In the present study, severe obesity was associated with increased EPO concentrations and iron dysregulation, with a positive correlation with basal weight, FM and FFM in the overall sample. After weight loss in patients with obesity, specifically that induced by a VLCKD, EPO circulating levels decreased coinciding with the moment of maximum ketosis, which was maintained over time and no effect on the EPO time-course was observed after LCD and bariatric surgery. In addition, high baseline EPO levels were correlated with higher impact on the course of weight loss and body composition changes in the first 2–3 months after the three different therapeutic weight-loss strategies. These results demonstrate that the increase in ketone bodies yielded by nutritional ketosis do not induce an increase in EPO secretion and they suggest a potential role of EPO on counteracting the metabolic stress associated to obesity and circulating levels of EPO could be a biomarker of the health status of patients under a weight-loss therapy.

In agreement with our results, EPO has previously been found to be increased in patients with obesity and anaemia [14], in subjects with metabolic syndrome and in individuals with abdominal obesity component [14, 15]. It is known that adipose tissue in patients with obesity produces an increased amount of proinflammatory cytokines, therefore contributing to the development of a low-grade systemic inflammation in these subjects [14, 33, 34]. The development of sideropenia could be a consequence of this obesity-associated low-grade inflammation, in addition to diets with low value of Index of Nutritional Quality for iron among other factors [33,34,35]. EPO levels have also been shown to be increased in this population, secondary to iron deficiency in its active metabolic pool [14]. However, in our population, clinical parameters of iron deficiency or anaemia were not observed, even though patients with obesity showed lower iron, TSI and haematocrit levels than normal-weight volunteers. On the other hand, growing evidence has also suggested that cellular hypoxia and reduced adipose tissue oxygenation may be an underlying trigger of adipose tissue dysfunction leading to metabolic variations associated with obesity and metabolic syndrome [15]. In this sense, hypoxia is a known stimulator of EPO production [3].

sTfR levels were also measured in the current study. Although previous studies have also shown higher sTfR values in subjects with obesity as well as in individuals with abdominal obesity component of metabolic syndrome [15, 36], no significant variations according to the degree of adiposity were observed in our population. In addition, it is known that sTfR levels may not only be increased in iron deficiency with inadequate iron supply for erythropoiesis [37] but also due to the use of erythropoiesis-stimulating agents such as EPO [38]. However, the fact that no differences were observed in these values according to adiposity degree and after weight-loss interventions, despite their positive correlation with basal EPO levels, leads us to think that the mechanisms causing the relationship between EPO and changes in weight and body composition in these patients are independent of sTfR levels.

In our study, EPO levels decreased precisely after VLCKD intervention, at the time of maximum ketosis as evidenced by β-OHB levels, and remained within that range during follow-up. The decrease in EPO levels seen after VLCKD was not observed after LCD or bariatric surgery, suggesting a potential role of the nutritional ketosis induced by the VLCKD in this effect. On the contrary, previous studies using SGLT2 inhibitors (which can also induce moderate weight loss) showed an increase in EPO levels along with an increase in haematocrit, proposing that the hyperketonaemia induced by this drug directly stimulates circulating EPO concentrations [18, 39]. The observed decrease in EPO levels in our population after VLCKD were accompanied by higher levels of β-OHB (~ 1.3 mmol/L) than is typically shown during SGLT2 inhibition (~ 0.6 mmol/L). Even higher levels of hyperketonaemia (4–5 mmol/L) were observed in a recent study, in which EPO concentrations were shown to be significantly greater after Na-3-βOHB administration in comparison with saline infusion [18]. This confirms the high complexity of the underlying mechanisms involved in EPO variations, which is why further research in this field is needed. On the other hand, apart from hyperketonaemia, another of the proposed theories is that the transfer of enhanced but less efficient oxygen-consuming active sodium reabsorption to the distal tubule results in the expression of hypoxia-inducible factors, which stimulate erythropoiesis [16]. Obesity-related adipose tissue is also characterised by a hypoxia status providing cellular mechanisms for chronic inflammation and mitochondrial dysfunction. Likewise, higher serum EPO concentrations may suggest underlying adipose tissue hypoxemia in patients with excess adiposity [15]. In this sense, the possible influence of adipose tissue-associated hypoxia and inflammation on the response of EPO to ketone bodies could also be clinically investigated by exploring the effect of VLCKD on EPO levels in a different population such as healthy competitive bodybuilders [40].

Beyond erythropoiesis, endogenous EPO protects against FM accumulation and systemic inflammation [41]. The increased EPO levels observed in obesity could suggest that EPO is secreted to counteract the metabolic deficiencies in these patients with a variable degree of systemic inflammation related with an increase in adipose tissue and cytokine production [14, 33, 34]. In this sense, it is known that many of the benefits of VLCKD in obesity are based on its ability to exert anti-inflammatory and antioxidant effects [24, 42], contrary to what occurs with bariatric surgery, which is related to metabolic stress during the weight-loss process [43]. This could also explain why EPO levels decrease and are maintained within that range after a weight-loss therapy that is capable of improving metabolic disorders and inflammation, such as VLCKD. In accordance with this proposal, the current results could also suggest, therefore, that under a healthy metabolic, hormonal and inflammatory profile, the secretion of protective proteins, such as EPO, is not needed. In addition, those patients with higher baseline EPO levels also showed a higher decrease in body weight and FM after weight-loss treatments, suggesting an additional capacity to predict response to a weight-loss therapy. Similar results were previously observed when studying other proteins with beneficial properties, such as FGF21 [43], irisin [44], betatrophin [45] and leptin [46], among others. These hormones were associated with beneficial metabolic effects and capacity to protect against obesity in preclinical models. All of them are increased in patients with obesity and their concentration in plasma decreases after a weight-loss therapy that improves the metabolic and inflammatory status of patients with obesity, as observed in EPO levels in the current study. In this regard, it has already been demonstrated that a VLCKD is able to reduce visceral FM, preserving muscle mass [22] and function [23], and that this beneficial effect on body composition was concomitant with an improvement in the inflammatory and oxidative stress status [24, 42] and a restoration of the obesity-related epigenome [47]. All of these effects were observed mainly in the first steps of this nutritional treatment when it induced a similar weight loss than that observed in patients undergoing bariatric surgery, which, as previously mentioned, is related to metabolic stress in the short-term [43]. The main difference between both weight-loss treatments regarding the effect on weight loss is the VLCKD-induced nutritional ketosis, which is the main player in the beneficial effect induced by this specific diet [24]. Not only has VLCKD also been linked to improving mitochondrial function and decreasing oxidative stress [24, 42], but also EPO promotes metabolic activity and adipocytes to increase mitochondrial function via Sirtuin 1, among other regulators of energy homeostasis [48]. Interestingly, some studies indicate that the anti-inflammatory effects of ketone bodies are actually mediated by Sirtuin 1 activity [49]. Thus, EPO variations and their metabolic effects seem to comprise a complex framework that needs to be examined in more depth to gain a greater understanding thereof.

The longitudinal design of the current study enables, for the first time, the evaluation of time-course changes in EPO levels in obesity after three different weight-loss therapies (VLCKD, LCD and bariatric surgery). The presence of a normal-weight control group also contributes to making the data more robust. Although it should be taken into account that, in the current study, the bariatric surgery group is mainly represented by women, no differences were found regarding EPO levels and gender. Moreover, the three different treatments were not matched for BMI because bariatric surgery is prescribed for patients with severe obesity. However, this issue does not invalidate the results because the effect of the three interventions were assessed respect to the baseline levels, comparing each patient with himself and after a statistical analysis adjusted for baseline BMI, differences in EPO levels between interventional groups were maintained. In addition, considering that this is an observational association study, and that, therefore, only hypotheses related to changes in erythropoiesis can be proposed, long-term prospective research is needed to examine causality.