it is possible to assume that CR has beneficial effects, i.e. the extension of the average and maximum life span and delaying the onset of age-associated changes. However, this has been proven only in animal models, such as yeast, worms, flies and some mammals (rats and mice), and some criticisms (as above suggested) lead to consider it as an artifact of domestication, particularly in rodents [
75,
83‐
85]. In higher mammals, CR delays many diseases associated with aging including cancer, diabetes, atherosclerosis, CVD and neurodegenerative diseases [
86,
87]. The incidence of these diseases increases with age and they contribute significantly to mortality. Therefore, CR could increase life span by increasing the body’s general state of health and providing a nonspecific, resistance to chronic diseases and metabolic derangements [
86,
87]. However, the ultimate question, how does CR effect the human body, was studied in a limited number of experiments [
88]. The study of CR effects on human longevity faces ethical and logistical challenges since the average life span is close to 80 years for the population in developed countries. Therefore, human studies are focused on measuring the CR-related changes that could slow the aging process and the progression of chronic diseases thus increasing life span. The most convincing evidence that CR could have a positive effect in humans was provided by experiments by Fontana and coworkers, by the Comprehensive Assessment of Long-Term Effects of Reducing Calorie Intake (CALERIE Phase 1), and by data obtained on the members of the Caloric Restriction Society [
89‐
93].
Fontana and coworkers [
89] assessed the effect of a 6-year long CR diet on risk factors for atherosclerosis in adult male and female adults (age range 35–82 years) and compared them to age-matched healthy individuals on typical American diets (control group). The total serum cholesterol level and low-density lipoprotein (LDL) cholesterol levels, the ratio of total cholesterol to high-density lipoprotein cholesterol (HDL), triglycerides, fasting glucose, fasting insulin, CRP, platelet-derived growth factor AB, and systolic and diastolic blood pressures were all markedly lower in the CR group. The HDL cholesterol was higher after CR. Medical records of individuals in the CR group indicated that, before they began CR, they had serum lipid-lipoprotein and blood pressure levels in the expected range for individuals on typical American diets, and similar to those of the comparison group. Thus, this study concluded that long-term CR can reduce the risk factors for atherosclerosis.
The effect of fat loss induced by either (a) a long-term 20% CR or (b) a 20% increased energy expenditure (IEE) by exercise on coronary heart disease (CHD) risk factors was detected in a one-year randomized, controlled trial on 48 non-obese male and female subjects. The CR or exercise induced reductions in body fat were quantitatively similar and were accompanied by similar reductions in most of the major CHD risk factors, including plasma LDL-cholesterol, total cholesterol/HDL ratio, and CRP concentrations. Thus, these data evidenced that long-term CR or IEE of the same magnitude lead to substantial and similar improvements in the major risk factors for CHD in normal-weight and overweight middle-aged adults [
90].
The effects of a 1-year, 20% CR regime or 20% IEE by exercise, on the oxidative damage of DNA and RNA, was evaluated by white blood cell and urine analyses in normal-to-overweight adults. Both interventions significantly reduced oxidative damage to both DNA and RNA in white blood cells compared to baseline. However, urinary levels of DNA and RNA oxidation products did not differ from baseline values following either 1-year intervention program. The conclusion of the study was that either CR or IEE by exercise reduce systemic oxidative stress which is reflected in a decreased DNA or RNA oxidative damage [
91].
CALERIE, a research program initiated by the National Institute on aging and involving three research centers, performed in the Phase 1 three pilot studies to determine whether long-term (6–12 months) effects of 20–25% CR in free-living, non-obese humans could be investigated and to evaluate the adaptive responses to CR. This randomized, controlled, clinical trial concluded that CR subjects had a lower body weight, a decreased whole body and visceral fat, a reduced activity energy expenditure, improved fasting insulin levels, improvements in cardiovascular disease markers (LDL, total cholesterol to HDL ratio, and CRP), and no change in bone density compared to controls [
88]. In the ongoing CALERIE Phase 2, the researchers are testing whether 2 years sustained 25% CR of
ad libitum energy intake results in beneficial effects, similar to those observed in animal studies [
92].
Members of the Caloric Restriction Society (CRS) restrict food intake with the expectation that this would delay the disease processes responsible for secondary aging and to slow the primary aging process. Compared to age-matched individuals eating typical American diets, CRS members (average age 50 ± 10 yr) had a lower body mass index, a reduced body fat, significantly lower values for total serum cholesterol, LDL cholesterol, total cholesterol/LDL, and higher HDL cholesterol. Also fasting plasma insulin and glucose values were significantly lower than in the age-matched control group. Left ventricular diastolic function in CRS members was similar to that of about 16 years younger individuals. Chronic inflammation was reduced by CR and this was reflected in significantly lower levels of plasma CRP and TNF-α [
88].
Aging is associated with a progressive reduction in heart-rate-variability (HRV)—a measure of declining autonomic function—and also a worse health outcome. The effect of a 30% CR on heart autonomic function was assessed by 24-hour monitoring of HRV in adults on self-imposed CR for 3 to 15 years and compared with an age-matched control eating a Western diet. The CR group had a significantly lower heart rate and significantly higher values for HRV. Also, HRV in the CR individuals was comparable to published norms for healthy individuals 20 years younger. The authors suggest that CR reset the balance between the sympathetic/parasympathetic modulation of heart frequency in favor of the parasympathetic drive thus increasing the circadian variability of heart rate [
93].
Thus, in humans CR could delay many diseases associated with aging including cancer, diabetes, atherosclerosis, cardiovascular disease, and neurodegenerative diseases. As an alternative to CR, several CR mimetics have been tested on animals and humans, as described below.