Background Diabetes accelerates atherosclerosis by driving persistent vascular inflammation. MicroRNA-155 (miR-155) is a post-transcriptional regulator of inflammatory genes, while suppressor of cytokine signaling 1 (Socs1) limits Janus kinase (JAK)/signal transducer and activator of transcription (STAT)-mediated cytokine responses. We explored how the imbalance between miR-155-5p and Socs1 contributes to atherosclerotic plaque progression in diabetes. Methods Apolipoprotein E knockout (ApoE-/-) mice were studied in two settings: age-dependent atherosclerosis progression under non-diabetic conditions, and streptozotocin-induced diabetes to model accelerated atherosclerosis. Diabetic mice received a miR-155-5p inhibitor, a Socs1-expressing adenovirus, or respective controls. Lesion size, composition, and gene expression were analyzed. Cultured vascular smooth muscle cells (VSMCs) and macrophages were transfected with miR-155-5p mimic/inhibitor and Socs1 siRNA/plasmid to assess inflammatory responses, phenotypes, and efferocytosis under diabetic-like conditions. Results During atherosclerosis progression, vascular miR-155-5p inversely correlated with Socs1 and positively with lesion size, while Socs1 correlated negatively with plaque burden. In diabetic mice, miR-155-5p inhibition reduced lesion area, lipid/collagen and macrophage/VSMC ratios, pro-inflammatory cytokines, M1 macrophages and synthetic VSMC markers, while increasing Socs1, M2 and contractile VSMC genes. Socs1 gene transfer reproduced these effects by reducing miR-155-5p and Stat1 expression, and lesion size. In vitro, miR-155-5p mimic suppressed Socs1, activated STAT1 and inflammatory phenotypes in macrophages and VSMCs, whereas miR-155-5p inhibition had opposite effects. Socs1 silencing amplified inflammation, and its overexpression counteracted miR-155-5p actions. Moreover, miR-155-5p inhibition reduced soluble Mer receptor tyrosine kinase (MerTK) in plaques and macrophages, indicating improved efferocytosis, whereas the mimic promoted macrophage MerTK shedding and impaired apoptotic cell clearance. Conclusion Reciprocal regulation between miR-155-5p and Socs1 influences vascular inflammation, phenotypic changes, and defective efferocytosis in a diabetic context. Targeting this axis may restore resolution mechanisms and enhance plaque stability in diabetes-associated vascular disease.

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is a prevalent chronic liver disease, encompassing a spectrum from simple steatosis to steatohepatitis (MASH), cirrhosis, and hepatocellular carcinoma. As part of metabolic syndrome, MASLD/MASH is characterized by inflammation, oxidative stress, and fibrosis, highlighting the need for targeted therapies. The dysregulation of Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and its negative regulators the suppressors of cytokine signaling (SOCS), plays a critical role in liver function and contributes to MASLD progression. Aim: Based on a SOCS1 functional domain, we developed mimetic peptides (linear and cyclic) targeting JAK activity and assessed their hepatoprotective potential in experimental MASLD/MASH. Results: In dietary mouse models of MASLD/MASH, the administration of peptides ameliorated liver damage at both early and advanced stages, as evidenced by significant decreases in serum transaminases and hepatic content of lipids, inflammatory cells, and collagen. Treatment attenuated hepatic STAT1/3 activation and downregulated genes involved in inflammation, fibrosis, and lipid metabolism. Livers from treated mice exhibited lower levels of oxidative damage markers, reduced expression of NADPH oxidase 1 (NOX1), and upregulation of the antioxidant genes catalase and superoxide dismutase. In vitro, the peptides were safe for hepatocytes at different doses and effectively counteracted palmitate-induced cytotoxicity, superoxide anion production, and cytokine and NOX1 expression, while increasing anti-inflammatory and antioxidant genes. Conclusions: SOCS1 mimetic peptides exhibit hepatoprotective effects in experimental MASLD/MASH by modulating lipotoxicity, inflammation, redox balance and fibrogenesis. This proof-of-concept supports their potential as candidates for preclinical MASLD therapy development.

Abstract Background and Aims Chronic kidney disease and atherosclerosis are common vascular complication of diabetes and a global health issue. Hyperglycemia, dyslipidemia, and cytokines activate Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway to induce mediators of inflammation and oxidative stress, which are critical events in the progression of renal and vascular damage in diabetic patients. MicroRNAs are small, non-coding molecules and key regulators of gene expression. There is evidence of microRNA-155 (miR-155) involvement in diabetes complications, but the underlying mechanisms are unclear. In this study, gain- and loss-of-function experiments were applied to investigate the interplay between miR-155-5p and suppressor of cytokine signaling 1 (SOCS1) in the regulation of JAK/STAT pathway during renal and vascular injury in diabetes. Method Mesangial, tubuloepithelial, and vascular smooth muscle cells were transfected with miR-155-5p mimic or inhibitor, SOCS1 expression plasmid or siRNA, before stimulation with cytokines or high-glucose. Apolipoprotein E deficient mice with streptozotocin-induced diabetes were treated with either SOCS1-encoding adenovirus or miR-155-5p inhibitor. Samples were analyzed for the expression of miR-155-5p, SOCS1, and mediators of renal and vascular damage. Results In diabetic mice, the expression of miR-155-5p correlated inversely with SOCS1 levels and positively with markers of renal dysfunction, atherosclerosis, inflammation and oxidative stress. In renal cells, transfection with miR-155-5p mimic downregulated SOCS1, activated STAT1 and cytokine expression, and stimulated cell proliferation and migration. Mimic transfection also promoted the transition from contractile to synthetic phenotype in vascular smooth muscle cells. Conversely, both miR-155-5p antagonism and SOCS1 overexpression protected cells from inflammatory damage. In vivo SOCS1 gene delivery in diabetic mice decreased miR-155-5p and prevented kidney and vascular injury. Finally, therapeutic inhibition of miR-155-5p alleviates albuminuria, renal inflammation and fibrosis in diabetic mice, and also reduced atherosclerosis burden and improved plaque stability. Conclusion The modulation of miR-155/SOCS1 axis protects against diabetic renal and vascular damage in mice, thus highlighting its potential as therapeutic target for chronic complications of diabetes.

Metabolic adaptations are a hallmark of cancer and may be exploited to develop novel diagnostic and therapeutic tools. Only about 50% of the patients who undergo thyroidectomy due to suspicion of thyroid cancer actually have the disease, highlighting the diagnostic limitations of current tools. We explored the possibility of using non-invasive blood tests to accurately diagnose thyroid cancer. We analyzed blood and thyroid tissue samples from two independent cohorts of patients undergoing thyroidectomy at the Hospital Universitario 12 de Octubre (Madrid, Spain). As expected, histological comparisons of thyroid cancer and hyperplasia revealed higher proliferation and apoptotic rates and enhanced vascular alterations in the former. Notably, they also revealed increased levels of membrane-bound phosphorylated AKT, suggestive of enhanced glycolysis, and alterations in mitochondrial sub-cellular distribution. Both characteristics are common metabolic adaptations in primary tumors. These data together with reduced mtDNA copy number and elevated levels of the mitochondrial antioxidant PRX3 in cancer tissue samples suggest the presence of mitochondrial oxidative stress. In plasma, cancer patients showed higher levels of cfDNA and mtDNA. Of note, mtDNA plasma levels inversely correlated with those in the tissue, suggesting that higher death rates were linked to lower mtDNA copy number. In PBMCs, cancer patients showed higher levels of PGC-1α, a positive regulator of mitochondrial function, but this increase was not associated with a corresponding induction of its target genes, suggesting a reduced activity in cancer patients. We also observed a significant difference in the PRDX3/PFKFB3 correlation at the gene expression level, between carcinoma and hyperplasia patients, also indicative of increased systemic metabolic stress in cancer patients. The correlation of mtDNA levels in tissue and PBMCs further stressed the interconnection between systemic and tumor metabolism. Evaluation of the mitochondrial gene ND1 in plasma, PBMCs and tissue samples, suggested that it could be a good biomarker for systemic oxidative metabolism, with ND1/mtDNA ratio positively correlating in PBMCs and tissue samples. In contrast, ND4 evaluation would be informative of tumor development, with ND4/mtDNA ratio specifically altered in the tumor context. Taken together, our data suggest that metabolic dysregulation in thyroid cancer can be monitored accurately in blood samples and might be exploited for the accurate discrimination of cancer from hyperplasia.

Diabetic kidney disease (DKD) is a common microvascular complication of diabetes, a global health issue. Hyperglycemia, in concert with cytokines, activates the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway to induce inflammation and oxidative stress contributing to renal damage. There is evidence of microRNA-155 (miR-155) involvement in diabetes complications, but the underlying mechanisms are unclear. In this study, gain- and loss-of-function experiments were conducted to investigate the interplay between miR-155-5p and suppressor of cytokine signaling 1 (SOCS1) in the regulation of the JAK/STAT pathway during renal inflammation and DKD. In experimental models of mesangial injury and diabetes, miR-155-5p expression correlated inversely with SOCS1 and positively with albuminuria and expression levels of cytokines and prooxidant genes. In renal cells, miR-155-5p mimic downregulated SOCS1 and promoted STAT1/3 activation, cytokine expression, and cell proliferation and migration. Conversely, both miR-155-5p antagonism and SOCS1 overexpression protected cells from inflammation and hyperglycemia damage. In vivo, SOCS1 gene delivery decreased miR-155-5p and kidney injury in diabetic mice. Moreover, therapeutic inhibition of miR-155-5p suppressed STAT1/3 activation and alleviated albuminuria, mesangial damage, and renal expression of inflammatory and fibrotic genes. In conclusion, modulation of the miR-155/SOCS1 axis protects kidneys against diabetic damage, thus highlighting its potential as therapeutic target for DKD.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, proteomic, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most obesity and overnutrition-related pathological features were successfully reverted, we observed a high degree of metabolic dysfunction in visceral white adipose tissue, characterized by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and an apparent global healthy phenotype, obesity triggered a cascade of events in visceral adipose tissue progressing from mitochondrial metabolic and proteostatic alterations to widespread cellular stress, which compromises its biosynthetic and recycling capacity. In humans, weight loss after bariatric surgery showed a transcriptional signature in visceral adipose tissue similar to our mouse model of obesity reversion. Overall, our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in visceral adipose tissue.

Remote renal ischemia–reperfusion injury (IRI) following infra-renal aortic occlusion leads to acute kidney injury and systemic inflammation. Hydrogen sulfide is a mediator of IRI and can ameliorate tissue injury in many organ systems. Its role in vascular surgery has yet to be established. We assessed the role of hydrogen sulfide in a rodent model of aortic occlusion.Wistar rats were divided into sham, control, and treatment groups (n = 6). Inflammation was assessed using a nonrecovery protocol. The infra-renal aorta was cross-clamped for 60 min and animals were reperfused for 120 min. Ten minutes before clamp release, treatment animals received hydrogen sulfide (10, 30, or 50 μg/kg) and control animals received 0.9% saline injected into the retroperitoneum. Renal injury and histology were assessed by a recovery protocol. The procedure was identical to the nonrecovery arm but with a single dose of hydrogen sulfide (30 μg/kg) and animals were recovered for 7 days.There was no difference in animal weight between the groups (P = 0.337). In the nonrecovery arm, there was a reduction in serum levels of tumor necrosis factor alpha in sulfide-treated animals compared with controls (909 ± 98 vs. 607 ± 159 pg/mL; P = 0.0038). There was also a reduction in myeloperoxidase-positive cells in renal tissue in the sulfide-treated animals compared with controls (8 ± 4 vs. 17 ± 9; P = 0.03). There was no difference in histological injury score or endothelin-1 levels. In the recovery arm, there was no difference in renal function, Kidney Injury Molecule-1 levels, or histological injury scores.Hydrogen sulfide has systemic and renal anti-inflammatory effects in remote IRI following aortic occlusion in rats.

The connection of oxidative stress with dyslipidemia creates a newly-emerging atherosclerosis risk factor involved in acute ischemic stroke development. This study analyzed the influence of oxidative stress on structural changes of high-density lipoprotein (HDL) particles connected with modification in protective paraoxonase 1 (PON1) activity.This study used 185 patients with acute ischemic stroke and 185 apparently healthy controls. Oxidative stress status, PON1 status, lipids and high-sensitivity C-reactive protein (hsCRP) were determined. In isolated HDL lipoprotein fraction we determined selected markers of oxidative stress (malondialdehyde, MDA) and the content of total sulfhydryl (SH) groups. The capability of oxidative and PON1 status parameters to discriminate patients according to survival status was evaluated.Stroke patients had lower HDL-cholesterol than controls and a remarkable fall in PON1 activity (control group-227 U/L, survivors-42 U/L, lethal outcome group-61 U/L, p < 0.001), along with more prominent inflammation. Pronounced oxidative stress and impaired antioxidative protection was present among patients. HDL fraction analysis revealed a significant decrease of SH groups content (control group vs. patients, p < 0.05) and increased in MDA content in patients (lethal outcome vs. control group, p < 0.05). According to logistic regression analysis, the best predictor of disease outcome was oxidative stress marker – prooxidative-antioxidative balance (PAB).Pronounced oxidative stress in this group of acute ischemic stroke patients probably led to HDL structural changes, which could further cause an alteration or decrease of PON1 activity. Evidence of increased prooxidant level associated with decreased protective, antioxidative factors suggests their mutual involvement in this complex pathology.

Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates dysfunctional mitochondria. Activated by inner membrane depolarization, it plays an important role during development and is fundamental in highly differentiated post-mitotic cells that are highly dependent on aerobic metabolism, such as neurons, muscle cells, and hepatocytes. Both defective and excessive mitophagy have been proposed to contribute to age-related neurodegenerative diseases, such as Parkinson's and Alzheimer's diseases, metabolic diseases, vascular complications of diabetes, myocardial injury, muscle dystrophy, and liver disease, among others. Pharmacological or dietary interventions that restore mitophagy homeostasis and facilitate the elimination of irreversibly damaged mitochondria, thus, could serve as potential therapies in several chronic diseases. However, despite extraordinary advances in this field, mainly derived from in vitro and preclinical animal models, human applications based on the regulation of mitochondrial quality in patients have not yet been approved. In this review, we summarize the key selective mitochondrial autophagy pathways and their role in prevalent chronic human diseases and highlight the potential use of specific interventions.

IntroductionThe proposed benefits of alkaline water generally lack experimental supporting evidence.Material and methodsWe tested the effects of alkaline water on tumor development in C57BL/6 mice. To recapitulate early tumor development, mice were fed a high-fat diet and treated with DEN (50 µg/l) and TCPOBOP (0.5 µg/g) for 24 weeks.ResultsCell proliferation was significantly lower in animals receiving alkaline water, and VEGFR2 staining was higher in the vasculature, suggesting less advanced disease stages. B16-V5 cells were injected subcutaneously or through the tail vein to generate primary tumors or lung metastatic nodules, respectively. Subcutaneous tumors of mice exposed to alkaline water showed a lower proliferative index and vasculature with a better-preserved intima layer and structure. The number of lung metastatic nodules was lower in mice exposed to filtered water. The vascular effects were tested in a rat model of hypertension (spontaneously hypertensive rat – SHR). Following 12 weeks of alkaline water consumption, the aortic rings had an enhanced vasodilatory response to a nitric oxide donor (nitroprusside – NTP), and several inflammatory markers were reduced in blood and heart tissue.ConclusionsThese results indicate that alkaline water could have a protective effect on vascular function, reduce inflammation, and in the context of tumor development, reduce the incidence of metastasis.

BACKGROUND AND PURPOSE:Abdominal aortic aneurysm (AAA) is a multifactorial disease characterized by chronic inflammation, oxidative stress and proteolytic activity in the aortic wall. Targeting JAK/signal transducer and activator of transcription (JAK/STAT) pathway is a promising strategy for chronic inflammatory diseases. We investigated the vasculo-protective role of suppressor of cytokine signalling-1 (SOCS1), the negative JAK/STAT regulator, in experimental AAA.EXPERIMENTAL APPROACH:A synthetic, cell permeable peptide (S1) mimic of SOCS1 kinase inhibitory domain to suppress STAT activation was evaluated in the well-established mouse model of elastase-induced AAA by monitoring changes in aortic diameter, cellular composition and gene expression in abdominal aorta. S1 function was further evaluated in cultured vascular smooth muscle cells (VSMC) and macrophages exposed to elastase or elastin-derived peptides.KEY RESULTS:S1 peptide prevented AAA development, evidenced by reduced incidence of AAA, aortic dilation and elastin degradation, partial restoration of medial VSMC and decreased inflammatory cells and oxidative stress in AAA tissue. Mechanistically, S1 suppressed STAT1/3 activation in aorta, down-regulated cytokines, metalloproteinases and altered the expression of cell differentiation markers by favouring anti-inflammatory M2 macrophage and contractile VSMC phenotypes. In vitro, S1 suppressed the expression of inflammatory and oxidative genes, reduced cell migration and reversed the phenotypic switch of macrophages and VSMC. By contrast, SOCS1 silencing promoted inflammatory response.CONCLUSION AND IMPLICATIONS:This preclinical study demonstrates the therapeutic potential of SOCS1-derived peptide to halt AAA progression by suppressing JAK/STAT-mediated inflammation and aortic dilation. S1 peptide may therefore be a valuable option for the treatment of AAA.

Abstract Background Abdominal aortic aneurysm (AAA), a degenerative vascular pathology characterized by permanent dilation of the aorta, is considered a chronic inflammatory disease involving innate/adaptive immunity. However, the functional role of antibody‐dependent immune response against antigens present in the damaged vessel remains unresolved. We hypothesized that engagement of immunoglobulin G (IgG) Fc receptors (FcγR) by immune complexes (IC) in the aortic wall contributes to AAA development. We therefore evaluated FcγR expression in AAA lesions and analysed whether inhibition of FcγR signaling molecules (γ‐chain and Syk kinase) influences AAA formation in mice. Methods FcγR gene/protein expression was assessed in human and mouse AAA tissues. Experimental AAA was induced by aortic elastase perfusion in wild‐type (WT) mice and γ‐chain knockout (γKO) mice (devoid of activating FcγR) in combination with macrophage adoptive transfer or Syk inhibitor treatment. To verify the mechanisms of FcγR in vitro, vascular smooth muscle cells (VSMC) and macrophages were stimulated with IgG IC. Results FcγR overexpression was detected in adventitia and media layers of human and mouse AAA. Elastase‐perfused γKO mice exhibited a decrease in AAA incidence, aortic dilation, elastin degradation, and VSMC loss. This was associated with (1) reduced infiltrating leukocytes and immune deposits in AAA lesions, (2) inflammatory genes and metalloproteinases downregulation, (3) redox balance restoration, and (4) converse phenotype of anti‐inflammatory macrophage M2 and contractile VSMC. Adoptive transfer of FcγR‐expressing macrophages aggravated aneurysm in γKO mice. In vitro, FcγR deficiency attenuated inflammatory gene expression, oxidative stress, and phenotypic switch triggered by IC. Additionally, Syk inhibition prevented IC‐mediated cell responses, reduced inflammation, and mitigated AAA formation. Conclusion Our findings provide insight into the role and mechanisms mediating IgG‐FcγR‐associated inflammation and aortic wall injury in AAA, which might represent therapeutic targets against AAA disease.

Abstract Background and Aims Diabetic nephropathy is the leading cause of chronic kidney disease worldwide. Hyperglycemia in concert with cytokines activate Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway and induce gene expression of many inflammatory and oxidative stress mediators, which are critical events at all stages of diabetic kidney disease. Suppressor of cytokine signaling 1 (SOCS1) is a STAT-inducible protein and a negative feedback regulator of JAK/STAT pathway. The microRNA-155 is an epigenetic modulator of SOCS1 gene by repressing its translation and, at the same time, is a transcriptional target of STAT, thus completing another regulatory loop of JAK/STAT pathway. Therefore, our aim was to explore the interplay between miR-155 and JAK/STAT/SOCS1 axis in experimental diabetic nephropathy. Method In streptozotocin-induced type 1 diabetic aged mice (wild-type and apolipoprotein E (apoE) knockout) we analyzed the kidney levels of miR-155 and markers of renal damage, inflammation and oxidative stress. In vitro, the expression of miR-155, SOCS1 and STAT1 in mesangial, tubuloepithelial and macrophage cell lines were modulated by silencing/inhibition or overexpression/mimicking experiments to further determine the JAK/STAT pathway activation and expression of downstream target genes. Results In vivo, type 1 diabetes significantly upregulated miR-155 expression in kidneys from both wild-type and apoE knockout mice (1.8- and 4.5-fold vs respective non-diabetic controls). The miR-155 levels directly correlated with parameters of renal damage (serum creatinine, albuminuria, kidney-to-body weight ratio and renal score) and the mRNA expression of chemokines (Ccl2 and Ccl5) and pro-oxidant enzymes (Nox2 and Nox4), but inversely with antioxidant genes (Sod1 and Cat). In vitro, the expression of miR-155 was increased in renal cells and macrophages exposed to hyperglycemia and/or inflammatory conditions. Overexpression of miR-155 reduced SOCS1 expression, enhanced STAT1 and STAT3 activation and pro-inflammatory cytokines and chemokines (Il6, Tnfa, Ccl2 and Cxcl10) expression. By contrast, miR-155 antagonist upregulated SOCS1 and had a protective effect on renal cells by decreasing STAT1/3 phosphorylation and pro-inflammatory gene expression. Additionally, loss- or gain-of function experiments indicate a direct implication of SOCS1 in the regulation of miR-155 expression by STAT transcription factors. Conclusion Our study indicates a pro-inflammatory role of miR-155 in diabetic kidney disease by downregulating renal expression of SOCS1. Therefore, antagonism of miR-155 may have a renoprotective effect in diabetic nephropathy through SOCS1-mediated feedback inhibition of JAK/STAT overactivation. Ongoing in vivo studies with miR-155 inhibitor in experimental diabetes will clarify its role in the development and progression of diabetic nephropathy.

Metabolic plasticity is the ability of a biological system to adapt its metabolic phenotype to different environmental stressors. We used a whole-body and tissue-specific phenotypic, functional, metabolomic and transcriptomic approach to systematically assess metabolic plasticity in diet-induced obese mice after a combined nutritional and exercise intervention. Although most pathological features were successfully reverted, we observed a high degree of metabolic dysfunction irreversibility in visceral white adipose tissue, characterised by abnormal mitochondrial morphology and functionality. Despite two sequential therapeutic interventions and apparent global phenotypic recovery, obesity specifically triggered in visceral adipose a cascade of events progressing from mitochondrial metabolic and proteostatic defects to widespread cellular stress, which compromises its biosynthetic and recycling capacity. Our data indicate that obesity prompts a lasting metabolic fingerprint that leads to a progressive breakdown of metabolic plasticity in white adipose tissue, becoming a significant milestone in disease progression. ### Competing Interest Statement The authors have declared no competing interest.

The chronic activation of the Janus kinase/signal transducer and activator of the transcription (JAK/STAT) pathway is linked to oxidative stress, inflammation and cell proliferation. Suppressors of cytokine signaling (SOCS) proteins negatively regulate the JAK/STAT, and SOCS1 possesses a small kinase inhibitory region (KIR) involved in the inhibition of JAK kinases. Several studies showed that KIR-SOCS1 mimetics can be considered valuable therapeutics in several disorders (e.g., diabetes, neurological disorders and atherosclerosis). Herein, we investigated the antioxidant and atheroprotective effects of PS5, a peptidomimetic of KIR-SOCS1, both in vitro (vascular smooth muscle cells and macrophages) and in vivo (atherosclerosis mouse model) by analyzing gene expression, intracellular O2•− production and atheroma plaque progression and composition. PS5 was revealed to be able to attenuate NADPH oxidase (NOX1 and NOX4) and pro-inflammatory gene expression, to upregulate antioxidant genes and to reduce atheroma plaque size, lipid content and monocyte/macrophage accumulation. These findings confirm that KIR-SOCS1-based drugs could be excellent antioxidant agents to contrast atherosclerosis.

PGC-1α controls, to a large extent, the capacity of cells to respond to changing nutritional requirements and energetic demands. The key role of metabolic reprogramming in tumor development has highlighted the potential role of PGC-1α in cancer. To investigate how loss of PGC-1α activity in primary cells impacts the oncogenic characteristics of spontaneously immortalized cells, and the mechanisms involved, we used the classic 3T3 protocol to generate spontaneously immortalized mouse embryonic fibroblasts (iMEFs) from wild-type (WT) and PGC-1α knockout (KO) mice and analyzed their oncogenic potential in vivo and in vitro. We found that PGC-1α KO iMEFs formed larger and more proliferative primary tumors than WT counterparts, and fostered the formation of lung metastasis by B16 melanoma cells. These characteristics were associated with the reduced capacity of KO iMEFs to respond to cell contact inhibition, in addition to an increased ability to form colonies in soft agar, an enhanced migratory capacity, and a reduced growth factor dependence. The mechanistic basis of this phenotype is likely associated with the observed higher levels of nuclear β-catenin and c-myc in KO iMEFs. Evaluation of the metabolic adaptations of the immortalized cell lines identified a decrease in oxidative metabolism and an increase in glycolytic flux in KO iMEFs, which were also more dependent on glutamine for their survival. Furthermore, glucose oxidation and tricarboxylic acid cycle forward flux were reduced in KO iMEF, resulting in the induction of compensatory anaplerotic pathways. Indeed, analysis of amino acid and lipid patterns supported the efficient use of tricarboxylic acid cycle intermediates to synthesize lipids and proteins to support elevated cell growth rates. All these characteristics have been observed in aggressive tumors and support a tumor suppressor role for PGC-1α, restraining metabolic adaptations in cancer.