Abbreviations

CD Standard control diet

CDE CD + empagliflozin

GIR Glucose infusion rate

NAFLD Non-alcoholic fatty liver disease

PDK4 Pyruvate dehydrogenase kinase 4

PGC1α

Peroxisome proliferator-activated receptor γ coac

tivator 1-α

SGLT2 Sodium–glucose cotransporter 2

T2 Transverse relaxation time

TEM Transmission electron microscopy

WD Western-type diet

WDE WD + empagliflozin

Introduction

      Obesity prevalence is dramatically increasing worldwide. Recently, overweight and obesity were estimated to account for 4 million deaths globally every year [1]. According to the WHO a vast 1.9 billion adults were overweight in 2016 and over 650 million were obese [2]. Chronic overfeeding, wrong choice of diet and an increasingly sedentary lifestyle are accepted as central contributing factors to the obesity pandemic [3]. There is a long and well-documented epidemiological and pathophysiological relationship between obesity and risk of type 2 diabetes. Despite extensive knowledge of the positive effects of a Mediterranean diet and physical activity on energy and glucose metabolism, sedentary lifestyle and intake of high-energy, fat- and sucrose-rich diets are still predominant in the western world [4].

      Sodium–glucose cotransporter 2 (SGLT2) inhibitors are now widely used for their specific beneficial effects in individuals with type 2 diabetes [5, 6]. Beside their glucoselowering properties these effects include cardio- and nephroprotection, and a modest reduction in BP and body weight [7, 8]. Interestingly, a weight loss of roughly 2–4 kg is usually only seen within the first 3 months of treatment despite ongoing glucosuria, probably explained by a compensatory increase in food intake [9]. Besides direct effects on hepatic inflammation, reactive oxygen species generation and mitochondrial function SGLT2 inhibitor-induced weight reduction might also contribute to beneficial effects on nonalcoholic fatty liver disease (NAFLD) [10, 11].

      While SGLT2 inhibitors are well established in the treatment of type 2 diabetes, heart failure and chronic kidney disease, their role in the primary prevention of metabolic dysfunction is unclear. Prospective studies utilising SGLT2 inhibitors in the primary prevention of obesity are scarce and to our knowledge have not been conducted in humans. In preclinical studies, empagliflozin, a specific SGLT2 inhibitor, showed increased energy expenditure, browning of adipose tissue and decreased adipose tissue inflammation in mice fed a high-fat diet [12].

      In this study we set out to test whether empagliflozin prevents weight gain and metabolic dysfunction in a mouse model of diet-induced obesity and insulin resistance.

Methods

      Animals A total of 140 male 6-week-old C57Bl/6 mice (Charles River Laboratories, Germany) were used in this study. Mice were kept under standard conditions of 12 h night–day cycle, 23±2–3°C and controlled humidity. After 1 week of acclimatisation, mice were fed four different diets ad libitum (SSNIFF Spezialdiäten, Germany): standard control diet (CD) (SSNIFF art. no. 1534-00); CD with added empagliflozin (CDE); western-type diet (WD) (21.2% fat and 33.3% sugar content, corresponding to 42% energy from fat and 43% energy from carbohydrates; SSNIFF art. no. E15721-34); and WD with added empagliflozin (WDE). Empagliflozin was provided by Boehringer Ingelheim and added directly to diets by SSNIFF to aim at an empagliflozin dose of 30 mg/kg body weight (260 mg empagliflozin/1000 g CD, 192 mg empagliflozin/1000 g WD). Dosing was based on previously published studies [13–15]. Group allocation was chosen randomly.

      Diets were fed to the mice for 10 weeks, with weekly measurements of capillary blood glucose via tail vein puncture and a handheld glucometer (Accu-Chek Performa Nano; Roche, Switzerland) and weekly measurements of body weight and water and food consumption. Before weekly measurements of blood glucose, mice were fasted for 6 h. At the end of study mice were anaesthetised after a 6 h fast. Mice were killed via a combination of central blood collection with cardiac puncture and cervical dislocation. Tissue samples were collected immediately afterwards, snap-frozen in liquid nitrogen and stored until further processing at −80°C. All animal procedures were performed in accordance with the guidelines of the Austrian Animal Testing Act of 1988. Approval for this animal study was granted by the Austrian Federal Ministry for Education, Science and Research (application no. BMWF-66.011/0066/ − V/3b/2018).

Hyperinsulinaemic–euglycaemic clamp

      After 10 weeks of diet and/or treatment with empagliflozin, a silicone catheter was surgically inserted into the right external jugular vein of the mice to provide i.v. access as described before [16]. The catheter was connected to an i.v. access point placed behind the neck of the mouse (VABM1BSM/25; Instech Laboratories, USA). During surgery, mice were anaesthetised using a combination of breathable isoflurane (5% [vol./vol.] for induction of anaesthesia and 2–3% [vol./vol.] as anaesthesia maintenance; Zoetis, USA) and given an s.c. injection of piritramide (0.1 mg/kg; Piramal, India). Mice were kept at 37°C via a heating pad during surgery. At 3–5 days after surgery, mice had regained their pre-surgery weight (±10%) and hyperinsulinaemic–euglycaemic clamping was performed as described [16]. In short, after a 6 h fast, i.v. access was established via connecting the i.v. access point. Capillary blood glucose levels were measured using the cut tail method and a handheld glucometer during the experiment. Readings were taken with mice in the fasted state, during the set-up phase, and every 5–10 min during the clamp until stable euglycaemia was reached with a glucose target of 5.55–6.66 mmol/l (Fig. 3e and f). Insulin (insulin aspart; Novo Nordisk, Denmark) was infused steadily at 8 mU kg−1 min−1 . A variable infusion of 20% (wt/vol.) glucose (Merck, USA) was given to reach and maintain the set glucose target.

Results

Empagliflozin treatment prevents diet-induced obesity, hyperglycaemia and hyperinsulinaemia

      After 10 weeks on the diets, WD-fed mice had a significantly higher body weight than CD-fed mice. Treatment with empagliflozin prevented diet-induced weight gain during the study, resulting in comparable body weights for WDE-fed and CD-fed mice (Fig. 1a). Accordingly, WDE-fed mice displayed a significantly lower total body fat content and smaller subcutaneous, visceral and retroperitoneal adipose tissue depots when compared with WD-fed mice (Fig. 2). WD-fed mice had elevated fasting blood glucose levels during the study period, with the difference being significant at week 10 when compared with CDfed mice (Fig. 1b). Empagliflozin prevented hyperglycaemia as well as the accompanying hyperinsulinaemia in WDE-fed mice (Fig. 1b, c). Glucagon, ketone body (β-hydroxybutyrate) and adiponectin levels were comparable between all groups (Fig. 1d–f). Empagliflozin treatment did not significantly affect daily food intake in CD-fed or WD-fed mice (Fig. 1g, h). Water intake was significantly increased upon addition of empagliflozin to the diet, irrespective of whether mice were fed CD or WD (Fig. 1i, j). Glucosuria was seen in all mice upon empagliflozin treatment, irrespective of the type of diet (ESM Fig. 2).

Fig. 1 Characteristics of mice during the study. (a) Body weight of mice„ over the course of the study (mean ± SEM, n=16 [WD=15]). Two-way repeated measures ANOVA was used (p for diet effects, p<0.0001; p for diet × time interaction, p<0.0001). ***p<0.001 WD vs WDE; † p<0.01 CD vs CDE at week 10. (b) Blood glucose levels during the study (mean ± SEM, n=16 [WD=15]). Two-way repeated measures ANOVA was used (p for diet effects <0.0001; p for diet × time interaction <0.0502). *p<0.05 WD vs WDE at week 10. (c–f) Plasma insulin (n=5–9) (c), glucagon (n=4–9) (d), β-hydroxybutyrate (n=8) (e) and adiponectin (n=8–10) (f) levels at week 10. (g, h) Daily food intake in CD and CDE mice (g) and WD and WDE mice (h) (n=4). (i, j) Daily water intake in CD and CDE mice (i) and WD and WDE mice (j) (n=4). Unless otherwise specified, data are presented as mean ± SD. Kruskal– Wallis test was performed for (c–f). Two-way ANOVA was performed for (a, b) and (g–j). Bars and asterisks (*p<0.05) indicate respective post hoc analysis. The key applies to (a, b and g–j). BSL, baseline

      Empagliflozin treatment prevents diet-induced insulin resistance After 10 weeks of being fed the diets, the whole-body insulin sensitivity of the mice was measured using hyperinsulinaemic–euglycaemic clamps. The glucose infusion rate (GIR) required to maintain stable euglycaemia under hyperinsulinaemic conditions was found to be significantly higher in WDE-fed mice than in WD-fed mice, indicating increased insulin sensitivity (Fig. 3a). Remarkably, the mean GIR (and thus insulin sensitivity) was comparable in WDE and CD-fed mice. When differences in plasma insulin concentration under clamp conditions and differences in lean body mass of mice were taken into consideration, there was an even greater difference seen when comparing insulin sensitivity in WD and WDE mice (Fig. 3b, c). The GIR was strongly correlated with body weight, suggesting that lack of excess weight gain significantly contributed to the improved insulin sensitivity in WDE mice (Fig. 3d).

      Empagliflozin provides protection against hepatic steatosis and improves hepatic insulin signalling The grade of steatosis was significantly lower in WDE-fed mice than in WD-fed mice and measurement of intrahepatic lipid content corroborated histological findings (Fig. 4a–c). Accordingly, hepatic insulin signalling as assessed by phosphorylated Akt/total Akt (p-Akt/tAkt) was significantly higher in WDE-fed mice than in WD-fed mice (Fig. 4d, e). Levels of insulin receptor expression were comparable between all groups (Fig. 4d, f).

      Empagliflozin supplementation affects hepatic energy metabolism mRNA expression of Pdk4, encoding pyruvate dehydrogenase kinase 4 (PDK4, an isoform known to regulate hepatic insulin signalling and energy metabolism), was significantly lower in the liver of WDE-fed mice compared with WD-fed mice (Fig. 5a). Increased expression of Adipor1 (encoding adiponectin receptor isoform I) and Cpt1a (encoding carnitine palmitoyltransferase I) mRNA suggested increased β-oxidation in livers of WDE-fed mice compared with WD-fed mice (Fig. 5b, c). On the other hand, liver expression of Pparγ (also known as Pparg, encoding peroxisome proliferator-activated receptor γ) and Cd36 mRNA was lower in WDE-fed mice than in WD-fed mice, suggesting lower hepatic fatty acid uptake (Fig. 5d, e). Glycogen levels (Fig. 5f) were not affected by empagliflozin, irrespective of the type of diet, while Pepck (encoding PEPCK) and G6pc (encoding glucose-6 phosphatase) mRNA levels were increased in WDE-fed mice when compared with WD-fed mice, indicative of higher gluconeogenesis (Fig. 5g, h). Gck (encoding glucokinase) mRNA expression was significantly increased in WD-fed mice compared with CD-fed mice (Fig. 5i); addition of empagliflozin to the WD led to a normalisation of Gck mRNA expression.

      Empagliflozin treatment affects mitochondrogenesis in skeletal muscle Citrate synthase activity, as a biomarker of cumulative mitochondrial mass, was increased by addition of empagliflozin to the WD (Fig. 6a). In line, expression levels of Pgc1α (also known as Ppargc1a, encoding peroxisome proliferator-activated receptor γ coactivator 1-α [PGC1α]) mRNA as well as Nrf1 (encoding nuclear respiratory factor 1) and Tfam (encoding mitochondrial transcription factor A) mRNA were increased in WDE-fed mice when compared with WD-fed mice (Fig. 6b–d). Irrespective of the type of diet, empagliflozin supplementation was associated with alterations of subsarcolemmal and intermyofibrillar mitochondrial size and morphology in both types of skeletal muscle fibres as assessed by TEM (Fig. 6e–h). Dietary empagliflozin supplementation led to larger mitochondria and significant changes in the aspect ratio and circularity of mitochondria (ESM Table 1). Skeletal muscle fibre type and myocellular location of mitochondria are known to affect size and shape of mitochondria. To account for this confounding effect, we performed multiple linear regression analysis after adjusting for fibre type and subcellular localisation, and confirmed a drugspecific effect (ESM Table 1). Compared with liver tissue, changes in the p-Akt/tAkt ratio were modest in skeletal muscle (ESM Fig. 3a, b). Skeletal muscle triacylglycerol content was higher in WD-fed mice than in WDE-fed mice, without the difference reaching statistical significance (ESM fig. 3c). Skeletal muscle glycogen content was unchanged with addition of empagliflozin (ESM Fig. 3d)

Discussion

      SGLT2 inhibitors have an indispensable role in the treatment of type 2 diabetes, chronic kidney disease and heart failure. Despite clinical evidence on cardio- and renoprotection the underlying beneficial mechanisms are still under discussion and include moderate weight loss, haemodynamic changes and cellular effects. While the glucose-lowering, cardioprotective and renoprotective properties of SGLT2 inhibitors are well established and these drugs are widely used in clinical practice, it is not clear whether SGLT2 inhibitors are capable of preventing metabolic disease in a high-risk scenario.

      The aim of this study was to investigate whether SGLT2 inhibition provides protection against diet-induced weight gain, disturbances in glucose metabolism and fatty liver disease. The study was performed in male C57BL/6 mice on a WD as this setting is known to induce a distinct phenotype of metabolic disease including obesity, insulin resistance and hepatic steatosis [22].

      Various mechanisms, especially in relation to possible benefits of SGLT2 inhibition regarding fatty liver disease, have been proposed and reviewed before [10, 23]. Mechanisms include attenuation of inflammation and macrophage polarisation [12, 24], autophagy [25], endoplasmic reticulum stress [25–27], and attenuation of steatosis and fibrosis [28] to name a few examples. However, whether SGLT2 inhibition is capable of preventing the development of obesity and insulin resistance is currently poorly studied. Therefore we chose to study empagliflozin in a diet-induced mouse model and to carry out extensive metabolic phenotyping to assess the metabolic health of the mice.

      Adipose tissue plays a key role in the pathophysiology of insulin resistance, type 2 diabetes and fatty liver disease mainly via the release of NEFA, proinflammatory cytokines and adipocytokines [29, 30]. In our study, we show that empagliflozin supplementation prevents excess weight gain in WD-fed mice while it had no relevant effect on adipose tissue depots in CD-fed mice. MRI studies revealed that empagliflozin supplementation reduced diet-induced expansion of subcutaneous, visceral and retroperitoneal adipose tissue depots. In a previous study by Vallon et al [31], epididymal fat adipocyte size was found to be reduced upon empagliflozin treatment in C57BL/6-background mice, while increased adipocyte size was found in insulindeficient Akita mice.

      In individuals with type 2 diabetes, SGLT2 inhibitor treatment is associated with a modest weight reduction, which is typically seen during the initial phase of treatment. As expected, in our study empagliflozin-treated mice displayed marked glycosuria irrespective of type of diet. The weight difference between empagliflozin-treated and control mice might be explained by urinary energy loss and increased energy expenditure upon SGLT2 inhibitor treatment, as reported previously [12]. Glucose excretion into the urine was measured in fasted mice only in this study. However, a contribution by urinary glucose excretion in empagliflozin-treated mice to overall GIR in mice from the hyperinsulinaemic–euglycaemic clamp part of the study cannot be ruled out by the study design.

      Interestingly, in humans, the initial weight loss is attenuated by a compensatory increase in energy intake [9]. In previous murine studies, both unchanged and increased food intake upon empagliflozin treatment was reported [31–33]. In our study, empagliflozin supplementation was associated with increased food intake in CD- and WD-fed mice, without the difference reaching statistical significance, as estimated by twice weekly determination of the food remaining in each cage. However, the sensitivity of this method might be too low to detect small differences in food intake.

      Importantly, in our model, empagliflozin protected the mice against WD-induced insulin resistance as shown by hyperinsulinaemic–euglycaemic clamp studies. When adjusting pure GIRs for lean body mass and increased insulin levels at clamp conditions the results indicate an even more drastic increase in insulin sensitivity upon addition of empagliflozin to WD. A full dataset for lean body mass, clamp data and insulin levels was only available in a small sample size, so results should be seen as hypothesis-generating despite being statistically significant.

      We found increased hepatic Akt phosphorylation, indicating increased hepatic insulin sensitivity with dietary empagliflozin supplementation. Akt phosphorylation in WD-fed compared with CD-fed mice was similar, although fasting insulin plasma levels were not. Mice on the WD were hyperinsulinaemic compared with CD-fed mice and this might explain similar Akt phosphorylation despite the presence of insulin resistance.

      Body weight is a major contributing factor to whole-body insulin sensitivity, with some authors arguing it is the most important predicting factor especially after weight loss [34, 35]. Here, we corroborate this association by correlating body weight with GIR (Fig. 3d). A limitation of the study is the relative per-body-weight dose adjustment of insulin for the clamp procedure instead of adjustment for lean body mass.

      Diet-induced obesity and insulin resistance are strongly associated with NAFLD. NAFLD is commonly associated with hepatic insulin resistance and results in further deterioration of systemic glucose metabolism [36, 37]. In our study, empagliflozin protected the WD-fed mice against dietinduced hepatic steatosis. In obese people with moderately controlled type 2 diabetes, empagliflozin treatment for 20 weeks resulted in improved blood glucose control, moderate weight reduction and significant improvement in liver triacylglycerol content [38]. In previous studies, significant weight loss of greater than 10% of initial body weight led to the resolution of steatosis in individuals with obesity [36, 37]. A study by Kahl et al showed that empagliflozin reduces liver fat content in individuals with well-controlled type 2 diabetes [11]. Interestingly, in that study no change in insulin sensitivity was observed, in contrast to findings of improved insulin sensitivity with empagliflozin in our study. In obese, insulinresistant C57BL/6J mice Xu and colleagues reported that empagliflozin diminished weight gain and reduced deteriorations of insulin sensitivity and hepatic steatosis of ongoing high-fat-diet feeding [12, 24]. Mechanistically, increased energy expenditure and browning of adipose tissue as well as reduced inflammation in adipose tissue and the liver were found upon SGLT2 inhibition.

      Here we show that empagliflozin not only exerts beneficial effects on overt NAFLD but also protects against diet-induced hepatic steatosis. Interestingly, we found reduced Pdk4 mRNA expression in WDE-fed mice. Hepatic PDK4 expression has been linked to impaired insulin sensitivity and fatty liver disease [39–41] through stimulation of fatty acid uptake and synthesis in the liver. Accordingly, in our study reduced Pdk4 expression was accompanied by decreased fatty acid uptake, estimated by expression of key regulators of cellular fatty acid metabolism. Barres et al [42] reported altered promotor methylation of PDK4 in obese individuals, with the alteration being restored by significant weight loss, suggesting that empagliflozin protects against obesityinduced alterations in PDK4 expression upon WD feeding.

      The p-Akt/tAkt ratio was increased in WDE-fed mice compared with WD-fed mice, suggesting enhanced hepatic insulin sensitivity. While empagliflozin supplementation did not affect hepatic glycogen content, expression data suggest increased gluconeogenesis and decreased glycolysis in WDEfed mice when compared with WD-fed mice. While increased gluconeogenesis and enhanced insulin signalling seems controversial, these data might be explained by the significantly reduced PDK4 expression in WDE-fed mice. PDK4 inhibits the pyruvate dehydrogenase complex, which links fatty acid and glucose metabolism by catalysing the oxidative decarboxylation of pyruvate [40, 43]. PDK4 deficiency was shown to prevent hepatic steatosis upon high-fat-diet feeding, probably due to increased PGC1⍺ activity. This decreased activity is associated with increased levels of PEPCK and reduced capacity for de novo fatty acid synthesis [44]. Importantly, PDK4 expression is increased and its methylation decreased in type 2 diabetes [45]. We hypothesise that decreased Pdk4 mRNA expression might underlie the increased hepatic gluconeogenesis observed in WDE-fed mice when compared with WD-fed mice. It might be speculated that the reduced Pdk4 mRNA expression in livers of WDE-fed mice might be associated with reduced oxidation of carbohydrates leading to a shift towards fatty acid oxidation and stimulation of gluconeogenesis. In contrast, reduced glycolysis, as indicated by decreased glucokinase mRNA expression levels, in WDE-fed mice in comparison with WD-fed mice might reflect improved hepatic insulin sensitivity.

      Unexpectedly, expression markers of key enzymes of gluconeogenesis were decreased in WD-fed mice when compared with CD-fed mice, suggesting preserved insulin action in insulin resistance partially compensated by hyperinsulinaemia. Further dynamic tests will be necessary to better understand the effects of SGLT2 inhibitors on hepatic glycogen metabolism.

      Mechanistically, high influx of adipose-tissue-derived fatty acids, diminished suppression of fatty acid synthesis in the liver, reduced hepatic insulin signalling, and impaired adiponectin signalling are major drivers of triacylglycerol accumulation in the liver. Our data suggest that empagliflozin supplementation reduces fatty acid uptake and synthesis (probably by affecting PDK4 expression), enhances adiponectin activity (due to increased adiponectin receptor expression) and improves hepatic insulin signalling (as shown by increasing Akt phosphorylation).

      Besides liver and adipose tissue, glucose disposal of skeletal muscle is another major determinant of systemic insulin sensitivity. Here, we did not see relevant changes in p-Akt/ tAkt ratio (ESM Fig. 3). However, assessment of Akt activaion was performed in fasted mice, whereas data from clamp studies were acquired under hyperinsulinaemic conditions. In skeletal muscle, abnormal mitochondrial function is thought to play a key role in pathophysiology of cellular insulin resistance [46, 47].

      Remarkably, our data suggest diet-independent effects on mitochondrial morphology and mass, as shown by increased citrate synthase activity and elevated mRNA expression levels of Pgc1α and downstream targets Nrf1 and Tfam upon empagliflozin supplementation. Finally, ultrastructural analysis revealed skeletal muscle mitochondria to be larger and have a more rounded shape after empagliflozin supplementation compared with no supplementation. Altered skeletal muscle mitochondrial morphology is a hallmark of type 2 diabetes and obesity [48]. Our group and others have shown weightindependent beneficial effects of empagliflozin on mitochondrial morphology [13] and improved mitochondrial respiration in cardiac muscle [15]. Additionally, empagliflozin treatment improved skeletal muscle mitochondrial function in a murine model of heart failure [32].

      Although previous studies have shown the beneficial effects of empagliflozin treatment in obese, insulin-resistant or diabetic humans or rodents, this is to our knowledge the first study that demonstrates preventative effects of empagliflozin in healthy mice. Our study suggests that empagliflozin treatment not only reverses obesity (or diabetes-specific alterations in metabolically important tissues such as adipose tissue, liver and skeletal muscle) but also protects against systemic and tissue-specific metabolic defects seen upon high-fat diet intake.

      In conclusion, empagliflozin treatment protects mice against obesity, insulin resistance and hepatic steatosis in a setting of high-energy, high-sucrose and high-fat intake. Although many metabolic effects of empagliflozin might predominantly result from prevention of excess weight gain, our data suggest that additional diet-independent benefits on mitochondrial structure and mass in skeletal muscle might also contribute to preservation of systemic insulin sensitivity.

Supplementary Information The online version contains peer-reviewed but unedited supplementary material available at https://doi.org/10.1007/ s00125-022-05851-x.

Acknowledgements Parts of this study were presented as a short oral discussion at the 58th annual meeting of the EASD 2022.

Data availability Original data are available upon reasonable request from the corresponding author.

Funding Open access funding provided by University of Innsbruck and Medical University of Innsbruck. The financial support by the Federal Ministry for Digital and Economic Affairs and the National Foundation for Research, Technology and Development is gratefully acknowledged. We would like to thank Boehringer Ingelheim for providing financial support for these studies (to SK).

Authors’ relationships and activities The authors declare that there are no relationships or activities that might bias, or be perceived to bias, their

Contribution statement BR, CR, SF, AL, BW, GS, KS, MG, JaH and JoH performed all experiments. WS and BR performed TEM sample preparation, image acquisition and data analysis. TJ contributed technical expertise for performing clamps in mice. CK and BR designed and performed MRI scans and subsequent image analysis. PM prepared H&E staining from tissue samples. BR, CR, MR and SK drafted an early version of the manuscript. All authors contributed to analysis and discussion of the data. All authors revised and approved the manuscript in its final form. SK is the guarantor of this work.

      Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/ .

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This article is excerpted from the Diabetologia (2023) 66:754–767 by Wound World.