SOCS1-based therapeutic peptides improve liver disease ...

Introduction

Non-alcoholic fatty liver disease (NAFLD), recently renamed steatotic liver disease, is a chronic condition characterised by an accumulation of ectopic lipids in the liver parenchyma, accounting for more than 5% of liver weight, regardless of its etiology1. The most prevalent subtype, metabolic dysfunction-associated steatotic liver disease (MASLD), is defined by hepatic steatosis in conjunction with cardiometabolic risk factors such as insulin resistance (IR), dyslipidemia, or hypertension2.

MASLD has become the leading chronic liver disease globally, affecting approximately 25–30% of the population, with its prevalence closely tied to rising rates of obesity and type 2 diabetes mellitus (T2DM)3,4. The disease spectrum ranges from simple steatosis to more severe forms characterized by lobular inflammation and hepatocyte ballooning, referred to as metabolic dysfunction-associated steatohepatitis (MASH). Progression to advanced fibrosis, cirrhosis, or hepatocellular carcinoma (HCC) can follow5,6,7.

The interaction between obesity, insulin resistance, and T2DM is central to MASLD progression, significantly increasing the risk of fibrotic and neoplastic complications8. Whether these metabolic disorders act as independent drivers of disease or share common pathogenic mechanisms with MASLD remains under investigation9.

In MASLD, chronic lipid accumulation sensitizes the liver to secondary insults, such as oxidative stress and inflammatory triggers, initiating a cycle of cell injury, inflammation, death and regeneration. As a result, the liver develops a pro-inflammatory environment that fuels disease progression10,11. The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays a central role in coordinating immune responses and inflammation in chronic metabolic diseases. In MASLD, JAK/STAT activation promotes hepatic inflammation and fibrogenesis12,13,14. In the context of obesity, JAK/STAT pathway activation is closely linked to the low-grade inflammation characteristic of obesity-related metabolic complications15. In the context of obesity, this pathway contributes to systemic low-grade inflammation, exacerbating insulin resistance and hepatic lipid accumulation15,16. Endogenous regulation of JAK/STAT pathway is mediated by suppressor of cytokine signaling (SOCS) proteins, with SOCS1 acting as a key negative feedback modulator17. Experimental and translational evidence indicates that SOCS proteins, particularly SOCS1 and SOCS3, act as critical gatekeepers of hepatic injury and carcinogenesis: their deficiency exacerbates inflammation, fibrosis, and tumorigenesis, whereas their preservation restrains pathological JAK/STAT signaling. These data position the JAK/STAT/SOCS axis as a central node in the pathogenesis of chronic liver disease and hepatocarcinogenesis in metabolic settings18,19,20.

Transcriptional inhibitory peptides, such as SOCS1 mimetics, have shown promise in modulating JAK/STAT signaling due to their specificity and favorable safety profiles. In our previous studies, a SOCS1-derived peptide effectively suppressed ameliorated renal pathology in diabetic nephropathy models21,22, and more recently improved liver injury in nutritional MASLD models not associated to diabetes23. However, the therapeutic potential of this strategy in liver disease linked to obesity and T2DM remained unexplored.

In this study, we employed the BTBR ob/ob mouse, a well-established model of severe obesity, hyperglycemia, and hepatic inflammation24 to evaluate the therapeutic efficacy of a SOCS1-mimetic peptide in MASLD. We investigated its impact on hepatic steatosis, lipid metabolism, and inflammation to assess its suitability as a treatment for metabolic liver disease.

Methods

Experimental model

This study utilized male BTBR ob/ob mice (BTBR.Cg-Lepob/WiscJ; RRID:IMSR_JAX:004824), a leptin-deficient strain characterized by obesity, severe T2DM, and associated complications including MASLD, to assess the hepatic effects of JAK/STAT pathway inhibition. BTBR heterozygous breeding pairs (BTBR ob + / −) were originally purchased from Jackson Laboratories (Bar Harbor, ME, USA), and the colony was expanded in-house in our animal facility. At 16 weeks of age (start of treatment), BTBR ob/ob mice had an average body weight of 64 ± 4 g. Mice were randomly allocated into three experimental groups: 1) Non-diabetic wild-type control mice (BTBR WT, BTBR T + Itpr3tf/J; RRID:IMSR_JAX:002282; n = 6); 2) Untreated diabetic and obese mice (BTBR ob/ob; n = 10); and 3) Diabetic and obese mice treated with SOCS1 mimetic peptide (MiS1), administered intraperitoneally thrice weekly at a dose of 10 μg/g/day (BTBR ob/ob + MiS1; n = 6) (Fig. 1).

Fig. 1

Experimental design of the main therapeutic study. BTBR WT, BTBR ob/ob, and BTBR ob/ob + MiS1 groups were studied. MiS1 was administered from week 16 to week 22, after which animals were sacrificed.

Treatment was initiated at 16 weeks of age and continued until 22 weeks. At the end of the study, mice were sacrificed by exsanguination under deep anesthesia induced by ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia for sample collection (blood and liver). Mice were housed in individually ventilated cages under controlled temperature (20–22 °C), with a 12-h light–dark cycle and 2–3 animals per cage. All mice had ad libitum access to standard chow (LASQCdiet Rod14–H, 3.5% fat) and water. Body weight and non-fasting glycemia (measured with a Nova Biomedical glucometer, Barcelona, Spain) were recorded weekly.

Prior to the main therapeutic efficacy study, a preliminary pilot validation experiment was performed to confirm the hepatic target engagement of MiS1. Twelve-week-old BTBR ob/ob mice were assigned to three groups: 1) untreated BTBR ob/ob controls, 2) mice treated with a single intraperitoneal dose of MiS1 (4 µg/g), and 3) mice treated with an inactive mutant peptide, used as a specificity control21,22.

Liver sections were collected and analyzed by immunohistochemistry (IHC) for phosphorylated STAT3 (p-STAT3) to assess MiS1-mediated modulation of the JAK/STAT pathway.

All animal procedures were approved by the Institutional Animal Care and Use Committee (Comité de Ética en Experimentación Animal, IIS-Fundación Jiménez Díaz) and the regional government of Madrid (Ref. PROEX 079/18) and conducted in accordance with Real Decreto 53/2013 and EU Directive 2010/63/EU on the protection of animals used for scientific purposes. All animal experiments were conducted and reported in accordance with the ARRIVE guidelines (https://arriveguidelines.org). All procedures complied with the institutional and regional regulations for animal research.

Peptidomimetic synthesis

The MiS1 peptide derived from the murine SOCS1 kinase inhibitory region (amino acids 53–68), was palmitoylated and synthesized by Proteogenix (Schiltigheim, France). It was dissolved in a vehicle containing 2% DMSO, 30% PEG 300, and 5% Tween 80 in 0.9% saline. A mutated version of the peptide was also synthesized and formulated in the same vehicle to validate the specificity and efficacy of the active compound.

Biochemical parameters

Aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (AP), albumin, total cholesterol, triglycerides (TGs), high-density lipoprotein cholesterol (HDL) and low-density lipoprotein cholesterol (LDL) according to the Friedwald formula were all measured in serum samples. Before animal sacrifice, blood was obtained from the femoral artery while animals were under ketamine (100 mg/kg) and xylazine (10 mg/kg) anesthesia, into Vacutainer ACD blood collection tubes (Becton Dickinson and Company, Plymouth, UK). These measurements were carried out in a Roche Cobas autoanalyzer at our institution’s central laboratories.

Liver lipids were extracted using the Folch method (chloroform–methanol extraction)25. Serum non-esterified fatty acids (NEFAs) were measured using NEFA C enzymatic assay kit (WAKO, Neuss, Germany) as described26. The liver triglyceride content was quantified by the GPO-Trinder colorimetric assay (Cayman Chemical Company; Ann Arbor, MI, USA).

Histological and immunohistochemistry analysis

Liver samples were fixed in 4% formaldehyde and embedded in paraffin. Tissue sections (4 µm) were stained with hematoxylin and eosin (H\&E) and Masson’s trichrome for histopathological analysis. Liver damage was semi-quantitatively assessed using the NAFLD Activity Score (NAS) by blinded expert observers.

Immunohistochemistry was performed using anti-F4/80 antibody (monocytes/macrophages; clone D2S9R; 1:300; #70076S, RRID: AB_2799771; Cell Signaling Technology, Danvers, MA, USA). Immunodetection was performed using an indirect immunoperoxidase method. After primary antibody incubations, samples were processed with a secondary antibody provided by the Vectastain Elite ABC HRP Kit, ready-to-use (PK-7100, RRID:AB_2336827; Vector Laboratories, Burlingame, CA, USA) and developed using DAB substrate (Abcam #ab64238). Quantification of positive staining was conducted using Image-Pro Plus software and reported as the percentage of stained area in eight randomly selected fields per sample.

Hepatic lipid profile determination

Fatty acid composition in liver triglycerides was analyzed using 20 mg of liver homogenate. Fatty acid methyl esters were generated and analyzed by gas chromatography–mass spectrometry (GC–MS) with electron ionization, following solid-phase extraction, as previously described24.

Protein studies

Liver and cultured cells were homogenized in BR20X lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 2 mM EDTA, 2 mM EGTA, 0.2% Triton X-100, 0.3% Igepal) with protease (Sigma CP8340) and phosphatase inhibitors (Sigma P0044) using zirconium oxide beads in a bullet blender (Next Advance, NY, USA).

Protein concentration was quantified via BCA assay (Thermo Fisher). Equal protein amounts (50 µg) were resolved by SDS-PAGE (8–12% acrylamide), transferred to PVDF membranes, blocked in TBS-T with 5% milk, and incubated overnight at 4 °C with primary antibodies: Phospho-STAT3 (Tyr705) (D3A7) (1:500, #9145S, RRID:AB_2491009, Cell Signaling Technology; Danvers, MA, USA), STAT3 (124H6) (1:1000, #9139S, RRID:AB_331757, Cell Signaling Technology; Danvers, MA, USA), and Fatty acid synthase (FASN) (C20G5) (1:1000, #3180S, RRID:AB_2100796, Cell Signaling Technology; Danvers, MA, USA). Secondary antibodies (HRP-conjugated; Invitrogen) were applied for 1 h, and signals were developed with ECL Luminata Crescendo (Millipore) and visualized using the iBright CL750 Imaging System (Thermo Fisher). Blots were reprobed with β-Actin (A2228, 1:5000, Sigma-Aldrich) for normalization. Densitometric analysis was performed using Quantity One software (Bio-Rad). All cropped gels and blots presented in the main figures are clearly delineated. Full-length, uncropped images of the original membranes, showing visible membrane edges and including all biological replicates, are provided in the Supplementary Fig. S1. Where indicated, membranes were cut prior to antibody incubation to allow simultaneous probing of proteins with different molecular weights.

Gene expression studies

Total RNA was isolated from liver tissue using TRIdity G reagent (Panreac AppliChem, Spain). First-strand cDNA synthesis was performed using the High-Capacity cDNA Archive Kit (Applied Biosystems) from 2 µg of RNA.

qRT-PCR was conducted on a 7500 Fast Real-Time PCR System (Applied Biosystems) using TaqMan gene expression assays (Thermo Fisher). Expression levels were normalized to 18S rRNA or Actb. Data are expressed as fold changes relative to the BTBR WT group (set as 1). Primer details are provided in Supplementary Table S1.

Cell cultures

Immortalized neonatal mouse hepatocyte cell lines were kindly provided by Dr. Ángela M. Valverde (Institute for Biomedical Research “Alberto Sols”—IIBM, CSIC–UAM, Madrid, Spain). These cell lines were originally generated from wild-type neonatal mice (3.5–5 days old) by collagenase dispersion and subsequent immortalization using the puromycin-resistant retroviral vector pBabe encoding SV40 large T antigen (LTAg), as described27,28 were cultured in DMEM medium (31885023; Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; F7524), 2 mM L-glutamine and 100 U/mL penicillin and 100 mg/mL streptomycin. Cells were serum-starved overnight and subsequently, stimulated with 100 nM insulin (Sigma) in the presence or absence of MiS1 (150 μg/mL).

Statistical analysis

Data are expressed as mean ± SEM (for graphs) or median ± IQR (for tables). Statistical significance was assessed using one-way or two-way ANOVA followed by Tukey’s or Bonferroni’s post hoc tests, or unpaired Mann–Whitney U-tests when appropriate. Analyses were performed with GraphPad Prism v8 (GraphPad Software, La Jolla, CA, USA). A p value < 0.05 was considered statistically significant.

Results

Pilot validation study: MiS1 inhibits hepatic STAT3 phosphorylation.

To assess direct hepatic effects of MiS1, we performed immunohistochemistry for phosphorylated STAT3 (p-STAT3) on liver sections from 12-week-old BTBR ob/ob mice. Three groups were analyzed: untreated BTBR ob/ob controls, mice treated with active MiS1 peptide (4 µg), and mice treated with a mutated version of the peptide (single amino acid modification). Histological evaluation revealed clear differences in staining patterns at both 100× and 200× magnification (Fig. 2A, B). Quantification of p-STAT3-positive area showed a significant reduction in mice treated with the active MiS1 peptide, while those treated with the mutated peptide showed levels comparable to untreated mice (Fig. 2C). These findings suggest a selective and specific inhibitory effect of the active MiS1 peptide on hepatic STAT3 activation.

Fig. 2

Histopathological assessment of p-STAT3 expression in the BTBR ob/ob model. (A) Representative images of p-STAT3 immunostaining in liver sections from untreated, MiS1-treated, and mutant peptide–treated mice at 200 ×  and (B) 100× magnification. (C) Quantification of p-STAT3-positive area (%). Data are presented as mean ± SEM for each group (n = 4–5 mice/group); ***p < 0.001 vs. BTBR ob/ob.

Metabolic, biochemical and liver morphological changes in obese diabetic mice

Changes in body weight and non-fasting glucose levels were monitored from the onset of treatment. BTBR ob/ob mice treated with MiS1 revealed a significant attenuation of the characteristic weight gain observed in this model of T2DM and obesity. By the end of the study, the treated group exhibited an 11% reduction in body weight compared to untreated BTBR ob/ob controls. Meanwhile, no significant changes in fasting glucose levels were observed between the control and treatment groups (Table 1).

Table 1 Metabolic and biochemical parameters in the experimental model. Data are shown as Median (IQR).

Full size table

The changes in serum metabolic parameters observed across the experimental groups are summarized in Table 1. BTBR ob/ob mice exhibited significant increases in ALT (276%), AP (252%), total cholesterol (154%), TGs (195%) and HDL (154%) compared to BTBR WT. In the MiS1-treated group, ALT levels were significantly reduced by 22% compared to untreated BTBR ob/ob mice, with no significant changes observed in AST, AP, or serum lipids.

To assess morphological changes associated with MASLD, liver samples from mice were evaluated at the end of the study period (22 weeks). Morphological analysis of BTBR ob/ob mice showed hallmark features of MASLD, including microvesicular and macrovesicular steatosis predominantly in zones 2 (transitional) and 3 (pericentral), along with inflammatory clusters and hepatocellular ballooning (Fig. 3A). However, no evidence of fibrosis was observed (Fig. 3B). The NAS score, a quantitative measure of liver damage, was assessed across the experimental groups. MiS1 treatment significantly reduced steatosis lesions (Fig. 3C) and hepatocellular ballooning (Fig. 3E), and showed a trend toward reduced inflammatory clusters (Fig. 3D). The total NAS score was significantly reduced (Fig. 3F). Quantification of Masson’s trichrome staining revealed no significant change in collagen fiber deposition in treated mice (Fig. 3G).

Fig. 3

Liver histopathological changes in the experimental model. Representative images of H&E staining (A) and Masson’s trichrome staining (B) (magnification 100x). (C–F) Quantification of histopathological characteristics of MASLD: (C) steatosis, (D) lobular inflammation, (E) hepatocellular ballooning, and (F) total NAS. (G) Quantification of positive Masson’s trichrome staining. Data are shown as scatter dot plots and mean ± SEM of each group (n = 6–10 mice/group). *p < 0.05, and ***p < 0.001 vs. BTBR WT or BTBR ob/ob.

Effect of MiS1 treatment on liver lipid profile and metabolism in obese diabetic mice

BTBR ob/ob mice displayed an approximately twofold increase in total TG content in the liver compared to BTBR WT, while MiS1 treatment resulted in a marked, significant reduction of hepatic TG levels (Fig. 4A). To further explore the link between MASLD and de novo lipogenesis, we analyzed the specific fatty acids comprising intrahepatic TG and NEFA fraction. In the hepatic TG fraction, BTBR ob/ob mice showed a significant increase in palmitic acid (C16:0; 737%), palmitoleic acid (C16:1n-7; 1072%), stearic acid (C18:0; 261%), and oleic acid (C18:1n-9cis; 940%), compared to BTBR WT (Fig. 4B). Interestingly, inhibition of the JAK-STAT pathway had a selective effect on lipid composition, with significantly decreases observed only in C16:0 and C16:1n-7 levels in the MiS1-treated group (55% and 63%, respectively) (Fig. 4B).

Fig. 4

Liver lipid composition and lipid metabolism genes in the experimental model. (A) Quantification of liver TGs. (B) GC − MS analysis of de novo lipogenesis fatty acids in liver TG fractions. Measurement of total NEFA in liver (C) and serum (D). (E) mRNA expression levels of lipogenic enzymes (Scd1, Fasn) were measured by RT-qPCR. Values were normalized by 18S rRNA and expressed as fold increases relative to BTBR WT group. (F) Western blot analysis of FASN in mouse liver. Proteins levels were normalized by β-Actin and expressed as fold changes. Representative immunoblot images are included. Data are shown as scatter dot plots and mean ± SEM for each group (n = 4–6 mice/group). *p < 0.05 and **p < 0.01 vs. BTBR ob/ob or BTBR WT.

Regarding to NEFAs, no significant changes were observed in total intrahepatic levels (Fig. 4C). By contrast, serum NEFA levels were notably elevated in the MiS1-treated group (Fig. 4D). Additional analyses of lipid fractions, including saturated, monounsaturated, and polyunsaturated fatty acids in both TG and NEFA fractions, are shown in Supplementary Fig. S2. These analyses revealed only minor treatment effects, with a general tendency toward increased monounsaturated and polyunsaturated fatty acids, while saturated fatty acids and de novo lipogenesis-related products remained virtually unchanged.

To validate the lipidomic findings, we examined the expression of key enzymes in de novo lipogenesis, specifically stearoyl-CoA desaturase (SCD1) and FASN. Gene expression analysis revealed a significant upregulation of both enzymes in untreated BTBR ob/ob mice, which was reversed by MiS1 treatment (Fig. 4E). These results were further confirmed at the protein level, where MiS1 treatment mitigated FASN overexpression observed in obese diabetic mice (Fig. 4F).

Effect of MiS1 administration on liver inflammatory status

Immunohistochemical staining for F4/80 in liver samples (Fig. 5A) revealed an increased content of macrophages in obese diabetic mice, although no significant differences were observed between untreated and MiS1-treated groups (Fig. 5B).

Fig. 5

Inflammatory profile in the experimental model. (A) Representative images of F4/80 immunohistochemistry. (B) Quantification of F4/80 + area (%). (C) Hepatic mRNA expression of pro-inflammatory cytokines (Tnf, Ccl2, Ccl5, Cxcl10, Ifng). (D) Expression of anti-inflammatory cytokines (Il10, Tgfb) and the M2 polarization marker Cd163. RT-qPCR expression values were normalized by 18S rRNA and expressed as fold changes relative to BTBR WT group. Data are shown as scatter dot plots and mean ± SEM for each group (n = 4–6 mice/group). *p < 0.05 vs. BTBR ob/ob.

RT-qPCR analysis showed increased hepatic expression of pro-inflammatory cytokines and chemokines (Tnf, Ccl2, Ccl5, Cxcl10, Ifng) in BTBR ob/ob mice (Fig. 5C). MiS1 treatment reduced the expression of these inflammatory genes, with significant decreases observed in Cxcl10 and Ifng (Fig. 5C). Although anti-inflammatory genes (Il10, Tgfb) did not show significant changes, MiS1 treatment was associated with upregulation of the M2 macrophage marker Cd163, indicating a modulation towards an anti-inflammatory profile. (Fig. 5D).

Direct effect of MiS1 on murine hepatocytes

Finally, to determine whether MiS1 exerts a direct effect on liver tissue, we conducted an experiment using an immortalized murine hepatocyte cell line. Insulin stimulus was applied to induce de novo lipogenesis and activate the JAK/STAT pathway, allowing us to evaluate the impact of MiS1 treatment on these processes.

Gene expression analysis revealed that insulin did not affect Stat3 gene expression (Fig. 6A), but notably upregulated the enzymes associated with de novo lipogenesis (Scd1 and Fasn), and effect significantly attenuated in the presence of MiS1 (Fig. 6B, C). Protein expression analysis confirmed the activation of JAK/STAT (STAT3 phosphorylation) and de novo lipogenesis (FASN) pathways in insulin-treated cells, which were notably reduced by MiS1 (Fig. 6D–F).

Fig. 6

Hepatocellular effects of MiS1 in vitro. mRNA expression levels of (A) the transcription factor Stat3 and the lipogenic enzymes (B) Scd1 and (C) Fasn. RT-qPCR values were normalized by Actb mRNA and expressed as fold increases relative to the control group. (D) Representative Western blot images and protein expression levels of (E) phospho-STAT3 (Tyr-705) (p-STAT3)/total STAT3 and (F) FASN were assessed by western blot. Proteins levels were normalized by β-Actin and expressed as fold changes. Data are shown as scatter dot plots and mean ± SEM for each group (n = 4–6 mice/group). *p < 0.05 and ***p < 0.001 vs. insulin group.

Discussion

Our study demonstrates the therapeutic potential of the SOCS1-derived peptidomimetic MiS1 to ameliorate MASLD in BTBR ob/ob mice, a model of obesity and T2DM, through inhibition of the JAK/STAT pathway. Key findings include significant reductions in hepatic steatosis, hepatocellular ballooning, and triglyceride accumulation, coupled with modulations in both pro-inflammatory and anti-inflammatory markers. These results highlight the ability of MiS1 to intervene in lipid dysregulation and inflammation thereby offering a targeted pharmacological approach for future disease management in patients with obesity and diabetes29.

Current conceptualizations of MASLD pathogenesis emphasize the ‘multiple parallel hits’ hypothesis, which posits that a combination of insults—including mitochondrial dysfunction, endoplasmic reticulum stress, lipotoxicity, and gut–liver axis dysregulation—act in concert to drive disease progression. Within this complex multifactorial network, the JAK/STAT signaling cascade operates as a critical signaling hub that integrates metabolic and inflammatory cues30. This pathway mediates cytokine-induced inflammatory responses in the liver10 and is intricately linked to lipid metabolism, immune regulation, and tumorigenesis31. Persistent activation of STAT3 has been implicated in liver fibrogenesis due to its dual role in hepatoprotection and proliferation32,33. This pathway is inherently regulated by SOCS proteins17. Evidence from SOCS1-deficienct mice has demonstrated that a lack of this regulatory protein promotes the accumulation of inflammatory macrophages and increases the expression of pro-inflammatory and fibrogenic markers during high-fat diet-induced liver fibrosis34.

Mechanistically, MiS1 compound exerts its effects by inhibiting JAK/STAT signaling, reducing STAT1/3 phosphorylation, and thereby modulating downstream inflammatory and metabolic responses21,22,35,36. The present study shows that this targeted inhibition by MiS1 downregulates the expression of lipogenic enzymes such as Scd1 and Fasn, effectively limiting hepatic TG accumulation, a key contributor to metabolic dysfunction29. De novo lipogenesis plays a central role in MASLD pathogenesis, and the observed reductions in lipogenesis-associated fatty acids such as palmitic and palmitoleic acid, together with FASN suppression following MiS1 treatment, highlight its therapeutic potential. By limiting de novo lipogenesis, MiS1 may also mitigate the harmful consequences of lipid accumulation, including inflammation and fibrosis37. Moreover, the clinical relevance of targeting FASN is underscored by the ongoing development of FASN inhibitors in clinical trials, offering promising strategies for the treatment of MASLD and related metabolic disorders38. Importantly, our in vitro findings in cultured hepatocytes demonstrate that MiS1 directly suppresses insulin-induced Scd1 and Fasn expression (Fig. 6). Since this effect occurs in a pure hepatocyte system devoid of immune cells, it confirms that the metabolic benefits of MiS1 are driven by a direct impact on hepatocyte JAK/STAT signaling, rather than being solely secondary to the reduced inflammatory milieu. This interpretation is further supported by previous studies demonstrating that STAT3 directly binds to the Fasn promoter and positively regulates its transcription, thereby promoting de novo lipogenesis; accordingly, JAK/STAT inhibition reduces Fasn expression and lipid synthesis39,40.

Another notable effect of MiS1 was the significant increase in CD163 expression without changes in total macrophage numbers, suggesting a modulation of hepatic macrophage phenotype rather than an expansion of macrophages. Importantly, hepatic macrophages represent highly heterogeneous populations that do not strictly conform to the classical M1/M2 paradigm, and CD163 has been described as a marker of a distinct hepatic macrophage subset with specific transcriptional and functional properties that do not necessarily overlap with canonical M2 markers41. In this context, the upregulation of CD163 in the MiS1-treated group likely reflects a shift toward a CD163⁺ hepatic macrophage subset rather than a definitive increase in conventional M2 macrophages. In early-stage MASLD with minimal fibrosis, a predominance of pro-resolving macrophage phenotypes may support inflammation resolution and tissue homeostasis42, although their role is complex and stage- and context-dependent. In our model, this phenotypic modulation coincided with reduced liver injury and inflammation, supporting a potentially beneficial role. In the context of MASH, macrophage phenotype modulation is crucial for resolving inflammation and promoting liver tissue regeneration43. Pro-resolving macrophage subsets enhance the clearance of inflammatory cells and facilitate the repair of damaged hepatic tissue, underscoring their essential role in restoring liver homeostasis44,45.

Besides inflammation, oxidative stress also plays an important role in MASLD/MASH contributing to liver fibrosis, which progresses to cirrhosis and HCC. In our previous study in nutritional NAFLD mouse models characterized by marked macrophage infiltration, administration of SOCS1-derived peptidomimetics reduced hepatic macrophages. In contrast, in the present BTBR ob/ob model, baseline infiltration was low and MiS1 treatment did not reduce macrophage content. These differences likely reflect distinct disease mechanisms, with lipotoxicity and metabolic dysfunction predominating over inflammatory cell infiltration in the obese diabetic model23.

The favorable pharmacokinetic profile of MiS1, including hepatic targeting and minimal systemic toxicity21,22,35, is critical for translating these results into potential clinical applications. Of interest, these peptides exerted remarkable beneficial effects on various diabetic complications, including diabetic nephropathy and retinopathy as well as atherosclerotic lesions46,47,48. Given the challenge of systemic toxicity in many therapeutic agents, MiS1’s ability to focus on the liver while minimizing broader adverse effects is a major advantage. Dose–response studies showing significant reductions in serum ALT levels and hepatic lipid content further reinforce its potential for therapeutic benefit.

Limitations and challenges

First, while a functionally inactive mutant peptide was included in a pilot validation experiment to confirm specificity, the long-term efficacy study focused exclusively on the active peptide. Nevertheless, the specificity of SOCS1-derived peptidomimetics has been extensively validated in our previous works across multiple organ and disease models21,23.

Second, this study relies on murine models. Given the heterogeneity of human MASLD, further validation in human hepatocytes and more advanced translational systems will strengthen clinical relevance. While the BTBR ob/ob mouse is a robust model for early metabolic liver disease, it does not fully recapitulate the full human disease spectrum (e.g., fibrosis progression and hepatocellular carcinoma), which represents a common limitation of preclinical MASLD research. Future studies using humanized models or long-term treatments will be essential for fully validating MiS1’s therapeutic potential in more advanced stages of MASLD.

Finally, while MiS1 treatment effectively improved hepatic parameters, a non-significant trend toward increased serum lipids, particularly NEFAs, was detected. This result is translationally relevant, as pharmacological JAK inhibition has been associated with dyslipidemia in patients with rheumatoid arthritis (e.g. increases in total cholesterol, triglycerides, and LDL and changes in HDL), underscoring the need for lipid monitoring during JAK inhibitor treatment49. In the context of the leptin-deficient ob/ob model, characterized by severe systemic metabolic dysregulation, this finding may reflect redistribution of lipids from the liver toward the circulation (‘liver unloading’), rather than a direct adverse systemic effect. Importantly, such lipid alterations were not observed in dietary MASLD models treated with SOCS1 mimetics23. This observation also highlights the dual, tissue-dependent role of STAT3 signaling in metabolism and the need for a comprehensive metabolic assessment in future translational studies involving systemic JAK/STAT modulation. Further research addressing MiS1 effects on additional metabolic tissues, such as adipose tissue and skeletal muscle, will be important to better delineate its systemic metabolic impact and possible broader therapeutic benefits.

Future directions

Long-Term Efficacy Studies: Extending treatment durations and incorporating models that represent more advanced stages of MASLD (such as fibrosis and HCC) will be important for assessing the long-term therapeutic potential of MiS1; Combination Therapy with GLP-1RA: The suggestion of combining MiS1 with GLP-1 receptor agonists (GLP-1RA) is intriguing. GLP-1RAs have shown promise in improving metabolic and hepatic outcomes, and combining them with MiS1’s targeted inhibition of JAK/STAT could provide complementary benefits. This could be an exciting avenue for future combination therapy studies.

Conclusion

This study provides robust evidence supporting the therapeutic potential of MiS1 in treating MASLD by targeting both lipid dysregulation and inflammation. The multifaceted approach, focusing on JAK/STAT signaling, macrophage polarization, and lipogenesis, positions MiS1 as a promising candidate for precision medicine in patients with comorbidities such as obesity and type 2 diabetes. While there are limitations, particularly in the animal model used, the findings set the stage for further preclinical and clinical investigations.

Data availability

The data that support the findings of this study are available from the corresponding authors upon reasonable request: Sebastian Mas-Fontao ([email protected]) and Jesús Egido ([email protected]).

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Funding

This research was supported by the Instituto de Salud Carlos III (PI20/00487, PI23/00119, PI24/01630, DTS25-00051), MICIU/AEI/10.13039/501100011033 (PID2021-127741OB-I00) and co-funded by the European Union. The research was also supported by the Spanish Biomedical Research Centre in Diabetes and Metabolic Diseases (CIBERDEM) and thank Conchita Rábago Foundation for M.S-C grant support.

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Author notes

1. M. Soto-Catalán and L. Opazo-Ríos are first authors contributed equally to the manuscript.

2. J. Egido and S. Mas-Fontao are senior authors contributed equally to the manuscript.

Authors and Affiliations

1. Renal, Vascular and Diabetes Research Laboratory, IIS-Fundación Jiménez Díaz, Spanish Biomedical Research Centre in Diabetes and Associated Metabolic Disorders (CIBERDEM), Universidad Autónoma de Madrid, Madrid, Spain

   M. Soto-Catalán, C. Espadas, M. Romero-Cote, C. Gómez-Guerrero, J. Egido & S. Mas-Fontao

2. Facultad de Ciencias de la Salud, Universidad de Las Américas, 4301099, Concepción-Talcahuano, Chile

   L. Opazo-Ríos

3. Cardiovascular Risk and Nutrition Research Group, Epidemiology and Public Health Program, Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain

   I. Lázaro

4. Department of Cell Biology, Physiology and Immunology, Maimónides Biomedical Research Institute of Cordoba (IMIBIC), UGC Nephrology, Hospital Universitario Reina Sofía, University of Cordoba, Córdoba, Spain

   J. A. Moreno

Authors

1. M. Soto-Catalán
2. L. Opazo-Ríos
3. C. Espadas
4. M. Romero-Cote
5. I. Lázaro
6. J. A. Moreno
7. C. Gómez-Guerrero
8. J. Egido
9. S. Mas-Fontao

Contributions

S.M.-F. and J.E. are the guarantors of this work and take responsibility for the integrity and the accuracy of the data analysis. L.O-R. and M.S-C. contributed to the conception, design, performance of the experiments and drafting of the manuscript for the study. I.L, M. R and C.E. contributed to the performance of the experiments and interpretation of data. JA.M., C.G-G, S.M.-F. and J.E., contributed to interpretation of the data, drafting and critical review of the manuscript, and in securing financial support for the study. The paper was written by the authors, with language polishing and corrections provided by AI. All the authors have reviewed the manuscript and approved the final version.

Corresponding authors

Correspondence to M. Soto-Catalán or J. Egido.

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Competing interests

MSC, LPR, SMF, JE and CGG are inventors on a patent application for SOCS peptidomimetics. The other authors declare no competing interests.

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Soto-Catalán, M., Opazo-Ríos, L., Espadas, C. et al. SOCS1-based therapeutic peptides improve liver disease and metabolic dysfunction in obesity and diabetes. Sci Rep 16, 16682 (2026). https://doi.org/10.1038/s41598-026-46312-4

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• Received: 11 November 2025

• Accepted: 25 March 2026

• Published: 09 April 2026

• Version of record: 29 May 2026

• DOI: https://doi.org/10.1038/s41598-026-46312-4

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