Regulation of cellular response to energy stress by RNF128 via modulation of AMPK activation

Regulation of cellular response to energy stress by RNF128 via modulation of AMPK activation | 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 Regulation of cellular response to energy stress by RNF128 via modulation of AMPK activation Ying-Chuan Chen, Pei-Yao Liu, Yu-Lung Lin, Yen-Chun Ho, Wen-Chiuan Tsai, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7469946/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 AMP-activated protein kinase (AMPK) is a highly conserved central regulator of metabolic processes, maintaining energy homeostasis under metabolic stress. Identifying new regulators of AMPK is critical to understanding its response to energy stress. This study reports the identification of RING finger protein 128 (RNF128), an AMPK-binding E3 ligase that physically and functionally interacts with AMPK. RNF128 facilitates the polyubiquitination and phosphorylation of AMPK, thereby modulating its activation in response to glucose deprivation. Overexpression of RNF128 enhanced cell death under energy stress conditions, whereas loss of RNF128 expression attenuated stress-induced cell death. RNF128 also regulated reactive oxygen species production and influenced the expression of glycolysis-related genes during glucose deprivation. In vivo, RNF128 deficiency reduced AMPK phosphorylation and alleviated fasting-induced liver injury, whereas adeno-associated virus serotype 8-mediated overexpression of RNF128 increased AMPK phosphorylation and exacerbated liver injury. In conclusion, this study demonstrates that RNF128 functions as an E3 ligase that promotes AMPK polyubiquitination and activation under energy stress, revealing a previously unrecognized role for RNF128. Biological sciences/Cell biology/Proteolysis/Ubiquitin ligases Biological sciences/Cell biology RNF128 AMPK cell death glucose starvation ubiquitination Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Cellular energy production is primarily mediated by glycolysis and oxidative phosphorylation. Glucose serves as the main substrate for glycolysis and facilitates ATP synthesis, the universal energy currency of the cell 1 . Under conditions of sufficient glucose availability, cells maintain a high ATP:AMP ratio. In contrast, periods of energetic stress—such as hypoxia, nutrient deprivation, or increased energy demand—are associated with a reduction in this ratio 2 , 3 , 4 . During glucose deprivation, cells initiate adaptive mechanisms to preserve energy homeostasis. Central to this response is AMP-activated protein kinase (AMPK), a key regulator of cellular energy balance. AMPK is a conserved serine/threonine kinase complex that functions as a cellular energy sensor 5 , 6 . The AMPK heterotrimer consists of a catalytic α subunit and regulatory β and γ subunits. It is broadly expressed in eukaryotes and regulates metabolism. AMPK is activated when cellular AMP:ATP and ADP:ATP ratios increase, reflecting energetic stress. Glucose deprivation, for example, reduces glycolytic ATP production, leading to elevated AMP and ADP levels. Once activated, AMPK initiates responses aimed at restoring energy balance 7 . AMPK serves as a central regulator of cellular energy homeostasis, playing a critical role in detecting cellular energy status and coordinating metabolic responses to nutrient fluctuations 5 , 6 . Glucose deprivation can also result in elevated levels of reactive oxygen species (ROS) and metabolic byproducts, which may further modulate AMPK activity. AMPK plays a critical role in promoting cellular survival by facilitating adaptation to energy stress 8 , 9 , 10 . However, when AMPK activation is prolonged or excessive and homeostasis cannot be restored, this may ultimately lead to cell death 11 , 12 . RING finger protein 128 (RNF128) is a type I transmembrane protein primarily localized to the transferrin-recycling endocytic pathway. It plays a critical role in inducing T cell anergy by suppressing cytokine expression 13 . RNF128 forms a ternary complex with Otub-1 and USP8, both of which contribute to the regulation of T cell anergy 14 , 15 . RNF128 is ubiquitously expressed, with particularly high levels in organs, such as the heart, kidney, and liver. It is involved in glucose and lipid metabolism, contributes to adipocyte differentiation, and has been implicated in diet-induced obesity 16 , 17 . Additionally, RNF128 may influence hepatic steatosis through inhibition of SIRT1 18 . RNF128 is also capable of influencing the progression of acute lung injury by modulating TLR4 and MPO expression in immune cells 19 . Collectively, these findings suggest that RNF128 participates in a wide spectrum of biological processes. In this study, we aimed to investigate the role of RNF128 in cellular responses to glucose deprivation under both in vitro and in vivo conditions. The rationale was to elucidate a previously unrecognized function of RNF128 in the context of energy stress. Results RNF128 was newly identified to interact with AMPK To elucidate the role of AMPK in cellular energy sensing, we employed mass spectrometry (MS) to identify novel AMPK regulators during energy stress. A total of 202 and 184 AMPK-interacting proteins were detected in glucose-deprived C2C12 and AML12 cells, respectively, with 90 proteins overlapping between the two cell types. Among the 10 most abundant proteins identified, the E3 ubiquitin ligase RNF128 (RING finger protein 128) was consistently enriched (Fig. 1 A). Using C2C12 cells, we confirmed the interaction between AMPK and RNF128 through co-immunoprecipitation assays, which demonstrated a reciprocal interaction between the two proteins (Fig. 1 B, 1 C). Similar interactions were confirmed in HEK293 cells (Fig. 1 D, 1 E). Immunofluorescence microscopy further revealed colocalization of AMPK and RNF128 in both C2C12 and AML12 cells (Fig. 1 F, 1 G), and confocal imaging yielded comparable results (Fig. S1 , S2). These findings establish RNF128 as a newly identified protein that interacts with AMPK. RNF128 promoted AMPK ubiquitination Since RNF128 functions as an E3 ubiquitin ligase targeting specific proteins 18 , 19 , 20 , 21 , 22 , we examined whether its E3 ligase activity regulates AMPK ubiquitination and activation. To address this, HEK293 cells were transfected with plasmids encoding RNF128, ubiquitin, and AMPK, followed by analysis of protein expression. As shown in Fig. 2 A, co-expression of RNF128 and AMPK resulted in increased AMPK polyubiquitination. Examination of endogenous AMPK ubiquitination further showed that RNF128 overexpression enhanced AMPK polyubiquitination compared with controls (Fig. 2 D), whereas RNF128 knockdown reduced AMPK polyubiquitination (Fig. 2 E). To investigate RNF128-mediated polyubiquitination of AMPK in further detail, we compared wild-type ubiquitin with mutant ubiquitin containing a single lysine residue (K48 or K63). RNF128 promoted AMPK polyubiquitination in the presence of hemagglutinin-tagged ubiquitin K48 (HA-Ub-K48) or K63 (HA-Ub-K63) (Fig. 2 B, 2 C). Increased K48- and K63-linked AMPK ubiquitination was also observed in C2C12 cells overexpressing RNF128 (Fig. 2 D), while silencing RNF128 decreased both K48- and K63-associated AMPK ubiquitination (Fig. 2 E). Together, these results indicate that RNF128 mediates K48- and K63-associated polyubiquitination of AMPK. RNF128 was involved in the regulation of AMPK activation In response to glucose deprivation, AMPK activation is tightly regulated to enable cellular adaptation to energy stress. Given that RNF128 interacts with and ubiquitinates AMPK, we examined whether RNF128 contributes to the regulation of AMPK activation under conditions of energy stress. To assess this, we examined the association between RNF128 and AMPK activation status, measured by AMPK T172 phosphorylation under 0 mM glucose. RNF128 expression was silenced in C2C12 (C2C12/shRNF128) cells, and downregulation was confirmed (Fig. S3A). Ablation of RNF128 resulted in reduced P-AMPK levels (Fig. 3 A). Consistently, expression of P-ACC, a direct AMPK downstream target, was also decreased in glucose-starved C2C12/shRNF128 cells compared to that in controls (Fig. 3 A). Similar results were observed in AML12/shRNF128 cells (Fig. S4A). To further assess the impact of RNF128 on AMPK activation, we generated C2C12 cell lines with stable RNF128 overexpression and confirmed increased RNF128 expression (Fig. S3B). In these cells, P-AMPK and P-ACC levels were significantly elevated under glucose deprivation compared to those in the control groups (Fig. 3 B). Consistent with these observations, RNF128 overexpression enhanced AMPK phosphorylation during glucose deprivation (Fig. 3 C– 3 F). Comparable results were also obtained in AML12/RNF128 cells (Fig. S4B). Collectively, these data demonstrate that RNF128 influences AMPK activation and may contribute to the regulation of AMPK-mediated responses to energy stress. RNF128 overexpression increased glucose deprivation-induced cell death Extended periods of glucose deprivation induce cellular energy stress, activating several pathways that ultimately lead to cell death. However, the mechanisms by which AMPK regulates cell death under sustained energy stress remain incompletely understood. To address this, we examined the influence of RNF128 on AMPK function during energy stress-induced cell death. The results showed that C2C12 cells overexpressing RNF128 (C2C12/RNF128) displayed reduced viability after glucose deprivation compared with control cells (Fig. 4 A). Similar results were observed in HEK293/RNF128, AML/RNF128, and HepG2/RNF128 cells (Fig. 4 B– 4 D). To further validate the cell viability findings, cell death under glucose deprivation was evaluated using PI staining. The data revealed a markedly higher death rate in C2C12/RNF128 cells (from 5.1–66.1%) than in C2C12/vector cells (from 5.2–19.6%) under glucose starvation (Fig. 4 E, 4 F). Additionally, energy stress led to elevated caspase-3 cleavage in C2C12/RNF128 cells relative to that in the control group (Fig. 4 G). Collectively, these findings indicate that RNF128 overexpression enhances cell death induced by glucose deprivation. Loss of RNF128 expression attenuated glucose deprivation-induced cell death To further verify the regulatory function of RNF128 in energy stress-induced cell death, we knocked down RNF128 expression in C2C12 (C2C12/shRNF128) cells. C2C12/shRNF128 cells exhibited higher viability following glucose deprivation than control cells (Fig. 5 A). Similar patterns were observed in HEK293/RNF128 knockout, AML/shRNF128, and HepG2/shRNF128 cell lines (Fig. 5 B– 5 D). PI staining further showed that C2C12/shRNF128 cells had lower cell death rates (from 4–13.5%) than C2C12/shluc cells (from 5–18.3%) under glucose starvation (Fig. 5 E, 5 F). Furthermore, energy stress was associated with reduced caspase-3 cleavage in C2C12/shRNF128 cells compared to that in the control group (Fig. 5 G). These results suggest that RNF128 ablation is associated with reduced cell death during glucose deprivation. Elevated RNF128 expression augmented cell death induced by AMPK activation To further elucidate the impact of RNF128 in AMPK-dependent cell death, we evaluated the effects of pharmacological AMPK activation in C2C12 and HEK293 cells overexpressing RNF128 (C2C12/RNF128 and HEK293/RNF128). Both cell types exhibited significantly reduced viability following treatment with 2-DG or phenformin compared to control cells (Fig. 6 A– 6 D). Consistently, C2C12/RNF128 cells displayed higher levels of cleaved caspase-3 than controls after 2-DG or phenformin treatment (Fig. 6 E, 6 F). Taken together, these results suggest that increased expression of RNF128 enhances cell death in response to AMPK activators. Lower RNF128 expression decreased AMPK activation-induced cell death We assessed the effect of RNF128 downregulation on cell death triggered by AMPK activators. The findings indicated that C2C12/shRNF128 cells exhibited increased viability after treatment with 2-DG or phenformin compared to control cells (Fig. 6 G, 6 H). Consistent patterns were also observed in HEK293/RNF128 knockout cell lines (Fig. 6 I, 6 J). In line with the hypothesis, C2C12/shRNF128 cells showed reduced cleaved caspase-3 levels compared to controls following 2-DG or phenformin treatment (Fig. 6 K, 6 L). These findings indicate that RNF128 downregulation reduces cell death in response to AMPK activators. AMPK inhibition attenuated the effect of RNF128 on glucose deprivation-induced cell death To further validate the role of AMPK in RNF128-mediated cell death during glucose deprivation, the established AMPK inhibitor Compound C was employed. Consistent with expectations, RNF128 overexpression resulted in increased cell death compared to controls under glucose-starved conditions (Fig. S5). Notably, treatment with Compound C attenuated RNF128-induced cell death under metabolic stress (Fig. S5). These findings indicate that RNF128 regulates AMPK-dependent cell death in response to glucose deprivation. RNF128 modulated ROS production under glucose deprivation Glucose deprivation can reduce ATP synthesis and increase ROS production, ultimately contributing to cell death. We therefore examined the effect of RNF128 on ROS generation. ROS levels were elevated in C2C12/RNF128 cells (from 0.11–37.7%) compared to those in C2C12/vector cells under glucose starvation conditions (from 0.1–11.7%) (Fig. 7 A, 7 B). In contrast, C2C12/shRNF128 cells showed reduced ROS levels (0.1–10.1%) compared to C2C12/shluc cells (0.17–16.3%) during glucose deprivation (Fig. 7 C, 7 D). Because glucose deprivation modulates the expression of major glycolytic enzymes and influences glycolytic activity, we evaluated the mRNA levels of genes associated with glycolysis 23 . Under glucose-starved conditions, C2C12/RNF128 and AML12/RNF128 cells exhibited increased mRNA expression of glycolysis-associated genes relative to control cells (Fig. 7 E, S4C). Conversely, C2C12/shRNF128 and AML12/shRNF128 cells showed reduced mRNA levels of these glycolysis-related genes compared to the controls (Fig. 7 F, S4D). Collectively, these results indicate that RNF128 may play a regulatory role in ROS production and glycolysis during glucose deprivation. RNF128 regulated AMPK activation and liver injury in response to fasting To elucidate the physiological role of RNF128 in modulating AMPK activation under fasting, we found that P-AMPK and P-ACC levels were significantly reduced in livers from fasted RNF128 KO mice compared to those in fasted WT mice (Fig. 8 A, 8 B). In contrast, P-AMPK and P-ACC levels were notably elevated in the livers of fasted AAV8-RNF128–treated mice relative to those in fasted AAV8-vector controls (Fig. 8 E, 8 F). Because liver injury is associated with elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) 24 , we measured these enzymes as markers of liver function. Fasted RNF128 KO mice displayed lower serum ALT and AST levels than fasted WT mice (Fig. 8 C, 8 D). Additionally, unlike RNF128 KO mice, AAV8-RNF128 mice demonstrated an increase in ALT and AST concentrations during fasting (Fig. 8 G, 8 H). Discussion AMPK functions as the principal regulator of cellular energy, coordinating adaptive responses to glucose deprivation. Identifying novel modulators of AMPK activity under energy stress is essential for a comprehensive understanding of these mechanisms. This study provides evidence that RNF128 is a critical regulator of cellular adaptation to glucose starvation. RNF128 modulated AMPK function in both in vitro and in vivo systems. Elevated RNF128 expression enhanced AMPK activation and increased cell death under glucose deprivation, whereas reduced RNF128 expression suppressed AMPK phosphorylation and attenuated glucose deprivation–induced cell death. In vivo, RNF128 deficiency diminished AMPK phosphorylation and mitigated fasting-induced liver injury, while AAV8-driven RNF128 overexpression produced the opposite effect. Mechanistically, RNF128 was found to regulate AMPK activity by promoting its polyubiquitination and phosphorylation (Fig. 8 I). Ubiquitination represents a key post-translational modification regulating diverse biological processes. K48-linked ubiquitin chains predominantly mediate protein degradation, whereas K63-linked ubiquitination generally influences signaling pathways and protein trafficking 25 , 26 . Under glucose deprivation, RNF128 facilitated both K63- and K48-linked ubiquitination of AMPK. Notably, RNF128 did not alter AMPK protein levels (Fig. 2 , 3 ). However, K48 ubiquitination of AMPK can promote its degradation under certain conditions 27 , likely reflecting differences in energy stress or cellular context. Further investigation is required to clarify this association. AMPK activation in the liver during fasting has context-dependent effects: it can protect against liver injury in conditions, such as NAFLD, but excessive or prolonged activation may also exacerbate liver damage 28 , 29 , 30 , 31 . Our findings indicate that RNF128 regulates AMPK polyubiquitination and thereby modulates its activation during glucose deprivation (Fig. 2 , 3 ). In contrast, prior work has demonstrated that USP10 interacts with and deubiquitinates AMPK, enhancing its phosphorylation and activity, which alleviates hepatic steatosis and metabolic dysfunction induced by high-fat diets in mice 32 . Together, these results suggest that distinct energy stress conditions may result in distinct patterns of AMPK regulation and function. Future studies exploring the interplay between USP10 and RNF128 in AMPK modulation could provide further mechanistic insights. As the principal energy substrate, glucose is essential for maintaining cellular function; its absence compromises key processes and may culminate in cell death. RNF128 was identified as a mediator of AMPK-dependent cell death under glucose deprivation. Previous studies have also implicated RNF128 in modulating p53-dependent cell death during DNA damage stress 20 . During glucose starvation, p53 can exert either pro- or anti-apoptotic effects depending on specific cellular conditions 33 , 34 . Further research is warranted to elucidate the direct interactions among RNF128, AMPK, and p53 in the regulation of cellular responses to energy stress. In conclusion, this study identified RNF128 as a regulator of AMPK in the context of energy stress. By delineating the mechanisms underlying AMPK activation and function, these findings advance the understanding of cellular adaptation to metabolic stress and provide a basis for developing therapeutic strategies for diseases associated with metabolic dysregulation. Materials and Methods Animal experiments The RNF128 KO mice, generated on a C57B/6J background using CRISPR–Cas9, carry a deletion at the exon 1 start codon. These mice were produced by the Transgenic Mouse Model Core (Taipei, Taiwan) 17 and maintained under a standard 12:12 h light/dark cycle at 22 ± 1°C. For fasting experiments, 8-week-old male WT and RNF128 KO mice were used. Fed mice had ad libitum access to food for 24 h, while fasted mice were deprived of food for 24 h and euthanized simultaneously with their fed counterparts. For in vivo AAV administration, AAV8-vector or AAV8-RNF128 was delivered via tail vein injection into 7-week-old male C57BL/6J mice at a dose of 1 × 10 12 vg/mouse in a total volume of 200 µL. Cells, plasmids, and transfection procedures C2C12, HEK293, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). AML12 cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium high glucose and Ham’s F-12 medium supplemented with 10% FBS. For glucose starvation, cells were washed twice with PBS and incubated in glucose- and pyruvate-free DMEM (0 g l − 1 glucose, 0 mM pyruvate; Invitrogen) supplemented with 10% dialyzed FBS (Invitrogen) 35 . RNF128 was cloned into a pCMV-TNT vector (Promega, CA, USA) using EcoRI and BamHI restriction sites. FLAG-AMPK, HA-ubiquitin-WT, HA-ubiquitin-48, and HA-ubiquitin-K63 plasmids were obtained from Addgene (Watertown, MA, USA). Transfections were performed using jetPRIME (New York, USA) according to the manufacturer’s protocol. Cells were seeded at a low density (approximately 1×10 5 cells/60 mm dish) and grown to 50–60% confluence before jetPRIME-mediated gene transfection. Transfected cells were lysed in radioimmunoprecipitation assay buffer for subsequent analyses. Immunoprecipitation and immunoblot analysis Cells were lysed in buffer containing 50 mM Tris (pH 8.0), 5 mM NaCl, 0.5% NP-40, and 1× protease inhibitor. Lysates underwent three freeze–thaw cycles, and proteins were extracted. Protein concentrations were determined using the Bradford method (Bio-Rad, CA, USA). Equal amounts of protein were immunoprecipitated in lysis buffer with antibodies against RNF128, AMPK, or FLAG at 4°C overnight. Dynabeads™ Protein G (Invitrogen, Waltham, MA, USA) were added to the immunoprecipitation mixture for 1 h, followed by three washes with SNNTE buffer (5% sucrose, 1% NP-40, 0.5 M NaCl, 50 mM Tris [pH 7.4], and 5 mM EDTA). The precipitates were suspended in SDS–polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled, and separated by SDS–PAGE. Proteins were transferred to nitrocellulose membranes and blocked for 1 h in buffer containing 10 mM Tris (pH 7.6), 137 mM NaCl, 0.1% (v/v) Tween 20, and 5% skimmed milk. Membranes were incubated overnight at 4°C with the indicated primary antibodies, followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies. Detection was performed using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA). Primary antibodies included anti-AMPK (10929-2-AP; Proteintech, Rosemount, IL, USA), anti-caspase 3 (19677-1-AP; Proteintech), anti-HA (51064-2-AP; Proteintech), anti-FLAG (F1804; MilliporeSigma, Burlington, MA, USA), anti-P-AMPK (Thr172) (40H9; Cell Signaling, Danvers, MA, USA), anti-cleaved caspase-3 (9661; Cell Signaling), anti-ACC (3662; Cell Signaling), anti-P-ACC (3661; Cell Signaling), anti-ubiquitin (P4D1; Cell Signaling), anti-K48 ubiquitin (D9D5; Cell Signaling), anti-K63 ubiquitin (D7A11; Cell Signaling), anti-AMPK (9G3; GeneTex; Irvine, CA, USA), anti-actin (MAb1501; Chemicon, Rolling Meadows, IL, USA), and anti-RNF128 (prepared in-house). In vivo ubiquitination assays C2C12/RNF128 or C2C12/shRNF128 cells were lysed in buffer (50 mM Tris [pH 8.0], 5 mM NaCl, 0.5% NP-40, and 1× protease inhibitor) and subjected to three freeze–thaw cycles to recover proteins. Equivalent amounts of protein were immunoprecipitated overnight at 4°C with an anti-AMPK antibody in the same buffer. Dynabeads Protein G (Invitrogen) were then added to the immunoprecipitation mixture and incubated at 4°C for 1 h. Samples were washed thrice with SNNTE buffer, resuspended in SDS–PAGE sample buffer, and separated by SDS–PAGE. Proteins were transferred to nitrocellulose membranes and probed with primary antibodies. Detection was performed using enhanced chemiluminescence reagents (GE Healthcare). Immunofluorescence staining Cells were cultured on glass coverslips, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. Following three PBS washes, slides were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min and incubated overnight at 4°C in 1% BSA in PBS with either rabbit polyclonal anti-RNF128 or mouse monoclonal anti-AMPK (9G3; GeneTex; Irvine, CA, USA) primary antibodies. Secondary antibodies Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) were applied for 1 h in 1% BSA in PBS at 37°C. Nuclei were counterstained with DAPI for 10 min. Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and immunofluorescence microscopy was performed using the THUNDER Imaging Systems. Virus particle production, viral transduction, and RNA interference RNF128 was cloned into the pQCXIP vector (Clontech Laboratories, Mountain View, CA, USA). The pQCXIP-RNF128 and empty pQCXIP plasmids were transfected into GP2-293 cells using jetPRIME® (Polyplus, NY, USA). Short hairpin RNA (shRNA) oligonucleotides were cloned into the expression vector, pSIREN-Retro-Q (Clontech Laboratories) (RNF128 shRNA target sequence 1: 5′-GAGGCATCCAAGTCACAATGG-3′; RNF128 shRNA target sequence 2: 5′-GCAGGAAGCAGAGGCAGTTAA-3′). Cells were infected with the specified retroviruses in a selection medium containing 2 µg/mL polybrene. After 48 h of infection, puromycin-resistant clones were selected by treating the cells with 2 µg/mL puromycin. The AAV expression vector was constructed using the Helper Free Expression System (Cell Biolabs Inc., San Diego, CA, USA). RNF128 was cloned into the pAAV-MCS vector, and AAV8-overexpressing RNF128 was produced following standard protocols. Cell viability assay Cell viability was assessed using the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich, USA). Cells were seeded into 96-well plates, and after adherence, 10 µL of CCK-8 solution was added to each well. Plates were incubated at 37°C in a 5% CO₂ atmosphere for 1 h. Optical density (OD) was measured at 450 nm. Each group was assayed in quintuplicate. Propidium iodide (PI) staining for flow cytometry PI fluorescent DNA-binding probes were utilized. Cells were harvested and aliquoted into FACS tubes (up to 1 x 10 6 cells per 100 µL), followed by washing with 2 mL of PBS, Subsequently, the cells were centrifuged at 300 x g for 5 min and the buffer was decanted from the pellet. This wash step was repeated twice. Cells were then placed in FACS tubes containing 1 mL PBS and 2 µg/mL PI. Flow cytometry was conducted in duplicate, and fluorescence emission was measured. ROS measurement ROS were detected using CellROX™ Green reagent (Invitrogen, Waltham, MA, USA) at a final concentration of 5 µM, following the manufacturer’s instructions. Cells were cultured in 0 mM glucose for 24 h, stained with CellROX reagent, and analyzed by flow cytometry. Flow cytometry was conducted across three independent experiments. Biochemical analysis Serum ALT and AST levels were quantified using commercial kits from FUJIFILM (GPT/ALT-P III and GOT/AST-P III, respectively). Quantitative reverse-transcription PCR RNA was isolated from cells and tissues using TRIzol reagent (Sigma, St. Louis, MO, USA). Complementary DNA was synthesized with Epicenter MMLV. Gene expression in cells was measured using the Applied Biosystems 7500 Real-Time PCR System and IQ2 FAST qPCR kit, whereas expression in tissues was quantified using the Roche LightCycler 480. Primers are listed in Supplementary Table 1. Statistical analysis Data were analyzed using GraphPad Prism 10 (GraphPad Software). Results are presented as mean ± standard deviation. One-way analysis of variance with multiple comparative analyses was used to compare multiple datasets. An unpaired two-tailed Student’s t -test was used for two-group comparisons. A P value < 0.05 was considered statistically significant. Abbreviations RNF128, RING finger protein 128; AMPK, AMP-activated protein kinase; QPCR, quantitative reverse-transcription polymerase chain reaction; shRNA, short hairpin RNA; AAV, adeno-associated virus Declarations Acknowledgements The authors thank the Instrument Center of National Defense Medical University for technical services, and BIOTOOLS Co., Ltd., Taiwan, for proteomics analysis support. This work was supported by grants from the National Science and Technology Council (NSTC 112-2320-B-016-006-, NSTC 113-2320-B-016-007-, NSTC 112-2314-B-016-032-MY3 and NSTC 113-2628-B-038-002-MY3), the National Defense Medical University (MND-MAB-C06-112023, MND-MAB-C05-113015 and MND-MAB-D-114184 ), Tri-Service General Hospital (TSGH-E-114227), the Taoyuan General Hospital, Ministry of Health and Welfare (PTH114008), and TMU Research Center of Cancer Translational Medicine from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) Conflict of Interest The authors declare no conflict of interest. Author Contribution Statement P.-Y.L., Y.-L.L., contributed to study design in vitro and in vivo experiments, result analysis, and the drafting of manuscript. Y.-C.H., W.-C.T., Y.-G.C., helped with in vivo experiment and data analysis. Y.-L.T., C.-M.L., J.-R.S., helped with data analysis and immunostaining. Y.-C.C., Y.-X.D., performed cell culture and immunostaining. C.-W.L., T.-J.L., and Y.-T.L., performed immunostaining and confocal imaging. Y.-C.C. contributed to the study, design, result interpretation, and manuscript writing. Ethics declarations All methods were performed in accordance with the relevant guidelines and regulations. All animal experiments were approved by the National Defense Medical Center Animal Experiment Ethics Committee (IACUC-23-058 and IACUC-24-026). This study does not directly involve human subjects or human data that requires ethical approval. Availability of Data and Materials The experimental data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. No applicable resources were generated during the current study. References Liu H, Wang S, Wang J, Guo X, Song Y, Fu K , et al. Energy metabolism in health and diseases. Signal Transduct Target Ther 2025, 10 (1) : 69. Hardie DG. 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Schuster K, Staffeld A, Zimmermann A, Böge N, Lang S, Kuhla A , et al. Starvation in Mice Induces Liver Damage Associated with Autophagy. Nutrients 2024, 16 (8). Clague MJ, Urbé S. Ubiquitin: same molecule, different degradation pathways. Cell 2010, 143 (5) : 682-685. Chen C, Qin H, Tan J, Hu Z, Zeng L. The Role of Ubiquitin-Proteasome Pathway and Autophagy-Lysosome Pathway in Cerebral Ischemia. Oxid Med Cell Longev 2020, 2020: 5457049. Pineda CT, Ramanathan S, Fon Tacer K, Weon JL, Potts MB, Ou YH , et al. Degradation of AMPK by a cancer-specific ubiquitin ligase. Cell 2015, 160 (4) : 715-728. An H, Jang Y, Choi J, Hur J, Kim S, Kwon Y. New Insights into AMPK, as a Potential Therapeutic Target in Metabolic Dysfunction-Associated Steatotic Liver Disease and Hepatic Fibrosis. Biomol Ther (Seoul) 2025, 33 (1) : 18-38. Zhao P, Saltiel AR. From overnutrition to liver injury: AMP-activated protein kinase in nonalcoholic fatty liver diseases. J Biol Chem 2020, 295 (34) : 12279-12289. Cai Y, Fang L, Chen F, Zhong P, Zheng X, Xing H , et al. Targeting AMPK related signaling pathways: A feasible approach for natural herbal medicines to intervene non-alcoholic fatty liver disease. J Pharm Anal 2025, 15 (1) : 101052. Karatas T, Onalan S, Yildirim S. Effects of prolonged fasting on levels of metabolites, oxidative stress, immune-related gene expression, histopathology, and DNA damage in the liver and muscle tissues of rainbow trout (Oncorhynchus mykiss). Fish Physiol Biochem 2021, 47 (4) : 1119-1132. Deng M, Yang X, Qin B, Liu T, Zhang H, Guo W , et al. Deubiquitination and Activation of AMPK by USP10. Mol Cell 2016, 61 (4) : 614-624. Zhou Y, Liu F. Coordination of the AMPK, Akt, mTOR, and p53 Pathways under Glucose Starvation. Int J Mol Sci 2022, 23 (23). Khan MR, Xiang S, Song Z, Wu M. The p53-inducible long noncoding RNA TRINGS protects cancer cells from necrosis under glucose starvation. Embo j 2017, 36 (23) : 3483-3500. Mo JS, Meng Z, Kim YC, Park HW, Hansen CG, Kim S , et al. Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. Nat Cell Biol 2015, 17 (4) : 500-510. Additional Declarations There is no duality of interest Supplementary Files Supplementaryinformation.docx Supplementary information Originalwesternblots.pdf Original western blots 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. 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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-7469946","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":511593653,"identity":"a36f85ce-2367-43f2-842e-ce0e4d80f96f","order_by":0,"name":"Ying-Chuan Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3ElEQVRIiWNgGAWjYFCCBAOGhAoJOX4wu4BYLR/OWBhLNkDYxGlhnNlWkbjhAIhDjBb+9uRt0rxtEsbG51cnfnhgwCDPL3YAvxaJM8/KpHnOSciZ3Xi7WQLoMMOZsxMIWHMjx0yap0zC2OzG2Q0gLQkGtwlokQdrYZNI3Dzj7OYfRGkxAGqRnNEmkbiBv3cbcbYYnnlWbPHhjISxxA3ebRYJBhKE/SJ3PHnjjYSKOjn+/rObb/6osJHnlyagBQhYJMCUBFilBEHlIMD8AUzxHyBK9SgYBaNgFIxAAAB0fUc/JG5+xQAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-8031-1893","institution":"College of Biomedical Sciences, National Defense Medical University","correspondingAuthor":true,"prefix":"","firstName":"Ying-Chuan","middleName":"","lastName":"Chen","suffix":""},{"id":511593654,"identity":"b1ec757a-e4e2-4e00-918d-89046e21152b","order_by":1,"name":"Pei-Yao Liu","email":"","orcid":"","institution":"University of Minnesota","correspondingAuthor":false,"prefix":"","firstName":"Pei-Yao","middleName":"","lastName":"Liu","suffix":""},{"id":511593655,"identity":"3dc19207-d5aa-443b-aaf2-36a653253e62","order_by":2,"name":"Yu-Lung Lin","email":"","orcid":"","institution":"University of Minnesota Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yu-Lung","middleName":"","lastName":"Lin","suffix":""},{"id":511593656,"identity":"fdfd62ea-c4f1-4a7d-afae-46e92753dd1e","order_by":3,"name":"Yen-Chun Ho","email":"","orcid":"https://orcid.org/0000-0001-9147-2502","institution":"Academia Sinica","correspondingAuthor":false,"prefix":"","firstName":"Yen-Chun","middleName":"","lastName":"Ho","suffix":""},{"id":511593657,"identity":"491d8304-f806-4a8c-893e-b4e7eff9a6e4","order_by":4,"name":"Wen-Chiuan Tsai","email":"","orcid":"https://orcid.org/0000-0003-1085-9014","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Wen-Chiuan","middleName":"","lastName":"Tsai","suffix":""},{"id":511593658,"identity":"4db79cd7-38f6-4b58-932f-842a07eab473","order_by":5,"name":"Yu-Guang Chen","email":"","orcid":"https://orcid.org/0000-0003-4379-5416","institution":"Tri-Service General Hospital, National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Yu-Guang","middleName":"","lastName":"Chen","suffix":""},{"id":511593659,"identity":"f6a8680c-6f05-417c-89e8-ee4c05641319","order_by":6,"name":"Yu-Ling Tsai","email":"","orcid":"https://orcid.org/0000-0001-6229-8189","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yu-Ling","middleName":"","lastName":"Tsai","suffix":""},{"id":511593660,"identity":"9d661269-8bbd-4902-887b-4b0bc970f7a3","order_by":7,"name":"Chien-Ming Lin","email":"","orcid":"https://orcid.org/0000-0001-7525-5743","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chien-Ming","middleName":"","lastName":"Lin","suffix":""},{"id":511593661,"identity":"d32cb303-ec5d-44c1-a27c-0bd75cd6723c","order_by":8,"name":"Jun-Ren Sun","email":"","orcid":"","institution":"National Defense Medical Center","correspondingAuthor":false,"prefix":"","firstName":"Jun-Ren","middleName":"","lastName":"Sun","suffix":""},{"id":511593662,"identity":"4db64dd1-50a4-40d5-82f7-9624c50806c0","order_by":9,"name":"Yu-Chan Chang","email":"","orcid":"https://orcid.org/0000-0003-0474-9935","institution":"National Yang Ming Chiao Tung University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Chan","middleName":"","lastName":"Chang","suffix":""},{"id":511593663,"identity":"2f012b46-55ee-4480-b276-336f69a4640b","order_by":10,"name":"Yi-Xuan Ding","email":"","orcid":"","institution":"Tri-Service General Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yi-Xuan","middleName":"","lastName":"Ding","suffix":""},{"id":511593664,"identity":"a05e864c-9497-4aee-950d-3e346540355b","order_by":11,"name":"Chi-Wei Liu","email":"","orcid":"","institution":"Taoyuan General Hospital, Ministry of Health and Welfare","correspondingAuthor":false,"prefix":"","firstName":"Chi-Wei","middleName":"","lastName":"Liu","suffix":""},{"id":511593665,"identity":"49a465e8-6a84-42bb-baae-2a22cdae1718","order_by":12,"name":"Te-Jung Liu","email":"","orcid":"","institution":"College of Biomedical Sciences, National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Te-Jung","middleName":"","lastName":"Liu","suffix":""},{"id":511593666,"identity":"7b821b73-4d09-4665-b1e9-7b4ec05fe958","order_by":13,"name":"Yi-Tian Lin","email":"","orcid":"","institution":"College of Biomedical Sciences, National Defense Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yi-Tian","middleName":"","lastName":"Lin","suffix":""}],"badges":[],"createdAt":"2025-08-27 09:10:47","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7469946/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7469946/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91206324,"identity":"16c777e4-5231-435a-951d-6289f6f6258d","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2004008,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 interacts with AMPK.\u003cbr\u003e\n \u003c/strong\u003e(A) Identification of AMPK-binding proteins by combining co-immunoprecipitation and mass spectrometry. (B–E) Endogenous RNF128 interacts with endogenous AMPK. Extracts from C2C12 or HEK293 cells were prepared, immunoprecipitated with anti-RNF128, anti-AMPK, or rabbit anti-immunoglobulin G antibodies, and analyzed using anti-RNF128 and anti-AMPK antibodies. (F, G) The subcellular localization of RNF128 and AMPK in C2C12 or AML-12 cells was assessed via immunofluorescence microscopy (THUNDER Imaging Systems).\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/36f30dace23a25d4292e2e17.png"},{"id":91206646,"identity":"7e75d756-7291-4ac1-b068-b79389001fe0","added_by":"auto","created_at":"2025-09-12 16:47:02","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":820209,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 enhances AMPK polyubiquitination.\u003cbr\u003e\n \u003c/strong\u003e(A–C) HEK293 cells were transiently transfected with hemagglutinin-ubiquitin (wild-type), hemagglutinin-ubiquitin (K48), hemagglutinin-ubiquitin (K63), Flag-AMPK, and RNF128. After 48 h, cell lysates were collected and subjected to immunoprecipitation with a Flag antibody. Ubiquitination was then assessed by immunoblotting with the indicated antibodies. (D, E) Lysates from C2C12 stable cell lines, either overexpressing or silencing RNF128, were immunoprecipitated using an AMPK antibody. Ubiquitination was subsequently analyzed by immunoblotting with the designated antibody.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/ba00bd776a97e1dd73899568.png"},{"id":91207335,"identity":"681f8563-9f40-49c8-ba54-fd53ad9d2415","added_by":"auto","created_at":"2025-09-12 16:55:02","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":647748,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 promotes AMPK phosphorylation under glucose starvation.\u003cbr\u003e\n\u003c/strong\u003e(A, B) Immunoblot analysis of P-AMPK, AMPK, P-ACC, and ACC expression in the indicated stable cell lines exposed to glucose-deprived medium. (C, D) HEK293/vector and HEK293/RNF128 cells were incubated in glucose-deprived medium and collected at specified time points. The resulting cell lysates were analysed by immunoblotting using the designated antibodies. (E, F) HEK293/shluc and HEK293/shRNF128 cells were exposed to glucose-deprived medium and harvested at the indicated intervals. Cell lysates were subjected to immunoblot analysis with the indicated antibodies. Data are presented as the mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance with the Newman–Keuls post hoc test or Student’s t-test. \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":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/b6e2442b1964a2f8d9f7e343.png"},{"id":91206332,"identity":"891c2baa-d688-425c-a437-2808825d06cf","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":691875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverexpression of RNF128 decreases cell viability during glucose deprivation.\u003cbr\u003e\n \u003c/strong\u003e(A–D) Cell viability was assessed in C2C12/RNF128, HEK293/RNF128, AML12/RNF128, and HepG2/RNF128 cell lines at the indicated time points post-induction. (E, F) Cell death was measured by PI staining in C2C12/vector and C2C12/RNF128 cells after 24 h of glucose starvation. (G) Cleaved caspase3, caspase3, and RNF128 protein levels in C2C12/vector and C2C12/RNF128 cells were measured following 24 h of glucose deprivation. Data are presented as the mean ± standard deviation. Statistical significancewas assessed using Student’s t-test. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/eb6345f40e8fad82b6918b92.png"},{"id":91206649,"identity":"31053fd5-d6b3-4336-914a-5f9c169d4380","added_by":"auto","created_at":"2025-09-12 16:47:02","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":728838,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 silencing is associated with increased cell viability during glucose deprivation.\u003cbr\u003e\n \u003c/strong\u003e(A–D) Cell viability was evaluated in C2C12/shRNF128, HEK293/RNF128 KO, AML12/shRNF128, and HepG2/shRNF128 cell lines at the indicated post-induction time points. (E, F) PI staining measured cell death in C2C12/shluc and C2C12/shRNF128 after 24 hof glucose starvation. (G) Levels of cleaved caspase3, caspase3, and RNF128 proteins were analyzed in C2C12/shluc and C2C12/shRNF128 cells following 24 h of glucose deprivation. Data are presented as the mean ± standard deviation. Statisticalsignificance was determined using Student’s t-test. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/309d8bac53c2c249b54ad43c.png"},{"id":91206339,"identity":"5d7d65a3-a68f-4fbf-941b-cd532f44e3d0","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":905704,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 modulates cell death induced by AMPK activators.\u003cbr\u003e\n \u003c/strong\u003e(A, B) Cell viability was assessed in C2C12/RNF128 and HEK293/RNF128 cell lines after 24 h of 2-DG treatment. (C, D) Viability was evaluated in these cell lines following 36 h of phenformin treatment. (E, F) Cleaved caspase3 and caspase3 levels were measured in C2C12/vector and C2C12/RNF128 cells after exposure to 2-DG or phenformin. (G, H) Cell viability of C2C12/shRNF128 and HEK293/RNF128 KO cell lines was determined after 24 h of 2-DG treatment. (I, J) Viability in these cell lines was also examined after 36 h of phenformin treatment. (K, L) Cleaved caspase3 and caspase3 were detected in C2C12/shluc and C2C12/shRNF128 cells after treatment with 2-DG or phenformin. Data are presented as the mean ± standard deviation. Statistical significance was determined using one-way analysis of variance with the Newman–Keuls post hoc test or Student’s t-test. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/1429643784bf06e91ff05a77.png"},{"id":91206651,"identity":"f1a95d5c-2d27-42ba-932d-5c671b8108a8","added_by":"auto","created_at":"2025-09-12 16:47:02","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":754727,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 regulates ROS levels and glycolysis gene expression during glucose starvation.\u003cbr\u003e\n \u003c/strong\u003e(A, B) ROS levels were assessed in C2C12/vector and C2C12/RNF128 cells after 24 h of glucose starvation. (C, D) ROS levels were also measured in C2C12/shluc and C2C12/shRNF128 cells under the same conditions. (E) The mRNA levels of hexokinase-2, phosphoglycerate mutase 2, pyruvate kinase M, aldolase A, phosphoglycerate kinase 1, triosephosphate isomerase 1, lactate dehydrogenase A, and phosphoglycerate mutase 1 were measured in C2C12/vector and C2C12/RNF128 cell lines, or (F) Expression of the same genes was assessed in C2C12/shluc and C2C12/shRNF128 cellsafter 6 h of glucose deprivation. Data are presented as the mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance with the Newman–Keuls post hoc test or Student’s t-test. ***P \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/5d59f72fce65390a7bd59733.png"},{"id":91206341,"identity":"57875765-390f-49de-9209-d73070bd79f6","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1075829,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRNF128 modulates AMPK activation and hepatic injury during fasting.\u003cbr\u003e\n\u003c/strong\u003e(A, B) Immunoblot analyses of P-AMPK, AMPK, P-ACC, and ACC were performed on the designated groups following a 24-h fasting period. (C, D) Serum AST and ALT levels were measured in each group after 24 h of fasting (n = 4 per group). (E, F) Immunoblot analyses for P-AMPK, AMPK, P-ACC, and ACC were conducted in AAV8-vector and AAV8-RNF128 mice after 24 h of fasting. (G, H) Serum AST and ALT concentrations were assessed in the indicated groups after 24 h of fasting (n = 4 per group). (I) Functional model of RNF128 in modulating the cellular response to energy stress. Figure created using elements from BioRender.com. Data are presented as the mean ± standard deviation. Statistical significance was assessed using one-way analysis of variance with the Newman–Keuls post hoc test or Student’s t-test. \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":"8.png","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/bc505eef67b165bdf9d5bad7.png"},{"id":91882661,"identity":"69047326-fd2f-4bcd-acc1-21f070344004","added_by":"auto","created_at":"2025-09-22 15:08:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10007554,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/e85b116d-299b-4b6f-bf48-2b75db112cd7.pdf"},{"id":91206335,"identity":"5be32c95-3430-4d22-96da-23a0a48dd3ab","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1369917,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"Supplementaryinformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/5535ec241b2a41430071fc6c.docx"},{"id":91206329,"identity":"30faef01-50ae-4317-a79b-4877b1349ff9","added_by":"auto","created_at":"2025-09-12 16:39:02","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1849636,"visible":true,"origin":"","legend":"Original western blots","description":"","filename":"Originalwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7469946/v1/d982c5eb575b0886cbdeffb5.pdf"}],"financialInterests":"There is no duality of interest","formattedTitle":"Regulation of cellular response to energy stress by RNF128 via modulation of AMPK activation","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCellular energy production is primarily mediated by glycolysis and oxidative phosphorylation. Glucose serves as the main substrate for glycolysis and facilitates ATP synthesis, the universal energy currency of the cell \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Under conditions of sufficient glucose availability, cells maintain a high ATP:AMP ratio. In contrast, periods of energetic stress\u0026mdash;such as hypoxia, nutrient deprivation, or increased energy demand\u0026mdash;are associated with a reduction in this ratio \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDuring glucose deprivation, cells initiate adaptive mechanisms to preserve energy homeostasis. Central to this response is AMP-activated protein kinase (AMPK), a key regulator of cellular energy balance. AMPK is a conserved serine/threonine kinase complex that functions as a cellular energy sensor \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The AMPK heterotrimer consists of a catalytic α subunit and regulatory β and γ subunits. It is broadly expressed in eukaryotes and regulates metabolism. AMPK is activated when cellular AMP:ATP and ADP:ATP ratios increase, reflecting energetic stress. Glucose deprivation, for example, reduces glycolytic ATP production, leading to elevated AMP and ADP levels. Once activated, AMPK initiates responses aimed at restoring energy balance \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. AMPK serves as a central regulator of cellular energy homeostasis, playing a critical role in detecting cellular energy status and coordinating metabolic responses to nutrient fluctuations \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eGlucose deprivation can also result in elevated levels of reactive oxygen species (ROS) and metabolic byproducts, which may further modulate AMPK activity. AMPK plays a critical role in promoting cellular survival by facilitating adaptation to energy stress \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. However, when AMPK activation is prolonged or excessive and homeostasis cannot be restored, this may ultimately lead to cell death \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRING finger protein 128 (RNF128) is a type I transmembrane protein primarily localized to the transferrin-recycling endocytic pathway. It plays a critical role in inducing T cell anergy by suppressing cytokine expression \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. RNF128 forms a ternary complex with Otub-1 and USP8, both of which contribute to the regulation of T cell anergy \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. RNF128 is ubiquitously expressed, with particularly high levels in organs, such as the heart, kidney, and liver. It is involved in glucose and lipid metabolism, contributes to adipocyte differentiation, and has been implicated in diet-induced obesity \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Additionally, RNF128 may influence hepatic steatosis through inhibition of SIRT1 \u003csup\u003e18\u003c/sup\u003e. RNF128 is also capable of influencing the progression of acute lung injury by modulating TLR4 and MPO expression in immune cells \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Collectively, these findings suggest that RNF128 participates in a wide spectrum of biological processes.\u003c/p\u003e\u003cp\u003eIn this study, we aimed to investigate the role of RNF128 in cellular responses to glucose deprivation under both in vitro and in vivo conditions. The rationale was to elucidate a previously unrecognized function of RNF128 in the context of energy stress.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eRNF128 was newly identified to interact with AMPK\u003c/h2\u003e\u003cp\u003eTo elucidate the role of AMPK in cellular energy sensing, we employed mass spectrometry (MS) to identify novel AMPK regulators during energy stress. A total of 202 and 184 AMPK-interacting proteins were detected in glucose-deprived C2C12 and AML12 cells, respectively, with 90 proteins overlapping between the two cell types. Among the 10 most abundant proteins identified, the E3 ubiquitin ligase RNF128 (RING finger protein 128) was consistently enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Using C2C12 cells, we confirmed the interaction between AMPK and RNF128 through co-immunoprecipitation assays, which demonstrated a reciprocal interaction between the two proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Similar interactions were confirmed in HEK293 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). Immunofluorescence microscopy further revealed colocalization of AMPK and RNF128 in both C2C12 and AML12 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG), and confocal imaging yielded comparable results (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, S2). These findings establish RNF128 as a newly identified protein that interacts with AMPK.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eRNF128 promoted AMPK ubiquitination\u003c/h3\u003e\n\u003cp\u003eSince RNF128 functions as an E3 ubiquitin ligase targeting specific proteins \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, we examined whether its E3 ligase activity regulates AMPK ubiquitination and activation. To address this, HEK293 cells were transfected with plasmids encoding RNF128, ubiquitin, and AMPK, followed by analysis of protein expression. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, co-expression of RNF128 and AMPK resulted in increased AMPK polyubiquitination. Examination of endogenous AMPK ubiquitination further showed that RNF128 overexpression enhanced AMPK polyubiquitination compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), whereas RNF128 knockdown reduced AMPK polyubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). To investigate RNF128-mediated polyubiquitination of AMPK in further detail, we compared wild-type ubiquitin with mutant ubiquitin containing a single lysine residue (K48 or K63). RNF128 promoted AMPK polyubiquitination in the presence of hemagglutinin-tagged ubiquitin K48 (HA-Ub-K48) or K63 (HA-Ub-K63) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB, \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Increased K48- and K63-linked AMPK ubiquitination was also observed in C2C12 cells overexpressing RNF128 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD), while silencing RNF128 decreased both K48- and K63-associated AMPK ubiquitination (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Together, these results indicate that RNF128 mediates K48- and K63-associated polyubiquitination of AMPK.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eRNF128 was involved in the regulation of AMPK activation\u003c/h3\u003e\n\u003cp\u003eIn response to glucose deprivation, AMPK activation is tightly regulated to enable cellular adaptation to energy stress. Given that RNF128 interacts with and ubiquitinates AMPK, we examined whether RNF128 contributes to the regulation of AMPK activation under conditions of energy stress. To assess this, we examined the association between RNF128 and AMPK activation status, measured by AMPK T172 phosphorylation under 0 mM glucose. RNF128 expression was silenced in C2C12 (C2C12/shRNF128) cells, and downregulation was confirmed (Fig. S3A). Ablation of RNF128 resulted in reduced P-AMPK levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Consistently, expression of P-ACC, a direct AMPK downstream target, was also decreased in glucose-starved C2C12/shRNF128 cells compared to that in controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Similar results were observed in AML12/shRNF128 cells (Fig. S4A). To further assess the impact of RNF128 on AMPK activation, we generated C2C12 cell lines with stable RNF128 overexpression and confirmed increased RNF128 expression (Fig. S3B). In these cells, P-AMPK and P-ACC levels were significantly elevated under glucose deprivation compared to those in the control groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Consistent with these observations, RNF128 overexpression enhanced AMPK phosphorylation during glucose deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC\u0026ndash;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). Comparable results were also obtained in AML12/RNF128 cells (Fig. S4B). Collectively, these data demonstrate that RNF128 influences AMPK activation and may contribute to the regulation of AMPK-mediated responses to energy stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eRNF128 overexpression increased glucose deprivation-induced cell death\u003c/h3\u003e\n\u003cp\u003eExtended periods of glucose deprivation induce cellular energy stress, activating several pathways that ultimately lead to cell death. However, the mechanisms by which AMPK regulates cell death under sustained energy stress remain incompletely understood. To address this, we examined the influence of RNF128 on AMPK function during energy stress-induced cell death. The results showed that C2C12 cells overexpressing RNF128 (C2C12/RNF128) displayed reduced viability after glucose deprivation compared with control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Similar results were observed in HEK293/RNF128, AML/RNF128, and HepG2/RNF128 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). To further validate the cell viability findings, cell death under glucose deprivation was evaluated using PI staining. The data revealed a markedly higher death rate in C2C12/RNF128 cells (from 5.1\u0026ndash;66.1%) than in C2C12/vector cells (from 5.2\u0026ndash;19.6%) under glucose starvation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). Additionally, energy stress led to elevated caspase-3 cleavage in C2C12/RNF128 cells relative to that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Collectively, these findings indicate that RNF128 overexpression enhances cell death induced by glucose deprivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003eLoss of RNF128 expression attenuated glucose deprivation-induced cell death\u003c/h3\u003e\n\u003cp\u003eTo further verify the regulatory function of RNF128 in energy stress-induced cell death, we knocked down RNF128 expression in C2C12 (C2C12/shRNF128) cells. C2C12/shRNF128 cells exhibited higher viability following glucose deprivation than control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). Similar patterns were observed in HEK293/RNF128 knockout, AML/shRNF128, and HepG2/shRNF128 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). PI staining further showed that C2C12/shRNF128 cells had lower cell death rates (from 4\u0026ndash;13.5%) than C2C12/shluc cells (from 5\u0026ndash;18.3%) under glucose starvation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE, \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Furthermore, energy stress was associated with reduced caspase-3 cleavage in C2C12/shRNF128 cells compared to that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG). These results suggest that RNF128 ablation is associated with reduced cell death during glucose deprivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eElevated RNF128 expression augmented cell death induced by AMPK activation\u003c/h2\u003e\u003cp\u003eTo further elucidate the impact of RNF128 in AMPK-dependent cell death, we evaluated the effects of pharmacological AMPK activation in C2C12 and HEK293 cells overexpressing RNF128 (C2C12/RNF128 and HEK293/RNF128). Both cell types exhibited significantly reduced viability following treatment with 2-DG or phenformin compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Consistently, C2C12/RNF128 cells displayed higher levels of cleaved caspase-3 than controls after 2-DG or phenformin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Taken together, these results suggest that increased expression of RNF128 enhances cell death in response to AMPK activators.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eLower RNF128 expression decreased AMPK activation-induced cell death\u003c/h3\u003e\n\u003cp\u003eWe assessed the effect of RNF128 downregulation on cell death triggered by AMPK activators. The findings indicated that C2C12/shRNF128 cells exhibited increased viability after treatment with 2-DG or phenformin compared to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH). Consistent patterns were also observed in HEK293/RNF128 knockout cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). In line with the hypothesis, C2C12/shRNF128 cells showed reduced cleaved caspase-3 levels compared to controls following 2-DG or phenformin treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eL). These findings indicate that RNF128 downregulation reduces cell death in response to AMPK activators.\u003c/p\u003e\n\u003ch3\u003eAMPK inhibition attenuated the effect of RNF128 on glucose deprivation-induced cell death\u003c/h3\u003e\n\u003cp\u003eTo further validate the role of AMPK in RNF128-mediated cell death during glucose deprivation, the established AMPK inhibitor Compound C was employed. Consistent with expectations, RNF128 overexpression resulted in increased cell death compared to controls under glucose-starved conditions (Fig. S5). Notably, treatment with Compound C attenuated RNF128-induced cell death under metabolic stress (Fig. S5). These findings indicate that RNF128 regulates AMPK-dependent cell death in response to glucose deprivation.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eRNF128 modulated ROS production under glucose deprivation\u003c/h2\u003e\u003cp\u003eGlucose deprivation can reduce ATP synthesis and increase ROS production, ultimately contributing to cell death. We therefore examined the effect of RNF128 on ROS generation. ROS levels were elevated in C2C12/RNF128 cells (from 0.11\u0026ndash;37.7%) compared to those in C2C12/vector cells under glucose starvation conditions (from 0.1\u0026ndash;11.7%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). In contrast, C2C12/shRNF128 cells showed reduced ROS levels (0.1\u0026ndash;10.1%) compared to C2C12/shluc cells (0.17\u0026ndash;16.3%) during glucose deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD). Because glucose deprivation modulates the expression of major glycolytic enzymes and influences glycolytic activity, we evaluated the mRNA levels of genes associated with glycolysis \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. Under glucose-starved conditions, C2C12/RNF128 and AML12/RNF128 cells exhibited increased mRNA expression of glycolysis-associated genes relative to control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE, S4C). Conversely, C2C12/shRNF128 and AML12/shRNF128 cells showed reduced mRNA levels of these glycolysis-related genes compared to the controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF, S4D). Collectively, these results indicate that RNF128 may play a regulatory role in ROS production and glycolysis during glucose deprivation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eRNF128 regulated AMPK activation and liver injury in response to fasting\u003c/h2\u003e\u003cp\u003eTo elucidate the physiological role of RNF128 in modulating AMPK activation under fasting, we found that P-AMPK and P-ACC levels were significantly reduced in livers from fasted RNF128 KO mice compared to those in fasted WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). In contrast, P-AMPK and P-ACC levels were notably elevated in the livers of fasted AAV8-RNF128\u0026ndash;treated mice relative to those in fasted AAV8-vector controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF). Because liver injury is associated with elevated serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e, we measured these enzymes as markers of liver function. Fasted RNF128 KO mice displayed lower serum ALT and AST levels than fasted WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). Additionally, unlike RNF128 KO mice, AAV8-RNF128 mice demonstrated an increase in ALT and AST concentrations during fasting (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eG, \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eH).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eAMPK functions as the principal regulator of cellular energy, coordinating adaptive responses to glucose deprivation. Identifying novel modulators of AMPK activity under energy stress is essential for a comprehensive understanding of these mechanisms. This study provides evidence that RNF128 is a critical regulator of cellular adaptation to glucose starvation. RNF128 modulated AMPK function in both in vitro and in vivo systems. Elevated RNF128 expression enhanced AMPK activation and increased cell death under glucose deprivation, whereas reduced RNF128 expression suppressed AMPK phosphorylation and attenuated glucose deprivation\u0026ndash;induced cell death. In vivo, RNF128 deficiency diminished AMPK phosphorylation and mitigated fasting-induced liver injury, while AAV8-driven RNF128 overexpression produced the opposite effect. Mechanistically, RNF128 was found to regulate AMPK activity by promoting its polyubiquitination and phosphorylation (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eI).\u003c/p\u003e\u003cp\u003eUbiquitination represents a key post-translational modification regulating diverse biological processes. K48-linked ubiquitin chains predominantly mediate protein degradation, whereas K63-linked ubiquitination generally influences signaling pathways and protein trafficking \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Under glucose deprivation, RNF128 facilitated both K63- and K48-linked ubiquitination of AMPK. Notably, RNF128 did not alter AMPK protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). However, K48 ubiquitination of AMPK can promote its degradation under certain conditions \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e, likely reflecting differences in energy stress or cellular context. Further investigation is required to clarify this association.\u003c/p\u003e\u003cp\u003eAMPK activation in the liver during fasting has context-dependent effects: it can protect against liver injury in conditions, such as NAFLD, but excessive or prolonged activation may also exacerbate liver damage \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Our findings indicate that RNF128 regulates AMPK polyubiquitination and thereby modulates its activation during glucose deprivation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). In contrast, prior work has demonstrated that USP10 interacts with and deubiquitinates AMPK, enhancing its phosphorylation and activity, which alleviates hepatic steatosis and metabolic dysfunction induced by high-fat diets in mice \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. Together, these results suggest that distinct energy stress conditions may result in distinct patterns of AMPK regulation and function. Future studies exploring the interplay between USP10 and RNF128 in AMPK modulation could provide further mechanistic insights.\u003c/p\u003e\u003cp\u003eAs the principal energy substrate, glucose is essential for maintaining cellular function; its absence compromises key processes and may culminate in cell death. RNF128 was identified as a mediator of AMPK-dependent cell death under glucose deprivation. Previous studies have also implicated RNF128 in modulating p53-dependent cell death during DNA damage stress \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. During glucose starvation, p53 can exert either pro- or anti-apoptotic effects depending on specific cellular conditions \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Further research is warranted to elucidate the direct interactions among RNF128, AMPK, and p53 in the regulation of cellular responses to energy stress.\u003c/p\u003e\u003cp\u003eIn conclusion, this study identified RNF128 as a regulator of AMPK in the context of energy stress. By delineating the mechanisms underlying AMPK activation and function, these findings advance the understanding of cellular adaptation to metabolic stress and provide a basis for developing therapeutic strategies for diseases associated with metabolic dysregulation.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eAnimal experiments\u003c/h2\u003e\u003cp\u003eThe RNF128 KO mice, generated on a C57B/6J background using CRISPR\u0026ndash;Cas9, carry a deletion at the exon 1 start codon. These mice were produced by the Transgenic Mouse Model Core (Taipei, Taiwan) \u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e and maintained under a standard 12:12 h light/dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C. For fasting experiments, 8-week-old male WT and RNF128 KO mice were used. Fed mice had ad libitum access to food for 24 h, while fasted mice were deprived of food for 24 h and euthanized simultaneously with their fed counterparts. For in vivo AAV administration, AAV8-vector or AAV8-RNF128 was delivered via tail vein injection into 7-week-old male C57BL/6J mice at a dose of 1 \u0026times; 10\u003csup\u003e12\u003c/sup\u003e vg/mouse in a total volume of 200 \u0026micro;L.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eCells, plasmids, and transfection procedures\u003c/h2\u003e\u003cp\u003eC2C12, HEK293, and HepG2 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). AML12 cells were maintained in a 1:1 mixture of Dulbecco's modified Eagle's medium high glucose and Ham\u0026rsquo;s F-12 medium supplemented with 10% FBS. For glucose starvation, cells were washed twice with PBS and incubated in glucose- and pyruvate-free DMEM (0 g l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e glucose, 0 mM pyruvate; Invitrogen) supplemented with 10% dialyzed FBS (Invitrogen) \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. RNF128 was cloned into a pCMV-TNT vector (Promega, CA, USA) using EcoRI and BamHI restriction sites. FLAG-AMPK, HA-ubiquitin-WT, HA-ubiquitin-48, and HA-ubiquitin-K63 plasmids were obtained from Addgene (Watertown, MA, USA). Transfections were performed using jetPRIME (New York, USA) according to the manufacturer\u0026rsquo;s protocol. Cells were seeded at a low density (approximately 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/60 mm dish) and grown to 50\u0026ndash;60% confluence before jetPRIME-mediated gene transfection. Transfected cells were lysed in radioimmunoprecipitation assay buffer for subsequent analyses.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eImmunoprecipitation and immunoblot analysis\u003c/h2\u003e\u003cp\u003eCells were lysed in buffer containing 50 mM Tris (pH 8.0), 5 mM NaCl, 0.5% NP-40, and 1\u0026times; protease inhibitor. Lysates underwent three freeze\u0026ndash;thaw cycles, and proteins were extracted. Protein concentrations were determined using the Bradford method (Bio-Rad, CA, USA). Equal amounts of protein were immunoprecipitated in lysis buffer with antibodies against RNF128, AMPK, or FLAG at 4\u0026deg;C overnight. Dynabeads\u0026trade; Protein G (Invitrogen, Waltham, MA, USA) were added to the immunoprecipitation mixture for 1 h, followed by three washes with SNNTE buffer (5% sucrose, 1% NP-40, 0.5 M NaCl, 50 mM Tris [pH 7.4], and 5 mM EDTA). The precipitates were suspended in SDS\u0026ndash;polyacrylamide gel electrophoresis (PAGE) sample buffer, boiled, and separated by SDS\u0026ndash;PAGE. Proteins were transferred to nitrocellulose membranes and blocked for 1 h in buffer containing 10 mM Tris (pH 7.6), 137 mM NaCl, 0.1% (v/v) Tween 20, and 5% skimmed milk. Membranes were incubated overnight at 4\u0026deg;C with the indicated primary antibodies, followed by horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies. Detection was performed using enhanced chemiluminescence reagents (GE Healthcare, Chicago, IL, USA). Primary antibodies included anti-AMPK (10929-2-AP; Proteintech, Rosemount, IL, USA), anti-caspase 3 (19677-1-AP; Proteintech), anti-HA (51064-2-AP; Proteintech), anti-FLAG (F1804; MilliporeSigma, Burlington, MA, USA), anti-P-AMPK (Thr172) (40H9; Cell Signaling, Danvers, MA, USA), anti-cleaved caspase-3 (9661; Cell Signaling), anti-ACC (3662; Cell Signaling), anti-P-ACC (3661; Cell Signaling), anti-ubiquitin (P4D1; Cell Signaling), anti-K48 ubiquitin (D9D5; Cell Signaling), anti-K63 ubiquitin (D7A11; Cell Signaling), anti-AMPK (9G3; GeneTex; Irvine, CA, USA), anti-actin (MAb1501; Chemicon, Rolling Meadows, IL, USA), and anti-RNF128 (prepared in-house).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e\u003cem\u003eIn vivo ubiquitination assays\u003c/em\u003e\u003c/h2\u003e\u003cp\u003eC2C12/RNF128 or C2C12/shRNF128 cells were lysed in buffer (50 mM Tris [pH 8.0], 5 mM NaCl, 0.5% NP-40, and 1\u0026times; protease inhibitor) and subjected to three freeze\u0026ndash;thaw cycles to recover proteins. Equivalent amounts of protein were immunoprecipitated overnight at 4\u0026deg;C with an anti-AMPK antibody in the same buffer. Dynabeads Protein G (Invitrogen) were then added to the immunoprecipitation mixture and incubated at 4\u0026deg;C for 1 h. Samples were washed thrice with SNNTE buffer, resuspended in SDS\u0026ndash;PAGE sample buffer, and separated by SDS\u0026ndash;PAGE. Proteins were transferred to nitrocellulose membranes and probed with primary antibodies. Detection was performed using enhanced chemiluminescence reagents (GE Healthcare).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e\u003cp\u003eCells were cultured on glass coverslips, fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.1% Triton X-100 for 10 min. Following three PBS washes, slides were blocked with 2% bovine serum albumin (BSA) in PBS for 30 min and incubated overnight at 4\u0026deg;C in 1% BSA in PBS with either rabbit polyclonal anti-RNF128 or mouse monoclonal anti-AMPK (9G3; GeneTex; Irvine, CA, USA) primary antibodies. Secondary antibodies Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen) were applied for 1 h in 1% BSA in PBS at 37\u0026deg;C. Nuclei were counterstained with DAPI for 10 min. Coverslips were mounted with Vectashield (Vector Laboratories, Burlingame, CA, USA), and immunofluorescence microscopy was performed using the THUNDER Imaging Systems.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eVirus particle production, viral transduction, and RNA interference\u003c/h2\u003e\u003cp\u003eRNF128 was cloned into the pQCXIP vector (Clontech Laboratories, Mountain View, CA, USA). The pQCXIP-RNF128 and empty pQCXIP plasmids were transfected into GP2-293 cells using jetPRIME\u0026reg; (Polyplus, NY, USA). Short hairpin RNA (shRNA) oligonucleotides were cloned into the expression vector, pSIREN-Retro-Q (Clontech Laboratories) (RNF128 shRNA target sequence 1: 5\u0026prime;-GAGGCATCCAAGTCACAATGG-3\u0026prime;; RNF128 shRNA target sequence 2: 5\u0026prime;-GCAGGAAGCAGAGGCAGTTAA-3\u0026prime;). Cells were infected with the specified retroviruses in a selection medium containing 2 \u0026micro;g/mL polybrene. After 48 h of infection, puromycin-resistant clones were selected by treating the cells with 2 \u0026micro;g/mL puromycin. The AAV expression vector was constructed using the Helper Free Expression System (Cell Biolabs Inc., San Diego, CA, USA). RNF128 was cloned into the pAAV-MCS vector, and AAV8-overexpressing RNF128 was produced following standard protocols.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eCell viability assay\u003c/h2\u003e\u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8; Sigma-Aldrich, USA). Cells were seeded into 96-well plates, and after adherence, 10 \u0026micro;L of CCK-8 solution was added to each well. Plates were incubated at 37\u0026deg;C in a 5% CO₂ atmosphere for 1 h. Optical density (OD) was measured at 450 nm. Each group was assayed in quintuplicate.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003ePropidium iodide (PI) staining for flow cytometry\u003c/h2\u003e\u003cp\u003ePI fluorescent DNA-binding probes were utilized. Cells were harvested and aliquoted into FACS tubes (up to 1 x 10\u003csup\u003e6\u003c/sup\u003e cells per 100 \u0026micro;L), followed by washing with 2 mL of PBS, Subsequently, the cells were centrifuged at 300 x \u003cem\u003eg\u003c/em\u003e for 5 min and the buffer was decanted from the pellet. This wash step was repeated twice. Cells were then placed in FACS tubes containing 1 mL PBS and 2 \u0026micro;g/mL PI. Flow cytometry was conducted in duplicate, and fluorescence emission was measured.\u003c/p\u003e\u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\u003ch2\u003eROS measurement\u003c/h2\u003e\u003cp\u003eROS were detected using CellROX\u0026trade; Green reagent (Invitrogen, Waltham, MA, USA) at a final concentration of 5 \u0026micro;M, following the manufacturer\u0026rsquo;s instructions. Cells were cultured in 0 mM glucose for 24 h, stained with CellROX reagent, and analyzed by flow cytometry. Flow cytometry was conducted across three independent experiments.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003eBiochemical analysis\u003c/h2\u003e\u003cp\u003eSerum ALT and AST levels were quantified using commercial kits from FUJIFILM (GPT/ALT-P III and GOT/AST-P III, respectively).\u003c/p\u003e\u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\u003ch2\u003eQuantitative reverse-transcription PCR\u003c/h2\u003e\u003cp\u003eRNA was isolated from cells and tissues using TRIzol reagent (Sigma, St. Louis, MO, USA). Complementary DNA was synthesized with Epicenter MMLV. Gene expression in cells was measured using the Applied Biosystems 7500 Real-Time PCR System and IQ2 FAST qPCR kit, whereas expression in tissues was quantified using the Roche LightCycler 480. Primers are listed in Supplementary Table\u0026nbsp;1.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData were analyzed using GraphPad Prism 10 (GraphPad Software). Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. One-way analysis of variance with multiple comparative analyses was used to compare multiple datasets. An unpaired two-tailed Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-test was used for two-group comparisons. A \u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eRNF128, RING finger protein 128;\u0026nbsp;AMPK, AMP-activated protein kinase; QPCR, quantitative reverse-transcription polymerase chain reaction; shRNA, short hairpin RNA; AAV, adeno-associated virus\u003c/p\u003e\n"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors thank the Instrument Center of National Defense Medical University for technical services, and BIOTOOLS Co., Ltd., Taiwan, for proteomics analysis support.\u003c/p\u003e\n\u003cp\u003eThis work was supported by grants from the National Science and Technology Council (NSTC 112-2320-B-016-006-, NSTC 113-2320-B-016-007-, NSTC 112-2314-B-016-032-MY3\u0026nbsp;and NSTC 113-2628-B-038-002-MY3), the National Defense Medical University (MND-MAB-C06-112023, MND-MAB-C05-113015 and MND-MAB-D-114184 ), Tri-Service General Hospital (TSGH-E-114227),\u0026nbsp;the Taoyuan General Hospital, Ministry of Health and Welfare (PTH114008), and TMU Research Center of Cancer Translational Medicine from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interest.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eP.-Y.L., Y.-L.L., contributed to study design in vitro and in vivo experiments, result analysis, and the drafting of manuscript. Y.-C.H., W.-C.T., Y.-G.C., helped with in vivo experiment and data analysis. Y.-L.T., C.-M.L., J.-R.S., helped with data analysis and immunostaining. Y.-C.C., Y.-X.D., performed cell culture and immunostaining. C.-W.L., T.-J.L., and Y.-T.L., performed immunostaining and confocal imaging. Y.-C.C. contributed to the study, design, result interpretation, and manuscript writing.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAll methods were performed in accordance with the relevant guidelines and regulations.\u0026nbsp;All animal experiments were approved by the National Defense Medical Center Animal Experiment Ethics Committee (IACUC-23-058 and IACUC-24-026).\u0026nbsp;This study does not directly involve human subjects or human data that requires ethical approval.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of Data and Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experimental data sets generated and/or analyzed during the current study are available from the corresponding author upon reasonable request. No applicable resources were generated during the current study.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eLiu H, Wang S, Wang J, Guo X, Song Y, Fu K\u003cem\u003e, et al.\u003c/em\u003e Energy metabolism in health and diseases. \u003cem\u003eSignal Transduct Target Ther\u003c/em\u003e 2025, \u003cstrong\u003e10\u003c/strong\u003e(1)\u003cstrong\u003e:\u003c/strong\u003e 69.\u003c/li\u003e\n \u003cli\u003eHardie DG. Sensing of energy and nutrients by AMP-activated protein kinase. \u003cem\u003eAm J Clin Nutr\u003c/em\u003e 2011, \u003cstrong\u003e93\u003c/strong\u003e(4)\u003cstrong\u003e:\u003c/strong\u003e 891s-896.\u003c/li\u003e\n \u003cli\u003eHardie DG. 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The p53-inducible long noncoding RNA TRINGS protects cancer cells from necrosis under glucose starvation. \u003cem\u003eEmbo j\u003c/em\u003e 2017, \u003cstrong\u003e36\u003c/strong\u003e(23)\u003cstrong\u003e:\u003c/strong\u003e 3483-3500.\u003c/li\u003e\n \u003cli\u003eMo JS, Meng Z, Kim YC, Park HW, Hansen CG, Kim S\u003cem\u003e, et al.\u003c/em\u003e Cellular energy stress induces AMPK-mediated regulation of YAP and the Hippo pathway. \u003cem\u003eNat Cell Biol\u003c/em\u003e 2015, \u003cstrong\u003e17\u003c/strong\u003e(4)\u003cstrong\u003e:\u003c/strong\u003e 500-510.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"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":"RNF128, AMPK, cell death, glucose starvation, ubiquitination","lastPublishedDoi":"10.21203/rs.3.rs-7469946/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7469946/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eAMP-activated protein kinase (AMPK) is a highly conserved central regulator of metabolic processes, maintaining energy homeostasis under metabolic stress. Identifying new regulators of AMPK is critical to understanding its response to energy stress. This study reports the identification of RING finger protein 128 (RNF128), an AMPK-binding E3 ligase that physically and functionally interacts with AMPK. RNF128 facilitates the polyubiquitination and phosphorylation of AMPK, thereby modulating its activation in response to glucose deprivation. Overexpression of RNF128 enhanced cell death under energy stress conditions, whereas loss of RNF128 expression attenuated stress-induced cell death. RNF128 also regulated reactive oxygen species production and influenced the expression of glycolysis-related genes during glucose deprivation. In vivo, RNF128 deficiency reduced AMPK phosphorylation and alleviated fasting-induced liver injury, whereas adeno-associated virus serotype 8-mediated overexpression of RNF128 increased AMPK phosphorylation and exacerbated liver injury. In conclusion, this study demonstrates that RNF128 functions as an E3 ligase that promotes AMPK polyubiquitination and activation under energy stress, revealing a previously unrecognized role for RNF128.\u003c/p\u003e","manuscriptTitle":"Regulation of cellular response to energy stress by RNF128 via modulation of AMPK activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-12 16:38:57","doi":"10.21203/rs.3.rs-7469946/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":"ca02f537-8577-4aad-aa96-279077d908bf","owner":[],"postedDate":"September 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54330572,"name":"Biological sciences/Cell biology/Proteolysis/Ubiquitin ligases"},{"id":54330573,"name":"Biological sciences/Cell biology"}],"tags":[],"updatedAt":"2025-09-22T15:12:20+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-12 16:38:57","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7469946","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7469946","identity":"rs-7469946","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}