Healthy human kidneys filter ~1.6 kg of salt and ~0.16 kg of glucose together with ~180 l of water per day; however, they only excrete 1–2 l of water while the rest is reabsorbed. The kidneys excrete surplus dietary salt (approximately 0.003–0.020 kg per day, depending on diet), but, under normal circumstances, reabsorb the glucose, rendering the urine nearly glucose- free1. Tubular reabsorption of filtered solutes, including glucose, Na+, K+ and Cl−, is a hallmark of body water conservation. Inhibition of Na+, K+ and Cl− reabsorption in the thick ascending loop of Henle (with loop diuretics), blockade of Na+ and Cl− reabsorption in the distal tubule (with thiazide diuretics), or inhibition of Na+ reabsorption more distally in the nephron (by blockade of epithelial Na+ channels or mineralocorticoid receptors) increases solute excretion in the urine2,3. The osmotic force resulting from the high excretion of solute particles increases urine volume by osmotic diuresis, which underlies the efficacy of

to be true and supported their therapeutic use in patients with type 2 diabetes mellitus (T2DM). Increased excretion of glucose reduced Hb1Ac levels by 0.5–1%4, led to a negative caloric balance with a sustainable reduction in body weight of 2–3 kg4, exploited glycogen reservoirs and increased gluconeogenesis5,6, and improved insulin sensitivity7,8. Also as expected, the osmotic–diuretic effect of increased Na+ and glucose excretion increased urine volume, resulting in a measurable reduction in extracellular volume9 and lowering of blood pressure by 3–6 mmHg4.

However, some effects of these agents were unexpected, such as the fact that the increases in urine volume during SGLT2 inhibition were transient10–12 despite the persistent glucosuria. This finding suggests that osmotic diuresis — and thereby water loss — is limited with long- term SGLT2 inhibition, despite the continued excretion of glucose solutes.

The big surprise, however, came with the results of large double- blind placebo- controlled randomized trials, which showed that the use of SGLT2 inhibitors in patients with T2DM reduced the progression of kidney disease by up to 40% and reduced the risk of hospitalization for heart failure by 30–40% within only a few months of therapy initiation13–16. Subsequent clinical trials and secondary data analyses that focused on patients with various degrees of kidney impairment confirmed the nephroprotective effects of these drugs16–18, and additional reductions in the risk of cardiovascular mortality and death from all causes were reported in a population with established cardiovascular disease13. Although some heterogeneity exists in terms of the reported effects of SGLT2 inhibitors on major cardiovascular outcomes, a meta- analysis found that SGLT2 inhibitors reduced the risk of a composite of cardiovascular death or hospitalization for heart failure by 23% and the risk of progression of kidney failure by 45%19. Of note, no other intervention — either pharmacological or lifestyle — that has achieved a reduction in blood pressure of 3–6 mmHg, a reduction in Hb1Ac level of 0.5–1%, or a reduction in body weight of 2–3 kg has resulted in such rapid improvements in cardiovascular and renal outcomes20–24.

diuretic drugs. Clinicians have used this osmotic–diuretic principle for decades to acutely reduce body water content in patients with fluid overload.

In the past few years, therapeutic inhibition of an additional tubular osmolyte transport system has emerged, with the development of renal sodium–glucose co- transporter 2 (SGLT2) inhibitors. These agents were originally developed as a new class of antidiabetic drugs under the premise that successful SGLT2 inhibition would significantly reduce glucose reabsorption by the tubules and thereby increase urinary glucose loss. This increase in glucose excretion was predicted to reduce daily calorie balance, promote the use of glycogen reservoirs in the liver and muscle, and improve blood glucose levels. As Na+ and glucose are co- transported in the proximal tubule, SGLT2 inhibitors would also inhibit Na+ reabsorption and thereby induce osmotic diuresis. Early clinical studies of SGLT2 inhibitors proved these hypotheses

Organ protection by SGLT2 inhibitors: role of metabolic energy and water conservationAdriana Marton , Tatsuroh Kaneko , Jean- Paul Kovalik , Atsutaka Yasui , Akira Nishiyama , Kento Kitada and Jens Titze

Abstract | Therapeutic inhibition of the sodium–glucose co- transporter 2 (SGLT2) leads to substantial loss of energy (in the form of glucose) and additional solutes (in the form of Na+ and its accompanying anions) in urine. However, despite the continuously elevated solute excretion, long- term osmotic diuresis does not occur in humans with SGLT2 inhibition. Rather, patients on SGLT2 inhibitor therapy adjust to the reduction in energy availability and conserve water. The metabolic adaptations that are induced by SGLT2 inhibition are similar to those observed in aestivation — an evolutionarily conserved survival strategy that enables physio logical adaptation to energy and water shortage. Aestivators exploit amino acids from muscle to produce glucose and fatty acid fuels. This endogenous energy supply chain is coupled with nitrogen transfer for organic osmolyte production, which allows parallel water conservation. Moreover, this process is often accompanied by a reduction in metabolic rate. By comparing aestivation metabolism with the fuel switches that occur during therapeutic SGLT2 inhibition, we suggest that SGLT2 inhibitors induce aestivation- like metabolic patterns, which may contribute to the improvements in cardiac and renal function observed with this class of therapeutics.

PERSPECTIVES

Nature reviews | Nephrology

Several mechanistic concepts have been proposed to explain the cardiorenal protective effects of SGLT2 inhibitors. One prominent hypothesis for the renal protective effects of SGLT2 inhibitors proposes that increased delivery of Na+ to the macula densa as a consequence of inhibited Na+ reabsorption activates tubuloglomerular feedback mechanisms to induce adenosine- mediated constriction of the afferent arteriole and reduce glomerular hyperfiltration, similar to the effects of angiotensin- converting enzyme (ACE) inhibitors and angiotensin- receptor blockers25–27. Alternatively, the cardioprotective effects have been suggested to be driven by compensatory changes in fuel consumption and myocardial energetics as a consequence of chronic urinary glucose loss, for example, through the preferential use of ketone bodies28–31, which produce ATP in a more oxygen- efficient manner than fatty acids, translating into a more energy- efficient cardiac workload. We suggest that it is not possible to separate the body’s response to therapeutic SGLT2 inhibition into its effects on ‘salt- and- water’ and ‘energy homeostasis’ and to study each category independently, because fuel production from endogenous protein stores is physiologically coupled with organic osmolyte production for body water conservation via a fundamental adaptation principle.

In this Perspective article, we describe how this evolutionarily conserved mechanism, termed ‘aestivation’, might underlie both the cardiovascular and renal protective effects of SGLT2 inhibitors. We summarize the complex interplay between renal and hepatic metabolic energy and water conservation systems that have evolved over the past 350 million years32 and explain how the process of fuel production from endogenous protein stores inevitably improves the organism’s ability to synthesize organic osmolytes and successfully conserve water. We then describe the specific metabolic adjustments that suggest that increased excretion of glucose and Na+ achieved by SGLT2 inhibition triggers an aestivation- like body water- conserving mechanism, and how these metabolic adjustments probably contribute to the favourable cardiovascular and renal outcomes of this class of therapeutics.

AestivationThe word ‘aestivation’ derives from the Latin word for ‘summer’ (aestas) or ‘heat’ (aestus) and describes a series of evolutionarily conserved metabolic switches that allow organisms to survive arid or hot conditions

with restricted water availability33–35. This physiological principle enabled the evolutionary transition of organisms from aquatic environments to arid land by allowing them to conserve body water, and has been extensively studied in lungfish and amphibians. Although the metabolic switches that occur during aestivation are known to be an integrative response for energy and water conservation in biology, they have not been considered in humans. Detailed descriptions of the molecular regulatory mechanisms of aestivation can be found elsewhere36–38. Here we focus on the metabolic adaptations that are key to the water conservation and maintenance of organ viability under conditions of dehydration stress.

Biological principle and consequences. All organisms on Earth are driven to develop, grow and reproduce. The primary environmental inputs needed to achieve these goals are water, nutrients and energy38. Restriction of any one of these primary inputs activates self- preservation strategies to avoid death. These mechanisms are probably best exemplified by animals known as aestivators. During periods of draught, aestivators reprioritize energy expenditure by suppressing the metabolic demands of sustained growth and reproduction and investing more energy into osmolyte production to conserve water and support the functions of vital organs39 (Fig. 1).

Many of the metabolic changes that occur in aestivation increase the body’s ability to conserve water, as demonstrated by studies in lungfish, frogs, toads and land snails38. Frogs and toads that enter brackish water with higher salinity than their usual environment are exposed to a hyperosmotic stress that results in the movement of water out of their body, predisposing them to dehydration40. In an effort to resist body water loss, aestivators need to produce and accumulate high levels of osmolytes in their bodies — the most studied of which is urea. Under conditions of dehydration, aestivators reduce renal and dermal excretion of urea and in parallel promote urea synthesis, thereby increasing urea levels in tissues and plasma41. In addition, they accumulate large amounts of Na+, Cl− and amino acid derivatives such as methylated glycine products (also known as organic osmolytes) in their tissues, which together with the high levels of urea counterbalance the high environmental osmotic pressure to maintain body hydration in a hostile ‘dehydrating’ environment40,42–58.

The metabolic adjustments that promote solute- driven water accumulation in the internal environment are, however,

energy- intensive. Water conservation in response to exposure to environmental hyperosmotic stress increases the metabolic rate of African clawed frogs by 80%59. The high metabolic rate required to achieve osmolyte- driven water conservation is accompanied by increased oxidative phosphorylation of fuels59, which provides the ATP necessary for the increased energy requirements (Fig. 1). However, as the fuels necessary to produce ATP by oxidative phosphorylation are not always readily available, additional compensatory processes have evolved. For example, during their air- breathing, terrestrial stage, lungfish bury themselves in mud and aestivate inside a cocoon, so they are unable to find food and fresh water60–62. The energy- intensive process of water conservation in this instance therefore initiates in a situation of fuel shortage63 — a problem that is solved by reducing ‘unnecessary’ energy expenditure in all organs to a minimum (Fig. 1). The resulting stupor and hypometabolic state does not support cell expansion and growth, but instead increases the functional lifespan of key survival organs. For example, the liver redistributes energy consumption to prioritize organic osmolyte production for water conservation, the heart reduces its energy expenditure and switches to the use of stored fuels as metabolic substrates to maintain blood circulation, and the kidneys use predominantly urea osmolytes for water reabsorption, which is the most energy- efficient way to concentrate the urine.

Thus, successful water conservation under conditions of dehydration stress require either an increase in fuel intake to provide the necessary energy for water conservation and sustained growth59, or a switch to a ‘survival metabolic’ or ‘aestivating’ state, which requires careful exploitation of endogenous fuels and a reduction in energy expenditure in case the energy needs cannot be satisfied by the available nutrients64,65 (Fig. 1). We propose that therapeutic interventions that stimulate similar metabolic changes and promote water conservation in conjunction with reduced energy expenditure might similarly increase the functional lifespan of the liver, heart and kidneys, with favourable effects on overall health.

Metabolic aestivation patterns in mammals. Mammals are considered to have developed such efficient salt and water- conserving mechanisms in the kidney that additional extrarenal osmolyte production is thought not to be necessary to maintain body

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osmolyte and water homeostasis32. However, studies in humans and mice have shown that, similar to amphibians66,67, mammals can also increase urea accumulation by reducing renal urea excretion and increasing de novo hepatic urea production to stabilize body fluid content when salt intake is high68,69.

In the mammalian kidney, urea has an essential role in the urine- concentrating mechanism70–74, which is fundamental for regulating body water excretion independently of changes in sodium excretion75,76. In the inner medulla — a major site of antidiuresis — the urea transporters UT- A1 and UT- A3 actively reabsorb urea to create the osmotic gradient

necessary for water reabsorption. In order to prevent body water loss during high salt intake, the osmotic force of the medullary interstitium needs to override the osmotic–diuretic force of tubular Na+ and Cl− solutes that are excreted. This natriuretic–ureotelic component of the water- conserving process is mediated by enhanced activity of UT- A1, which significantly increases urea reabsorption in the inner medulla resulting in urea- driven water reabsorption (Fig. 2). Therefore, enhanced urea reabsorption within the urine- concentrating mechanism couples the renal elimination of surplus dietary salt with water conservation, enabling urea- driven water reabsorption

to counterbalance the otherwise osmotic–diuretic effect of increased solute excretion into the urine.

In addition, mice with high salt intake, and therefore high urinary Na+ and Cl− solute excretion, also produce urea in their liver and in their skeletal muscle (Fig. 2). Whether this stimulation of urea production happens at the same time as UT- A1 activation, or later owing to an increased demand for urea in the inner medulla, is not yet known. However, it is clear that like amphibians under conditions of dehydration stress77,78, mice invest energy into urea production to activate and support the water- conserving process.

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Fig. 1 | levels of physiological adaptation to arid environments. All organisms strive to grow and reproduce. Growth is an anabolic state that is characterized by increased cell and energy turnover, which requires suffi-cient fuels and water supply (baseline). In the context of dehydration stress, which can occur as a result of water loss across biological barriers (for example, skin) in aquatic environments with high solute concentrations (brackish water), in the heat of arid environments, or with osmotic diuresis as a result of excess salt intake, additional organic osmolyte production is necessary to maintain a constant body fluid content. This water- conserving process is energy- intense. Thus, to sustain growth, an increase in fuel intake is necessary (compensatory energy loading). Increased nitrogen utilization for the production of organic osmolytes such as urea will be observed, which requires a fuel switch in favour of protein catabolism. If the need to increase energy intake cannot be satisfied during dehydration stress, the organism will rapidly exploit endogenous energy and nitrogen reservoirs, and muscle mass loss will occur (use of stored fuels). Life on Earth has evolved under environmental conditions under which dehydration stress typically occurred in combination with reduced availability of fuels and

water. These hostile environments required activation of adaptive aestiva-tion metabolism as a self- preservation strategy to avoid extinction. This physiological survival response requires: water conservation in the biolog-ical barriers kidney, skin, gut and lung; efficient use of stored nitrogen- rich fuels; and global suppression of metabolic rate with reprioritization of energy use, thereby combining maintenance of vital organ function with sustainable exploitation of endogenous fuels. In contrast to organisms that can survive large variations in body temperature (poikilothermic organ-isms), homeothermic organisms need to constantly burn fuels to produce the thermic energy necessary to keep their body temperature constant. Amphibians and fish therefore can suppress their metabolic rate to greater extremes than mammals. In extreme aestivation states, the organism goes into partial metabolic arrest, coupled with various cell- preservation strat-egies that prolong functional cellular lifespan until the water and fuel nec-essary for the growth phase become available. We propose that glucose loss into the urine with SGLT2 inhibitor therapy induces a combined energy and dehydration stress that triggers evolutionarily preserved aestivation- like adaptation responses.

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As long as food is freely available, mice on a high- salt diet increase their calorie intake by 20–30%, which provides the surplus fuel necessary to sustain both normal growth and energy- intensive water- conserving mechanisms. However, if the additional energy necessary for successful water conservation is not available in the diet, mice fed a high- salt diet enter a catabolic state in

which the energy and nitrogen necessary for urea osmolyte production are taken from muscle stores by breaking down muscle protein, leading to muscle wasting68 (Fig. 1). The available data suggest that mammals have preserved and fine- tuned some of the water- conserving mechanisms used by aestivators as part of their adaptation to life on land, and use concerted renal and

extrarenal actions to limit water loss in response to a high urine solute load. A 2020 study demonstrated that similar protein catabolism- dependent water- conserving mechanisms occur under real- life conditions in humans with high Na+ solute excretion in the urine79.

We propose that SGLT2 inhibitors, which markedly increase the excretion of Na+ and glucose solutes in urine, similarly induce aestivation- like water- conserving responses to limit Na+ and glucose- driven osmotic diuresis. In response, energy expenditure in the liver, heart and kidney may be reduced to compensate for the loss of glucose solutes and fuels into the urine.

SGLT2 inhibition and water conservationA negative energy balance occurs not only because of reduced energy intake or increased energy loss, but also in response to increased energy expenditure during states of increased osmotic diuresis68. SGLT2 inhibition combines energy loss into the urine with an initial increase in urine volume due to glucose- driven osmotic diuresis. Here, we describe the metabolic and physiological adaptations that counterbalance the osmotic–diuretic effect of sustainably increased glucosuria with SGLT2 inhibition. We note in this context that any state of compensatory de novo glucose production (that is, gluconeogenesis) will couple endogenous fuel production with ureagenesis and thereby automatically facilitate renal water conservation.

Renal water conservation. The plasma concentration of urea in mammals is relatively low (4–10 mmol/l) compared with that of other solutes such as sodium (140 mmol/l). Urea represents <2% of filtered solutes, but about 40–50% of all solutes in the urine. These values indicate that the concentration of urea in urine is up to 100 times higher than in plasma, that the bulk of water reabsorbed by the kidney to concentrate the urine is attributable to the high concentration of urea and that renal water conservation in mammals relies on urea metabolism76,80.

During states of high Na+ and Cl− solute excretion, urea- driven concentration of urine is an important mechanism to limit osmotic diuresis and prevent dehydration68,69. Various reports suggest that SGLT2 inhibition similarly induces renal urea reabsorption to limit Na+- driven and glucose- driven osmotic diuresis (Fig. 2). Treatment of diabetic rats with dapagliflozin induces expression of the

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Fig. 2 | Water and energy conservation metabolism under dehydration stress. Situations of dehy-dration stress, such as that resulting from osmotic diuresis triggered by a high- salt diet, induce activa-tion of a glucose–alanine shuttle to transfer amino acids (AAs), particularly branched- chain amino acids (BCAAs), as a nitrogen source from muscle to liver. Generation of two pyruvate metabolites from glucose generates two ATP molecules. Additional transamination of pyruvate results in alanine, which is transferred to the liver. Transamination of two alanine to glutamate ultimately results in nitrogen transfer and urea osmolyte production, which costs three ATP molecules for one urea osmolyte. The kidney then utilizes the urea to enhance water reabsorption in the renal medulla (hepatorenal regulation of the renal concentration mechanism) and counterbalance the osmotic–diuretic effect of salt in order to prevent body water loss and dehydration. We hypothesize that the same sequence of events occurs to limit glucose- driven osmotic diuresis during therapeutic SGLT2 inhibition. By investing four more ATP and two more GTP molecules (depicted as –6 ATP molecules), the liver can synthesize glucose from the remaining two pyruvate metabolites. Alternatively, the liver can generate acetyl- CoA from the surplus pyruvate, which in case of parallel high fatty- acid oxidation will result in ketogenesis. Note that hepatic gluconeogenesis from muscle alanine results in a negative ATP balance. Any chronic ‘starvation state’ will result in exploitation of glycogen and fatty acids reservoirs from muscle and liver. Patients on SGLT2 inhibitor treatment have a reduction in glycogen and fat stores in the liver and skeletal muscle and show signs of increased energy and nitrogen transfer from muscle to liver. Note that increased anti- diuretic hormone (ADH, also known as vasopressin) action not only supports free- water reabsorption across water channels in the collecting duct, but in parallel promotes the transamination of amino acids, which increases the biosynthesis of the urea solutes that will ultimately generate the osmotic driving force necessary for this urine- concentrating process. GG, glucogenic; Glc, glucose; KG, ketogenic; UT- A1, urea transporter A1.

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urea transporter UT- A1 in the renal medulla, indicating that attenuation of Na+ and glucose reabsorption in the proximal tubule induces compensatory urea- driven water reabsorption in the collecting duct81. This drug- induced effect has also been demonstrated in rats without diabetes82. Of note, this urea- driven strengthening of the renal concentration mechanism resembles the water- conserving mechanisms that are activated in aestivating lungfish or cartilaginous fishes in high- salinity marine environments, which rely predominantly on urea- driven water reabsorption without a notable contribution of Na+ transport for water reabsorption83–86.

The physiological mechanisms of renal water conservation within the urine- concentrating mechanism rely not only on active urea transport to increase the concentration of osmolytes in the interstitium, but also on free- water reabsorption in the renal medulla, regulated by the anti- diuretic hormone vasopressin, and mediated via water transport across aquaporin 2 (AQP2) channels (Fig. 2). Initial studies on the effect of SGLT2 inhibitors on the urine- concentrating mechanism suggested that SGLT2 inhibition does not increase vasopressin release in patients with T2DM11, or the expression of AQP2 in diabetic rats81. However, a 2019 study that measured levels of copeptin — a surrogate marker of vasopressin, which is more stable in plasma87–89 — showed that dapagliflozin not only increases copeptin levels — indicative of increased vasopressin release — but also activates renal water- conserving mechanisms as evidenced by increased urine osmolality and decreased free- water clearance in humans89. This finding is supported by a 2020 study in diabetic rats, which suggested that the osmotic diuresis induced by SGLT2 inhibition is indeed limited by vasopressin- mediated phosphorylation and activation of water transport across AQP2, and that this water- conserving strategy contributes to successful maintenance of body hydration90. In addition to promoting water reabsorption through medullary AQP2 channels, vasopressin also facilitates UT- A1- driven and UT- A3- driven urea transport and accumulation in the renal medulla, most likely by vasopressin V2 receptor- mediated increases in UT- A1 protein expression in the collecting duct74,91,92. Together these findings suggest that SGLT2 inhibition triggers vasopressin- mediated renal water conservation by increasing urea- solute and water reabsorption during the urine- concentrating process in the inner medulla.

Beyond vasopressin- mediated regulation of AQPs and urea transporters, it is tempting to speculate that the increased Na+ and glucose solute load to the distal tubule resulting from successful SGLT2 inhibition might also directly enhance UT- A1- driven urea transport through activation of the osmoregulatory transcription factor tonicity- enhancer binding protein (TonEBP, encoded by NFAT5)93, which initiates promoter- driven increases in UT- A1 mRNA and protein expression in the collecting duct94–96. The renin–angiotensin–aldosterone system also limits renal water loss by increasing Na+ reabsorption in the distal segments of the kidneys. Patients with T2DM showed no increase in plasma renin or aldosterone levels following treatment with multiple doses of empagliflozin10, although patients treated with canagliflozin or dapagliflozin showed slight increases in plasma renin activity without detectable changes in aldosterone levels11,89.

Thus, available evidence suggests that SGLT2 inhibition triggers a predominantly vasopressin- driven water- conserving response in the renal medulla. This water- conserving response is time- dependent. Within the first day of therapeutic SGLT2 inhibition with empagliflozin12 or dapagliflozin97, patients show an increase in urine volume of 0.5–1.0 l per day. However, this osmotic–diuretic effect disappears within 72 hours97, after which the urine volume returns to baseline levels, and remains normal over the following 4 weeks despite persistently increased (primarily glucose- driven) solute excretion12. Patients on long- term SGLT2 inhibitor treatment therefore overcome the osmotic–diuretic effect of sustained glucosuria by strengthening the urine- concentrating mechanism and reducing renal free- water clearance89. We propose that these time- course observations are indicative of aestivation- like water- conserving motifs, which we interpret as a physiological adaptive response to limit osmotic diuresis and conserve body water during long- term SGLT2 inhibition.

Hepatic support of water conservation. The metabolism of inorganic Na+ and K+ solutes in the kidney relies solely on transporter- mediated electrolyte reabsorption and recycling. By contrast, urea- driven water- conserving mechanisms do not rely on urea recycling alone, but also involve the synthesis of additional urea osmolytes through intensification of the hepatic urea cycle. Therefore, in mice fed a high- salt diet, the osmotic–diuretic effect of high

urinary Na+ and Cl− solute excretion (in the absence of accompanying glucosuria) is counterbalanced by hepatic urea synthesis and enhanced renal urea reabsorption. The energy- intensive nature of this metabolic water- conserving process requires either increased food intake, or, in the absence of sufficient food, exploitation of endogenous protein reservoirs from skeletal muscle for urea production68 (Fig. 1). The resulting hepatorenal regulation of fluid balance intimately links body fluid homeostasis with systemic fuel and energy metabolism.

In addition to requiring energy, the generation of urea also requires nitrogen, which is not present in fat and glycogen. Only dietary protein, or exploitation of endogenous protein reservoirs such as skeletal muscle, can deliver both the energy and the nitrogen necessary for the increased metabolic demands of glucose and organic osmolyte production in the liver. The transfer of amino acids from skeletal muscle to liver via the glucose–alanine shuttle (also known as the Cahill cycle) was initially discovered in humans during prolonged fasting (that is, in individuals with a negative energy balance due to reduced energy intake)98–100. However, the protein catabolic Cahill cycle is similarly activated in the absence of a reduced fuel intake in the context of chronic salt- driven osmotic diuresis (that is, in situations of negative energy balance as a consequence of increased energy expenditure for water conservation)68. The same metabolic process seems to be triggered in humans on SGLT2 inhibitor therapy during the night, when energy intake is reduced, but the loss of glucose fuels into the urine persists101. This persistent glucosuria, however, additionally requires an efficient anti- diuresis mechanism to prevent glucose- driven osmotic diuresis. It is therefore likely that, as in aestivators61,64,102–105, in patients on SGLT2 inhibitor therapy the Cahill cycle is activated not only to metabolically compensate for the reduced availability of glucose, but also to prevent renal water loss (Fig. 2).

The catabolism of amino acids from skeletal muscle for use as fuel also yields free ammonium ions, which are transferred by transamination to pyruvate to form alanine. The alanine is then released into the systemic circulation and transported to the liver, where the reverse process (deamination) takes place. In the liver, the pyruvate can be used as fuel or as a substrate for gluconeogenesis, and the nitrogen is transferred via transamination of α- ketoglutarate to glutamate into the hepatic urea cycle for ureagenesis.

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The production of one urea molecule after transamination of the nitrogen from two alanine molecules requires three ATP molecules, whereas hepatic de novo synthesis of glucose from two pyruvate molecules requires another six ATP molecules (Fig. 2) so the process of hepatic gluconeogenesis and ureagenesis results in a negative energy balance. Therefore, to save energy during periods of negative energy balance, the liver will produce more ketone fuels from fatty acids, resulting in parallel exploitation of

body triglyceride stores. This metabolic switch is an energy- efficient strategy to enable de novo fuel synthesis in the liver during periods of nutrient deprivation. The production of ketone bodies from acetyl- CoA, in contrast to the production of glucose from pyruvate, allows the liver to produce fuel for the brain, heart, kidney, muscle and other organs, without investing ATP into this process. Such energy- efficient fuel production may be further supported by the increased breakdown of ketogenic amino acids from dietary or endogenous

protein sources, the catabolism of which results in the production of short- chain free fatty acids (FFAs) and ketone intermediates. This alternative variant of extrahepatic metabolic ketone production, which should not be confused with de novo ketogenesis from acetyl- CoA, occurs in all tissues that are able to catabolize the ketogenic amino acids phenylalanine and leucine.

The fates of the carbon and nitrogen components of dietary or muscle- derived amino acids during their catabolism in various organs highlight the intimate connection between body fluid and energy homeostasis (Fig. 3). The carbon components will ultimately end up as pyruvate, oxaloacetate, α- ketoglutarate and succinate (via catabolism of glucogenic amino acids) or as short- chain FFAs and/or ketones (via catabolism of ketogenic amino acids), which ultimately generate acetyl- CoA for ATP generation via oxidative phosphorylation. The nitrogen component will eventually result in the generation of an osmolyte (urea) for body water conservation, which in situations of adequate hydration is excreted into the urine. Given the connectivity of these two homeostatic systems, it is not surprising that some of the key hormones involved in the regulation of energy metabolism have parallel water- conserving properties, and that central hormonal regulators of water homeostasis can induce the transamination machinery to promote amino acid catabolism. For example, insulin, which is secreted to induce the storage of energy when glucose levels are high, decreases hepatic ureagenesis106, whereas glucagon, which promotes the breakdown of glucose and thus the generation of pyruvate (Fig. 2), also stimulates the transamination of the carbonic acids pyruvate (to alanine), oxaloacetate (to aspartate) and α- ketoglutarate (to glutamate). The resulting transamination cascade promotes protein catabolism for gluconeogenesis88,107–110. Increased endogenous glucose fuel generation in the context of reduced insulin levels and increased glucagon action will therefore inevitably be paralleled by increased urea osmolyte generation, which in turn promotes osmolyte- driven body water conservation. Of note, SGLT2 inhibitors increase glucagon levels and the glucagon- to- insulin ratio7,8.

Similarly, the effects of vasopressin on water conservation extend beyond the above mentioned effects on UT- A1- mediated and AQP2- mediated transport111,112. Synergistically with glucagon, vasopressin promotes the breakdown of glycogen to

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Fig. 3 | reprioritization of fuel utilization pathways during hypometabolism and water conser-vation. Aestivators induce hypometabolism to ensure maintenance of essential cellular processes and the survival of essential organs during periods of dehydration stress. This hypometabolic response is characterized by a number of metabolic changes, including reduced glucose fuel utilization by reduc-ing hexokinase activity (1), and reduced oxidative phosphorylation, achieved through a reduction in the activity of citrate synthase (2) and/or cytochrome c (3). This reduction in carbon oxidation results in lower levels of reactive oxygen species and thereby protects cells. Aestivators rely on the utilization of stored fuels. Increased activity of alanine aminotransferase (4), aspartate aminotransferase (5) and glutamate aminotransferase (6) initiates catabolism of glucogenic amino acids (AAs) alanine (Ala), aspartate (Asp) and glutamate (Glu) to form the carbonic acids pyruvate (Pyr), oxaloacetate (OxA) and α- ketoglutarate (α- KG), respectively. The catabolism of glucogenic AAs such as valine or alanine pro-duces succinate and/or pyruvate or the catabolism of ketogenic AAs such as leucine (Leu) or lysine produces ketones and/or acetyl- CoA from the catabolism of amino acid- derived short- chain free fatty acids (SCFFAs). The catabolism of branched- chain amino acids (BCAA) is particularly noteworthy, because hepatocytes do not express BCAA transferase (7), leaving BCAA catabolism of leucine (Leu) to ketones, valine (Val) to succinate, and iso- leucine (I- Leu) to acetyl- CoA, ketones or succinate to occur in specifically the heart and kidney. By contrast, the production of ketones from triglyceride- derived long chain fatty acids (LCFAs) is limited to the liver (L). Of note, AA catabolism promotes cytoplasmatic gluconeogenesis via increased activity of phosphoenolpyruvate (PEP) carboxy kinase (PEPCK) (8), and this process is further driven by the reduction in mitochondrial oxida-tive phosphorylation and reduced generation of citrate from OxA. Finally, catabolism of AAs by a transamination cascade ultimately triggers the generation of organic osmolytes for body water conser-vation, for example by nitrogen transfer into the urea cycle driven by Ala, Asp and Glu. Many of the above depicted metabolic changes are not restricted to aestivators, but similarly occur in experimental animals and patients with SGLT2 inhibition. Note that gluconeogenesis from many glucogenic amino acids, such as alanine, is initiated in the mitochondrion by carboxylation of their intermediate pyruvate to oxaloacetate (not shown). Fructose-1,6- BP, fructose-1,6- bisphosphate; TCA, tricarboxylic acid.

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stimulate gluconeogenesis, and increases ureagenesis in the liver113–118. Therefore, the observed increase in vasopressin levels achieved with SGLT2 inhibitor therapy will induce protein catabolism, and activate the glucose–alanine shuttle for increased energy and nitrogen transfer to the liver, ultimately generating more urea osmolytes for water conservation.

We propose that SGLT2 inhibition initially couples the sustained urinary loss of fuel in the form of glucose with body water loss via its osmotic diuretic effect. This results in a physiological adaptation process that successfully combines fuel generation with efficient water conservation, relying on the exploitation and recycling of amino acids by transamination to satisfy the increased metabolic demands of glucose and urea production. The relative contribution of the energy- conserving versus the water- conserving components to this adaptive physiological response is difficult to assess. However, adequate hydration prevents the development of ketoacidosis in SGLT2 inhibitor- treated rats, suggesting that the osmotic–diuretic effect contributes markedly to the increased energy expenditure during states of SGLT2 inhibitor- driven glucosuria119. We hypothesize that the need to simultaneously conserve energy and water triggers adaptive aestivation- like metabolic survival motifs60,61,64,102–105,120, which protect organ function under conditions that mimic dehydration stress (Fig. 1).

Reprioritization of energy expenditure. As described above, our view is that SGLT2 inhibition activates hepatorenal water- conserving mechanisms not only to compensate for the urinary loss of glucose (through glycogen breakdown and de novo glucose synthesis from amino acids), but also to provide the urea needed to strengthen the renal concentration mechanism and thereby limit the osmotic–diuretic driving force resulting from increased glucose excretion (Fig. 2). We have previously demonstrated that activation of water- conserving mechanisms to counterbalance osmotic diuresis, without a parallel loss of fuel into the urine, is sufficient to induce systemic metabolic aestivation motifs in the liver, skeletal muscle and kidney. Mice on a high- salt diet that were not permitted to increase their calorie intake demonstrated activation of the glucose–alanine–nitrogen shuttle with increased expression of ornithine aminotransferase (OAT), another transamination enzyme that is required for the generation of alanine in muscle;

increased expression of the hepatic sodium- coupled neutral amino acid transporters SLC38A1 and SLC38A2, which promote alanine uptake by liver; and increased hepatic arginase activity to promote ureagenesis in the liver68 (Fig. 2).

The metabolic consequence of this energy and nitrogen transfer to liver, triggered solely by increased Na+ and Cl− intake and excretion without parallel caloric loss into the urine, is the mobilization of muscle- derived amino acids in parallel with exploitation of muscle glycogen for transamination reactions (Fig. 1). The resulting depletion of glycogen stores stimulates skeletal muscle to prioritize β- oxidation for ATP generation. The increased reliance of muscle on fatty acids as a fuel supply is regulated by the phosphorylation of acetyl- CoA carboxylase by phosphorylated AMP- activated protein kinase (pAMPK). The liver acts as an acceptor of amino acid- derived energy and nitrogen from muscle, and invests energy to produce the urea osmolytes that are necessary for successful hepatorenal water conservation.

Ketogenesis to conserve muscle mass. Patients receiving SGLT2 inhibitor therapy demonstrate increased gluconeogenesis, which is necessary to replace the glucose

that is lost into the urine. The extent to which this replacement relies on increased utilization of dietary protein sources, and/or on the exploitation of endogenous protein sources is, however, unclear (Fig. 4).

Gluconeogenesis in combination with increased ketogenesis ultimately conserves muscle mass under conditions that exploit endogenous energy. If all glucose lost in the urine was replaced by de novo glucose production from amino acids, skeletal muscle would ultimately disappear. Therefore, parallel exploitation of energy stored in body fat by increased β- oxidation of long- chain fatty acids from triglycerides occurs, which increases the availability of acetyl- CoA for energy- neutral ketone body formation in the liver. This switch towards the production of ketone fuels not only reduces the hepatic burden for de novo glucose production, but also supports the switch from glucose to fatty acid and ketone body utilization for ATP production that is observed in peripheral organs, such as skeletal muscle.

This switch from glucose to fatty acid utilization by peripheral organs for acetyl- CoA and ultimately ATP generation enables more efficient glucose recycling between muscle and liver via the Cahill cycle (Fig. 2), albeit at the expense of a negative

Energy and nitrogen supply

• Glucogenic AAs• Ketogenic AAs• BCAAs

Circulationpump

Osmolyte and fuel production

Water conservationand gluconeogenesis

Ureasupply

Fig. 4 | hypothesized key organ-specific metabolic changes in patients on SglT2 inhibitor therapy. In response to acute renal fuel and water loss due to glucosuria, internal organs activate evolu-tionarily conserved metabolic aestivation patterns to stabilize their function. The resulting metabolic survival pattern includes reprioritization of metabolic processes in the liver, kidney and heart in an effort to economize organ workload and compensate for the loss of fuel and water over a prolonged period. The function of these key survival organs is supported by skeletal muscle, which serves as fuel and nitrogen reservoir through catabolic processes, which provides the nutrients necessary for successful physiological adaptation to the renal glucose leak during times when dietary protein is not available (for example, during sleep). AAs, amino acids; BCAAs, branched- chain amino acids.

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energy balance of −7 ATP molecules per recycled glucose molecule. This process does not require any further exploitation of muscle protein, as long as the nitrogen necessary for the transamination of glucose- derived pyruvate to alanine is available. Therefore, the retention of body nitrogen, which can be depicted as UT- A1- mediated reabsorption of urea in the kidney (Fig. 2), not only couples gluconeogenesis with water conservation, but also preserves muscle mass during this endogenous glucose recycling process. Our view is that body nitrogen retention for successful water conservation, combined with preferential use of fatty acid fuels for successful ATP generation, will ultimately reduce the need to generate glucose from amino acids and thereby prevent muscle mass loss during states of increased gluconeogenesis in patients treated with SGLT2 inhibitors. Of note, in this context the resulting liver- driven and kidney- driven increase in plasma urea concentration, which is a physiological response necessary for body water conservation68, should not be mistaken for impairment of kidney function.

The retention of urea is, however, not a very useful example of body nitrogen conservation in humans, because humans do not express urease in their tissues, and therefore cannot breakdown urea into nitrogen and CO2 without the help of urease- positive bacteria in their gut. It is therefore tempting to speculate that, in addition to inducing accumulation of urea, SGLT2 inhibitor therapy might also trigger the production and retention of methylated glycine products (methylamines) (Fig. 3), such as 1- methyl glycine (sarcosine), 2- methyl glycine, 3- methyl- glycine (betaine) or 3- methylamine- N- oxides (TMAO). Increased levels of these organic osmolytes would not only improve the ability of the organism to conserve water in the intracellular space121, but also generate an easily exploitable nitrogen reservoir in all body cells. Although such organ- specific metabolic fluxes are difficult to study in humans, an initial description of the metabolomic signature in patients treated with SGLT2 inhibitors showed reduced blood glucose levels along with evidence of increased catabolism of ketogenic and glucogenic amino acids, activation of urea cycle components122 and elevated serum urea levels10,12,89,122, lending support to the hypothesis that metabolic adjustments to SGLT2 inhibition are a response to both chronic urinary caloric loss and renal water loss.

Diurnal effects. Although food intake seems to be increased during SGLT2 inhibition123,124, the observed weight loss of 2–3 kg in patients receiving these drugs suggests that this increased calorie load is not sufficient123,125 to fully compensate for the urinary loss of fuel as well as for the energy investment necessary to limit glucose- driven osmotic diuresis. SGLT2 inhibitors have been suggested to induce gluconeogenesis and ketogenesis when food intake is reduced during sleep, suggesting that daily exploitation of endogenous energy (and nitrogen) reservoirs may predominantly occur during the physiologically inactive period, during which urine production is also low101. The need to metabolically replace the glucose lost into the urine, combined with the increased need for energy and nitrogen for water conservation to prevent osmotic diuresis might explain a number of metabolic observations associated with SGLT2 inhibitor therapy, including: a rapid increase in the ratio of glucagon to insulin7,8; stimulation of amino acid catabolism; activation of the urea cycle122; exploitation of glycogen and fat reservoirs in muscle and in liver5,126–133, resulting in improved insulin sensitivity134–136; a shift in fuel utilization to the preferential use of fatty acids7,28; and the promotion of hepatic ketogenesis while preserving renal gluconeogenesis5,6.

Fuel switches with SGLT2 inhibitionMuscle: an energy and nitrogen reservoir. The strategies used by aestivators to prolong lifespan under conditions of dehydration stress (Fig. 1) include a reliance on stored reserves of body fuels together with reprioritization of fuel use in favour of those which can most efficiently support vital functions, suppression of cell functions with a reduction of cell ‘workload’ and the implementation of cell preservation mechanisms such as antioxidant defences38. A comparison of these three components in aestivators, in animals that limit osmotic diuresis by natriuretic–ureotelic regulation, and in humans or animals receiving SGLT2 inhibitors, reveals interesting insights into the evolutionarily preserved nature of these aestivation patterns and how the resulting metabolic switches might contribute to the cardiorenal protective effects of SGLT2 inhibition.

pAMPK acts as a cellular energy sensor and is a key modulator of catabolic versus anabolic metabolism in aestivators38. Similar to aestivators, mice on a high- salt diet that cannot increase calorie intake phosphorylate AMPK (pAMPK) in skeletal muscle, which reduces muscle protein

synthesis and promotes muscle degradation via increased autophagy and proteasomal degradation38,68,137,138. Degradation of muscle protein increases the availability of glucogenic and ketogenic amino acids, which serve as precursors for pyruvate and/or acetyl- CoA and, as discussed above, results in a net flux of energy and nitrogen substrate from skeletal muscle to the liver61,68,103,120. This protein catabolic water- conserving response, which can occur in the absence of reduced energy intake or increased energy loss68, also involves exploitation of energy from triglyceride stores, as discussed above.

This process becomes more complex in the context of SGLT2 inhibition, during which osmotic diuresis is induced by loss of glucose fuels that need to be metabolically replaced. The extent to which the use of stored energy sources during SGLT2 inhibition is driven by the need to restore lost fuels, or, alternatively, to support energy- intense water- conserving processes, is unclear. Catabolism of protein reservoirs might also be involved in the metabolic adaptation process associated with SGLT2 inhibition, as fat and glycogen stores can replace energy deficits but cannot provide the nitrogen necessary for urea osmolyte production (Fig. 3). Therefore, in addition to depleting glycogen and fat energy stores in the liver and skeletal muscle127,128,130,132,135,136, SGLT2 inhibition would be expected to exploit muscle energy and nitrogen reservoirs — a proposal that is supported by several body composition studies that have shown reductions in lean body mass during SGLT2 inhibition126,139. Furthermore, an analysis of the plasma metabolome of patients with T2DM on SGLT2 inhibitor therapy demonstrated that urea cycle activation occurs in parallel with increased catabolism of the branched- chained amino acids (BCAAs) valine, leucine and isoleucine122 (Fig. 3).

BCAAs are essential amino acids that act as a source of nitrogen for the synthesis of alanine and glutamine, which are subsequently released into the blood and used in visceral tissues, especially as gluconeogenic substrates140. We have long known that plasma concentrations of BCAAs increase during starvation141. BCAAs are a special source of energy because, unlike most other amino acids, their catabolism does not start in the liver as the BCAA transaminases (BCATs) — which are necessary to convert muscle BCAAs into their corresponding branched- chain α- keto acids and initiate their catabolism for energy generation — are not expressed in

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hepatocytes. Rather, BCATs are expressed in visceral tissues, such as the kidney and heart. After their BCAT- dependent transamination, these peripheral organs catabolize the BCAAs to their corresponding branched- chain α- keto acids and short- chain FFAs (Fig. 3), resulting in the generation of succinyl- CoA from valine (a glucogenic BCAA) and acetyl- CoA and/or ketone bodies from leucine (a ketogenic BCAA); while iso- leucine has both glucogenic and ketogenic properties142. An increasing body of evidence supports the notion that BCAAs and their corresponding branched- chain α- keto acids cycle between organs140. The use of in vivo isotopic tracing to study whole- body BCAA metabolism has shown that organs such as the heart and the kidneys take up and catabolize BCAAs within minutes after injection, suggesting that BCAAs are readily used as a fuel in these organs when they are available143.

Given that BCAAs represent a source of energy and nitrogen that can be easily transferred between different organs, we hypothesize that SGLT2 inhibition not only promotes the use of diet- derived or muscle- derived energy and nitrogen substrates to help the liver counterbalance urinary glucose and water loss68,122 (Fig. 4), but also triggers the transfer of BCAAs from muscle to the heart and kidneys to support their function30.

Fuel switch patterns in the heart. When treated with SGLT2 inhibitors, both patients with T2DM and healthy individuals show increased fatty acid oxidation and elevated ketone body production23,28,125. The ‘thrifty substrate’ hypothesis29 suggests that during persistent hyperketonaemia (such as that induced by SGLT2 inhibitor therapy), liver- derived ketone bodies are taken up by the heart and oxidized in favour of fatty acids, thereby improving myocardial energetics31,144,145.

The suggestion that SGLT2 inhibitor therapy triggers adaptive aestivation motifs for energy and water conservation provides insights into the possible fuel switches that occur with increased glycosuria. As described earlier, the water- conserving component in this aestivation response requires utilization of energy and nitrogen from the diet or from body protein stores. This need cannot be satisfied by exploitation of glycogen or fat reservoirs, which will readily supply high amounts of energy, but are nitrogen- free (Fig. 3). Metabolomic analyses122 suggest that SGLT2 inhibition promotes protein and BCAA catabolism for adaptive aestivation- like water conservation.

Available evidence also suggests that in addition to energy and nitrogen transfer from body triglyceride and protein stores to liver for simultaneous ketogenesis, gluconeogenesis and ureagenesis (Fig. 2), SGLT2 inhibitors might also trigger the transfer of muscle BCAAs to the heart30. One metabolomic analysis of heart tissue from pigs after myocardial infarction showed that SGLT2 inhibitor therapy not only increases cardiac BCAA content, but also facilitates their catabolism to short- chain fatty acids and/or ketone bodies by increasing the activity of branched- chain α- keto acid dehydrogenase complex (BCKD) in the myocardium30 (Fig. 3). The consequence of this process is increased use of cardiac ketones and FFAs for ATP generation and a reduced reliance on glycolysis for energy production. In addition to this switch in metabolic fuel, administration of SGLT2 inhibitors to pigs with myocardial infarction improved the cardiac efficiency and reduced sympathetic drive, thereby reducing heart rate and cardiac afterload30, again similar to the findings in aestivating lungfish144. According to our scheme of muscle amino acid exploitation for energy and nitrogen transfer to key aestivation organs (Fig. 3), these combined physiological responses probably occurred as a result of reduced myocardial glucose uptake, increased myocardial BCAA uptake and increased activity of BCAT to generate de novo short- chain α- keto FFAs from BCAA catabolism. Further catabolism of these amino acid- derived myocardial FFAs would in turn increase the production of myocardial ketone bodies (acetoacetate and/or β- hydroxybutyrate) and ultimately promote FFA and ketone body oxidation. In other words, BCAAs as a fuel that originates from skeletal muscle or from the diet will be catabolized into FFAs and ketone bodies locally in the cardiomyocyte, satisfying the cardiac fuel need by switching from glycolysis to β- oxidation (Fig. 4). Except for the increase in BCAT activity, which was unmeasured, all the above- described metabolic changes necessary for successful amino acid energy transfer from muscle to heart were observed in pigs with myocardial infarction treated with SGLT2 inhibitors30.

Fuel switch patterns in the kidney. The beneficial effects of SGLT2 inhibitors on kidney function have primarily been attributed to a reduction in glomerular hyperfiltration due to increased solute delivery to the macula densa9,25 and possibly improved energetics and ATP utilization in the renal medulla146. Therefore, changes

in both intrarenal haemodynamics and renal energy metabolism probably underlie the nephroprotective effects of SGLT2 inhibition.

Administration of SGLT2 inhibitors to cultured human renal proximal tubule cells induced activation of gluconeogenesis with increased expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6- phosphatase (G6Pase)147. These in vitro findings were reproduced in healthy mice without diabetes, in which SGLT2 inhibition also increased PEPCK and G6Pase protein expression in the renal cortex (Fig. 3). This increase in the key enzymes for de novo glucose generation was paralleled by elevated protein expression of phosphorylated cyclic- AMP response element binding protein (pCREB), indicating glucagon- driven activation of gluconeogenesis in the kidney147. A separate study further showed that SGLT2 inhibitor- mediated stimulation of gluconeogenesis in healthy mice without diabetes is kidney- specific. Treatment of non- diabetic mice with ipragliflozin increased mRNA expression of the gluconeogenic genes Foxo1, Creb, Pepck and G6pc (which encodes G6Pase) in the renal cortex (Fig. 3), but not in the liver5. Moreover, SGLT2 inhibitor treatment also induced an accumulation of glycogen in the kidney5. This finding is remarkable, because it suggests that SGLT2 inhibition shifts the balance from pyruvate oxidation (that is, the use of glucose for ATP production by oxidative phosphorylation) towards phosphoenolpyruvate generation (that is, the use of pyruvate for the de novo generation and storage of glucose) (Fig. 3). This seemingly paradoxical increase in de novo glucose production and storage during SGLT2 inhibition occurred in the kidney, but not in the liver, suggesting that induction of gluconeogenesis by SGLT2 inhibition might be a kidney- specific effect. Furthermore, the same mice showed increased mRNA levels of the genes encoding the glycogenolysis- inducing enzyme glycogen phosphorylase (Pygl) and unchanged levels of the glycogenesis enzyme glycogen synthase (Gys1) in the renal cortex5.

These findings indicate increased glycogen mobilization for glucose generation, which would predict a decrease in renal glycogen content. The fact that renal glycogen levels are instead elevated suggests that the kidneys, similar to the heart and skeletal muscle, may reduce their use of glucose, and use more fatty acids and/or amino acids for oxidative

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phosphorylation during SGLT2 inhibition. In line with this hypothesis, non- diabetic mice treated with ipragliflozin showed increased mRNA expression of the genes that encode acyl- CoA dehydrogenase and fatty acid synthase in the renal cortex, which suggests a switch towards the preferential use of short- chain fatty acids for oxidative phosphorylation5. Whether these renal metabolic changes in mice are comparable to the fuel switches observed in the heart of pigs with empagliflozin treatment30 is currently unclear. Additional studies that include kidney- specific metabolomic profiling and metabolic flux analysis are required to test this hypothesis.

In contrast to the above- described studies in healthy animals, the metabolic fuel switches that occur in response to SGLT2 inhibition in animals or patients with diabetes are probably more complex and difficult to understand. For example, Akita mice, which are a genetic model of type 1 diabetes mellitus, show marked catabolism at baseline with stunted growth despite significantly increased food intake148. Given the existing energy deficit at baseline and the lack of insulin necessary for tissue glucose utilization, one would predict that SGLT2 inhibition would predispose these animals to increased ketogenesis instead of gluconeogenesis. Indeed, mRNA expression of Pepck is reduced in the kidneys of empagliflozin- treated Akita mice; however, whether such markers of reduced renal gluconeogenesis are coupled with increased hepatic ketogenesis is unknown. Similarly, leptin receptor- deficient db/db mice149, which are massively obese and are used as a genetic model of T2DM, did not show increased mRNA expression of gluconeogenic enzymes in their kidneys. Although the reason for this finding is unclear, it is possible that the immense disbalance in body composition as well as the array of endocrine abnormalities present in this model of T2DM might circumvent the urinary glucose fuel loss that occurs with SGLT2 inhibition by preferentially increasing β- oxidation and exploiting their vast triglyceride stores, rather than increasing amino acid utilization for gluconeogenesis149. By contrast, wild- type mice that are rendered insulin- resistant by a long- term high- fat diet recapitulate the increased renal gluconeogenesis observed in humans in response to SGLT2 inhibition147 (Fig. 3).

Hints of renal hypometabolism. Beyond the above- described exploitation of endogenous energy stores and successful water conservation, a key survival strategy

of aestivating organisms is their efficient use of stored body fuels combined with a reduction in ATP usage, which they achieve by entering a hypometabolic state. Most organisms in which the aestivation adaptations have been described are poikilothermic animals such as amphibia and fish, whose internal body temperatures can vary considerably. By contrast, mammals are homeothermic animals that need to constantly oxidize fuels in order to produce the heat necessary to keep their body temperature constant. This requirement results in a major metabolic difference in fuel utilization: in contrast to mammals, poikilothermic animals can massively reduce levels of oxidative phosphorylation to enter a hypometabolic state, as they do not require the same rate of fuel- driven thermogenesis37,38,150. However, these survival mechanisms, while more obvious in poikilothermic animals due to the massive changes in energy expenditure, are not entirely absent in homeothermic organisms. Mammals are also able to use some of the key processes by which aestivators extend cellular lifespan under conditions of stress, albeit to a lesser extent.

In aestivating lungfish, increased nitrogen transfer for urea osmolyte production is accompanied by reduced mitochondrial oxidative phosphorylation and reduced activity of citrate synthase and cytochrome c oxidase in the liver (Fig. 3), muscle and kidney. These changes result in a hypometabolic state with reduced levels of mitochondrial fuel and electron transfer for ATP production64. The decrease in oxidative phosphorylation reduces fuel requirements, as evidenced by a lower rate of fatty acid transfer into the mitochondria of aestivating compared with non- aestivating lungfish64. Similarly, a reduction in the activity of the glycolytic enzyme, hexokinase, in the heart (Fig. 3) — indicative of reduced levels of glucose entering the tricarboxylic acid (TCA) cycle — is coupled with maintained or increased ATP generation from skeletal muscle- derived glucogenic and ketogenic amino acids151, suggesting that skeletal muscle is an amino acid reservoir that constantly supports and supplies the heart with energy. The above- described increase in BCAA catabolism in the heart of SGLT2- treated pigs thus could be interpreted as an aestivation- like fuel switch that is beneficial for cardiac energy metabolism30.

SGLT2 inhibition may exert a comparable effect in the kidney. A 2018 study of fuel utilization in the kidneys showed that leptin- deficient ob/ob mice have reduced

glucose levels in their renal cortex, but elevated levels of the TCA metabolites citrate, cis- aconitate and α- ketoglutarate, and generation of reactive oxygen species, as estimated by glutathione and oxidized glutathione levels. This sequence of events indicates that ob/ob mice (which, in contrast to db/db mice, are hyperinsulinaemic) have an increased metabolic rate and higher levels of oxidative phosphorylation in their kidneys6. Treatment of these mice with SGLT2 inhibitors reduced TCA metabolite and reactive oxygen species levels, suggesting that SGLT2 inhibition suppresses the metabolic rate in the renal cortex. It is tempting to speculate that SGLT2 inhibition, similar to aestivation, reduces the enzyme activity of citrate synthase and/or cytochrome c oxidase in ob/ob mice (Fig. 3). Similar to the metabolic changes that occur in aestivators37, the local beneficial effect of metabolic suppression was reduced oxidative phosphorylation, which reduced the generation of reactive oxygen species and thereby preserved organ function in these mice.

However, even though a reduction in oxidative phosphorylation increases cellular longevity by generating fewer reactive oxygen species, the beneficial effect of metabolic rate suppression comes at the price of reduced ATP production. Thus, a hypometabolic state also requires a reduction in cellular workload to maintain viability. Increased generation of urea osmolytes and parallel UT- A1- driven accumulation of urea in the renal medulla with SGLT2 inhibitor therapy can be viewed as a prototype of renal workload reduction88. Increased hepatic and extrahepatic production of urea production leads to increased filtration of urea into the proximal tubule, which improves the ability of the kidney to conserve water within the renal concentration mechanism152,153. Additional UT- A1- driven reabsorption of urea also improves the ‘efficiency of water conservation’73,154, by supporting and enhancing the urea- driven countercurrent system of the renal medulla155 (Fig. 2). We therefore propose that SGLT2 inhibitors induce metabolic changes similar to those seen in aestivation, which combine organ- protective hypometabolic responses in the renal cortex (Fig. 3) with haemodynamic

glossary

LungfishFreshwater fish belonging to the subclass Dipnoi, which have lungs and a specialized respiratory system. During periods of draught, lungfish bury themselves into the ground and survive by breathing atmospheric air and encasing themselves in a sheath to avoid dehydration.

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adjustments and energy- efficient urea- driven water conservation in the renal medulla (Fig. 2), and thereby preserve renal function.

ConclusionsPatients and experimental animals treated with SGLT2 inhibitors experience an acute loss of energy in the form of glucose into the urine, which results in glucose- driven osmotic diuresis and water loss, predisposing the individual to dehydration (Fig. 2). We hypothesize that this combination of acute energy and water loss triggers evolutionarily preserved motifs of aestivation metabolism, whereby skeletal muscle serves as an energy and nitrogen reservoir that supports key survival organs such as the kidney, liver and heart by supplying energy and nitrogen to maintain essential metabolic processes when dietary protein is not available (Fig. 4). The metabolism of glucogenic and ketogenic amino acids is of particular importance in this context of energy and water loss. Glycogen and fat stores contain a lot of energy- rich fuels, which can compensate for an energy deficit, but cannot provide the nitrogen necessary to produce organic osmolytes for water conservation. By contrast, amino acids can provide both the energy and the nitrogen necessary to compensate for the glucosuria- induced energy and water loss (Fig. 3).

In the context of SGLT2 inhibition, several metabolic switches occur in different organs. Reprioritization of metabolic processes in the liver leads to a focus on organic osmolyte production, with available evidence suggesting that the liver might preferentially produce de novo ketones as a fuel source over glucose from gluconeogenesis, owing to the energy- intense nature of de novo glucose production from pyruvate.

In the heart, the increased use of short- chain fatty acids and ketone bodies as fuel sources will be supported by the increased catabolism of the BCAAs. These BCAAs represent a special fuel, because they specifically supply the heart and the kidney with energy, but not the liver, which lacks the transaminase necessary to initiate their catabolism (Fig. 3).

The kidneys promote and increase urea- driven water- reabsorption, which is the most energy- efficient way to concentrate the urine. This economy of water conservation allows the kidneys to reduce in parallel their metabolic rate, which in turn reduces the workload needed to successfully prevent dehydration and simultaneously improves their ability to generate more glucose fuels in the proximal tubular cells

through de novo glucose production from amino acids (renal gluconeogenesis) (Fig. 3).

We hypothesize that liver, heart and kidney cells can adapt to systemic energy and/or dehydration stress via activation of aestivation- like hypometabolism motifs, which economizes their function and supports their longevity. We propose that the activation of such metabolic processes with SGLT2 inhibitor treatment improves the cellular lifespan of these organs and, at least in part, underlies the hepatocardiorenal protection observed with this class of therapeutic agents.Adriana Marton 1,6, Tatsuroh Kaneko 2,6, Jean- Paul Kovalik 1, Atsutaka Yasui 2, Akira Nishiyama 3, Kento Kitada1,3 and Jens Titze 1,4,5 ✉1Programme in Cardiovascular and Metabolic Disorders, Duke- NUS Medical School, Singapore, Singapore.2Medicine Division, Nippon Boehringer Ingelheim Co., Ltd, Tokyo, Japan.3Department of Pharmacology, Faculty of Medicine, Kagawa University, Kagawa, Japan.4Division of Nephrology and Hypertension, University Clinic Erlangen, Erlangen, Germany.5Division of Nephrology, Duke University Medical Center, Durham, NC, USA.6These authors contributed equally: Adriana Marton, Tatsuroh Kaneko. ✉e- mail: jens.titze@duke- nus.edu.sg

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Author contributionsA.M., T.K. and J.T. wrote the article. All authors researched data for the article, contributed substantially to discussion of the article’s content and reviewed/edited the manuscript before submission.

Competing interestsT.K. and A.Y. are employees of Nippon Boehringer Ingelheim Co. Ltd. J.T. and A.N. have received research support from Boehringer Ingelheim International GmbH for a collaborative preclinical study. A.N. has also received speaker honoraria from Taisho Pharmaceutical and Daiichi- Sankyo. J.T. has also received research support from AstraZeneca and received travel support and speaker honoraria from Boehringer Ingelheim International GmbH and AstraZeneca. All other authors declare no competing interests.

Peer review informationNature Reviews Nephrology thanks L. Gallo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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