Reinterpreting Cardiorenal Protection of Renal Sodium–Glucose Cotransporter 2 Inhibitors via Cellular Life History Programming

Biological programs can be divided into growth, reproduction, and maintenance. The choice among these programs is mainly determined by the environment. A favorable environment promotes growth and reproduction, whereas a hostile one favors maintenance and survival (11,12). The final scope is to maximize reproduction. Switching toward maintenance and survival programs, as occurs in response to nutrient scarcity, psychosocial stress, and toxins, can threaten health status. As an example of this, low birth weight, as a proxy of in utero nutrient scarcity, not only predicts cardiovascular disease, but also obesity and type 2 diabetes (13). Furthermore, this condition may also reduce cortisol and hypothalamic–pituitary–adrenal axis response to stress in adulthood (14,15). Psychosocial stress itself may profoundly increase cardiovascular risk. In a population-based study with a follow-up of 27 years, exposure to stress was associated with a hazard ratio for any cardiovascular disease of 1.64, with stress-related disorders being more strongly associated with early-onset cardiovascular disease (16). Interestingly, in mouse models, chronic variable stress accelerates atherosclerosis by inducing inflammatory leucocytosis (17).

Stressful conditions are common among people with diabetes, and a strong association between glucose control and stress has been reported (18).

Hyperglycemia, and, consequently, diabetes, can be considered a toxic determinant able to interfere with the basic programs of cell evolution. The consequence of chronic exposure to hyperglycemia is that people affected by diabetes have shorter life expectancy as compared with those without (19,20). Moreover, fertility, as a proxy of reproduction, is reduced in subjects with diabetes. As it has been shown by the Type 1 Diabetes Genetics Consortium (T1DGC), subjects with type 1 diabetes have fewer children than their unaffected siblings (21).

In summary, the diabetic condition is paradigmatic of the response to a hostile metabolic environment with the activation of a maintenance program. The latter can be directed toward the defense program or the dormancy program, the former being characterized by energy consumption and anabolic metabolism, the latter by deferral in nonessential functions, energy conservation, and reliance on catabolic metabolism (22).

The defense program is characterized by activation of the immune response and anabolic metabolism. This program mainly activates intracellular pathways, which mediate cell proliferation of proto-oncogenes and transcription factors such as phosphoinositide 3-kinases (PI3K), mammalian target of rapamycin (mTOR), c-Myc, and mitogen-activated protein kinase (MAPK). The consequence of this activation is (stem) cell proliferation and activation of the immune system.

The clinical implication of switching toward a defense program is threefold. First, an increased level of white blood cells, an independent predictor of incident type 2 diabetes (23,24), as well as acute coronary syndrome, is frequently observed in individuals with obesity (25). Second, in obese subjects, especially those with visceral or ectopic adiposity, there is a condition of subclinical inflammation and immune activation, which is not just the biologic feature of an expanded adipose tissue, but an important process leading to the atherosclerotic lesion (26). In the atherosclerotic plaque, the concerted action of M1-polarized monocytes, T lymphocytes, B lymphocytes, mast cells, and dendritic cells supports inflammation, which is implicated in the pathogenesis of acute coronary syndromes. In the Canakinumab Anti-inflammatory Thrombosis Outcome Study (CANTOS), the use of canakinumab, a monoclonal antibody against interleukin-1β, decreased the rate of recurrent cardiovascular events by 15% in patients with previous myocardial infarction and elevated levels of C-reactive protein (27). Similarly, reduction of circulating levels of C-reactive protein with high-intensity statin decreased the incidence of major cardiovascular events by 44% (28). Third, activation of the immune system after an acute myocardial infarction triggers an increased inflammatory response, which further enhances the risk of a recurrent event, an effect mediated by stem cell activation and propagation of inflammatory leukocytes to the atherosclerotic plaques (29).

Activation of the immune system is an exquisite anabolic process. For example, in activated T cells, the PI3K–protein kinase B (Akt) axis promotes plasma membrane translocation of glucose transporter 1 (GLUT1) (30) and increases the activity of the glycolytic enzyme hexokinase so that glucose can fuel several intracellular metabolic pathways (31,32).

In summary, in the context of the defense program, glucose utilization in leukocytes is tightly linked to the systemic proinflammatory response. In addition, increased glycolysis in M1‐polarized macrophages within the atherosclerotic plaque allows amplification of the inflammatory response (33).

The dormancy program is an energy-preserving state with high resistance to environmental stressors. It has strong analogy with the situation of animal hibernation where fuel is stored, metabolic rate is suppressed, and insulin secretion is reduced (34). In humans, a switch toward the dormancy program occurs in the myocardium in response to chronic ischemia, where a persistently impaired function of viable cardiac muscle cells is observed in response to chronic starvation due to reduction of coronary blood flow (35). At variance with the defense mechanism, catabolism and stem cell quiescence prevail and the immune system is suppressed. Several pathways mediate this condition, the principal one being the activation of fatty acid oxidation with increased production of ketone bodies, increase of circulating levels of fibroblast growth factor (FGF) 21, and activation of Forkhead box protein (FOXO) and 5′ AMP-activated protein kinase (AMPK) (11).

A key element in the dormancy program is the role of the transcription factor and tumor-suppressor gene p53, which not only promotes cell cycle arrest, senescence, or apoptosis, but also plays a significant role in the development of metabolic diseases. For instance, in diet-induced insulin resistance, p53 represses the transcription of GLUT1 and GLUT4, downregulates glycolysis, and promotes gluconeogenesis (36). The activity of p53 is strictly related to autophagy, an evolutionarily conserved lysosomal degradation process, linked to the dormancy program. While autophagy may promote cancer by repressing p53, p53, in turn, activates autophagy-related genes, thus leading to neoplastic cell apoptosis (37).

Although there is still no evidence that SGLT2i can promote the dormancy program, we wish to note that achievement of such effect with antidiabetic therapy is feasible. Peroxisome proliferator–activated receptor (PPAR)-γ agonists used in the treatment of type 2 diabetes recapitulate some features of the dormancy program. Glitazones can stimulate lipid synthesis, counteract inflammation, favor adipose tissue browning, and improve insulin sensitivity. Moreover, p53 is a cofactor of the PPAR-γ coactivator-1 alpha (PGC-1α) in regulating cell cycle arrest, while PPAR-α itself can induce autophagy and apoptosis, thus protecting against immune activation in atherosclerosis (38). PPAR-α has been shown to be an important transcriptional activator of FGF21 expression, a potent regulator with physiological effects on glucose and lipid metabolism that also exerts important antiatherosclerotic and anti-inflammatory effects (39). Circulating levels of FGF21 are elevated in patients with obesity and type 2 diabetes, and FGF21 mRNA expression is twofold higher in the visceral fat of obese individuals as compared with healthy subjects (40). Nonetheless, FGF21 can exert beneficial effects, including weight loss and improved insulin sensitivity, in subjects with obesity or type 2 diabetes (41). Though Eriksson et al. (42) found that dapagliflozin reduced the levels of FGF21 in patients with type 2 diabetes and nonalcoholic fatty liver disease, other studies clearly show that SGLT2i increase both hepatic and plasma FGF21 (43,44). However, it should be emphasized that these last two reports were obtained in mice; therefore, their results should be interpreted with caution, in the light of species-related differences.

Interestingly, the response of FGF21 to SGLT2i appears significantly related to BMI (45).

These findings as well as those from the study by Eriksson et al. (42) performed in humans indicate a decrease rather than an increase in FGF21 levels. Furthermore, the rise in body weight over time was accompanied by an FGF21 elevation, implying that an increase in FGF21 levels may not be beneficial in general.

As mentioned, all responses activated by switching cell programming toward a dormancy program are typically occurring in hibernating animals. Under this condition, ketone bodies become key metabolic substrates favoring glucose sparing. Therefore, in the context of fuel selection, one of the most relevant adaptive mechanism in the dormancy program is the rise of 3-hydroxybutyrate (46).

If we consider the metabolic changes that follow treatment with SGLT2i, these are much reminiscent of the metabolic picture characteristic of the dormancy program. Therefore, we hypothesize that the beneficial cardioprotective effects of SGLT2i may be related to their ability to switch cell life programs from defense, as triggered by the metabolically toxic environment (i.e., diabetic hyperglycemia), to dormancy, thus lending additional benefit.

Type 2 diabetes and obesity are conditions characterized by chronic exposure to high-energy substrates and high plasma insulin conditions, disrupting the physiologic diurnal cycling between catabolic and anabolic phases. A corollary condition of this loss of energy fluctuations is the development of metabolic inflexibility (47,48). As a consequence, amino acid concentrations are consistently augmented, resulting in stimulation of mTOR, a central regulator of cell proliferation and of the defense program through regulation of GATOR1 and 2 complexes (49). mTOR also plays a crucial role in innate as well as adaptive immune responses (50). Moreover, mTOR signaling can be activated by a variety of immunological stimuli, including signaling through the Toll-like receptor family of receptors (51). The role of mTOR is also central in macrophage polarization to the proinflammatory M1 state, thus potentiating inflammation (52). As mentioned, the dormancy program foresees activation of several pathways including AMPK (11). Osataphan et al. (44) showed that the SGLT2i canagliflozin increased AMPK activity in obese mice independently of insulin or glucagon sensitivity or signaling. This SGLT2i-mediated activation of AMPK has been confirmed at the cellular level in mesangial (53) and myocardial cells (54). At variance with AMPK, activation of mTOR is essential in the defense mechanism and in mediating the immune response (55). In the work of Osataphan et al., canagliflozin significantly repressed mTOR. In another study, empagliflozin was shown to increase autophagic activities in renal tubular cells under high glucose environment with concomitant mTOR inhibition (56). No data are so far available on the effect of SGLT2i on FOXO.

Globally, these studies showed that the SGLT2i can cause modifications in different pathways involved in the switching of cellular reprogramming from defense to dormancy.

Data obtained in rodent models (43,57–59) and in humans (60,61) consistently indicate that SGLT2i blunt inflammation with a specific protective effect exerted in the kidney and the liver (62). This effect has been shown also in humans, where canagliflozin was able to reverse molecular processes related to inflammation by decreasing interleukin-1, interleukin-6, and tumor necrosis factor 1 receptor (63). In line with these effects, there is mounting evidence showing that SGLT2i can improve vascular stiffness (64,65) and endothelial function (66) and reduce microvascular injury (67), possibly as a result of their anti-inflammatory properties (68). SGLT2i have the ability to modify macrophage polarization from proinflammatory M1 to anti-inflammatory M2 and to stimulate whole-body energy expenditure via activation of AMPK and increased expression of the uncoupling protein 1 in brown and white adipose tissue (43,69). This is reminiscent of the decrease of the number of leukocytes occurring in European ground squirrels during hibernation (70). In conclusion, there is ample evidence that SGLT2i have the ability to decrease the cellular inflammatory responses; however, we acknowledge that the robust data on the potential anti-inflammatory actions of these agents in the human are yet to be obtained.

A feature of the dormancy program is the activation of a catabolic state. SGLT2i therapy is associated with an increase in ketone body concentrations (71,72) that occurs, at least according to some reports, mainly under conditions of impaired glucose regulation, i.e., a toxic environment. Stimulation of fatty acid oxidation is due to a switch in energy metabolism from carbohydrate to lipid sources (73). The glucose-lowering effect of SGLT2i results in a reduction of the circulating plasma insulin concentration paralleled by an increase of glucagon secretion. This metabolic adaptation becomes more apparent during interprandial and nocturnal fasting, resulting in the improvement, if not restoration, of the circadian alternation of anabolic (postprandial) and catabolic condition, recovery of metabolic flexibility, and liver–brain–adipose neurocircuitry (74).

The increased availability of ketone bodies has been suggested as a main mechanism of cellular protection, due to a highly favorable ratio between oxygen consumption and energy production (75). As mentioned previously, ketone bodies are key in the metabolic reprogramming during hibernation and play a fundamental role in the organ-specific switches to tissue-protective, dormant metabolic states (46). During hibernation, metabolic fluxes limit carbohydrate oxidation and increase free fatty acid availability and oxidation. The latter is more efficient as, on a per-gram basis, it provides more than twice as much energy as carbohydrate oxidation (76). These metabolic differences are similar to the ones promoted by SGLT2i. Ketone bodies may also link SGLT2i to their potential anti-inflammatory action. High ketone body levels, as occurs for diabetic ketoacidosis, can trigger a proinflammatory condition, whereas at levels commonly reached during SGLT2i treatment, they can repress inflammation (77,78).

Obata et al. (79) have shown that the SGLT2i tofogliflozin increased the phosphorylation of hormone-sensitive lipase and adipose triglyceride lipase protein levels in white adipose tissue, with concomitant increase of the expressions of genes related to β-oxidation, such as CPT1α, in the liver. It is not readily intuitive to reconcile the increased lipid oxidation triggered by SGLT2i and their positive cardiovascular action, as lipid oxidation has been regarded as a main source of oxidative stress paralleled by increased atherogenic oxidized lipoproteins (80,81). However, the increase in lipid oxidation accompanying SGLT2i therapy is similar to that observed with low-intensity endurance exercise or daily physical exercise, which facilitate weight loss, increase lean body mass, and ameliorate all traits of the metabolic syndrome (82,83).

On a more chronic basis, the increased fat oxidation associated with SGLT2i therapy may prevent the impaired oxidation of these substrates occurring with aging (84).

Finally, increased lipid oxidation is the main source of the energy necessary for M2 macrophage polarization that confers anti-inflammatory properties. On the other hand, inhibition of fatty acid oxidation impairs polarization to M2 and, consequently, reduces their anti-inflammatory response (85).

In summary, SGLT2i can offset the toxic insult of hyperglycemia in a direct manner by increasing elimination of glucose excess through the urine and in an indirect manner by modifying the metabolic milieu of the cell, switching from a negative, defense program to a positive, dormancy one. Then, this set of actions can play a positive effect in the biology of the cellular phenotype of the arterial wall, thus preventing atherosclerotic process (86).

The role of hormones is of paramount importance for the response to a toxic environment and hence for the maintenance of cellular homeostasis. Hormones play a fundamental role in growth and reproduction, with the GH–IGF axis and sex hormones promoting anabolism and maintenance and glucocorticoids being the main activators of catabolism (11).

Therapy with SGLT2i is associated with increased plasma glucagon concentrations, which, at the organismal level, promote maintenance and dormancy. Of interest, the dormancy program activated at the time of hibernation of the golden-mantled ground squirrel is characterized by a reduction in the circulating levels of insulin, pancreatic polypeptide, and somatostatin but not of glucagon, suggesting an important role for this hormone in the dormancy/hibernation programs (87). Similar findings have been observed in the seasonal adaptation of the little brown bat (88). Unfortunately, no data are available on direct effects of SGLT2i on GH–IGF axis, sex hormones, and glucocorticoids. However, it was shown that canagliflozin can attenuate proinflammatory gene expression in the hypothalamus, an integration sensor of environmental quality and glucose metabolism (89,90).

We have screened the available literature to identify arguments supporting the hypothesis that, in the presence of pathologic elevation of plasma glucose levels, glycosuria develops as the clinical expression of glucose concentrations above the renal threshold, and, teleologically, as a way to remove cellular (glucose) toxins. SGLT2i increases glucosuria by lowering the renal glucose threshold: this effect positively modifies both hemodynamic and metabolic milieu, through which cardiovascular protection is conferred.

Based on experimental data, largely obtained in animal models, we now offer the hypothesis that these effects are, at least in part, the result of cellular life history programming induced by SGLT2i, i.e., deviation of cellular life trajectory in response to the toxic environment, to a dormancy rather than a defense program (Fig. 1). Although we acknowledge that most of the data presented herein are largely derived from animal studies, these modifications remain highly reminiscent of those occurring in hibernating animals as well as those in the hibernation-like state observed in chronically ischemic myocardium and in the brain after stroke (91), all paradigmatic conditions of a dormancy program.

A consistently emerging effect of SGLT2i is represented by kidney protection, which is believed to be accounted for largely by restoration of the tubular-glomerular feedback and constriction of the afferent arteriole, with reduction of intraglomerular pressure. Of interest, in hibernating animals, even in the presence of perfectly preserved glomerular structure, a significant reduction in renal blood flow and glomerular filtration rate develops (92). Finally, in keeping with our hypothesis also is the potential oxygen sparing effect (93).

An important point is whether this purported effect of SGLT2i on cellular reprogramming may occur with other non–insulin dependent reductions in plasma glucose concentration such as exercise or very-low calorie diet. Low-intensity endurance exercise or daily physical exercise may induce cellular reprogramming by increasing lipid oxidation (82). Very-low calorie diet reduces the proinflammatory state, i.e., defense program, in right ventricular function in healthy men (94) and overall in obese subjects (95). Recently, Jordan et al. (96) have shown that caloric restriction not only activates AMPK and PPAR-α but also reduces monocyte polarization, a paramount condition of switching from a defense to a dormancy program. However, the effects on cellular reprogramming are probably not only related to the scarcity of caloric intake, per se, but also to the nutritional composition of the diet (97).

In conclusion, we notice a remarkable resemblance of the metabolic changes occurring in subjects with diabetes in response to treatment with SGLT2i and conditions dictated by activation of the cell dormancy program and speculate it is through such a switch that SGLT2i may provide cardiorenal protection. Dedicated studies will be needed to test this hypothesis experimentally and gather further support from human studies.

Funding. This work was funded by Università degli Studi di Padova.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.