Protective Effects of ACF210, a Dual GLP-1/APJ Receptor Agonist, against Cardiovascular-Kidney-Metabolic Syndrome Induced by T2D

Protective Effects of ACF210, a Dual GLP-1/APJ Receptor Agonist, against Cardiovascular-Kidney-Metabolic Syndrome Induced by T2D | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Protective Effects of ACF210, a Dual GLP-1/APJ Receptor Agonist, against Cardiovascular-Kidney-Metabolic Syndrome Induced by T2D Qingbin Zhao, Qingfeng Wu, Huiyi Wei, Yuanyuan Du, Huifen Zhou This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7782667/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Current therapies cannot simultaneously address the interconnected metabolic, cardiac, and renal damage in Cardiovascular-Kidney-Metabolic (CKM) syndrome. This study investigated ACF210, a novel long-acting dual agonist targeting both GLP-1 and APJ receptors, as a potential treatment for type 2 diabetes (T2D)-induced CKM syndrome. ACF210 was synthesized by fusing the human IgG4 Fc fragment to the C-terminus of GLP-1 and the N-terminus of Elabela-21 (ELA). In vitro receptor activation assays confirmed that ACF210 effectively activated the GLP-1 receptor while inhibiting APJ-related cAMP signaling. db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced T2D were treated with dulaglutide, Fc-ELA, or ACF210 for 12 weeks. Observation of organizational structure, functional assessment and serum analyses were conducted. ACF210 outperformed other treatments, showing superior improvements in blood glucose control, β-cell function, and reduction of hepatic steatosis. Crucially, it provided significant multi-organ protection: enhancing cardiac diastolic function, reducing biomarkers of heart failure (NT-proBNP), and lessening mitochondrial damage and fibrosis, and inducing the microangiogenesisl in the heart. In the kidneys, it improved function, indicated by lower cystatin C levels, and mitigated podocyte damage. In conclusion, ACF210 not only effectively alleviates dysglycemia by enhancing β-cell function but also significantly protects against diabetes-associated hepatic, cardiac, and renal damage, supporting its further exploration for the clinical management of CKM syndrome. Health sciences/Medical research/Drug development Biological sciences/Drug discovery/Target validation GLP-1 receptor agonist Elabela dual receptor agonist CKM syndrome type 2 diabetes multiorgan protection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Cardiovascular-kidney-metabolic (CKM) syndrome has emerged as a critical paradigm for understanding the interconnected complications of type 2 diabetes (T2D), characterized by a vicious cycle of metabolic dysfunction, cardiovascular damage, and renal impairment—together contributing to significantly elevated morbidity and mortality and posing a major public health challenge(1, 2). The pathophysiological mechanism of CKM syndrome is multifactorial(3), T2D-induced CKM syndrome is characterized by chronic hyperglycaemia or advanced glycation end product (AGE) production resulting from insulin resistance and impaired insulin secretion due to pancreatic β-cell dysfunction(4). This triggers a cascade of pathological changes including cardiac remodelling, renal glomerulosclerosis, and hepatic steatosis through a mechanism involving excessive activation of RAAS, endoplasmic reticulum stress, abnormal calcium processing, mitochondrial dysfunction and energy metabolism disorders, oxidative stress and chronic inflammation(5), these processes collectively lead to multiorgan damage, imposing a substantial burden on healthcare systems(6). Although single-target glucose-lowering agents have demonstrated certain cardiovascular and renal protective effects in clinical studies of T2D, the complex pathophysiology of CKM syndrome induced by T2D necessitates multitarget therapeutic strategies that address the underlying disease mechanisms and provide protection across various organ systems(7). Recent therapeutic advances have focused on glucagon-like peptide-1 receptor agonists (GLP-1RAs), a class of agents that mimic the action of GLP-1, a hormone secreted by L cells in the intestinal mucosa(8). GLP-1RAs increase glucose-dependent insulin secretion, suppress glucagon release, delay gastric emptying, and reduce appetite, thereby contributing to weight loss and ameliorating T2D-associated metabolic symptoms(9, 10). However, despite these advantages, native GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) in circulation(11), which limits its therapeutic application. To address this limitation, GLP-1 analogues such as dulaglutide have been developed, in which a GLP-1 peptide is fused to the Fc fragment of human IgG4, thereby prolonging the half-life and enabling less frequent dosing(12).In addition, clinical studies have demonstrated that GLP-1RAs provide cardiovascular benefits and reduce the risk of heart failure(13, 14). Nevertheless, the underlying molecular mechanisms through which GLP-1RAs improve cardiovascular outcomes remain poorly understood. Consequently, the cardiorenal protective effects of GLP-1RAs in T2D warrant further mechanistic exploration. Elabela (ELA), also known as Toddler, is an endogenous ligand that specifically binds to the APJ receptor and is further enzymatically processed in the endoplasmic reticulum and Golgi apparatus into smaller molecular weight isoforms, including ELA-32, ELA-21, and ELA-11(15-17), with ELA-32 improving cardiac dysfunction in sepsis, pressure overload, preeclampsia, and pulmonary hypertension(18), ELA-21 protecting against cardiac injury, and ELA-11 preventing renal ischaemia‒reperfusion–induced acute kidney injury(19, 20). In brief, ELA has demonstrated therapeutic potential in cardiorenal disease. However, its clinical application is limited by a short circulating half-life of approximately 13 min(21, 22). To address this issue, we previously successfully generated the Fc-ELA fusion protein to extend the half-life of ELA; only Fc-ELA-21 remained intact without cleavage, and Fc-ELA-21 significantly mitigated cardiac dysfunction and pathological changes in myocardial infarction (MI) model rats in our study(23). However, there has been no research on metabolic syndrome. In this study, we developed ACF210, a dual receptor agonist designed to simultaneously activate both the GLP-1 and APJ receptors. By fusing GLP-1 and ELA-21 peptides to the Fc fragment of human IgG4, ACF210 exerts both the metabolism-improving effects of GLP-1 and the cardiorenal protective effects of ELA. The primary objective of this study was to evaluate the therapeutic potential of ACF210 in a mouse model of T2D-induced CKM syndrome, with a particular focus on glycemic homeostasis and cardiorenal protection. Materials and Methods 2.1 Synthesis of ACF210 ACF210 and Fc-ELA-21(23) were synthesized by Wuxi Biologic and provided by Wuxi AccufusionBiotech Co., Ltd. (Wuxi, China). As shown in Fig. 1A, the Fc fragment of human IgG4 was employed as a structural scaffold. A GLP-1 analogue derived from dulaglutide (Eli Lilly, USA) and the APJ receptor ligand ELA-21 were fused to the N- and C-termini of the Fc fragment via linker peptides. The biofunctional fusion protein also incorporated a signal peptide to facilitate expression in Chinese hamster ovary (CHO) cells. The protein was subsequently expressed and purified. The full amino acid sequence of ACF210 is presented in Supplementary Table 1. A three-dimensional schematic representation of the ACF210 molecular structure is shown in Fig. 1B. 2.2 Receptor Activation Assay To evaluate the receptor activity of ACF210 on GLP-1R and APJ, a receptor activation assay was conducted by Codex (Maryland, USA). The assay employed BD ACTOne™ high-throughput screening technology, which measures cyclic cAMP levels in living cells(24). HEK293T cells stably expressing either GLP-1R or APJ were treated with various agonists, and intracellular cAMP levels were quantified using fluorescent membrane potential dyes. The half-maximal effective concentration (EC 50 ) values after ACF210 treatment were determined for both receptors and compared with those after dulaglutide treatment and Fc-ELA treatment. The HEK293-CNG-APJ and HEK293-CNG-GLP-1R cell lines produced by BD Bioscience (Erembodegem, Belgium) are based on the BD ACTOne™ parental cell line (HEK293T cells) stably expressing a mutated CNG gene. The APJ or GLP-1R gene in the retroviral vector pBabe was introduced by retroviral infection from the culture supernatant of transfected phoenix-ampho cells, followed by the selection of puromycin-resistant clones of virus-infected cells and the measurement of membrane potential changes triggered by receptor activation. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated foetal bovine serum, 1 µg/mL puromycin, and 250 µg/mL G418 at 37°C and 5% CO2. The cells were harvested by gentle trypsinization, seeded aseptically into poly-D-lysine-coated 384-well cell culture plates (BD Biocoat) at a density of 10,000 cells per well and cultured overnight at 37°C. The membrane potential was measured with a BD ACTOne™ fluorescent membrane potential assay kit (Erembodegem, Belgium). The membrane potential dye was diluted 8-fold with dye dilution buffer and added to the cells without removing the medium. The cells were then incubated at 37°C for 75 min. Baseline fluorescence was subsequently determined with a POLARstar Optima multimode microplate reader (BMG Labtech, Offenburg, Germany) with 544-nm excitation and 590-nm emission filters. Following baseline measurements, the cells were treated with different concentrations of agonist solution and incubated for an additional 60 min, followed by a second round of fluorescence measurements. 2.3 Animal Experiments The animal experiments were approved by the Institutional Animal Care and Use Committee at Xi'an Jiaotong University (XJTUAE2024-2652). C57BL/6J male mice were fed a high-fat diet (HFD) (D12492, Reagy Dietech Co., China) for 8 weeks, followed by the intraperitoneal injection of streptozotocin (STZ, S0130, Sigma, USA; 40 mg/kg) for 3 consecutive days to induce T2D. In parallel, db/db mice (Cyagen Biosciences, Suzhou, China), a leptin receptor-deficient genetic model of T2D, were also used. The mice were randomly assigned to receive ACF210, Trulicity/dulaglutide (Eli Lilly and Company), Fc-ELA, or saline (vehicle control) at a dose of 1 mg/kg for 12 weeks. All the treatments were administered subcutaneously every other day. Body weight and fasting blood glucose levels (Contour TS, Bayer Co., Germany) were monitored biweekly. After 12 weeks of treatment, the mice were euthanized, and tissues were harvested for further analysis. 2.4 Glucose and Insulin Tolerance Tests At baseline and after 12 weeks of treatment, glucose tolerance was assessed using intraperitoneal glucose tolerance tests (IPGTTs), and insulin sensitivity was evaluated through intraperitoneal insulin tolerance tests (IPITTs). For IPGTTs, the mice were fasted for 6 hours, followed by the intraperitoneal injection of glucose (2 g/kg). For IPITTs, the mice received an intraperitoneal injection of insulin (0.7 U/kg). Blood glucose levels were measured at multiple time points (0, 30, 60, 90, and 120 min), and the area under the curve (AUC) was calculated. Pancreatic β-cell function and insulin resistance were estimated using homeostatic model assessment (HOMA) indices, which were calculated as follows(19): HOMA-β=20*Fasting insulin (mU/L)/[Fating glucose (mmol/L)*18.018-63] HOMA-IR=Fasting insulin (mU/L)*Fasting glucose (mmol/L)*18.018/405 2.5 Biochemical Assays Serum levels of insulin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and cystatin C were measured using enzyme-linked immunosorbent assays (ELISAs) (TW8514 and TW9810, TW-REAGENT, China) or an automated blood biochemical analyser (HITACHI, LABOSPECT008). Hepatic triglyceride content in homogenized liver tissue was measured (#74385, Sigma) followed by lipid quantification using a commercial triglyceride assay kit (#MAK266, Sigma‒Aldrich). 2.6 Echocardiography and Haemodynamic Measurements Echocardiography was performed using a Vevo LAZR-X ultrasound system (VisualSonics, Toronto, Canada) equipped with a 40 MHz transducer. Left ventricular (LV) dimensions, including left ventricular internal diameters at diastole and systole (LVIDd and LVIDs), were measured in M-mode. Additionally, tissue Doppler imaging was employed to evaluate myocardial velocities (E' and A'), and pulsed-wave Doppler was used to assess diastolic filling patterns. 2.7 Histological and Ultrastructural Analyses Tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for histological staining. Haematoxylin and eosin (H&E) staining was performed to evaluate general morphology, and Masson’s trichrome staining was used to assess tissue fibrosis (BA4079A, Baso, China). Kidney sections were stained with periodic acid-Schiff (PAS) reagent to examine glomerular alterations (BA4080A, Baso, China). Using a microscope (Leica Olympus IX-51), 3 nonoverlapping images were obtained per slide. For ultrastructural analysis, fresh heart, kidney and pancreas tissue samples (approximately 1 mm 3 ) in size were immediately placed in 2.5% glutaraldehyde fixative solution at 4°C for more than 2 h, followed by soaking in 0.1 M phosphoric acid buffer for 30 min, 1% osmium tetroxide fixative solution at 4°C for 2 h, and 0.1 M phosphoric acid buffer for 10 min and ethanol gradient dehydration. Then, the tissues were stained with 70% uranium dioxane acetate for 2 h, soaked in 90% ethanol for 10 min twice, soaked in 100% ethanol for 10 min 3 times, and incubated with oxypropane for 10 min. The tissue samples were embedded in Epon812 epoxy resin, and 1–2 µm semiultra-thin sections were obtained after tissue polymerization. The sections were then stained with Meilan and visualized under a light microscope. Ultrathin slicing (50–70 nm) was carried out using an UC7 ultrathin slicer from Leica, Germany. Finally, the sections were stained with uranium acetate and lead citrate and observed and photographed with a Hitachi H-7650 transmission electron microscope. Paraffin-embedded cardiac tissue sections (5 μm) were heated at 60°C for 3 h, deparaffinized in xylene and dehydrated in gradient concentrations of ethanol. Antigen repair of the tissue sections was subsequently performed in Tris-EDTA (pH = 9.0) in a microwave oven. Endogenous peroxidases were then deactivated with 3% hydrogen peroxide for 15 min. Subsequently, the sections were incubated in 10% goat serum for 30 min at room temperature to block nonspecific binding and then with a primary antibody against CD31 (Abcam, No. ab28364, 1:50) overnight at 4°C. On the second day, the sections were incubated with the corresponding secondary antibody at room temperature for 1 h. Then, diaminobenzidine (DAB) staining, haematoxylin staining, dehydration in gradient ethanol and clearing in xylene were performed. In each section, 3–5 high-magnification fields were randomly selected to count the number of CD31-positive microvessels. 2.8 Statistical Analysis All the data are presented as means ± SDs. Statistical analysis was performed using GraphPad Prism software (v6.0). EC 50 values for receptor activation were calculated using nonlinear regression analysis. Comparisons between two groups of independent samples were performed using unpaired 2-tailed Student’s t tests. For comparisons of more than three groups of independent samples, one-way ANOVA or two-way ANOVA was used. P ≤0.05 was considered significant. Results 3.1 Dual-receptor Targeting by ACF210 To validate the dual receptor agonist activity of ACF210, we assessed, using cAMP assays, the effects of ACF210 on the GLP-1R and APJ receptors in HEK293T cells. ACF210 effectively enhanced GLP-1R signalling, as evidenced by a dose-dependent increase in cAMP, with a calculated EC 50 value of 2.8 µg/ml (Fig. 2A). In contrast, ACF210 inhibited APJ receptor signalling, leading to a decrease in the intracellular cAMP level, with an EC 50 value of 75.47 µg/ml (Fig. 2B). Dulaglutide, a known GLP-1R agonist, activated GLP-1R but had no effect on the APJ receptor (Fig. 2C, D), whereas Fc-ELA exclusively activated the APJ receptor without affecting GLP-1R signalling (Fig. 2E, F). These results demonstrate that ACF210 functions as a dual agonist capable of modulating both the GLP-1R pathway and the APJ pathway. 3.2 ACF210 Improves Glucose Metabolism in T2D Mice To investigate the effects of ACF210 on glucose metabolism, we first established a T2D model by feeding mice a HFD combined with multiple injections of low-dose STZ, hereafter referred to as HFD+STZ mice. In parallel, we employed leptin receptor-deficient db/db mice, a well-established genetic model of T2D. The experimental design is illustrated in Fig. 3A and 3B. Compared with vehicle treatment, 12 weeks of treatment with ACF210 significantly reduced fasting blood glucose levels in both HFD+STZ mice (Fig. 3C) and db/db mice (Fig. 3D) ( p < 0.01). Glucose tolerance, as evaluated by IPGTTs and IPITTs, was markedly improved in both models following ACF210 treatment, with results comparable to those observed with dulaglutide (Fig. 3E-L). Notably, Fc-ELA did not alter blood glucose levels (Supplementary Fig. 2A-B), and ACF210-treated mice maintained stable body weight. For diabetic controls, significant weight reduction was observed in the dulaglutide-treated group (Supplementary Fig. 2C-D). In summary, the results suggest that the hypoglycaemic effect of ACF210 is mediated predominantly through GLP-1R activation. 3.3 ACF210 Enhances Islet β-Cell Function TEM revealed that ACF210 treatment preserved the ultrastructure of pancreatic islet β-cells in T2D mice. In contrast to the swollen mitochondria and dilated rough endoplasmic reticulum observed in β-cells from untreated diabetic mice, β-cells from ACF210-treated mice presented no significant organelle abnormalities (Fig. 4A). These morphological improvements were supported by HOMA-β analysis, which revealed enhanced β-cell function in ACF210-treated mice compared with diabetic controls (Fig. 4B). However, no significant difference in insulin resistance, as assessed by HOMA-IR, was observed between the ACF210 and Fc-ELA groups (Fig. 4C). These results indicate that ACF210 treatment may ameliorate hyperglycaemia and metabolic dysfunction primarily through the preservation of islet β-cell function without affecting systemic insulin sensitivity. 3.4 ACF210 Ameliorates Hepatic Steatosis In liver tissue, ACF210 treatment markedly attenuated hepatic steatosis, as evidenced by histological analysis results (Fig. 5A) and a reduction in lipid droplet size (Fig. 5B). Liver weight, hepatic triglyceride content, and serum ALT levels were significantly lower in the ACF210-treated group than in the diabetic control group (Fig. 5C-E). However, there was no significant difference in the serum AST levels between the different groups (Fig. 5F). Those effects were comparable to the effects of dulaglutide, whereas Fc-ELA had no significant effect on liver steatosis. These findings suggest that ACF210 ameliorates hepatic steatosis, possibly through GLP-1 receptor activation. 3.5 ACF210 Protects Cardiac Function in Mice with CKM induced by Diabetes Echocardiographic analysis revealed that ACF210 treatment improved cardiac structure in diabetic mice, as evidenced by reductions in left ventricular mass (Fig. 6A) and relative wall thickness (Fig. 6B). No significant differences in LVIDd or LVEF were observed among the groups (Fig. 6C, D and Supplementary Fig. 3A). Doppler ultrasound analysis revealed improved E/A and E/E' ratios in ACF210-treated mice, indicating enhanced diastolic function (Fig. 6E-G). Furthermore, ACF210 treatment significantly decreased the serum levels of NT-proBNP, a biomarker of heart failure (Fig. 6H). TEM analysis revealed a preserved myocardial ultrastructure in ACF210-treated mice, with cardiomyocytes displaying intact mitochondrial morphology (Fig. 6I). Compared with those in untreated diabetic mice, Masson’s trichrome staining revealed reduced perivascular and interstitial fibrosis in mice in the ACF210 group (Fig. 6J, L). Additionally, ACF210 treatment promoted angiogenesis, as evidenced by increased CD31-positive microvessel density (Fig. 6K, M). In brief, these findings provide evidence that ACF210 restores cardiac function and structure in diabetic mice. 3.6 ACF210 Protects against Diabetic Kidney Damage We next evaluated the effects of ACF210 on renal structure and function in T2D mice. ACF210 treatment significantly reduced mesangial cell proliferation and glomerular basement membrane (GBM) thickening, as demonstrated by PAS staining (Fig. 7A). TEM analysis revealed that diabetic mice without treatment presented uniform GBM thickening, irregular podocyte foot processes, and foot process effacement, which are hallmarks of diabetic nephropathy. Those pathological alterations were markedly alleviated in ACF210-treated mice (Fig. 7B). Although no significant differences in serum creatinine levels were observed among the groups (Fig. 7C), serum cystatin C levels were significantly lower in ACF210-treated mice than in diabetic control mice (Fig. 7D), suggesting improved renal function. Collectively, these results indicate that ACF210 exerts renoprotective effects in diabetic mice. Discussion In this study, we investigated the therapeutic potential of ACF210, a dual receptor agonist that targets both GLP-1R and the APJ receptor, in a mouse model of CKM induced by T2D. Our findings demonstrate that ACF210 improves glucose metabolism by enhancing pancreatic β-cell function and more significantly provides substantial protection against cardiorenal injury in mice with T2D-induced metabolic syndrome. With the global burden of T2D—which accounts for approximately 90–95% of all diagnosed diabetes cases—continuing to rise, the incidence and mortality of CKM syndrome, which was newly conceptualized by the AHA in 2023(5), have also increased, making cardiorenal disease a major contributor to morbidity and mortality in T2D patients(25, 26). Traditional treatments focus primarily on glycaemic control in patients with CKM induced by T2D(27); however, these approaches often fail to address the underlying pathophysiology or prevent cardiorenal complications because they involve the use of a single-target drug(28). GLP-1 receptor agonists (GLP-1RAs) represent an important therapeutic advancement in T2D management, offering not only improved glycaemic control but also cardiovascular benefits(29). However, their clinical efficacy is constrained by rapid degradation in the circulation(30). The fusion of GLP-1 analogues with IgG Fc fragments, as seen in drugs such as dulaglutide, has been shown to prolong the half-life of these drugs and improve patient compliance(31, 32). Nevertheless, the specific mechanisms underlying their organ-protective effects remain incompletely understood. Elabela (ELA), an endogenous ligand for the APJ receptor(33), has been shown to exert protective effects across multiple organ systems, including the heart and kidneys(21, 34, 35), but its clinical application is limited by its short plasma half-life. ACF210 was designed to overcome these limitations by combining the glucose-lowering effects of GLP-1R activation with the cardioprotective and renoprotective actions of ELA. Dual-receptor targeting by ACF210 was confirmed through in vitro assays, which demonstrated enhanced GLP-1R activation and the inhibition of APJ receptor signalling in this study. In vivo, ACF210 treatment significantly improved glucose metabolism in both the HFD+STZ-induced and db/db mouse models of T2D. Fasting blood glucose levels were reduced, and glucose tolerance was markedly increased, with ACF210 exhibiting efficacy comparable to that of dulaglutide. These findings suggest that the hypoglycaemic effect of ACF210 is primarily mediated via GLP-1R activation, a mechanism that has been well documented for GLP-1RAs(36). Notably, Fc-ELA, which lacks GLP-1R agonist activity, did not affect blood glucose levels, further supporting the pivotal role of GLP-1R in mediating the glucose-lowering effects of ACF210. Interestingly, in addition to its metabolic benefits, ACF210 also had protective effects on key diabetes-affected organs, including the heart, kidneys and liver. These pleiotropic effects suggest that ACF210 may offer broader therapeutic benefits than conventional monotherapies by targeting both metabolic and organ-specific pathologies. Furthermore, we found that ACF210 ameliorated cardiac diastolic dysfunction in T2D mice, alleviated cardiac remodelling and fibrosis, and reduced serum NT-proBNP levels. Previous studies have explored the cardioprotective role of metformin(37); although it has been shown to effectively improve glycaemic control in mice fed a high-fat, high-sugar diet, it fails to preserve endoplasmic reticulum–mitochondrial calcium coupling or prevent the development of cardiac hypertrophy and dysfunction, highlighting its limited efficacy in early diabetic cardiomyopathy(38-40). The SGLT2 inhibitor empagliflozin exerts some beneficial effects against heart failure with preserved ejection fraction (HFpEF), primarily by improving cardiac electrophysiological function; however, its effects exhibit sex-specific variability(41). Currently, some emerging therapies are under investigation and may offer improved outcomes for HFpEF patients in the future(42, 43). Importantly, our findings suggest that ACF210 has great therapeutic potential for diabetes-induced HFpEF. Electron microscopy revealed that ACF210 more effectively preserved the cardiomyocyte ultrastructure than both dulaglutide and Fc-ELA did. Previous studies have shown that dulaglutide activates GLP-1R, triggering the cAMP/PKA pathway, enhancing nitric oxide production, and activating AMPK signalling, thereby improving cardiac metabolism and function(44, 45); it also exerts anti-inflammatory and antioxidant effects(46, 47). Fc-ELA, on the one hand, activates the APJ receptor and modulates ERK1/2 and p38 MAPK signalling to promote vascular repair and regeneration(48-50). Additionally, it improves endothelial function through the PI3K/Akt pathway, reduces oxidative stress, and has antifibrotic effects(51, 52). Our results indicated that ACF210 exerts an anti-HFpEF effect likely through the dual activation of the GLP-1R and APJ dual receptor signalling pathways. Nonetheless, additional studies are warranted to further elucidate the precise mechanisms underlying its cardioprotective actions. Current treatment guidelines recommend sodium‒glucose cotransporter 2 inhibitors (SGLT2 inhibitors) as first-line therapies for diabetic kidney disease(53); however, their use is associated with a relatively high risk of genitourinary tract infections(54). The emergence of nonsteroidal mineralocorticoid receptor antagonists (MRAs) offers an alternative therapeutic approach by reducing sodium retention, potassium excretion, and inflammatory and fibrotic processes, thereby demonstrating cardiovascular and renoprotective effects(55, 56). However, these agents require the close monitoring of serum potassium levels, and further evidence is needed to confirm their long-term efficacy and safety(57). GLP-1RAs may exert renoprotective effects not only through glycaemic control, blood pressure reductions and weight loss but also through direct actions on endothelial function and inflammation(58, 59). In addition, ELA has been shown to preserve glomerular architecture, with high kidney ELA expression protecting against AKI by preventing renal cell damage and apoptosis(34). Therefore, this study also evaluated the renoprotective potential of ACF210 in diabetic mice. Both ACF210 and dulaglutide significantly attenuated diabetic kidney injury, as evidenced by reduced serum cystatin C levels and the improved structural integrity of the renal filtration barrier. These findings support the therapeutic potential of ACF210 in preserving renal function under diabetic conditions. GLP-1 receptor agonists, such as liraglutide and semaglutide, have been shown to alleviate hepatic steatosis and reduce serum AST and ALT levels, potentially through weight loss mechanisms(60). In our study, ACF210 treatment produced more significant improvements in hepatic steatosis and liver enzyme profiles than did dulaglutide, despite the absence of a substantial weight reduction. These findings suggest that the hepatoprotective effects of ACF210 may not be solely attributable to weight loss. Thus, ACF210 may represent a more suitable therapeutic option for elderly or malnourished patients with type 2 diabetes who are unable to tolerate weight loss–based interventions. Several limitations of this study should be considered. First, the animal models employed may not fully replicate the complexity of CKM induced by T2D in humans, particularly in terms of metabolic and genetic heterogeneity. Although the findings are encouraging, clinical trials are essential to confirm the safety and therapeutic efficacy of ACF210 in humans. Furthermore, the long-term effects of ACF210—especially its influence on organ function during prolonged treatment—remain to be clarified. Finally, while ACF210 has significant organ-protective effects, the precise molecular mechanisms responsible for these effects require further elucidation. In conclusion, ACF210 represents a promising dual receptor agonist that not only improves glucose metabolism but also provides significant protection against diabetes-associated organ damage. These findings highlight the therapeutic potential of ACF210 as a novel double-target drug for treating CKM induced by T2D. Declarations DATA AVAILABILITY All the data generated from this study are presented in the main manuscript and supplementary information. The raw data are available upon request. ACKNOWLEDGEMENTS The authors thank Jimmy Lu (CodexBiosolutions, MD, USA) for providing support with the ACTOne™ high-throughput screening technology. FUNDING This work was supported by grants from the National Natural Science Foundation of China [81970329] and the Key Research and Development Program Project of Shaanxi Province, China [2025GH-YBXM-072]. AUTHOR CONTRIBUTIONS QZ contributed to the research design. QW, YD and HW conducted the experiments, analysed the data and interpreted the data. QW and HZ drafted the manuscript. QZ contributed to the discussion and revised the manuscript. QZ was responsible for obtaining financial support and study supervision. All the authors have read and approved the final version of this manuscript. COMPETING INTERESTS The authors declare that they have no competing interests. ETHICS APPROVAL AND CONSENT TO PARTICIPATE Ethics approval for animal work was provided by the Institutional Animal Care and Use Committee of The First Affiliated Hospital of Xi'an Jiaotong University (Approval Number: XJTUAE2024-2652). All methods in this study were conducted in accordance with the relevant institutional guidelines and regulations. This study did not involve human participants and therefore did not require any related ethics approval or consent to participate. References Xu Y, Lu J, Li M, Wang T, Wang K, Cao Q, et al. Diabetes in China part 1: epidemiology and risk factors. Lancet Public Health 9 , e1089-e1097 (2024). Zhou XD, Zheng MH. Cardiovascular-kidney-metabolic syndrome and MASLD: integrating medical perspectives. Nat Rev Cardiol (2025). https://doi.org/10.1038/s41569-025-01199-y Zhou M, Sun W, Gao Y, Jiang B, Sun T, Xu R, et al. Metabolomic profiling reveals interindividual metabolic variability and its association with cardiovascular-kidney-metabolic syndrome risk. Cardiovasc Diabetol 24, 315-329 (2025). Liu Y, Miao Q, Liu Y, Jiang M. Effects of chitosan guanidine on blood glucose regulation and gut microbiota in T2DM. Int J Biol Macromol 279 , 135422 (2024). Ndumele CE, Neeland IJ, Tuttle KR, Chow SL, Mathew RO, Khan SS, et al. A Synopsis of the Evidence for the Science and Clinical Management of Cardiovascular-Kidney-Metabolic (CKM) Syndrome: A Scientific Statement From the American Heart Association. Circulation 148, 1636-1664 (2023). Gray V, Chen W, Tan RJY, Teo JMN, Huang Z, Fong CH, et al. Hyperglycemia-triggered lipid peroxidation destabilizes STAT4 and impairs anti-viral Th1 responses in type 2 diabetes. Cell Metab 36, 2511-2527 (2024). Chan FL, Li YC, Chen XRC. Therapeutic inertia in proteinuria management among type 2 diabetes (T2DM) patients in primary care settings: prevalence and associated risk factors. BMC Fam Pract 22, 118-128 (2021). Das S, Ravi H, Babu A, Banerjee M, Kanagavalli R, Dhanasekaran S, et al. Therapeutic potentials of glucose-dependent insulinotropic polypeptide (GIP) in T2DM: Past, present, and future. Adv Protein Chem Struct Biol 142, 293-328 (2024). Willard FS, Ho JD, Sloop KW. Discovery and pharmacology of the covalent GLP-1 receptor (GLP-1R) allosteric modulator BETP: A novel tool to probe GLP-1R pharmacology. Adv Pharmacol 88, 173-191 (2020). Withaar C, Meems LMG, Nollet EE, Schouten EM, Schroeder MA, Knudsen LB, et al. The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF. JACC Basic Transl Sci 8, 1298-1314 (2023). Gallwitz B. GLP-1 agonists and dipeptidyl-peptidase IV inhibitors. Handb Exp Pharmacol 1 . 53-74 (2011). Zheng Z, Zong Y, Ma Y, Tian Y, Pang Y, Zhang C, et al. Glucagon-like peptide-1 receptor: mechanisms and advances in therapy. Signal Transduct Target Ther 9 , 234-263 (2024). Caruso I, Cignarelli A, Natalicchio A, Perrini S, Laviola L, Giorgino F. Commentary: Glucose control: Not just a bystander in GLP-1RA-mediated cardiovascular protection. Metabolism 109, 154272 (2020). Pagidipati NJ, Nelson AJ, Kaltenbach LA, Leyva M, McGuire DK, Pop-Busui R, et al. Coordinated Care to Optimize Cardiovascular Preventive Therapies in Type 2 Diabetes: A Randomized Clinical Trial. JAMA 329 , 1261-1270 (2023). Liu Y, Jiang M, Li Y, Chen P, Chen X. Advances in the study of ELABELA in renal physiological functions and related diseases. Front Pharmacol 14, 1276488-1276496 (2023). Ason B, Chen Y, Guo Q, Hoagland KM, Chui RW, Fielden M, et al. Cardiovascular response to small-molecule APJ activation. JCI Insight 5, e132898-e134904 (2020) Li C, Cheng H, Adhikari BK, Wang S, Yang N, Liu W, et al. The Role of Apelin-APJ System in Diabetes and Obesity. Front Endocrinol (Lausanne) 9 , 820002-820013 (2022). Nyimanu D, Kay RG, Kuc RE, Brown AJH, Gribble FM, Maguire JJ, et al. In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA. Peptides 145, 170642-170654 (2021). Wang X, Zhang L, Li P, Zheng Y, Yang Y, Ji S. Apelin/APJ system in inflammation. Int Immunopharmacol 109 , 108822-108832 (2022). Xu F, Wu M, Lu X, Zhang H, Shi L, Xi Y, et al. Effect of Fc-Elabela-21 on renal ischemia/reperfusion injury in mice: Mediation of anti-apoptotic effect via Akt phosphorylation. Peptides 147, 170682-170691 (2022). Gao S, Chen H. Therapeutic potential of apelin and Elabela in cardiovascular disease. Biomed Pharmacother 166, 115268-115282 (2023). Sharma M, Prabhavalkar KS, Bhatt LK. Elabela Peptide: An Emerging Target in Therapeutics. Curr Drug Targets 23 , 1304-1318 (2022). Wang Y. Recombinant Elabela-Fc fusion protein has extended plasma half-life and mitigates post-infarct heart dysfunction in rats. Int J Cardiol 300, 217-225 (2020). Visegrady A, Boros A, Nemethy Z, Kiss B, Keseru GM. Application of the BD ACTOne technology for the high-throughput screening of Gs-coupled receptor antagonists. J Biomol Screen 12, 1068-1073 (2007). Collaborators GBDD. Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021. Lancet 402 , 203-234 (2023). Claudel SE, Schmidt IM, Waikar SS, Verma A. Cumulative Incidence of Mortality Associated with Cardiovascular-Kidney-Metabolic (CKM) Syndrome. J Am Soc Nephrol 36 , 1343-1351 (2025). Moghissi ES, Korytkowski MT, DiNardo M, Einhorn D, Hellman R, Hirsch IB, et al. American Association of Clinical Endocrinologists and American Diabetes Association consensus statement on inpatient glycemic control. Diabetes Care 32 , 1119-1131 (2009). Ritchie RH, Abel ED. Basic Mechanisms of Diabetic Heart Disease. Circ Res 126 , 1501-1525 (2020). Ma X, Liu Z, Ilyas I, Little PJ, Kamato D, Sahebka A, et al. GLP-1 receptor agonists (GLP-1RAs): cardiovascular actions and therapeutic potential. Int J Biol Sci 17 , 2050-68 (2021). Kutz A, Kim DH, Wexler DJ, Liu J, Schneeweiss S, Glynn RJ, et al. Comparative Cardiovascular Effectiveness and Safety of SGLT-2 Inhibitors, GLP-1 Receptor Agonists, and DPP-4 Inhibitors According to Frailty in Type 2 Diabetes. Diabetes Care 46 , 2004-2014 (2023). Gerstein HC, Colhoun HM, Dagenais GR, Diaz R, Lakshmanan M, Pais P, et al. Dulaglutide and cardiovascular outcomes in type 2 diabetes (REWIND): a double-blind, randomised placebo-controlled trial. Lancet 394 , 121-30 (2019). Pratley RE, Aroda VR, Lingvay I, Ludemann J, Andreassen C, Navarria A, et al. Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes (SUSTAIN 7): a randomised, open-label, phase 3b trial. Lancet Diabetes Endocrinol 6 , 275-286 (2018). Monastero R, Magro D, Venezia M, Pisano C, Balistreri CR. A promising therapeutic peptide and preventive/diagnostic biomarker for age-related diseases: The Elabela/Apela/Toddler peptide. Ageing Res Rev 91, 102076-102085 (2023). Xiong M, Chen H, Fan Y, Jin M, Yang D, Chen Y, et al. Tubular Elabela-APJ axis attenuates ischemia-reperfusion induced acute kidney injury and the following AKI-CKD transition by protecting renal microcirculation. Theranostics 13 , 3387-3401 (2023). Ma Z, Song JJ, Martin S, Yang XC, Zhong JC. The Elabela-APJ axis: a promising therapeutic target for heart failure. Heart Fail Rev 26 , 1249-1258 (2021). Akoumianakis I, Zagaliotis A, Konstantaraki M, Filippatos TD. GLP-1 analogs and regional adiposity: A systematic review and meta-analysis. Obes Rev 24 , e13574-e13584 (2023). Yao L, Wang L, Zhang R, Soukas AA, Wu L. The direct targets of metformin in diabetes and beyond. Trends Endocrinol Metab 36 , 364-372 (2025). Dia M, Leon C, Chanon S, Bendridi N, Gomez L, Rieusset J, et al. Effect of Metformin on T2D-Induced MAM Ca(2+) Uncoupling and Contractile Dysfunction in an Early Mouse Model of Diabetic HFpEF. Int J Mol Sci 23 , 3569-3577 (2022). Chen YC, Chung CH, Weng TH, Tsai CT, Cheng LH, Chien WC. Metformin Use Reduces the Risk of Olfactory Dysfunction in Diabetic Patients: A 22-Year Follow-Up Study. Int Forum Allergy Rhinol 15, 658-661 (2025). Chan DC, Lin YC, Tzeng HP, Yang RS, Chiang MT, Liu SH. Exendin-4, a glucagon-like peptide-1 receptor agonist, alleviates muscular dysfunction and wasting in a streptozotocin-induced diabetic mouse model compared to metformin. Tissue Cell 89 , 102479- 102488 (2024). Mira Hernandez J, Shen EY, Ko CY, Hourani Z, Spencer ER, Smoliarchuk D, et al. Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model. Cardiovasc Res 121, 254-266 (2024). Campbell P, Rutten FH, Lee MM, Hawkins NM, Petrie MC. Heart failure with preserved ejection fraction: everything the clinician needs to know. Lancet 403 , 1083-1092 (2024). Paterek A, Zaleska-Kociecka M, Surzykiewicz M, Wojdynska Z, Leszek P, Maczewski M. Non-coding RNA therapeutics in the treatment of heart failure. Eur Heart J Cardiovasc Pharmacother 10 , 353-360 (2024). Darwish AB, El Sayed NS, Salama AAA, Saad MA. Dulaglutide impedes depressive-like behavior persuaded by chronic social defeat stress model in male C57BL/6 mice: Implications on GLP-1R and cAMP/PKA signaling pathway in the hippocampus. Life Sci 320 , 121546 (2023). Lee J, Hong SW, Kim MJ, Moon SJ, Kwon H, Park SE, et al. Glucagon-Like Peptide Receptor Agonist Inhibits Angiotensin II-Induced Proliferation and Migration in Vascular Smooth Muscle Cells and Ameliorates Phosphate-Induced Vascular Smooth Muscle Cells Calcification. Diabetes Metab J 48 , 83-96 (2024). Kamiya M, Mizoguchi F, Yasuda S. Amelioration of inflammatory myopathies by glucagon-like peptide-1 receptor agonist via suppressing muscle fibre necroptosis. J Cachexia Sarcopenia Muscle 13 , 2118-2131 (2022). Meurot C, Martin C, Sudre L, Breton J, Bougault C, Rattenbach R, et al. Liraglutide, a glucagon-like peptide 1 receptor agonist, exerts analgesic, anti-inflammatory and anti-degradative actions in osteoarthritis. Sci Rep 12 , 1567-1582 (2022). Luo X, Liu J, Zhou H, Chen L. Apelin/APJ system: A critical regulator of vascular smooth muscle cell. J Cell Physiol 233 , 5180-5188 (2018). Su J, Zhang Y, Kumar SA, Sun M, Yao Y, Duan Y. APJ/apelin: A promising target for the treatment of retinopathy of prematurity. Drug Discov Today 27 , 2342-2352 (2022). Wang X, Zhang L, Feng M, Xu Z, Cheng Z, Qian L. ELA-11 protects the heart against oxidative stress injury induced apoptosis through ERK/MAPK and PI3K/AKT signaling pathways. Front Pharmacol 13 , 873614--873627 (2022). Ye L, Huang Y, Liu X, Zhang X, Cao Y, Kong X, et al. Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia. Free Radic Biol Med 208 , 759-770 (2023). Fu J, Chen X, Liu X, Xu D, Yang H, Zeng C, et al. ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways. Stem Cell Res Ther 11 , 541-553 (2020). Czupryniak L, Mosenzon O, Rychlik I, Clodi M, Ebrahimi F, Janez A, et al. Barriers to early diagnosis of chronic kidney disease and use of sodium-glucose cotransporter-2 inhibitors for renal protection: A comprehensive review and call to action. Diabetes Obes Metab 26 , 4165-4177 (2024). Aboukaoud M, Morhi Y, Osher E. Sodium-Glucose Co-Transporter 2 Inhibitors and Severe Urinary Tract Infections: Real-World Meta-Analysis of Cohort Studies. Ann Pharmacother ( 2025). https://doi.org/10.1177/10600280241312432 Kintscher U, Bakris GL, Kolkhof P. Novel non-steroidal mineralocorticoid receptor antagonists in cardiorenal disease. Br J Pharmacol 179 , 3220-3234 (2022). Huart J, Jouret F. Non-steroidal mineralocorticoid receptor antagonists: a paradigm shift in the management of diabetic nephropathy. Kidney Blood Press Res 50, 267-275 (2025). Jiang X, Zhang Z, Li C, Zhang S, Su Q, Yang S, et al. Efficacy and Safety of Non-Steroidal Mineralocorticoid Receptor Antagonists in Patients With Chronic Kidney Disease and Type 2 Diabetes: A Systematic Review Incorporating an Indirect Comparisons Meta-Analysis. Front Pharmacol 13 , 896947-896961 (2022). Koh G. Enhancing Patient Outcomes: Prioritizing SGLT2is and GLP-1RAs in Diabetes with CVD. Diabetes Metab J 48 , 208-220 (2024) . Kunutsor SK, Seidu S, Dey RS, Baidoo IK, Oulhaj A. Cardiovascular and kidney benefits of SGLT-2is and GLP-1RAs according to baseline blood pressure in type 2 diabetes: a systematic meta-analysis of cardiovascular outcome trials. Scand Cardiovasc J 58 , 2418086-2418098 (2024). Zhang Q, Wang J, Hu X, Lu W, Cao Y, Niu C, et al. GLP-1RAs regulate lipid metabolism and induce autophagy through AMPK/SIRT1 pathway to improve NAFLD. Prostaglandins Other Lipid Mediat 178 , 106987-106998 (2025). Additional Declarations There is NO Competing Interest. Supplementary Files Supplementarymaterial.docx Supplementary material. supplementaryfigure1.tif supplementary figure1.tif supplementaryfigure2.tif supplementary figure 2 supplementaryfigure3.tif supplementary figure3 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7782667","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":530437383,"identity":"d94af41a-14e2-4552-93f3-4af1d29a0942","order_by":0,"name":"Qingbin Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIiWNgGAWjYDACCRBhAETMDAyHf1RIyMmTooXxMcMZC2PDBqK0QHQxGzO2VSQyHCCgQ35288MHbwru2G1n5z0mXThPIoGxgfnhoxt4tDDOOWZsOMfgWfLOZr406ZnbJPLYGdiMjXPwaGGWSDCT5jE4nGxwmMdMgnebRDFjAw+bND4tbBLp35C0zJFIbDhAQAuPRA7YFjugFmNj3gYitEhI5BQD/XI4wbKZx/DhjGMSxobNBPwiPyN944M3fw7bm/OfMTjwoaZOTp69+eFjfFogrmNgSGyA85gJKYdqsSdG3SgYBaNgFIxQAABQlkaE16B8oAAAAABJRU5ErkJggg==","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":true,"prefix":"","firstName":"Qingbin","middleName":"","lastName":"Zhao","suffix":""},{"id":530437384,"identity":"2177783c-73af-4d8d-81eb-8c2c1017d715","order_by":1,"name":"Qingfeng Wu","email":"","orcid":"https://orcid.org/0000-0002-6687-307X","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Qingfeng","middleName":"","lastName":"Wu","suffix":""},{"id":530437385,"identity":"1fba59f1-97f5-4e0e-ba1b-551fe970e928","order_by":2,"name":"Huiyi Wei","email":"","orcid":"","institution":"School of Medicine, Yan'an University","correspondingAuthor":false,"prefix":"","firstName":"Huiyi","middleName":"","lastName":"Wei","suffix":""},{"id":530437386,"identity":"ff85a1e5-3552-470d-b25e-79c86f7c9f45","order_by":3,"name":"Yuanyuan Du","email":"","orcid":"","institution":"The First Affiliated Hospital of Xi'an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yuanyuan","middleName":"","lastName":"Du","suffix":""},{"id":530437387,"identity":"a3b8d8c5-bab4-40d1-ba72-414c24137135","order_by":4,"name":"Huifen Zhou","email":"","orcid":"","institution":"Hubei University of Science and Technology, Hubei, Xianning","correspondingAuthor":false,"prefix":"","firstName":"Huifen","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-10-05 04:15:31","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7782667/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7782667/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":93736648,"identity":"21aaab0c-65d3-4901-a8a6-aab63f1f712e","added_by":"auto","created_at":"2025-10-17 03:39:06","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":340697,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic representation of the ACF210 fusion protein design.\u003c/p\u003e\n\u003cp\u003e(A) Overview of the ACF210 fusion protein structure, linking the human IgG4 Fc fragment with GLP-1 and ELA-21. (B) 3D structure of ACF210. SEQ ID NO.1: Linker 1; SEQ ID NO.2: Linker 2; SEQ ID NO.3: IgG4 Fc fragment; SEQ ID NO.4: GLP-1 analogue; SEQ ID NO.5: Elabela; SEQ ID NO.6: Signal peptide; SEQ ID NO.7: Full ACF210 fusion protein sequence. The detailed amino acid sequences are provided in the Supplementary Materials.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/aac8108b712ecd6635f15ca0.png"},{"id":93736038,"identity":"a944d549-2410-43b6-b01c-942e41b019ad","added_by":"auto","created_at":"2025-10-17 03:31:06","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":386397,"visible":true,"origin":"","legend":"\u003cp\u003eDose‒response analysis of the effects of ACF210 on the GLP-1 and APJ receptors.\u003c/p\u003e\n\u003cp\u003eHEK293T-APJ and HEK293T-GLP-1R cells were treated with various concentrations of agonists. The fluorescenceratio (Ft/F0) correlates with cAMP levels measured 60 min posttreatment. (A, B) Effects of ACF210 on the GLP-1 and APJ receptors. (C, D) Effects of Trulicity on the GLP-1 and APJ receptors. (E-F) Effects of Fc-ELA21 on the GLP-1 and APJ receptors. The data are presented as the mean ± SD of multiple experiments.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/91b1da9b1608e961dd3fe1a5.png"},{"id":93736040,"identity":"b25b3125-b185-4842-99b0-7c413a6e2c1e","added_by":"auto","created_at":"2025-10-17 03:31:06","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1150106,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of ACF210 on glucose metabolism in T2D mice.\u003c/p\u003e\n\u003cp\u003e(A) Experimental protocol for C57BL/6J mice: high-fat diet (HFD) for 8 weeks followed by injection with STZ and treatment with ACF210, dulaglutide, or Fc-ELA. (B) Protocol for db/db mice. (C, D) Fasting blood glucose levels in C57BL/6J (C) and db/db (D) mice after 12 weeks of treatment. (E, G) IPGTT curves for C57BL/6J (E) and db/db mice (G). (F, H) Areas under the IPGTT curve (AUCs) for C57BL/6J (F) and db/db (H) mice. The data are presented as the mean ± SD, with statistical significance indicated.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/1325af2cb6d8bcbe5e2f8a01.png"},{"id":93736042,"identity":"f46aba99-4d3c-4288-8022-4f19cf5ab017","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2063616,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACF210 on islet β-cells.\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy images of islet β-cells in treated mice. (B) Posttreatment HOMA-β values. (C) Posttreatment HOMA-IR values. The data are shown as the mean ± SD and were analysed via one-way ANOVA. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/9eafdc64e40005dba25ab6a5.png"},{"id":93736041,"identity":"c45151e7-bf8b-4cad-b2ca-e4626ad1ee1f","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3265513,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACF210 on hepatic steatosis in diabetic mice.\u003c/p\u003e\n\u003cp\u003e(A) Representative images of H\u0026amp;E-stained liver tissue showing hepatic steatosis. (B) Fat droplet area ratio in liver tissue was quantified using ImageJ. (C) Liver weight changes after treatment. (D) Liver triglyceride (TG) content posttreatment. (E, F) Serum ALT and AST levels. The data are presented as the mean ± SD and were analysed via one-way ANOVA. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/7e39954f54c23fd1679aadc7.png"},{"id":93736043,"identity":"1293f65b-7435-4697-ac54-a42e3b5fdbe0","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":11809989,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACF210 on cardiac function in diabetic mice.\u003c/p\u003e\n\u003cp\u003e(A) Comparison of LV mass. (B) Comparison of relative wall thickness (RWT). (C) Representative M-mode echocardiographic images of the left ventricle (LV) in mice in each group. (D) Comparison of LVIDd. (E, F) Doppler flow and tissue Doppler ultrasound showing LV diastolic function. (H) Serum NT-proBNP levels. (I) TEM images of the left ventricular myocardium. (J) Masson’s staining for myocardial fibrosis. (K) Immunohistochemical staining of CD31-positive microvessels. (L) Fibrosis area analysis via Masson’s trichrome staining. (M) Microvessel density quantified from CD31-positive vessels. The data are presented as the mean ± SD and were analysed via one-way ANOVA. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/5b69436c6c2c2565601735e5.png"},{"id":93736044,"identity":"f1b43591-10c4-45e6-a049-d2fbb156c7bc","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":5751694,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ACF210 on renal function in diabetic mice.\u003c/p\u003e\n\u003cp\u003e(A) Images of PAS-stained kidney tissues showing mesangial cell proliferation and basement membrane thickening. (B) TEM images of the glomerular filtration barrier. (C) Serum creatinine levels. (D) Serum cystatin C levels posttreatment. The data are presented as the mean ± SD and were analysed by one-way ANOVA. \u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01; and \u003csup\u003e***\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/0e2025bcfee9b8f7b6ea3218.png"},{"id":95312119,"identity":"6dacbd7d-f10f-4559-8535-6b2f4649a5c8","added_by":"auto","created_at":"2025-11-06 15:47:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":29698234,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/dc538aab-5f1b-48b4-b302-9a2307335e22.pdf"},{"id":93736037,"identity":"fbf82cc0-d3fe-46cc-b0bd-dd53199a43fb","added_by":"auto","created_at":"2025-10-17 03:31:06","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":18485,"visible":true,"origin":"","legend":"Supplementary material.","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/55f4bf5aa66d946de32fd2aa.docx"},{"id":93736649,"identity":"df7fec01-2345-4cee-b402-44c9ad2f28b9","added_by":"auto","created_at":"2025-10-17 03:39:07","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":30141716,"visible":true,"origin":"","legend":"supplementary figure1.tif","description":"","filename":"supplementaryfigure1.tif","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/124361f2941af807586b4633.tif"},{"id":93736046,"identity":"9fd3562e-28fa-4bb0-944e-b4d419a26cc0","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":31382412,"visible":true,"origin":"","legend":"supplementary figure 2","description":"","filename":"supplementaryfigure2.tif","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/17ebb9b6443d1b8f23596825.tif"},{"id":93736047,"identity":"65353e52-e622-40eb-9296-86d7ec539549","added_by":"auto","created_at":"2025-10-17 03:31:07","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":27623452,"visible":true,"origin":"","legend":"supplementary figure3","description":"","filename":"supplementaryfigure3.tif","url":"https://assets-eu.researchsquare.com/files/rs-7782667/v1/e48fd45e42ebefa1e04c2a6e.tif"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Protective Effects of ACF210, a Dual GLP-1/APJ Receptor Agonist, against Cardiovascular-Kidney-Metabolic Syndrome Induced by T2D","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular-kidney-metabolic (CKM) syndrome has emerged as a critical paradigm for understanding the interconnected complications of type 2 diabetes (T2D), characterized by a vicious cycle of metabolic dysfunction, cardiovascular damage, and renal impairment—together contributing to significantly elevated morbidity and mortality and posing a major public health challenge(1, 2). The pathophysiological mechanism of CKM syndrome is multifactorial(3), T2D-induced CKM syndrome is characterized by chronic hyperglycaemia or advanced glycation end product (AGE) production resulting from insulin resistance and impaired insulin secretion due to pancreatic β-cell dysfunction(4). This triggers a cascade of pathological changes including cardiac remodelling, renal glomerulosclerosis, and hepatic steatosis through a mechanism involving excessive activation of RAAS, endoplasmic reticulum stress, abnormal calcium processing, mitochondrial dysfunction and energy metabolism disorders, oxidative stress and chronic inflammation(5), these processes collectively lead to multiorgan damage, imposing a substantial burden on healthcare systems(6). Although single-target glucose-lowering agents have demonstrated certain cardiovascular and renal protective effects in clinical studies of T2D, the complex pathophysiology of CKM syndrome induced by T2D necessitates multitarget therapeutic strategies that address the underlying disease mechanisms and provide protection across various organ systems(7).\u003c/p\u003e\n\u003cp\u003eRecent therapeutic advances have focused on glucagon-like peptide-1 receptor agonists (GLP-1RAs), a class of agents that mimic the action of GLP-1, a hormone secreted by L cells in the intestinal mucosa(8). GLP-1RAs increase glucose-dependent insulin secretion, suppress glucagon release, delay gastric emptying, and reduce appetite, thereby contributing to weight loss and ameliorating T2D-associated metabolic symptoms(9, 10). However, despite these advantages, native GLP-1 is rapidly degraded by dipeptidyl peptidase-4 (DPP-4) in circulation(11), which limits its therapeutic application. To address this limitation, GLP-1 analogues such as dulaglutide have been developed, in which a GLP-1 peptide is fused to the Fc fragment of human IgG4, thereby prolonging the half-life and enabling less frequent dosing(12).In addition, clinical studies have demonstrated that GLP-1RAs provide cardiovascular benefits and reduce the risk of heart failure(13, 14). Nevertheless, the underlying molecular mechanisms through which GLP-1RAs improve cardiovascular outcomes remain poorly understood. Consequently, the cardiorenal protective effects of GLP-1RAs in T2D warrant further mechanistic exploration.\u003c/p\u003e\n\u003cp\u003eElabela (ELA), also known as Toddler, is an endogenous ligand that specifically binds to the APJ receptor and is further enzymatically processed in the endoplasmic reticulum and Golgi apparatus into smaller molecular weight isoforms, including ELA-32, ELA-21, and ELA-11(15-17), with ELA-32 improving cardiac dysfunction in sepsis, pressure overload, preeclampsia, and pulmonary hypertension(18), ELA-21 protecting against cardiac injury, and ELA-11 preventing renal ischaemia‒reperfusion–induced acute kidney injury(19, 20). In brief, ELA has demonstrated therapeutic potential in cardiorenal disease. However, its clinical application is limited by a short circulating half-life of approximately 13 min(21, 22). To address this issue, we previously successfully generated the Fc-ELA fusion protein to extend the half-life of ELA; only Fc-ELA-21 remained intact without cleavage, and Fc-ELA-21 significantly mitigated cardiac dysfunction and pathological changes in myocardial infarction (MI) model rats in our study(23). However, there has been no research on metabolic syndrome.\u003c/p\u003e\n\u003cp\u003eIn this study, we developed ACF210, a dual receptor agonist designed to simultaneously activate both the GLP-1 and APJ receptors. By fusing GLP-1 and ELA-21 peptides to the Fc fragment of human IgG4, ACF210 exerts both the metabolism-improving effects of GLP-1 and the cardiorenal protective effects of ELA. The primary objective of this study was to evaluate the therapeutic potential of ACF210 in a mouse model of T2D-induced CKM syndrome, with a particular focus on glycemic homeostasis and cardiorenal protection.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Synthesis of ACF210\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eACF210 and Fc-ELA-21(23) were synthesized by Wuxi Biologic and provided by Wuxi AccufusionBiotech Co., Ltd. (Wuxi, China). As shown in Fig. 1A, the Fc fragment of human IgG4 was employed as a structural scaffold. A GLP-1 analogue derived from dulaglutide (Eli Lilly, USA) and the APJ receptor ligand ELA-21 were fused to the N- and C-termini of the Fc fragment via linker peptides. The biofunctional fusion protein also incorporated a signal peptide to facilitate expression in Chinese hamster ovary (CHO) cells. The protein was subsequently expressed and purified. The full amino acid sequence of ACF210 is presented in Supplementary Table 1. A three-dimensional schematic representation of the ACF210 molecular structure is shown in Fig. 1B.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Receptor Activation Assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate the receptor activity of ACF210 on GLP-1R and APJ, a receptor activation assay was conducted by Codex (Maryland, USA). The assay employed BD ACTOne™ high-throughput screening technology, which measures cyclic cAMP levels in living cells(24). HEK293T cells stably expressing either GLP-1R or APJ were treated with various agonists, and intracellular cAMP levels were quantified using fluorescent membrane potential dyes. The half-maximal effective concentration (EC\u003csub\u003e50\u003c/sub\u003e) values after ACF210 treatment were determined for both receptors and compared with those after dulaglutide treatment and Fc-ELA treatment.\u003c/p\u003e\n\u003cp\u003eThe HEK293-CNG-APJ and HEK293-CNG-GLP-1R cell lines produced by BD Bioscience (Erembodegem, Belgium) are based on the BD ACTOne™ parental cell line (HEK293T cells) stably expressing a mutated CNG gene. The APJ or GLP-1R gene in the retroviral vector pBabe was introduced by retroviral infection from the culture supernatant of transfected phoenix-ampho cells, followed by the selection of puromycin-resistant clones of virus-infected cells and the measurement of membrane potential changes triggered by receptor activation. The cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated foetal bovine serum, 1 µg/mL puromycin, and 250 µg/mL G418 at 37°C and 5% CO2. The cells were harvested by gentle trypsinization, seeded aseptically into poly-D-lysine-coated 384-well cell culture plates (BD Biocoat) at a density of 10,000 cells per well and cultured overnight at 37°C. The membrane potential was measured with a BD ACTOne™ fluorescent membrane potential assay kit (Erembodegem, Belgium). The membrane potential dye was diluted 8-fold with dye dilution buffer and added to the cells without removing the medium. The cells were then incubated at 37°C for 75 min. Baseline fluorescence was subsequently determined with a POLARstar Optima multimode microplate reader (BMG Labtech, Offenburg, Germany) with 544-nm excitation and 590-nm emission filters. Following baseline measurements, the cells were treated with different concentrations of agonist solution and incubated for an additional 60 min, followed by a second round of fluorescence measurements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Animal\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eExperiments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe animal experiments were approved by the Institutional Animal Care and Use Committee at Xi'an Jiaotong University (XJTUAE2024-2652). C57BL/6J male mice were fed a high-fat diet (HFD) (D12492, Reagy Dietech Co., China) for 8 weeks, followed by the intraperitoneal injection of streptozotocin (STZ, S0130, Sigma, USA; 40 mg/kg) for 3 consecutive days to induce T2D. In parallel, db/db mice (Cyagen Biosciences, Suzhou, China), a leptin receptor-deficient genetic model of T2D, were also used. The mice were randomly assigned to receive ACF210, Trulicity/dulaglutide (Eli Lilly and Company), Fc-ELA, or saline (vehicle control) at a dose of 1 mg/kg for 12 weeks. All the treatments were administered subcutaneously every other day. Body weight and fasting blood glucose levels (Contour TS, Bayer Co., Germany) were monitored biweekly. After 12 weeks of treatment, the mice were euthanized, and tissues were harvested for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Glucose and Insulin Tolerance Tests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt baseline and after 12 weeks of treatment, glucose tolerance was assessed using intraperitoneal glucose tolerance tests (IPGTTs), and insulin sensitivity was evaluated through intraperitoneal insulin tolerance tests (IPITTs). For IPGTTs, the mice were fasted for 6 hours, followed by the intraperitoneal injection of glucose (2 g/kg). For IPITTs, the mice received an intraperitoneal injection of insulin (0.7 U/kg). Blood glucose levels were measured at multiple time points (0, 30, 60, 90, and 120 min), and the area under the curve (AUC) was calculated. Pancreatic β-cell function and insulin resistance were estimated using homeostatic model assessment (HOMA) indices, which were calculated as follows(19):\u003c/p\u003e\n\u003cp\u003eHOMA-β=20*Fasting insulin (mU/L)/[Fating glucose (mmol/L)*18.018-63]\u003c/p\u003e\n\u003cp\u003eHOMA-IR=Fasting insulin (mU/L)*Fasting glucose (mmol/L)*18.018/405\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Biochemical Assays\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSerum levels of insulin, N-terminal pro-B-type natriuretic peptide (NT-proBNP), alanine transaminase (ALT), aspartate transaminase (AST), creatinine, and cystatin C were measured using enzyme-linked immunosorbent assays (ELISAs) (TW8514 and TW9810, TW-REAGENT, China) or an automated blood biochemical analyser (HITACHI, LABOSPECT008). Hepatic triglyceride content in homogenized liver tissue was measured (#74385, Sigma) followed by lipid quantification using a commercial triglyceride assay kit (#MAK266, Sigma‒Aldrich).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Echocardiography and Haemodynamic Measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEchocardiography was performed using a Vevo LAZR-X ultrasound system (VisualSonics, Toronto, Canada) equipped with a 40 MHz transducer. Left ventricular (LV) dimensions, including left ventricular internal diameters at diastole and systole (LVIDd and LVIDs), were measured in M-mode. Additionally, tissue Doppler imaging was employed to evaluate myocardial velocities (E' and A'), and pulsed-wave Doppler was used to assess diastolic filling patterns.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Histological and Ultrastructural Analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned for histological staining. Haematoxylin and eosin (H\u0026amp;E) staining was performed to evaluate general morphology, and Masson’s trichrome staining was used to assess tissue fibrosis (BA4079A, Baso, China). Kidney sections were stained with periodic acid-Schiff (PAS) reagent to examine glomerular alterations (BA4080A, Baso, China). Using a microscope (Leica Olympus IX-51), 3 nonoverlapping images were obtained per slide. For ultrastructural analysis, fresh heart, kidney and pancreas tissue samples (approximately 1 mm\u003csup\u003e3\u003c/sup\u003e) in size were immediately placed in 2.5% glutaraldehyde fixative solution at 4°C for more than 2 h, followed by soaking in 0.1 M phosphoric acid buffer for 30 min, 1% osmium tetroxide fixative solution at 4°C for 2 h, and 0.1 M phosphoric acid buffer for 10 min and ethanol gradient dehydration. Then, the tissues were stained with 70% uranium dioxane acetate for 2 h, soaked in 90% ethanol for 10 min twice, soaked in 100% ethanol for 10 min 3 times, and incubated with oxypropane for 10 min. The tissue samples were embedded in Epon812 epoxy resin, and 1–2 µm semiultra-thin sections were obtained after tissue polymerization. The sections were then stained with Meilan and visualized under a light microscope. Ultrathin slicing (50–70 nm) was carried out using an UC7 ultrathin slicer from Leica, Germany. Finally, the sections were stained with uranium acetate and lead citrate and observed and photographed with a Hitachi H-7650 transmission electron microscope.\u003c/p\u003e\n\u003cp\u003eParaffin-embedded cardiac tissue sections (5 μm) were heated at 60°C for 3 h, deparaffinized in xylene and dehydrated in gradient concentrations of ethanol. Antigen repair of the tissue sections was subsequently performed in Tris-EDTA (pH = 9.0) in a microwave oven. Endogenous peroxidases were then deactivated with 3% hydrogen peroxide for 15 min. Subsequently, the sections were incubated in 10% goat serum for 30 min at room temperature to block nonspecific binding and then with a primary antibody against CD31 (Abcam, No. ab28364, 1:50) overnight at 4°C. On the second day, the sections were incubated with the corresponding secondary antibody at room temperature for 1 h. Then, diaminobenzidine (DAB) staining, haematoxylin staining, dehydration in gradient ethanol and clearing in xylene were performed. In each section, 3–5 high-magnification fields were randomly selected to count the number of CD31-positive microvessels.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.8 Statistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data are presented as means ± SDs. Statistical analysis was performed using GraphPad Prism software (v6.0). EC\u003csub\u003e50\u003c/sub\u003e values for receptor activation were calculated using nonlinear regression analysis. Comparisons between two groups of independent samples were performed using unpaired 2-tailed Student’s \u003cem\u003et\u003c/em\u003e tests. For comparisons of more than three groups of independent samples, one-way ANOVA or two-way ANOVA was used. \u003cem\u003eP\u003c/em\u003e≤0.05 was considered significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Dual-receptor Targeting by ACF210\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo validate the dual receptor agonist activity of ACF210, we assessed, using cAMP assays, the effects of ACF210 on the GLP-1R and APJ receptors in HEK293T cells. ACF210 effectively enhanced GLP-1R signalling, as evidenced by a dose-dependent increase in cAMP, with a calculated EC\u003csub\u003e50\u003c/sub\u003e value of 2.8 µg/ml (Fig. 2A). In contrast, ACF210 inhibited APJ receptor signalling, leading to a decrease in the intracellular cAMP level, with an EC\u003csub\u003e50\u003c/sub\u003e value of 75.47 µg/ml (Fig. 2B). Dulaglutide, a known GLP-1R agonist, activated GLP-1R but had no effect on the APJ receptor (Fig. 2C, D), whereas Fc-ELA exclusively activated the APJ receptor without affecting GLP-1R signalling (Fig. 2E, F). These results demonstrate that ACF210 functions as a dual agonist capable of modulating both the GLP-1R pathway and the APJ pathway.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 ACF210 Improves Glucose Metabolism in T2D Mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo investigate the effects of ACF210 on glucose metabolism, we first established a T2D model by feeding mice a HFD combined with multiple injections of low-dose STZ, hereafter referred to as HFD+STZ mice. In parallel, we employed leptin receptor-deficient \u003cem\u003edb/db\u003c/em\u003e mice, a well-established genetic model of T2D. The experimental design is illustrated in Fig. 3A and 3B. Compared with vehicle treatment, 12 weeks of treatment with ACF210 significantly reduced fasting blood glucose levels in both HFD+STZ mice (Fig. 3C) and \u003cem\u003edb/db\u0026nbsp;\u003c/em\u003emice (Fig. 3D) (\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01). Glucose tolerance, as evaluated by IPGTTs and IPITTs, was markedly improved in both models following ACF210 treatment, with results comparable to those observed with dulaglutide (Fig. 3E-L). Notably, Fc-ELA did not alter blood glucose levels (Supplementary Fig. 2A-B), and ACF210-treated mice maintained stable body weight. For diabetic controls, significant weight reduction was observed in the dulaglutide-treated group (Supplementary Fig. 2C-D). In summary, the results suggest that the hypoglycaemic effect of ACF210 is mediated predominantly through GLP-1R activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 ACF210 Enhances Islet β-Cell Function\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTEM revealed that ACF210 treatment preserved the ultrastructure of pancreatic islet β-cells in T2D mice. In contrast to the swollen mitochondria and dilated rough endoplasmic reticulum observed in β-cells from untreated diabetic mice, β-cells from ACF210-treated mice presented no significant organelle abnormalities (Fig. 4A). These morphological improvements were supported by HOMA-β analysis, which revealed enhanced β-cell function in ACF210-treated mice compared with diabetic controls (Fig. 4B). However, no significant difference in insulin resistance, as assessed by HOMA-IR, was observed between the ACF210 and Fc-ELA groups (Fig. 4C). These results indicate that ACF210 treatment may ameliorate hyperglycaemia and metabolic dysfunction primarily through the preservation of islet β-cell function without affecting systemic insulin sensitivity.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 ACF210 Ameliorates Hepatic Steatosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn liver tissue, ACF210 treatment markedly attenuated hepatic steatosis, as evidenced by histological analysis results (Fig. 5A) and a reduction in lipid droplet size (Fig. 5B). Liver weight, hepatic triglyceride content, and serum ALT levels were significantly lower in the ACF210-treated group than in the diabetic control group (Fig. 5C-E). However, there was no significant difference in the serum AST levels between the different groups (Fig. 5F). Those effects were comparable to the effects of dulaglutide, whereas Fc-ELA had no significant effect on liver steatosis. These findings suggest that ACF210 ameliorates hepatic steatosis, possibly through GLP-1 receptor activation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 ACF210 Protects Cardiac Function in Mice with CKM induced by Diabetes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEchocardiographic analysis revealed that ACF210 treatment improved cardiac structure in diabetic mice, as evidenced by reductions in left ventricular mass (Fig. 6A) and relative wall thickness (Fig. 6B). No significant differences in LVIDd or LVEF were observed among the groups (Fig. 6C, D and Supplementary Fig. 3A). Doppler ultrasound analysis\u0026nbsp;revealed improved E/A and E/E' ratios in ACF210-treated mice, indicating enhanced diastolic function (Fig. 6E-G). Furthermore, ACF210 treatment significantly decreased the serum levels of NT-proBNP, a biomarker of heart failure (Fig. 6H). TEM analysis revealed a preserved myocardial ultrastructure in ACF210-treated mice, with cardiomyocytes displaying intact mitochondrial morphology (Fig. 6I). Compared with those in untreated diabetic mice, Masson’s trichrome staining revealed reduced perivascular and interstitial fibrosis in mice in the ACF210 group (Fig. 6J, L). Additionally, ACF210 treatment promoted angiogenesis, as evidenced by increased CD31-positive microvessel density (Fig. 6K, M). In brief, these findings provide evidence that ACF210 restores cardiac function and structure in diabetic mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 ACF210 Protects against Diabetic Kidney Damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe next evaluated the effects of ACF210 on renal structure and function in T2D mice. ACF210 treatment significantly reduced mesangial cell proliferation and glomerular basement membrane (GBM) thickening, as demonstrated by PAS staining (Fig. 7A). TEM analysis revealed that diabetic mice without treatment presented uniform GBM thickening, irregular podocyte foot processes, and foot process effacement, which are hallmarks of diabetic nephropathy. Those pathological alterations were markedly alleviated in ACF210-treated mice (Fig. 7B). Although no significant differences in serum creatinine levels were observed among the groups (Fig. 7C), serum cystatin C levels were significantly lower in ACF210-treated mice than in diabetic control mice (Fig. 7D), suggesting improved renal function. Collectively, these results indicate that ACF210 exerts renoprotective effects in diabetic mice.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the therapeutic potential of ACF210, a dual receptor agonist that targets both GLP-1R and the APJ receptor, in a mouse model of CKM induced by T2D. Our findings demonstrate that ACF210 improves glucose metabolism by enhancing pancreatic β-cell function and more significantly provides substantial protection against cardiorenal injury in mice with T2D-induced metabolic syndrome.\u003c/p\u003e\n\u003cp\u003eWith the global burden of T2D—which accounts for approximately 90–95% of all diagnosed diabetes cases—continuing to rise, the incidence and mortality of CKM syndrome, which was newly conceptualized by the AHA in 2023(5), have also increased, making cardiorenal disease a major contributor to morbidity and mortality in T2D patients(25, 26). Traditional treatments focus primarily on glycaemic control in patients with CKM induced by T2D(27); however, these approaches often fail to address the underlying pathophysiology or prevent cardiorenal complications because they involve the use of a single-target drug(28). GLP-1 receptor agonists (GLP-1RAs) represent an important therapeutic advancement in T2D management, offering not only improved glycaemic\u0026nbsp;control but also cardiovascular benefits(29). However, their clinical efficacy is constrained by rapid degradation in the circulation(30). The fusion of GLP-1 analogues with IgG Fc fragments, as seen in drugs such as dulaglutide, has been shown to prolong the half-life of these drugs and improve patient compliance(31, 32). Nevertheless, the specific mechanisms underlying their organ-protective effects remain incompletely understood. Elabela (ELA), an endogenous ligand for the APJ receptor(33), has been shown to exert protective effects across multiple\u0026nbsp;organ systems, including the heart and kidneys(21, 34, 35), but its clinical application is limited by its\u0026nbsp;short plasma half-life. ACF210 was designed to overcome these limitations by combining the glucose-lowering effects of GLP-1R activation with the cardioprotective and renoprotective actions of ELA. Dual-receptor targeting by ACF210 was confirmed through in vitro assays, which demonstrated enhanced GLP-1R\u0026nbsp;activation\u0026nbsp;and the inhibition\u0026nbsp;of\u0026nbsp;APJ receptor signalling in this study.\u003c/p\u003e\n\u003cp\u003eIn vivo, ACF210 treatment significantly improved glucose metabolism in both the HFD+STZ-induced and \u003cem\u003edb/db\u0026nbsp;\u003c/em\u003emouse models of T2D. Fasting blood glucose levels were reduced, and glucose tolerance was markedly increased, with ACF210 exhibiting efficacy comparable to that of dulaglutide. These findings suggest that the hypoglycaemic effect of ACF210 is primarily mediated via GLP-1R activation, a mechanism that has been well documented for GLP-1RAs(36). Notably, Fc-ELA, which lacks GLP-1R agonist activity, did not affect blood glucose levels, further supporting the pivotal role of GLP-1R in mediating the glucose-lowering effects of ACF210. Interestingly, in addition to its metabolic benefits, ACF210 also had protective effects on key diabetes-affected organs, including the heart, kidneys and liver. These pleiotropic effects suggest that ACF210 may offer broader therapeutic benefits than conventional monotherapies by targeting both metabolic and organ-specific pathologies.\u003c/p\u003e\n\u003cp\u003eFurthermore, we found that ACF210 ameliorated cardiac diastolic dysfunction in T2D mice, alleviated cardiac remodelling and fibrosis, and reduced serum NT-proBNP levels. Previous studies have explored the cardioprotective role of metformin(37); although it has been shown to effectively improve glycaemic control in mice fed a high-fat, high-sugar diet, it fails to preserve endoplasmic reticulum–mitochondrial calcium coupling or prevent the development of cardiac hypertrophy and dysfunction, highlighting its limited efficacy in early diabetic cardiomyopathy(38-40). The SGLT2 inhibitor empagliflozin exerts some beneficial effects against heart failure with preserved ejection fraction (HFpEF), primarily by improving cardiac electrophysiological function; however, its effects exhibit sex-specific variability(41). Currently, some emerging therapies are under investigation and may offer improved outcomes for HFpEF patients in the future(42, 43). Importantly, our findings suggest that ACF210 has great therapeutic potential for diabetes-induced HFpEF. Electron microscopy revealed that ACF210 more effectively preserved the cardiomyocyte ultrastructure than both dulaglutide and Fc-ELA did. Previous studies have shown that dulaglutide activates GLP-1R, triggering the cAMP/PKA pathway, enhancing nitric oxide production, and activating AMPK signalling, thereby improving cardiac metabolism and function(44, 45); it also exerts anti-inflammatory and antioxidant effects(46, 47). Fc-ELA, on the one hand, activates the APJ receptor and modulates ERK1/2 and p38 MAPK signalling to promote vascular repair and regeneration(48-50). Additionally, it improves endothelial function through the PI3K/Akt pathway, reduces oxidative stress, and has antifibrotic effects(51, 52). Our results indicated that ACF210 exerts an anti-HFpEF effect likely through the dual activation of the GLP-1R and APJ dual receptor signalling pathways. Nonetheless, additional studies are warranted to further elucidate the precise mechanisms underlying its cardioprotective actions. Current treatment guidelines recommend sodium‒glucose cotransporter 2 inhibitors (SGLT2 inhibitors) as first-line therapies for diabetic kidney disease(53); however, their use is associated with a relatively high risk of genitourinary tract infections(54). The emergence of nonsteroidal mineralocorticoid receptor antagonists (MRAs) offers an alternative therapeutic approach by reducing sodium retention, potassium excretion, and inflammatory and fibrotic processes, thereby demonstrating cardiovascular and renoprotective effects(55, 56). However, these agents require the close monitoring of serum potassium levels, and further evidence is needed to confirm their long-term efficacy and safety(57). GLP-1RAs may exert renoprotective effects not only through glycaemic control, blood pressure reductions and weight loss but also through direct actions on endothelial function and inflammation(58, 59). In addition, ELA has been shown to preserve glomerular architecture, with high kidney ELA expression protecting against AKI by preventing renal cell damage and apoptosis(34). Therefore, this study also evaluated the renoprotective potential of ACF210 in diabetic mice. Both ACF210 and dulaglutide significantly attenuated diabetic kidney injury, as evidenced by reduced serum cystatin C levels and the improved structural integrity of the renal filtration barrier. These findings support the therapeutic potential of ACF210 in preserving renal function under diabetic conditions. GLP-1 receptor agonists, such as liraglutide and semaglutide, have been shown to alleviate hepatic steatosis and reduce serum AST and ALT levels, potentially through weight loss mechanisms(60). In our study, ACF210 treatment produced more significant improvements in hepatic steatosis and liver enzyme profiles than did\u0026nbsp;dulaglutide, despite\u0026nbsp;the\u0026nbsp;absence\u0026nbsp;of a\u0026nbsp;substantial weight reduction. These findings suggest that the hepatoprotective effects of ACF210 may not be solely attributable to weight loss. Thus,\u0026nbsp;ACF210 may represent a more suitable therapeutic option for elderly\u0026nbsp;or malnourished patients with type 2 diabetes who are unable to tolerate weight loss–based interventions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSeveral limitations of this study should be considered. First, the animal models employed may not fully replicate the complexity of\u0026nbsp;CKM induced by T2D in humans, particularly in terms of metabolic and genetic heterogeneity. Although the findings are encouraging, clinical trials are essential to confirm the safety and therapeutic efficacy of ACF210 in humans. Furthermore, the long-term effects of ACF210—especially its influence on organ function during prolonged treatment—remain to be clarified. Finally, while ACF210 has significant organ-protective effects, the precise molecular mechanisms responsible for these effects require further elucidation.\u003c/p\u003e\n\u003cp\u003eIn conclusion, ACF210 represents a promising dual receptor agonist that not only improves glucose metabolism but also provides significant protection against diabetes-associated organ damage. These findings highlight the therapeutic potential of ACF210 as a novel double-target drug for treating CKM induced by T2D.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll the data generated from this study are presented in the main manuscript and\u003c/p\u003e\n\u003cp\u003esupplementary information. The raw data are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank Jimmy Lu (CodexBiosolutions, MD, USA) for providing support with the ACTOne™ high-throughput screening technology.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Natural Science Foundation of China [81970329] and the Key Research and Development Program Project of Shaanxi Province, China [2025GH-YBXM-072].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eQZ contributed to the research design. QW, YD and HW conducted the experiments, analysed the data and interpreted the data. QW and HZ drafted the manuscript. QZ\u0026nbsp;contributed to the discussion and revised the manuscript. QZ was responsible for obtaining financial support and study supervision. All the authors have read and approved the final version of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS APPROVAL AND CONSENT TO PARTICIPATE\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval for animal work was provided by the Institutional Animal Care and\u003c/p\u003e\n\u003cp\u003eUse Committee of The First Affiliated Hospital of Xi'an Jiaotong University (Approval Number: XJTUAE2024-2652). All methods in this study were conducted in accordance with the relevant institutional guidelines and regulations. This study did not involve human participants and therefore did not require any related ethics approval or consent to participate.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eXu Y, Lu J, Li M, Wang T, Wang K, Cao Q, et al. Diabetes in China part 1: epidemiology and risk factors. \u003cem\u003eLancet Public Health\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e\u003cem\u003e, \u003c/em\u003e\u003c/strong\u003ee1089-e1097 (2024).\u003c/li\u003e\n\u003cli\u003eZhou XD, Zheng MH. Cardiovascular-kidney-metabolic syndrome and MASLD: integrating medical perspectives. \u003cem\u003eNat Rev Cardiol\u003c/em\u003e (2025). https://doi.org/10.1038/s41569-025-01199-y\u003c/li\u003e\n\u003cli\u003eZhou M, Sun W, Gao Y, Jiang B, Sun T, Xu R, et al. Metabolomic profiling reveals interindividual metabolic variability and its association with cardiovascular-kidney-metabolic syndrome risk. \u003cem\u003eCardiovasc Diabetol\u003c/em\u003e \u003cstrong\u003e24,\u003c/strong\u003e 315-329 (2025).\u003c/li\u003e\n\u003cli\u003eLiu Y, Miao Q, Liu Y, Jiang M. Effects of chitosan guanidine on blood glucose regulation and gut microbiota in T2DM. \u003cem\u003eInt J Biol Macromol\u003c/em\u003e \u003cstrong\u003e279\u003c/strong\u003e\u003cem\u003e,\u003c/em\u003e135422 (2024).\u003c/li\u003e\n\u003cli\u003eNdumele CE, Neeland IJ, Tuttle KR, Chow SL, Mathew RO, Khan SS, et al. A Synopsis of the Evidence for the Science and Clinical Management of Cardiovascular-Kidney-Metabolic (CKM) Syndrome: A Scientific Statement From the American Heart Association. \u003cem\u003eCirculation\u003c/em\u003e \u003cstrong\u003e148,\u003c/strong\u003e 1636-1664 (2023).\u003c/li\u003e\n\u003cli\u003eGray V, Chen W, Tan RJY, Teo JMN, Huang Z, Fong CH, et al. Hyperglycemia-triggered lipid peroxidation destabilizes STAT4 and impairs anti-viral Th1 responses in type 2 diabetes. \u003cem\u003eCell Metab\u003c/em\u003e \u003cstrong\u003e36, \u003c/strong\u003e2511-2527 (2024).\u003c/li\u003e\n\u003cli\u003eChan FL, Li YC, Chen XRC. Therapeutic inertia in proteinuria management among type 2 diabetes (T2DM) patients in primary care settings: prevalence and associated risk factors. \u003cem\u003eBMC Fam Pract\u003c/em\u003e \u003cstrong\u003e22,\u003c/strong\u003e 118-128 (2021).\u003c/li\u003e\n\u003cli\u003eDas S, Ravi H, Babu A, Banerjee M, Kanagavalli R, Dhanasekaran S, et al. Therapeutic potentials of glucose-dependent insulinotropic polypeptide (GIP) in T2DM: Past, present, and future. \u003cem\u003eAdv Protein Chem Struct Biol\u003c/em\u003e \u003cstrong\u003e142,\u003c/strong\u003e 293-328 (2024).\u003c/li\u003e\n\u003cli\u003eWillard FS, Ho JD, Sloop KW. Discovery and pharmacology of the covalent GLP-1 receptor (GLP-1R) allosteric modulator BETP: A novel tool to probe GLP-1R pharmacology. \u003cem\u003eAdv Pharmacol\u003c/em\u003e \u003cstrong\u003e88, \u003c/strong\u003e173-191 (2020).\u003c/li\u003e\n\u003cli\u003eWithaar C, Meems LMG, Nollet EE, Schouten EM, Schroeder MA, Knudsen LB, et al. The Cardioprotective Effects of Semaglutide Exceed Those of Dietary Weight Loss in Mice With HFpEF. \u003cem\u003eJACC Basic Transl Sci\u003c/em\u003e \u003cstrong\u003e8, \u003c/strong\u003e1298-1314 (2023).\u003c/li\u003e\n\u003cli\u003eGallwitz B. GLP-1 agonists and dipeptidyl-peptidase IV inhibitors.\u003cem\u003e Handb Exp Pharmacol\u003c/em\u003e \u003cstrong\u003e1\u003c/strong\u003e. 53-74 (2011).\u003c/li\u003e\n\u003cli\u003eZheng Z, Zong Y, Ma Y, Tian Y, Pang Y, Zhang C, et al. Glucagon-like peptide-1 receptor: mechanisms and advances in therapy. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e234-263 (2024).\u003c/li\u003e\n\u003cli\u003eCaruso I, Cignarelli A, Natalicchio A, Perrini S, Laviola L, Giorgino F. Commentary: Glucose control: Not just a bystander in GLP-1RA-mediated cardiovascular protection. \u003cem\u003eMetabolism\u003c/em\u003e \u003cstrong\u003e109,\u003c/strong\u003e 154272 (2020).\u003c/li\u003e\n\u003cli\u003ePagidipati NJ, Nelson AJ, Kaltenbach LA, Leyva M, McGuire DK, Pop-Busui R, et al. Coordinated Care to Optimize Cardiovascular Preventive Therapies in Type 2 Diabetes: A Randomized Clinical Trial. \u003cem\u003eJAMA\u003c/em\u003e \u003cstrong\u003e329\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1261-1270 (2023).\u003c/li\u003e\n\u003cli\u003eLiu Y, Jiang M, Li Y, Chen P, Chen X. Advances in the study of ELABELA in renal physiological functions and related diseases. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e14,\u003c/strong\u003e 1276488-1276496 (2023).\u003c/li\u003e\n\u003cli\u003eAson B, Chen Y, Guo Q, Hoagland KM, Chui RW, Fielden M, et al. Cardiovascular response to small-molecule APJ activation. \u003cem\u003eJCI Insight\u003c/em\u003e \u003cstrong\u003e5,\u003c/strong\u003e e132898-e134904 (2020)\u003c/li\u003e\n\u003cli\u003eLi C, Cheng H, Adhikari BK, Wang S, Yang N, Liu W, et al. The Role of Apelin-APJ System in Diabetes and Obesity. \u003cem\u003eFront Endocrinol (Lausanne)\u003c/em\u003e \u003cstrong\u003e9\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 820002-820013 (2022).\u003c/li\u003e\n\u003cli\u003eNyimanu D, Kay RG, Kuc RE, Brown AJH, Gribble FM, Maguire JJ, et al. In vitro metabolism of synthetic Elabela/Toddler (ELA-32) peptide in human plasma and kidney homogenates analyzed with mass spectrometry and validation of endogenous peptide quantification in tissues by ELISA. \u003cem\u003ePeptides\u003c/em\u003e \u003cstrong\u003e145,\u003c/strong\u003e 170642-170654 (2021).\u003c/li\u003e\n\u003cli\u003eWang X, Zhang L, Li P, Zheng Y, Yang Y, Ji S. Apelin/APJ system in inflammation. \u003cem\u003eInt Immunopharmacol\u003c/em\u003e \u003cstrong\u003e109\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 108822-108832 (2022).\u003c/li\u003e\n\u003cli\u003eXu F, Wu M, Lu X, Zhang H, Shi L, Xi Y, et al. Effect of Fc-Elabela-21 on renal ischemia/reperfusion injury in mice: Mediation of anti-apoptotic effect via Akt phosphorylation. \u003cem\u003ePeptides\u003c/em\u003e \u003cstrong\u003e147,\u003c/strong\u003e 170682-170691 (2022).\u003c/li\u003e\n\u003cli\u003eGao S, Chen H. Therapeutic potential of apelin and Elabela in cardiovascular disease. \u003cem\u003eBiomed Pharmacother\u003c/em\u003e \u003cstrong\u003e166,\u003c/strong\u003e 115268-115282 (2023).\u003c/li\u003e\n\u003cli\u003eSharma M, Prabhavalkar KS, Bhatt LK. Elabela Peptide: An Emerging Target in Therapeutics. \u003cem\u003eCurr Drug Targets\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e1304-1318 (2022).\u003c/li\u003e\n\u003cli\u003eWang Y. Recombinant Elabela-Fc fusion protein has extended plasma half-life and mitigates post-infarct heart dysfunction in rats. \u003cem\u003eInt J Cardiol\u003c/em\u003e \u003cstrong\u003e300,\u003c/strong\u003e 217-225 (2020).\u003c/li\u003e\n\u003cli\u003eVisegrady A, Boros A, Nemethy Z, Kiss B, Keseru GM. Application of the BD ACTOne technology for the high-throughput screening of Gs-coupled receptor antagonists. \u003cem\u003eJ Biomol Screen\u003c/em\u003e \u003cstrong\u003e12,\u003c/strong\u003e 1068-1073 (2007).\u003c/li\u003e\n\u003cli\u003eCollaborators GBDD. Global, regional, and national burden of diabetes from 1990 to 2021, with projections of prevalence to 2050: a systematic analysis for the Global Burden of Disease Study 2021.\u003cem\u003e Lancet\u003c/em\u003e \u003cstrong\u003e402\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 203-234 (2023).\u003c/li\u003e\n\u003cli\u003eClaudel SE, Schmidt IM, Waikar SS, Verma A. Cumulative Incidence of Mortality Associated with Cardiovascular-Kidney-Metabolic (CKM) Syndrome. \u003cem\u003eJ Am Soc Nephrol\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e1343-1351 (2025).\u003c/li\u003e\n\u003cli\u003eMoghissi ES, Korytkowski MT, DiNardo M, Einhorn D, Hellman R, Hirsch IB, et al. American Association of Clinical Endocrinologists and American Diabetes Association consensus statement on inpatient glycemic control. \u003cem\u003eDiabetes Care\u003c/em\u003e \u003cstrong\u003e32\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1119-1131 (2009).\u003c/li\u003e\n\u003cli\u003eRitchie RH, Abel ED. Basic Mechanisms of Diabetic Heart Disease. \u003cem\u003eCirc Res\u003c/em\u003e \u003cstrong\u003e126\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1501-1525 (2020).\u003c/li\u003e\n\u003cli\u003eMa X, Liu Z, Ilyas I, Little PJ, Kamato D, Sahebka A, et al. GLP-1 receptor agonists (GLP-1RAs): cardiovascular actions and therapeutic potential. \u003cem\u003eInt J Biol Sci\u003c/em\u003e \u003cstrong\u003e17\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e2050-68 (2021).\u003c/li\u003e\n\u003cli\u003eKutz A, Kim DH, Wexler DJ, Liu J, Schneeweiss S, Glynn RJ, et al. Comparative Cardiovascular Effectiveness and Safety of SGLT-2 Inhibitors, GLP-1 Receptor Agonists, and DPP-4 Inhibitors According to Frailty in Type 2 Diabetes. \u003cem\u003eDiabetes Care\u003c/em\u003e \u003cstrong\u003e46\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e2004-2014 (2023).\u003c/li\u003e\n\u003cli\u003eGerstein HC, Colhoun HM, Dagenais GR, Diaz R, Lakshmanan M, Pais P, et al. Dulaglutide and cardiovascular outcomes in type 2 diabetes (REWIND): a double-blind, randomised placebo-controlled trial. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e394\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 121-30 (2019).\u003c/li\u003e\n\u003cli\u003ePratley RE, Aroda VR, Lingvay I, Ludemann J, Andreassen C, Navarria A, et al. Semaglutide versus dulaglutide once weekly in patients with type 2 diabetes (SUSTAIN 7): a randomised, open-label, phase 3b trial. \u003cem\u003eLancet Diabetes Endocrinol\u003c/em\u003e \u003cstrong\u003e6\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 275-286 (2018).\u003c/li\u003e\n\u003cli\u003eMonastero R, Magro D, Venezia M, Pisano C, Balistreri CR. A promising therapeutic peptide and preventive/diagnostic biomarker for age-related diseases: The Elabela/Apela/Toddler peptide. \u003cem\u003eAgeing Res Rev\u003c/em\u003e \u003cstrong\u003e91,\u003c/strong\u003e 102076-102085 (2023).\u003c/li\u003e\n\u003cli\u003eXiong M, Chen H, Fan Y, Jin M, Yang D, Chen Y, et al. Tubular Elabela-APJ axis attenuates ischemia-reperfusion induced acute kidney injury and the following AKI-CKD transition by protecting renal microcirculation. \u003cem\u003eTheranostics\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 3387-3401 (2023).\u003c/li\u003e\n\u003cli\u003eMa Z, Song JJ, Martin S, Yang XC, Zhong JC. The Elabela-APJ axis: a promising therapeutic target for heart failure. \u003cem\u003eHeart Fail Rev\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e1249-1258 (2021).\u003c/li\u003e\n\u003cli\u003eAkoumianakis I, Zagaliotis A, Konstantaraki M, Filippatos TD. GLP-1 analogs and regional adiposity: A systematic review and meta-analysis. \u003cem\u003eObes Rev\u003c/em\u003e \u003cstrong\u003e24\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e e13574-e13584 (2023).\u003c/li\u003e\n\u003cli\u003eYao L, Wang L, Zhang R, Soukas AA, Wu L. The direct targets of metformin in diabetes and beyond. \u003cem\u003eTrends Endocrinol Metab\u003c/em\u003e \u003cstrong\u003e36\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 364-372 (2025).\u003c/li\u003e\n\u003cli\u003eDia M, Leon C, Chanon S, Bendridi N, Gomez L, Rieusset J, et al. Effect of Metformin on T2D-Induced MAM Ca(2+) Uncoupling and Contractile Dysfunction in an Early Mouse Model of Diabetic HFpEF. \u003cem\u003eInt J Mol Sci\u003c/em\u003e \u003cstrong\u003e23\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 3569-3577 (2022).\u003c/li\u003e\n\u003cli\u003eChen YC, Chung CH, Weng TH, Tsai CT, Cheng LH, Chien WC. Metformin Use Reduces the Risk of Olfactory Dysfunction in Diabetic Patients: A 22-Year Follow-Up Study. \u003cem\u003eInt Forum Allergy Rhinol\u003c/em\u003e \u003cstrong\u003e15,\u003c/strong\u003e 658-661 (2025).\u003c/li\u003e\n\u003cli\u003eChan DC, Lin YC, Tzeng HP, Yang RS, Chiang MT, Liu SH. Exendin-4, a glucagon-like peptide-1 receptor agonist, alleviates muscular dysfunction and wasting in a streptozotocin-induced diabetic mouse model compared to metformin. \u003cem\u003eTissue Cell\u003c/em\u003e \u003cstrong\u003e89\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 102479- 102488 (2024).\u003c/li\u003e\n\u003cli\u003eMira Hernandez J, Shen EY, Ko CY, Hourani Z, Spencer ER, Smoliarchuk D, et al. Differential sex-dependent susceptibility to diastolic dysfunction and arrhythmia in cardiomyocytes from obese diabetic HFpEF model. \u003cem\u003eCardiovasc Res\u003c/em\u003e \u003cstrong\u003e121, \u003c/strong\u003e254-266 (2024).\u003c/li\u003e\n\u003cli\u003eCampbell P, Rutten FH, Lee MM, Hawkins NM, Petrie MC. Heart failure with preserved ejection fraction: everything the clinician needs to know. \u003cem\u003eLancet\u003c/em\u003e \u003cstrong\u003e403\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 1083-1092 (2024).\u003c/li\u003e\n\u003cli\u003ePaterek A, Zaleska-Kociecka M, Surzykiewicz M, Wojdynska Z, Leszek P, Maczewski M. Non-coding RNA therapeutics in the treatment of heart failure. \u003cem\u003eEur Heart J Cardiovasc Pharmacother\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 353-360 (2024).\u003c/li\u003e\n\u003cli\u003eDarwish AB, El Sayed NS, Salama AAA, Saad MA. Dulaglutide impedes depressive-like behavior persuaded by chronic social defeat stress model in male C57BL/6 mice: Implications on GLP-1R and cAMP/PKA signaling pathway in the hippocampus. \u003cem\u003eLife Sci\u003c/em\u003e \u003cstrong\u003e320\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 121546 (2023).\u003c/li\u003e\n\u003cli\u003eLee J, Hong SW, Kim MJ, Moon SJ, Kwon H, Park SE, et al. Glucagon-Like Peptide Receptor Agonist Inhibits Angiotensin II-Induced Proliferation and Migration in Vascular Smooth Muscle Cells and Ameliorates Phosphate-Induced Vascular Smooth Muscle Cells Calcification. \u003cem\u003eDiabetes Metab J\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 83-96 (2024).\u003c/li\u003e\n\u003cli\u003eKamiya M, Mizoguchi F, Yasuda S. Amelioration of inflammatory myopathies by glucagon-like peptide-1 receptor agonist via suppressing muscle fibre necroptosis. \u003cem\u003eJ Cachexia Sarcopenia Muscle\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e2118-2131 (2022).\u003c/li\u003e\n\u003cli\u003eMeurot C, Martin C, Sudre L, Breton J, Bougault C, Rattenbach R, et al. Liraglutide, a glucagon-like peptide 1 receptor agonist, exerts analgesic, anti-inflammatory and anti-degradative actions in osteoarthritis. \u003cem\u003eSci Rep\u003c/em\u003e \u003cstrong\u003e12\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e1567-1582 (2022).\u003c/li\u003e\n\u003cli\u003eLuo X, Liu J, Zhou H, Chen L. Apelin/APJ system: A critical regulator of vascular smooth muscle cell. \u003cem\u003eJ Cell Physiol\u003c/em\u003e \u003cstrong\u003e233\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 5180-5188 (2018).\u003c/li\u003e\n\u003cli\u003eSu J, Zhang Y, Kumar SA, Sun M, Yao Y, Duan Y. APJ/apelin: A promising target for the treatment of retinopathy of prematurity. \u003cem\u003eDrug Discov Today\u003c/em\u003e \u003cstrong\u003e27\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 2342-2352 (2022).\u003c/li\u003e\n\u003cli\u003eWang X, Zhang L, Feng M, Xu Z, Cheng Z, Qian L. ELA-11 protects the heart against oxidative stress injury induced apoptosis through ERK/MAPK and PI3K/AKT signaling pathways. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e873614--873627 (2022).\u003c/li\u003e\n\u003cli\u003eYe L, Huang Y, Liu X, Zhang X, Cao Y, Kong X, et al. Apelin/APJ system protects placental trophoblasts from hypoxia-induced oxidative stress through activating PI3K/Akt signaling pathway in preeclampsia. \u003cem\u003eFree Radic Biol Med\u003c/em\u003e \u003cstrong\u003e208\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e759-770 (2023).\u003c/li\u003e\n\u003cli\u003eFu J, Chen X, Liu X, Xu D, Yang H, Zeng C, et al. ELABELA ameliorates hypoxic/ischemic-induced bone mesenchymal stem cell apoptosis via alleviation of mitochondrial dysfunction and activation of PI3K/AKT and ERK1/2 pathways. \u003cem\u003eStem Cell Res Ther\u003c/em\u003e \u003cstrong\u003e11\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 541-553 (2020).\u003c/li\u003e\n\u003cli\u003eCzupryniak L, Mosenzon O, Rychlik I, Clodi M, Ebrahimi F, Janez A, et al. Barriers to early diagnosis of chronic kidney disease and use of sodium-glucose cotransporter-2 inhibitors for renal protection: A comprehensive review and call to action. \u003cem\u003eDiabetes Obes Metab\u003c/em\u003e \u003cstrong\u003e26\u003c/strong\u003e\u003cstrong\u003e, \u003c/strong\u003e4165-4177 (2024).\u003c/li\u003e\n\u003cli\u003eAboukaoud M, Morhi Y, Osher E. Sodium-Glucose Co-Transporter 2 Inhibitors and Severe Urinary Tract Infections: Real-World Meta-Analysis of Cohort Studies. \u003cem\u003eAnn Pharmacother\u003c/em\u003e\u003cem\u003e (\u003c/em\u003e2025). https://doi.org/10.1177/10600280241312432\u003c/li\u003e\n\u003cli\u003eKintscher U, Bakris GL, Kolkhof P. Novel non-steroidal mineralocorticoid receptor antagonists in cardiorenal disease. \u003cem\u003eBr J Pharmacol\u003c/em\u003e \u003cstrong\u003e179\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 3220-3234 (2022).\u003c/li\u003e\n\u003cli\u003eHuart J, Jouret F. Non-steroidal mineralocorticoid receptor antagonists: a paradigm shift in the management of diabetic nephropathy. \u003cem\u003eKidney Blood Press Res\u003c/em\u003e \u003cstrong\u003e50, \u003c/strong\u003e267-275 (2025).\u003c/li\u003e\n\u003cli\u003eJiang X, Zhang Z, Li C, Zhang S, Su Q, Yang S, et al. Efficacy and Safety of Non-Steroidal Mineralocorticoid Receptor Antagonists in Patients With Chronic Kidney Disease and Type 2 Diabetes: A Systematic Review Incorporating an Indirect Comparisons Meta-Analysis. \u003cem\u003eFront Pharmacol\u003c/em\u003e \u003cstrong\u003e13\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 896947-896961 (2022).\u003c/li\u003e\n\u003cli\u003eKoh G. Enhancing Patient Outcomes: Prioritizing SGLT2is and GLP-1RAs in Diabetes with CVD. \u003cem\u003eDiabetes Metab J\u003c/em\u003e \u003cstrong\u003e48\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 208-220 (2024) .\u003c/li\u003e\n\u003cli\u003eKunutsor SK, Seidu S, Dey RS, Baidoo IK, Oulhaj A. Cardiovascular and kidney benefits of SGLT-2is and GLP-1RAs according to baseline blood pressure in type 2 diabetes: a systematic meta-analysis of cardiovascular outcome trials. \u003cem\u003eScand Cardiovasc J\u003c/em\u003e \u003cstrong\u003e58\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 2418086-2418098 (2024).\u003c/li\u003e\n\u003cli\u003eZhang Q, Wang J, Hu X, Lu W, Cao Y, Niu C, et al. GLP-1RAs regulate lipid metabolism and induce autophagy through AMPK/SIRT1 pathway to improve NAFLD. \u003cem\u003eProstaglandins Other Lipid Mediat\u003c/em\u003e \u003cstrong\u003e178\u003c/strong\u003e\u003cstrong\u003e,\u003c/strong\u003e 106987-106998 (2025).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"GLP-1 receptor agonist, Elabela, dual receptor agonist, CKM syndrome, type 2 diabetes, multiorgan protection","lastPublishedDoi":"10.21203/rs.3.rs-7782667/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7782667/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Current therapies cannot simultaneously address the interconnected metabolic, cardiac, and renal damage in Cardiovascular-Kidney-Metabolic (CKM) syndrome. This study investigated ACF210, a novel long-acting dual agonist targeting both GLP-1 and APJ receptors, as a potential treatment for type 2 diabetes (T2D)-induced CKM syndrome. ACF210 was synthesized by fusing the human IgG4 Fc fragment to the C-terminus of GLP-1 and the N-terminus of Elabela-21 (ELA). In vitro receptor activation assays confirmed that ACF210 effectively activated the GLP-1 receptor while inhibiting APJ-related cAMP signaling. db/db leptin receptor-deficient mice and high-fat diet/streptozotocin-induced T2D were treated with dulaglutide, Fc-ELA, or ACF210 for 12 weeks. Observation of organizational structure, functional assessment and serum analyses were conducted. ACF210 outperformed other treatments, showing superior improvements in blood glucose control, β-cell function, and reduction of hepatic steatosis. Crucially, it provided significant multi-organ protection: enhancing cardiac diastolic function, reducing biomarkers of heart failure (NT-proBNP), and lessening mitochondrial damage and fibrosis, and inducing the microangiogenesisl in the heart. In the kidneys, it improved function, indicated by lower cystatin C levels, and mitigated podocyte damage. In conclusion, ACF210 not only effectively alleviates dysglycemia by enhancing β-cell function but also significantly protects against diabetes-associated hepatic, cardiac, and renal damage, supporting its further exploration for the clinical management of CKM syndrome.","manuscriptTitle":"Protective Effects of ACF210, a Dual GLP-1/APJ Receptor Agonist, against Cardiovascular-Kidney-Metabolic Syndrome Induced by T2D","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-17 03:31:02","doi":"10.21203/rs.3.rs-7782667/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0099bec3-81e7-429f-9e80-665cc06db9ab","owner":[],"postedDate":"October 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":56387513,"name":"Health sciences/Medical research/Drug development"},{"id":56387514,"name":"Biological sciences/Drug discovery/Target validation"}],"tags":[],"updatedAt":"2025-11-04T03:01:08+00:00","versionOfRecord":[],"versionCreatedAt":"2025-10-17 03:31:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7782667","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7782667","identity":"rs-7782667","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}