Prediabetes and type 2 diabetes (T2D) are metabolic disorders characterized by insulin resistance and β-cell dysfunction. To understand the molecular mechanisms driving the transition from prediabetes to T2D, we performed a longitudinal proteogenomic analysis on 458 participants from the Prediabetes Lifestyle Intervention Study (PLIS). We identified 185 plasma proteins to be differentially expressed between conditions, 36 of which predict future T2D-onset. Integrating genetic data from 321 individuals, we generated a genome-wide protein quantitative trait loci (pQTL) map, identifying 86 differential and 700 shared cis-pQTLs between prediabetes and T2D. Mediation analysis revealed 60 putative causal links connecting allele-driven plasma protein expression to clinical traits, identifying body fat distribution, insulin resistance, and β-cell function as central drivers of pathogenesis. Collectively, these findings highlight specific proteins underlying disease progression and substantiate the view that prediabetes and T2D are not distinct conditions, but rather stages on a unified metabolic spectrum. ### Competing Interest Statement The authors have declared no competing interest. ### Clinical Trial NCT01947595 ### Funding Statement Archit Singh, Dr Mauro Tutino and Dr Ozvan Bocher have received funding from the European Union's Horizon 2020 research and innovation program under Grant Agreement No 101017802 (OPTOMICS). PLIS and this post hoc analysis were supported by the German Center for Diabetes Research, which is funded by the German Federal Ministry for Education and Research and the German states where its partner institutions are located (01GI0925). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The study protocol was approved by the ethics committee of the University Clinic of Tübingen (Tübingen, 55/2012; ClinicalTrials.gov registration: [NCT01947595][1]). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present study are available upon reasonable request to the authors. [1]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT01947595&atom=%2Fmedrxiv%2Fearly%2F2026%2F02%2F16%2F2026.02.13.26346161.atom

Although Western diet (WD) rich in saturated fat is regarded as a trigger of metabolic disease and inflammation, the role of this diet in the absence of obesity is unclear. We studied the metabolic, inflammatory, anthropometric, and adipose tissue transcriptomic responses of 92 twins to 6 weeks of WD following 6 weeks of a healthy low-fat diet while maintaining stable body weight. The WD increased total, LDL and HDL cholesterol but not triglycerides or free fatty acids and induced transient and moderate increases of insulin resistance and some inflammatory markers after 1 week, which disappeared after 6 weeks. Extensive transcriptomic changes reflected restructuring of subcutaneous abdominal adipose tissue. Olfactory receptor (OR) mRNAs were increased in correlation with insulin resistance, liver, and visceral fat, cytokines like IL-18 and VEGF, but inversely related to adiponectin and extracellular NAMPT. In conclusion, WD triggers metabolic and transcriptomic reprogramming closely linked to ectopic OR responses.

OBJECTIVES:Skeletal muscle is a central regulator of metabolic health, serving as the primary site of postprandial glucose uptake and playing a critical role in whole-body insulin sensitivity. Despite its importance, the molecular mechanisms governing muscle differentiation (myogenesis) and their modulation by metabolic interventions remain poorly defined. This study identifies the clathrin adaptor protein Picalm (phosphatidylinositol-binding clathrin assembly protein) as a novel regulator of myogenesis and investigates its regulation in response to exercise training and intermittent fasting. METHODS:Functional characterization of Picalm was conducted in C2C12 myoblasts and primary myocytes using siRNA-mediated knockdown. Clathrin-mediated endocytosis was performed using dynamin inhibition (Dyngo-4a) and via an EGF internalization assay. Surface proteome alterations were analyzed by plasma membrane proteomics, and autophagy dynamics were assessed via immunoblotting and fluorescence imaging. Jasplakinolide was used to rescue differentiation defects by enhancing actin polymerization. RESULTS:Picalm-depleted C2C12 myoblasts exhibited impaired differentiation, presumably due to diminished intracellular trafficking dynamics of cell surface proteins. Inhibition of dynamin-dependent endocytosis phenocopied the differentiation defect and further aggravated myogenesis in Picalm-depleted cells, indicating that Picalm-dependent endocytic function is required for efficient differentiation. Consistent with this, Picalm knockdown significantly decreased clathrin-dependent uptake of EGF. Proteome analysis of a plasma membrane-enriched fraction revealed increased abundance of over 100 proteins after Picalm knockdown, particularly candidates involved in vesicular trafficking (Vamp3, Vamp5), actin remodeling (Actn1, Actn4, Rhog, Rock1, Rock2) and cell adhesion (integrin receptors). In line with this, Picalm knockdown resulted in impaired maturation and lysosomal degradation of autophagic vesicles. Remarkably, pharmacological stabilization of actin filaments with Jasplakinolide restored myogenic differentiation in Picalm-deficient cells, highlighting a functional link between actin remodeling and myogenesis. CONCLUSIONS:Picalm regulates skeletal muscle differentiation by supporting clathrin-mediated endocytosis and plasma membrane remodeling, thereby maintaining trafficking-dependent control of actin organization. Its expression is responsive to metabolic cues such as exercise and intermittent fasting. These findings reveal a novel molecular link between nutrient signaling and myogenesis, with implications for metabolic disease and muscle regeneration.

metaKEGG is a comprehensive software package designed to streamline the visualization and integration of pathway enrichment results from multi-omics data, providing accessible and detailed insights into the molecular mechanisms driving health and disease. Unlike standard pipeline approaches, metaKEGG incorporates novel concepts allowing for clear, granular representation of gene-level or transcript-level expression changes. Beyond transcriptomic analysis, metaKEGG also supports epigenetic and regulatory metadata layers, such as methylation profiles and miRNA target annotations, offering users a versatile solution to depict complex regulatory interactions within a single pathway map. Its modular architecture provides nine analysis pipelines to suit various experimental designs, from comparing gene expression across multiple conditions to the integration of compound-based metabolomics data. Its implementation in Python ensures easy adoption and reproducibility, while a user-friendly web app allows researchers with limited bioinformatics expertise to harness metaKEGG's full potential.

Golgi-associated membrane scaffolds, or tethers, have broad roles in membrane-bound protein and lipid trafficking and in maintaining Golgi architecture. Accordingly, they exert strong influence over cellular development, signalling, cargo modification and transport. An ever-expanding group of Golgins and multi-subunit tethering complexes assumes distinct functions in specific Golgi subcompartments in close partnership with Rab and ARL family GTPases. Their dysregulation or mutation impairs glycosylation, vesicle trafficking, and cytoskeletal dynamics, thereby contributing to a spectrum of human pathologies ranging from neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease) to cancers (e.g. lung, breast, colon) and metabolic defects (impaired insulin secretion and lipid droplet formation). Here, we review these diverse roles across molecular, cellular and organismal physiology.

Previously, we identified six prediabetes clusters, three at moderate and three at high-risk for type 2 diabetes and/or complications. While this novel classification could enable earlier and improved disease prevention, it relies on intensive clinical phenotyping. Here, we developed a machine learning workflow to identify blood-based epigenetic markers to distinguish between prediabetes clusters. DNA methylation was profiled in blood cells of different cohorts including individuals that belong to clusters 2 (low-risk), 3, 5, and 6 (each high-risk) and data was subjected to a machine learning workflow. In a discovery cohort (n = 187), we identified 1,557 CpG sites as predictors for clusters 2, 3, 5, and 6. These CpGs were sufficient to distinguish between individuals belonging to the high-risk clusters 3, 5 and 6 in an independent replication cohort (n = 146) with an accuracy of 92

Restoration of organellar membrane integrity is critical for maintaining cellular homeostasis. Lysosomal membrane damage activates local repair machineries and global stress responses, but how signaling lipid metabolism is engaged by damage sensors to support and mechanistically link these processes remains poorly understood. Here we show that the phosphoinositide 3-phosphatase MTMR14 is recruited to damaged lysosomes through calcium-dependent binding to sphingomyelin. At these sites, MTMR14 promotes local PI(3)P hydrolysis and supports PI(4)P accumulation, thereby facilitating formation of ER–lysosome contact sites associated with membrane repair, without affecting ESCRT recruitment. MTMR14-dependent lipid remodelling causes reduced mTORC1 signalling and a decrease in global protein synthesis, consistent with an acute proteostatic adaptation to lysosomal injury. Cells lacking MTMR14 display impaired damage-induced lipid remodelling, altered repair-associated structures, sustained protein synthesis, and increased sensitivity to lysosomal injury, all of which can be mitigated by mTORC1/S6K inhibition. Our findings identify damage-sensing recruitment of MTMR14 and local PI(3)P turnover on damaged lysosomes as a phosphoinositide module that promotes lysosomal membrane integrity and homeostasis while functionally linking nutrient signalling to proteostasis under membrane stress.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a widespread chronic liver disorder spanning simple steatosis to metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis. Suitable animal models are crucial for therapeutic development, yet many fail to reproduce progression to fibrosing MASH. Here, we employed the STAM model in male C57BL/6J mice to simulate MASLD and investigated whether a high-fat, high-cholesterol (HFHC) diet would intensify disease progression compared with a standard high-fat (HF) diet. HFHC feeding in the STAM model accelerated hepatic lipid accumulation, crown-like structure formation, inflammation, and fibrosis, as demonstrated by histological analyses. Transcriptomic profiling and pathway enrichment confirmed activation of lipid and cholesterol metabolism, regulation of TNF production, and inflammatory signaling. Reactome analysis further indicated alterations in extracellular matrix formation and immune system pathways, consistent with advanced fibrotic progression. In summary, integration of HFHC feeding with the STAM model produced a more severe liver phenotype that better mirrors the pathological spectrum of MASLD. This optimized model offers a valuable platform for preclinical testing of therapeutic candidates targeting MASH and fibrosis, with potential to accelerate drug development and to improve treatment strategies for MASLD.

Clinical practice guidelines recommend defined weight loss goals for the prevention of type 2 diabetes (T2D) in those individuals with increased risk, such as prediabetes. However, achieving prediabetes remission, that is, reaching normal glucose regulation according to American Diabetes Association criteria, is more efficient in preventing T2D than solely reaching weight loss goals. Here we present a post hoc analysis of the large, multicenter, randomized, controlled Prediabetes Lifestyle Intervention Study (PLIS), demonstrating that prediabetes remission is achievable without weight loss or even weight gain, and that it also protects against incident T2D. The underlying mechanisms include improved insulin sensitivity, β-cell function and increments in β-cell-GLP-1 sensitivity. Weight gain was similar in those achieving prediabetes remission (responders) compared with nonresponders; however, adipose tissue was differentially redistributed in responders and nonresponders when compared against each other-while nonresponders increased visceral adipose tissue mass, responders increased adipose tissue in subcutaneous depots. The findings were reproduced in the US Diabetes Prevention Program. These data uncover essential pathways for prediabetes remission without weight loss and emphasize the need to include glycemic targets in current clinical practice guidelines to improve T2D prevention.

Time-restricted eating (TRE) is a promising strategy against metabolic disorders, but its effects on lipid metabolism remain controversial. The present research assesses and compares the impact of early (eTRE) versus late (lTRE) TRE on the plasma lipidomic profile. This is an exploratory outcome of the previously published randomized crossover trial, which examines 31 women with overweight or obesity who follow a two-week eTRE and a two-week lTRE in an intended isocaloric setting. Blood plasma and subcutaneous adipose tissue biopsies are analyzed using shotgun lipidomics and transcriptomics, respectively. Between interventions and within the lTRE, lipid species and classes, as well as enzyme activity indices, are not substantially changed. Within the eTRE, changes are observed for 103 lipid species, including a reduction of ceramide and phosphatidylcholine classes, and for the desaturation indices D5D, D6D, and D9D, as well as the elongation index ELOVL6. Combined analysis of plasma lipidome and adipose tissue reveals alterations in the glycerophospholipid pathway and in the expression of phospholipase enzymes PLB1, PLA2G6, and PLAG4B, dependent on TRE timing. These results suggest that eating timing during TRE may be crucial for remodeling the plasma lipidome and adipose tissue transcriptome and highlight the need of future lipidomic research in TRE.

Introduction and Objective: Prediabetes remission has beneficial effects for type 2 diabetes (T2D) prevention. However, it is unknown whether early remission is superior to late remission. Thus, we investigated if reaching prediabetes remission early during a lifestyle intervention is associated with lower T2D risk compared to reaching remission later on. Methods: We studied 865 individuals with prediabetes from the German multi-center Prediabetes Lifestyle Intervention Study (PLIS) who could be classified into early remission at 6 months of a lifestyle intervention (ER, n = 217), late remission at 12 months (LR, n = 110), or no remission (NR, n = 538). Prediabetes remission was defined as return to normal glucose regulation and normalized HbA1c according to ADA criteria. Cox regression models were fit with age, sex, intervention intensity, T2D risk and weight loss as covariates. Results: The ER group (n=217) was comparable in age (p=0.24), sex distribution (p = 0.07), BMI (p > 0.99), insulin sensitivity (p = 0.2) and insulin secretion (p = 0.48) vs the LR group (n=110). Fasting glucose (5.70 ±0.43 mmol/L vs 5.83 ±0.44, p = 0.016) and HbA1c (5.53 ±0.29 % vs 5.68 ±0.31, p < 0.001) was slightly lower in ER compared to LR, while 2h glucose was similar (p = 0.77). Overall, T2D risk was lower in both ER and LR compared to NR (n=538; RR 0.15 [95% CI: 0.07-0.31], p < 0.001 and 0.44 [0.23-0.82], p = 0.009, respectively). However, ER provided a more pronounced T2D risk reduction than LR (0.31 [0.12-0.79], p = 0.01). Conclusion: Achieving early remission of prediabetes to NGR during lifestyle intervention may provide additional benefits for T2D prevention compared with late remission. A. Sandforth: None. L. Sandforth: None. S. Katzenstein: None. J. Seissler: None. N. Perakakis: Other Relationship; Novo Nordisk, Lilly Diabetes. Advisory Panel; Bayer Pharmaceuticals, Inc. Other Relationship; APOGEPHA, Transmedac Innovations AG, GWT-TUD, Elbe-Gesundsheintszentrum GmbH, Open Exploration. R. Wagner: Speaker's Bureau; Boehringer-Ingelheim, Novo Nordisk. Advisory Panel; Sanofi. Speaker's Bureau; Sanofi. Advisory Panel; Lilly Diabetes. A. Peter: None. R. Lehmann: None. H. Preissl: None. I. Yurchenko: None. J. Szendroedi: Advisory Panel; Novo Nordisk, Lilly Diabetes, Novartis AG, Boehringer-Ingelheim. M. Blüher: Advisory Panel; AstraZeneca. Speaker's Bureau; Amgen Inc. Advisory Panel; Bayer Pharmaceuticals, Inc, Boehringer-Ingelheim. Speaker's Bureau; Daiichi Sankyo. Advisory Panel; Eli Lilly and Company, Novo Nordisk, Nestlé Health Science, Sanofi-Aventis Deutschland GmbH. A. Schürmann: None. S. Kabisch: Research Support; Almond Board California, California Walnut Commission. Other Relationship; JuZo-Akademie, Boehringer-Ingelheim. Research Support; J. Rettenmaier & Söhne. Other Relationship; Lilly Diabetes. Research Support; Wilhelm-Doerenkamp-Foundation. K. Mai: None. P.E. Schwarz: None. M. Heni: Advisory Panel; Amryt Pharma. Speaker's Bureau; Amryt Pharma, AstraZeneca, Boehringer-Ingelheim. Advisory Panel; Boehringer-Ingelheim. Speaker's Bureau; Lilly Diabetes, Novartis AG, Novo Nordisk, Sanofi. M. Roden: Research Support; Boehringer-Ingelheim. Advisory Panel; Echosens. Speaker's Bureau; Madrigal Pharmaceuticals, Inc. Advisory Panel; MSD Life Science Foundation. Board Member; Novo Nordisk. Advisory Panel; TARGET PharmaSolutions, Inc. N. Stefan: Speaker's Bureau; AstraZeneca, Boehringer-Ingelheim. Consultant; Lilly Diabetes. Speaker's Bureau; Lilly Diabetes. Consultant; Pfizer Inc. Speaker's Bureau; Sanofi. Research Support; Sanofi. Speaker's Bureau; Novo Nordisk, GlaxoSmithKline plc. Consultant; GlaxoSmithKline plc. A. Fritsche: Advisory Panel; Abbott. Speaker's Bureau; AstraZeneca. A.L. Birkenfeld: None. R. Jumpertz von Schwartzenberg: None.

White adipose tissue (WAT) dysfunction including an aberrant expression of miRNAs is strongly associated with the risk of developing type 2 diabetes (T2D), with limited evidence linking early changes in the WAT-derived miRNAs and T2D. The present study aims to identify early miRNome changes prognostic for T2D in mice and humans. Gonadal (g) WAT of diabetes-resistant and diabetes-prone mice were subjected to multi-omics analyses (transcriptome, miRNome, methylome, proteome). Metabolic phenotypes linked with T2D were correlated with adipose tissue miRNA expression and DNA methylation from 14 monozygotic twin pairs discordant for T2D. Plasma miRNA levels from females at high risk of developing T2D (TÜF study) were included. Adipose tissue of the diabetes-susceptible mice was less insulin sensitive with 200 differentially expressed mature miRNAs compared to diabetes-resistant mice. Integrative analysis of miRNome-transcriptome-proteome identified 227 proteins involved in amino acid metabolism, inflammation, signalling pathways, and insulin resistance. More than 20 differentially expressed miRNAs are located in the imprinted region Dlk1-Gtl2 and Mest (miR-335) potentially regulated by DNA methylation. Imprinted miRNAs also exhibited similar alterations in adipose tissue from monozygotic twin pairs discordant for T2D, with miR-335 expression altered only in females. Moreover, plasma levels of miR-335-5p were negatively correlated with fasting blood glucose in females at high risk of developing T2D. Early alterations of WAT-derived miRNAs such as miR-335-5p could contribute to systemic metabolic changes associated with the risk of developing T2D.

Introduction and Objective: Current guidelines recommend weight loss targets for individuals at risk for type 2 diabetes (T2D). Prediabetes is a high-risk state for T2D, and remission of prediabetes during weight loss has additional benefits for T2D prevention. Thus, we hypothesized that reaching glycemic targets is a more effective strategy for T2D prevention than weight loss targets. Methods: We studied 903 individuals with prediabetes from the German Prediabetes Lifestyle Intervention Study for whom data for weight loss and glycemic category classification was available. Glucose regulation was assessed by a 75 g oral glucose tolerance test. Prediabetes remission was defined as return to normal glucose regulation and normalized HbA1c according to ADA criteria. T2D risk was compared between responders and non-responders (R and NR) who lost weight (WL, n=298; < -5% of initial body weight), remained weight stable (WS, n=371; -5-0%) and gained weight (WG, n=234; >0%). Cox regression models were fit with age, sex and intervention intensity as covariates. Results: At baseline, age (p=0.11), fasting glucose (p=0.09), 2-hour glucose (p=0.98) and beta cell function were comparable between all three responder groups. WL-, WS- and WG-response was similarly protective from developing future T2D (HR for WL R vs. WL NR 0.11 [95 CI: 0.03-0.36], p = 0.00026, HR for WS R vs. WS NR 0.40 [95 CI: 0.17-0.93], p = 0.033, HR for WG R vs. WG NR 0.25 [95 CI: 0.09 -0.69], p = 0.0072,). T2D risk did not differ between weight loss strata (HR 0.82 [95 CI: 0.54-1.25], p = 0.36 for WS-R vs WG-R; and HR 0.91 [0.64-1.30], p = 0.61 for WL-R vs WG-R). Conclusion: Prediabetes remission, i.e. glycemic targets rather than weight loss targets, should be the primary treatment goal for T2D prevention. A. Sandforth: None. L. Sandforth: None. S. Katzenstein: None. J. Seissler: None. N. Perakakis: Other Relationship; Novo Nordisk, Lilly Diabetes. Advisory Panel; Bayer Pharmaceuticals, Inc. Other Relationship; APOGEPHA, Transmedac Innovations AG, GWT-TUD, Elbe-Gesundsheintszentrum GmbH, Open Exploration. R. Wagner: Speaker's Bureau; Boehringer-Ingelheim, Novo Nordisk. Advisory Panel; Sanofi. Speaker's Bureau; Sanofi. Advisory Panel; Lilly Diabetes. A. Peter: None. R. Lehmann: None. H. Preissl: None. I. Yurchenko: None. J. Szendroedi: Advisory Panel; Novo Nordisk, Lilly Diabetes, Novartis AG, Boehringer-Ingelheim. M. Blüher: Advisory Panel; AstraZeneca. Speaker's Bureau; Amgen Inc. Advisory Panel; Bayer Pharmaceuticals, Inc, Boehringer-Ingelheim. Speaker's Bureau; Daiichi Sankyo. Advisory Panel; Eli Lilly and Company, Novo Nordisk, Nestlé Health Science, Sanofi-Aventis Deutschland GmbH. A. Schürmann: None. S. Kabisch: Research Support; Almond Board California, California Walnut Commission. Other Relationship; JuZo-Akademie, Boehringer-Ingelheim. Research Support; J. Rettenmaier & Söhne. Other Relationship; Lilly Diabetes. Research Support; Wilhelm-Doerenkamp-Foundation. K. Mai: None. P.E. Schwarz: None. M. Heni: Advisory Panel; Amryt Pharma. Speaker's Bureau; Amryt Pharma, AstraZeneca, Boehringer-Ingelheim. Advisory Panel; Boehringer-Ingelheim. Speaker's Bureau; Lilly Diabetes, Novartis AG, Novo Nordisk, Sanofi. M. Roden: Research Support; Boehringer-Ingelheim. Advisory Panel; Echosens. Speaker's Bureau; Madrigal Pharmaceuticals, Inc. Advisory Panel; MSD Life Science Foundation. Board Member; Novo Nordisk. Advisory Panel; TARGET PharmaSolutions, Inc. N. Stefan: Speaker's Bureau; AstraZeneca, Boehringer-Ingelheim. Consultant; Lilly Diabetes. Speaker's Bureau; Lilly Diabetes. Consultant; Pfizer Inc. Speaker's Bureau; Sanofi. Research Support; Sanofi. Speaker's Bureau; Novo Nordisk, GlaxoSmithKline plc. Consultant; GlaxoSmithKline plc. A. Fritsche: Advisory Panel; Abbott. Speaker's Bureau; AstraZeneca. R. Jumpertz von Schwartzenberg: None. A.L. Birkenfeld: None.