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Metabolic dysfunction-associated steatotic liver disease (MASLD) and its advanced subtype, metabolic dysfunction-associated steatohepatitis (MASH), have emerged as the most common chronic liver disease worldwide, yet there is no targeted pharmacotherapy presently available. This study aimed to investigate the possible in vivo function of STE20-type protein kinase MST4, which was earlier implicated in the regulation of hepatocellular lipotoxic milieu in vitro, in the control of the diet-induced impairment of systemic glucose and insulin homeostasis as well as MASLD susceptibility. Whole-body and liver-specific Mst4 knockout mice were generated by crossbreeding conditional Mst4fl/fl mice with mice expressing Cre recombinase under the Sox2 or Alb promoters, respectively. To replicate the environment in high-risk subjects, Mst4-/- mice and their wild-type littermates were fed a high-fat or a methionine-choline-deficient (MCD) diet. Different in vivo tests were conducted in obese mice to describe the whole-body metabolism. MASLD progression in the liver and lipotoxic damage to adipose tissue, kidney, and skeletal muscle were analyzed by histological and immunofluorescence analysis, biochemical assays, and protein and gene expression profiling. In parallel, intracellular fat storage and oxidative stress were assessed in primary mouse hepatocytes, where MST4 was silenced by small interfering RNA. We found that global MST4 depletion had no effect on body weight or composition, locomotor activity, whole-body glucose tolerance or insulin sensitivity in obese mice. Furthermore, we observed no alterations in lipotoxic injuries to the liver, adipose, kidney, or skeletal muscle tissue in high-fat diet-fed whole-body Mst4-/- vs. wild-type mice. Liver-specific Mst4-/- mice and wild-type littermates displayed a similar severity of MASLD when subjected to an MCD diet, as evidenced by equal levels of steatosis, inflammation, hepatic stellate cell activation, fibrosis, oxidative/ER stress, and apoptosis in the liver. In contrast, the in vitro silencing of MST4 effectively protected primary mouse hepatocytes against ectopic lipid accumulation and oxidative cell injury triggered by exposure to fatty acids. In summary, these results suggest that the genetic ablation of MST4 in mice does not mitigate the initiation or progression of MASLD and has no effect on systemic glucose or insulin homeostasis in the context of nutritional stress. The functional compensation for the genetic loss of MST4 by yet undefined mechanisms may contribute to the apparent discrepancy between in vivo and in vitro phenotypic consequences of MST4 silencing.
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Hígado Graso , Enfermedades Metabólicas , Enfermedad del Hígado Graso no Alcohólico , Humanos , Ratones , Animales , Ratones Obesos , Hepatocitos/metabolismo , Hígado Graso/metabolismo , Hígado/metabolismo , Glucosa/metabolismo , Dieta Alta en Grasa/efectos adversos , Ratones Noqueados , Colina/metabolismo , Insulina/metabolismo , Enfermedades Metabólicas/metabolismo , Ratones Endogámicos C57BL , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND: Despite therapeutic advances, the prognosis of pancreatic ductal adenocarcinoma (PDAC) remains extremely poor. Metabolic reprogramming is increasingly recognized as a key contributor to tumor progression and therapy resistance in PDAC. One of the main metabolic changes essential for tumor growth is altered cholesterol flux. Targeting cholesterol flux appears an attractive therapeutic approach, however, the complex regulation of cholesterol balance in PDAC cells remains poorly understood. METHODS: The lipid content in human pancreatic duct epithelial (HPDE) cells and human PDAC cell lines (BxPC-3, MIA PaCa-2, and PANC-1) was determined. Cells exposed to eight different inhibitors targeting different regulators of lipid flux, in the presence or absence of oleic acid (OA) stimulation were assessed for changes in viability, proliferation, migration, and invasion. Intracellular content and distribution of cholesterol was assessed. Lastly, proteome profiling of PANC-1 exposed to the sterol O-acyltransferase 1 (SOAT1) inhibitor avasimibe, in presence or absence of OA, was performed. RESULTS: PDAC cells contain more free cholesterol but less cholesteryl esters and lipid droplets than HPDE cells. Exposure to different lipid flux inhibitors increased cell death and suppressed proliferation, with different efficiency in the tested PDAC cell lines. Avasimibe had the strongest ability to suppress proliferation across the three PDAC cell lines. All inhibitors showing cell suppressive effect disturbed intracellular cholesterol flux and increased cholesterol aggregation. OA improved overall cholesterol balance, reduced free cholesterol aggregation, and reversed cell death induced by the inhibitors. Treatment with avasimibe changed the cellular proteome substantially, mainly for proteins related to biosynthesis and metabolism of lipids and fatty acids, apoptosis, and cell adhesion. Most of these changes were restored by OA. CONCLUSIONS: The study reveals that disturbing the cholesterol flux by inhibiting the actions of its key regulators can yield growth suppressive effects on PDAC cells. The presence of fatty acids restores intracellular cholesterol balance and abrogates the alternations induced by cholesterol flux inhibitors. Taken together, targeting cholesterol flux might be an attractive strategy to develop new therapeutics against PDAC. However, the impact of fatty acids in the tumor microenvironment must be taken into consideration.
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BACKGROUND: Profound resistance to chemotherapy remains a major challenge in achieving better clinical outcomes for patients with pancreatic ductal adenocarcinoma (PDAC). Recent studies indicate that gemcitabine (GEM) resistance is promoted both by pancreatic stellate cells (PSCs) and through increased glycolysis. However, it remains unknown whether PSCs affect GEM sensitivity via glycolytic regulation. METHODS: Human pancreatic cancer cell (PCC) lines (BxPC-3, Capan-2, HPAF-II, Mia PaCa-2, Panc-1, SW-1990) were exposed to three different PSC-conditioned media (PSC-CM; PSC-1, PSC-2, HPaSteC), following either pre-treatment with glycolysis inhibitor NV-5440 or transfection for transient silencing of key glycolytic regulators (LDHA and MCT4). Proliferation, glucose transport, extracellular lactate, and GEM sensitivity were assessed. Protein expression was determined by Western blot and immunostaining. Moreover, secreted proteins in PSC-CMs were profiled by mass spectrometry (MS). RESULTS: While exposure to PSC-CMs did not affect glucose transport in PCCs, it increased their lactate release and proliferation, and reduced the sensitivity for GEM. Both NV-5440 treatment and transient silencing of LDHA and MCT4 inhibited these PSC-induced changes in PCCs. MS analysis identified 688 unique proteins with differential expression, of which only 87 were common to the three PSC-CMs. Most PSC-secreted proteins were extracellular matrix-related, including SPARC, fibronectin, and collagens. Moreover, exposure to PSC-CMs increased the phosphorylation of ERK in PCCs, but the treatment of PCCs with the MEK/ERK inhibitor PD98059 resulted in a reduction of PSC-CM-induced glycolysis and improved GEM sensitivity. CONCLUSIONS: The study findings suggest that PSC-secreted factors promote both glycolysis and GEM resistance in PCCs, and that glycolysis inhibition by NV-5440 and blocking of ERK phosphorylation by PD98059 protect PCCs from PSC-CM-induced loss of GEM sensitivity. Taken together, PSCs appear to promote GEM resistance in PDAC via glycolysis. Thus, targeting glycolysis may improve the effect of chemotherapy in PDAC.
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Nonalcoholic fatty liver disease (NAFLD) is emerging as a leading cause of chronic liver disease worldwide. Despite intensive nonclinical and clinical research in this field, no specific pharmacological therapy is currently approved to treat NAFLD, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies have identified STE20-type kinase MST3, which localizes to intracellular lipid droplets, as a critical regulator of ectopic fat accumulation in human hepatocytes. Here, we explored whether treatment with Mst3-targeting antisense oligonucleotides (ASOs) can promote hepatic lipid clearance and mitigate NAFLD progression in mice in the context of obesity. We found that administration of Mst3-targeting ASOs in mice effectively ameliorated the full spectrum of high-fat diet-induced NAFLD including liver steatosis, inflammation, fibrosis, and hepatocellular damage. Mechanistically, Mst3 ASOs suppressed lipogenic gene expression, as well as acetyl-CoA carboxylase (ACC) protein abundance, and substantially reduced lipotoxicity-mediated oxidative and endoplasmic reticulum stress in the livers of obese mice. Furthermore, we found that MST3 protein levels correlated positively with the severity of NAFLD in human liver biopsies. In summary, this study provides the first in vivo evidence that antagonizing MST3 signaling is sufficient to mitigate NAFLD progression in conditions of excess dietary fuels and warrants future investigations to assess whether MST3 inhibitors may provide a new strategy for the treatment of patients with NAFLD.
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Dieta Alta en Grasa/efectos adversos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Obesidad/complicaciones , Oligonucleótidos Antisentido/genética , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Animales , Lipogénesis , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Serina-Treonina Quinasas/genética , Transducción de SeñalRESUMEN
Ectopic lipid storage in the liver is considered the main risk factor for nonalcoholic steatohepatitis (NASH). Understanding the molecular networks controlling hepatocellular lipid deposition is therefore essential for developing new strategies to effectively prevent and treat this complex disease. Here, we describe a new regulator of lipid partitioning in human hepatocytes: mammalian sterile 20-like (MST) 3. We found that MST3 protein coats lipid droplets in mouse and human liver cells. Knockdown of MST3 attenuated lipid accumulation in human hepatocytes by stimulating ß-oxidation and triacylglycerol secretion while inhibiting fatty acid influx and lipid synthesis. We also observed that lipogenic gene expression and acetyl-coenzyme A carboxylase protein abundance were reduced in MST3-deficient hepatocytes, providing insight into the molecular mechanisms underlying the decreased lipid storage. Furthermore, MST3 expression was positively correlated with key features of NASH (i.e., hepatic lipid content, lobular inflammation, and hepatocellular ballooning) in human liver biopsies. In summary, our results reveal a role of MST3 in controlling the dynamic metabolic balance of liver lipid catabolism vs. lipid anabolism. Our findings highlight MST3 as a potential drug target for the prevention and treatment of NASH and related complex metabolic diseases.-Cansby, E., Kulkarni, N. M., Magnusson, E., Kurhe, Y., Amrutkar, M., Nerstedt, A., Ståhlman, M., Sihlbom, C., Marschall, H.-U., Borén, J., Blüher, M., Mahlapuu, M. Protein kinase MST3 modulates lipid homeostasis in hepatocytes and correlates with nonalcoholic steatohepatitis in humans.
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Hepatocitos/metabolismo , Proteínas Asociadas a Gotas Lipídicas/fisiología , Gotas Lipídicas/metabolismo , Metabolismo de los Lípidos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Animales , Compartimento Celular , Células Cultivadas , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/farmacocinética , Femenino , Técnicas de Silenciamiento del Gen , Homeostasis , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Mitocondrias Hepáticas/metabolismo , Especificidad de Órganos , Oxidación-Reducción , Estrés Oxidativo , Proteínas Serina-Treonina Quinasas/análisis , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacología , Ratas , Triglicéridos/metabolismoRESUMEN
BACKGROUND: Gemcitabine remains a cornerstone in chemotherapy of pancreatic ductal adenocarcinoma (PDAC) despite suboptimal clinical effects that are partly due to the development of chemoresistance. Pancreatic stellate cells (PSCs) of the tumor stroma are known to interact with pancreatic cancer cells (PCCs) and influence the progression of PDAC through a complex network of signaling molecules that involve extracellular matrix (ECM) proteins. To understand tumor-stroma interactions regulating chemosensitivity, the role of PSC-secreted fibronectin (FN) in the development of gemcitabine resistance in PDAC was examined. METHODS: PSC cultures obtained from ten different human PDAC tumors were co-cultured with PCC lines (AsPC-1, BxPC-3, Capan-2, HPAF-II, MIA PaCa-2, PANC-1 and SW-1990) either directly, or indirectly via incubation with PSC-conditioned medium (PSC-CM). Gemcitabine dose response cytotoxicity was determined using MTT based cell viability assays. Protein expression was assessed by western blotting and immunofluorescence. PSC-CM secretome analysis was performed by proteomics-based LC-MS/MS, and FN content in PSC-CM was determined with ELISA. Radiolabeled gemcitabine was used to determine the capacity of PCCs to uptake the drug. RESULTS: In both direct and indirect co-culture, PSCs induced varying degrees of resistance to the cytotoxic effects of gemcitabine among all cancer cell lines examined. A variable degree of increased phosphorylation of ERK1/2 was observed across all PCC lines upon incubation with PSC-CM, while activation of AKT was not detected. Secretome analysis of PSC-CM identified 796 different proteins, including several ECM-related proteins such as FN and collagens. Soluble FN content in PSC-CM was detected in the range 175-350 ng/ml. Neither FN nor PSC-CM showed any effect on PCC uptake capacity of gemcitabine. PCCs grown on FN-coated surface displayed higher resistance to gemcitabine compared to cells grown on non-coated surface. Furthermore, a FN inhibitor, synthetic Arg-Gly-Asp-Ser (RGDS) peptide significantly inhibited PSC-CM-induced chemoresistance in PCCs via downregulation of ERK1/2 phosphorylation. CONCLUSIONS: The findings of this study suggest that FN secreted by PSCs in the ECM plays a key role in the development of resistance to gemcitabine via activation of ERK1/2. FN-blocking agents added to gemcitabine-based chemotherapy might counteract chemoresistance in PDAC and provide better clinical outcomes.
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Carcinoma Ductal Pancreático/patología , Desoxicitidina/análogos & derivados , Resistencia a Antineoplásicos/efectos de los fármacos , Fibronectinas/metabolismo , Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Desoxicitidina/farmacología , Relación Dosis-Respuesta a Droga , Matriz Extracelular/metabolismo , Fibronectinas/antagonistas & inhibidores , Flavonoides/farmacología , Humanos , Sistema de Señalización de MAP Quinasas , Oligopéptidos/farmacología , Fosforilación , Proteoma/análisis , Proteínas Proto-Oncogénicas c-akt/metabolismo , GemcitabinaRESUMEN
Objective- Recent cohort studies have shown that nonalcoholic fatty liver disease (NAFLD), and especially nonalcoholic steatohepatitis (NASH), associate with atherosclerosis and cardiovascular disease, independently of conventional cardiometabolic risk factors. However, the mechanisms underlying the pathophysiological link between NAFLD/NASH and cardiovascular disease still remain unclear. Our previous studies have identified STK25 (serine/threonine protein kinase 25) as a critical determinant in ectopic lipid storage, meta-inflammation, and progression of NAFLD/NASH. The aim of this study was to assess whether STK25 is also one of the mediators in the complex molecular network controlling the cardiovascular disease risk. Approach and Results- Atherosclerosis was induced in Stk25 knockout and transgenic mice, and their wild-type littermates, by gene transfer of gain-of-function mutant of PCSK9 (proprotein convertase subtilisin/kexin type 9), which induces the downregulation of hepatic LDLR (low-density lipoprotein receptor), combined with an atherogenic western-type diet. We found that Stk25-/- mice displayed reduced atherosclerosis lesion area as well as decreased lipid accumulation, macrophage infiltration, collagen formation, and oxidative stress in aortic lesions compared with wild-type littermates, independently from alterations in dyslipidemia. Reciprocally, Stk25 transgenic mice presented aggravated plaque formation and maturation compared with wild-type littermates despite similar levels of fasting plasma cholesterol. We also found that STK25 protein was expressed in all layers of the aorta, suggesting a possible direct role in cardiovascular disease. Conclusions- This study provides the first evidence that STK25 plays a critical role in regulation of cardiovascular disease risk and suggests that pharmacological inhibition of STK25 function may provide new possibilities for prevention/treatment of atherosclerosis.
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Aorta/enzimología , Enfermedades de la Aorta/enzimología , Aterosclerosis/enzimología , Hipercolesterolemia/enzimología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Placa Aterosclerótica , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Aorta/patología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Técnicas de Transferencia de Gen , Hipercolesterolemia/genética , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/enzimología , Hígado/patología , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Proproteína Convertasa 9/genética , Proproteína Convertasa 9/metabolismo , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de SeñalRESUMEN
AIMS/HYPOTHESIS: Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator of liver steatosis, hepatic lipid metabolism and whole body glucose and insulin homeostasis. Here, we assessed the role of STK25 in control of ectopic fat storage and insulin responsiveness in skeletal muscle. METHODS: Skeletal muscle morphology was studied by histological examination, exercise performance and insulin sensitivity were assessed by treadmill running and euglycaemic-hyperinsulinaemic clamp, respectively, and muscle lipid metabolism was analysed by ex vivo assays in Stk25 transgenic and wild-type mice fed a high-fat diet. Lipid accumulation and mitochondrial function were also studied in rodent myoblasts overexpressing STK25. Global quantitative phosphoproteomics was performed in skeletal muscle of Stk25 transgenic and wild-type mice fed a high-fat diet to identify potential downstream mediators of STK25 action. RESULTS: We found that overexpression of STK25 in transgenic mice fed a high-fat diet increases intramyocellular lipid accumulation, impairs skeletal muscle mitochondrial function and sarcomeric ultrastructure, and induces perimysial and endomysial fibrosis, thereby reducing endurance exercise capacity and muscle insulin sensitivity. Furthermore, we observed enhanced lipid accumulation and impaired mitochondrial function in rodent myoblasts overexpressing STK25, demonstrating an autonomous action for STK25 within cells. Global phosphoproteomic analysis revealed alterations in the total abundance and phosphorylation status of different target proteins located predominantly to mitochondria and sarcomeric contractile elements in Stk25 transgenic vs wild-type muscle, respectively, providing a possible molecular mechanism for the observed phenotype. CONCLUSIONS/INTERPRETATION: STK25 emerges as a new regulator of the complex interplay between lipid storage, mitochondrial energetics and insulin action in skeletal muscle, highlighting the potential of STK25 antagonists for type 2 diabetes treatment.
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Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos/fisiología , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Western Blotting , Cromatografía Liquida , Dieta Alta en Grasa , Resistencia a la Insulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Metabolismo de los Lípidos/genética , Espectrometría de Masas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mitocondrias/patología , Proteínas Serina-Treonina Quinasas/genética , Proteómica , Ratas , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and 10-20% of patients with NAFLD progress to nonalcoholic steatohepatitis (NASH) with a high risk of cirrhosis, liver failure, and hepatocellular carcinoma. Despite its high medical importance, the molecular mechanisms controlling progression from simple liver steatosis to NASH remain elusive. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of ectopic lipid deposition, systemic glucose, and insulin homeostasis. To elucidate the role of STK25 in the development of NASH, we challenged Stk25-knockout and transgenic mice with a methionine and choline-deficient (MCD) diet. We show that Stk25-/- mice are protected against MCD-diet-induced NASH, as evidenced by repressed liver steatosis, oxidative damage, inflammation, and fibrosis, whereas Stk25 transgenic mice developed a more severe NASH phenotype, compared with corresponding wild-type littermates. Consistently, NASH features were suppressed in STK25-deficient human hepatocytes cultured in MCD medium, and reciprocally enhanced in STK25-overexpressing cells. We also found a significant positive correlation in human liver biopsies between STK25 expression and NASH development. The study provides evidence for multiple roles of STK25 in NASH pathogenesis and future investigations to address the potential therapeutic relevance of pharmacological STK25 inhibitors in prevention and treatment of NASH are warranted.-Amrutkar, M., Chursa, U., Kern, M., Nuñez-Durán, E., Ståhlman, M., Sütt, S., Borén, J., Johansson, B. R., Marschall, H.-U., Blüher, M., Mahlapuu, M. STK25 is a critical determinant in nonalcoholic steatohepatitis.
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Deficiencia de Colina/metabolismo , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Deficiencia de Colina/complicaciones , Modelos Animales de Enfermedad , Metabolismo de los Lípidos/genética , Ratones Transgénicos , Triglicéridos/metabolismoRESUMEN
AIMS/HYPOTHESIS: Type 2 diabetes is closely associated with pathological lipid accumulation in the liver, which is suggested to actively contribute to the development of insulin resistance. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of liver steatosis, whole-body glucose tolerance and insulin sensitivity in a mouse model system. The aim of this study was to assess the role of STK25 in the control of lipid metabolism in human liver. METHODS: Intracellular fat deposition, lipid metabolism and insulin sensitivity were studied in immortalised human hepatocytes (IHHs) and HepG2 hepatocellular carcinoma cells in which STK25 was overexpressed or knocked down by small interfering RNA. The association between STK25 mRNA expression in human liver biopsies and hepatic fat content was analysed. RESULTS: Overexpression of STK25 in IHH and HepG2 cells enhanced lipid deposition by suppressing ß-oxidation and triacylglycerol (TAG) secretion, while increasing lipid synthesis. Conversely, knockdown of STK25 attenuated lipid accumulation by stimulating ß-oxidation and TAG secretion, while inhibiting lipid synthesis. Furthermore, TAG hydrolase activity was repressed in hepatocytes overexpressing STK25 and reciprocally increased in cells with STK25 knockdown. Insulin sensitivity was reduced in STK25-overexpressing cells and enhanced in STK25-deficient hepatocytes. We also found a statistically significant positive correlation between STK25 mRNA expression in human liver biopsies and hepatic fat content. CONCLUSIONS/INTERPRETATION: Our data suggest that STK25 regulates lipid partitioning in human liver cells by controlling TAG synthesis as well as lipolytic activity and thereby NEFA release from lipid droplets for ß-oxidation and TAG secretion. Our findings highlight STK25 as a potential drug target for the prevention and treatment of type 2 diabetes.
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Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , Metabolismo de los Lípidos/genética , Hígado/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Adiposidad , Animales , Transporte Biológico/genética , Células Cultivadas , Femenino , Células Hep G2 , Humanos , Movilización Lipídica/genética , Masculino , Ratones , Ratones Noqueados , Triglicéridos/metabolismoRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is the most common form of liver disease, and 10% to 20% of NAFLD patients progress to nonalcoholic steatohepatitis (NASH). The molecular pathways controlling progression to NAFLD/NASH remain poorly understood. We recently identified serine/threonine protein kinase 25 (STK25) as a regulator of whole-body insulin and glucose homeostasis. This study investigates the role of STK25 in liver lipid accumulation and NASH. Stk25 transgenic mice challenged with a high-fat diet displayed a dramatic increase in liver steatosis and hepatic insulin resistance compared to wild-type siblings. Focal fibrosis, hepatocellular damage, and inflammation were readily seen in transgenic but not wild-type livers. Transgenic livers displayed reduced ß-oxidation and triacylglycerol secretion, while lipid uptake and synthesis remained unchanged. STK25 was associated with lipid droplets, colocalizing with the main hepatic lipid droplet-coating protein adipose differentiation-related protein, the level of which was increased 3.8 ± 0.7-fold in transgenic livers (P < 0.01), while a key hepatic lipase, adipose triacylglycerol lipase, was translocated from the lipid droplets surface to the cytoplasm, providing the likely mechanism underlying the effect of STK25. In summary, STK25 is a lipid droplet-associated protein that promotes NAFLD through control of lipid release from the droplets for ß-oxidation and triacylglycerol secretion. STK25 also drives pathogenesis of NASH.
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Péptidos y Proteínas de Señalización Intracelular/metabolismo , Metabolismo de los Lípidos , Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/etiología , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a la Insulina , Péptidos y Proteínas de Señalización Intracelular/genética , Metabolismo de los Lípidos/genética , Lipoproteínas VLDL/metabolismo , Hígado/patología , Masculino , Ratones , Ratones Transgénicos , Modelos Biológicos , Enfermedad del Hígado Graso no Alcohólico/patología , Proteínas Serina-Treonina Quinasas/genética , Triglicéridos/metabolismo , Regulación hacia ArribaRESUMEN
Partial depletion of serine/threonine protein kinase 25 (STK25), a member of the Ste20 superfamily of kinases, increases lipid oxidation and glucose uptake in rodent myoblasts. Here we show that transgenic mice overexpressing STK25, when challenged with a high-fat diet, develop reduced glucose tolerance and insulin sensitivity compared to wild-type siblings, as evidenced by impairment in glucose and insulin tolerance tests as well as in euglycemic-hyperinsulinemic clamp studies. The fasting plasma insulin concentration was elevated in Stk25 transgenic mice compared to wild-type littermates (4.9±0.8 vs. 2.6±0.4 ng/ml after 17 wk on high-fat diet, P<0.05). Overexpression of STK25 decreased energy expenditure during the dark phase of observation (P<0.05), despite increased spontaneous activity. The oxidative capacity of skeletal muscle of transgenic carriers was reduced, as evidenced by altered expression of Cpt1, Acox1, and ACC. Hepatic triglycerides and glycogen were elevated (1.6- and 1.4-fold, respectively; P<0.05) and expression of key enzymes regulating lipogenesis (Fasn), glycogen synthesis (Gck), and gluconeogenesis (G6pc, Fbp1) was increased in the liver of the transgenic mice. Our findings suggest that overexpression of STK25 in conditions of excess dietary fuels associates with a shift in the metabolic balance in peripheral tissues from lipid oxidation to storage, leading to a systemic insulin resistance.
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Dieta Alta en Grasa/efectos adversos , Glucosa/metabolismo , Resistencia a la Insulina/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Adipocitos/metabolismo , Animales , Composición Corporal/genética , Composición Corporal/fisiología , Calorimetría Indirecta , Células Cultivadas , Prueba de Tolerancia a la Glucosa , Inmunohistoquímica , Resistencia a la Insulina/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Serina-Treonina Quinasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The modest clinical benefits of neoadjuvant chemotherapy (NAT) in pancreatic ductal adenocarcinoma (PDAC) are associated with a lack of robust data on treatment-induced changes in the tumor. To this end, comparative proteomic profiling of tumor tissue samples from treatment-naïve (TN, n = 20) and NAT-treated (n = 22) PDACs was performed. Differentially expressed proteins were identified and correlation with overall survival (OS) was performed. Tumors were also examined for histopathological changes and expression of cancer stem cell (CSC) markers. Serum from 33 matched patients was analyzed for metabolic markers. Cytotoxicity, proliferation, and expression of CSC markers were assessed in chemoresistant Panc-1 and Mia PaCa-2 cells. Of the 2265 proteins identified, 227 and 144 proteins showed significantly altered expression and differential phosphorylation, respectively, in NAT compared with TN samples. The majority of these were metabolism-related proteins, and 14 of these correlated moderately with OS. NAT-treated tumors and chemoresistant cancer cells showed increased expression of CSC markers. Serum ALDH1A1 was higher in NAT compared with TN. Differentially phosphorylated proteins were mainly involved in cytoskeleton organization, cell locomotion, motility, and migration, and 17 of these showed a strong positive correlation with OS. This study provides evidence of the effects of NAT on PDAC metabolism at both the tumor and the systemic levels. NAT-treated tumors showed significantly lower expression of metabolic proteins, and patients who underwent NAT showed reduced serum lactate and high-density lipoprotein-cholesterol. Lastly, cancer cells that survived cytotoxic treatment expressed higher CSC markers, both in vivo and in vitro.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Terapia Neoadyuvante , Proteómica , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/patología , Metaboloma , Estudios Retrospectivos , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC), also known as pancreatic cancer (PC), is characterized by an overall poor prognosis and a five-year survival that is less than 10%. Characteristic features of the tumor are the presence of a prominent desmoplastic stromal response, an altered metabolism, and profound resistance to cancer drugs including gemcitabine, the backbone of PDAC chemotherapy. The pancreatic stellate cells (PSCs) constitute the major cellular component of PDAC stroma. PSCs are essential for extracellular matrix assembly and form a supportive niche for tumor growth. Various cytokines and growth factors induce activation of PSCs through autocrine and paracrine mechanisms, which in turn promote overall tumor growth and metastasis and induce chemoresistance. To maintain growth and survival in the nutrient-poor, hypoxic environment of PDAC, tumor cells fulfill their high energy demands via several unconventional ways, a process generally referred to as metabolic reprogramming. Accumulating evidence indicates that activated PSCs not only contribute to the therapy-resistant phenotype of PDAC but also act as a nutrient supplier for the tumor cells. However, the precise molecular links between metabolic reprogramming and an acquired therapy resistance in PDAC remain elusive. This review highlights recent findings indicating the importance of PSCs in aiding growth-permissive metabolic reprogramming and gemcitabine chemoresistance in PDAC.
RESUMEN
Inter- and intratumor heterogeneity is an important cause of treatment failure. In human pancreatic cancer (PC), heterogeneity has been investigated almost exclusively at the genomic and transcriptional level. Morphological heterogeneity, though prominent and potentially easily assessable in clinical practice, remains unexplored. This proof-of-concept study aims at demonstrating that morphological heterogeneity reflects structural and functional divergence. From the wide morphological spectrum of conventional PC, four common and distinctive patterns were investigated in 233 foci from 39 surgical specimens. Twenty-six features involved in key biological processes in PC were analyzed (immuno-)histochemically and morphometrically: cancer cell proliferation (Ki67) and migration (collagen fiber alignment, MMP14), cancer stem cells (CD44, CD133, ALDH1), amount, composition and spatial arrangement of extracellular matrix (epithelial proximity, total collagen, collagen I and III, fibronectin, hyaluronan), cancer-associated fibroblasts (density, αSMA), and cancer-stroma interactions (integrins α2, α5, α1; caveolin-1). All features differed significantly between at least two of the patterns. Stromal and cancer-cell-related features co-varied with morphology and allowed prediction of the morphological pattern. In conclusion, morphological heterogeneity in the cancer-cell and stromal compartments of PC correlates with structural and functional diversity. As such, histopathology has the potential to inform on the operationality of key biological processes in individual tumors.
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Gemcitabine resistance in pancreatic ductal adenocarcinoma (PDAC) is attributed to cancer cell-intrinsic drug processing and the impact of the tumor microenvironment, especially pancreatic stellate cells (PSCs). This study uses human PDAC-derived paired primary cancer cells (PCCs) and PSCs from four different tumors, and the PDAC cell lines BxPC-3, Mia PaCa-2, and Panc-1, to assess the fate of gemcitabine by measuring its cellular uptake, cytotoxicity, and LC-MS/MS-based metabolite analysis. Expression analysis and siRNA-mediated knockdown of key regulators of gemcitabine (hENT1, CDA, DCK, NT5C1A) was performed. Compared to PSCs, both the paired primary PCCs and cancer cell lines showed gemcitabine-induced dose-dependent cytotoxicity, high uptake, as well as high and variable intracellular levels of gemcitabine metabolites. PSCs were gemcitabine-resistant and demonstrated significantly lower drug uptake, which was not influenced by co-culturing with their paired PCCs. Expression of key gemcitabine regulators was variable, but overall strong in the cancer cells and significantly lower or undetectable in PSCs. In cancer cells, hENT1 inhibition significantly downregulated gemcitabine uptake and cytotoxicity, whereas DCK knockdown reduced cytotoxicity. In conclusion, heterogeneity in gemcitabine processing among different pancreatic cancer cells and stellate cells results from the differential expression of molecular regulators which determines the effect of gemcitabine.
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Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extremely poor prognosis, and its treatment remains a challenge. As the existing in vitro experimental models offer only a limited resemblance to human PDAC, there is a strong need for additional research tools to better understand PDAC tumor biology, particularly the impact of the tumor stroma. Here, we report for the first time the establishment and characterization of human PDAC-derived paired primary monolayer cultures of (epithelial) cancer cells (PCCs) and mesenchymal stellate cells (PSCs) derived from the same tumor by the outgrowth method. Characterization of cell morphology, cytostructural, and functional profiles and proteomics-based secretome analysis were performed. All PCCs harbored KRAS and TP53 mutations, and expressed cytokeratin 19, ki-67, and p53, while the expression of EpCAM and vimentin was variable. All PSCs expressed α-smooth muscle actin (α-SMA) and vimentin. PCCs showed a significantly higher growth rate and proliferation than PSCs. Secretome analysis confirmed the distinct nature of PCCs as compared to PSCs and allowed identification of potential molecular regulators of PSC-conditioned medium (PSC-CM)-induced migration of PCCs. Paired primary cultures of PCCs and PSCs derived from the same tumor specimen represent a novel experimental model for basic research in PDAC tumor biology.
Asunto(s)
Neoplasias Pancreáticas/patología , Células Estrelladas Pancreáticas/patología , Adenocarcinoma/genética , Adenocarcinoma/patología , Alelos , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proliferación Celular/genética , Forma de la Célula/efectos de los fármacos , Forma de la Célula/genética , Medios de Cultivo Condicionados/farmacología , ADN de Neoplasias/biosíntesis , Humanos , Mutación/genética , Neoplasias Pancreáticas/genética , Células Estrelladas Pancreáticas/efectos de los fármacos , Fenotipo , Proteoma/metabolismo , Células Tumorales CultivadasRESUMEN
Activated pancreatic stellate cells (PSCs) play a central role in the tumor stroma of pancreatic ductal adenocarcinoma (PDAC). Given the limited availability of patient-derived PSCs from PDAC, immortalized PSC cell lines of murine and human origin have been established; however, it is not elucidated whether differences in species, organ disease status, donor age, and immortalization alter the PSC phenotype and behavior compared to that of patient-derived primary PSC cultures. Therefore, a panel of commonly used PSC cultures was examined for important phenotypical and functional features: three primary cultures from human PDAC, one primary from normal human pancreas, and three immortalized (one from human, two from murine pancreas). Growth rate was considerably lower in primary PSCs from human PDAC. Basal collagen synthesis varied between the PSC cultures, and TGF-ß stimulation increased collagen synthesis only in non-immortalized cultures. Differences in secretome composition were observed along with a divergence in the DNA synthesis, migration, and response to gemcitabine of PDAC cell lines that were grown in conditioned medium from the various PSC cultures. The findings reveal considerable differences in features and functions that are key to PSCs and in the interactions with PDAC. These observations may be relevant to researchers when selecting the most appropriate PSC culture for their experiments.
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Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patología , Células Estrelladas Pancreáticas/metabolismo , Células Estrelladas Pancreáticas/patología , Animales , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Colágeno/metabolismo , Humanos , Fenotipo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
BACKGROUND & AIMS: Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are emerging as leading causes of liver disease worldwide. Currently, no specific pharmacologic therapy is available for NAFLD/NASH, which has been recognized as one of the major unmet medical needs of the 21st century. Our recent studies in genetic mouse models, human cell lines, and well-characterized patient cohorts have identified serine/threonine protein kinase (STK)25 as a critical regulator of hepatic lipid partitioning and NAFLD/NASH. Here, we studied the metabolic benefit of liver-specific STK25 inhibitors on NAFLD development and progression in a mouse model of diet-induced obesity. METHODS: We developed a hepatocyte-specific triantennary N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide (ASO) targeting Stk25 and evaluated its effect on NAFLD features in mice after chronic exposure to dietary lipids. RESULTS: We found that systemic administration of hepatocyte-targeting GalNAc-Stk25 ASO in obese mice effectively ameliorated steatosis, inflammatory infiltration, hepatic stellate cell activation, nutritional fibrosis, and hepatocellular damage in the liver compared with mice treated with GalNAc-conjugated nontargeting ASO, without any systemic toxicity or local tolerability concerns. We also observed protection against high-fat-diet-induced hepatic oxidative stress and improved mitochondrial function with Stk25 ASO treatment in mice. Moreover, GalNAc-Stk25 ASO suppressed lipogenic gene expression and acetyl-CoA carboxylase protein abundance in the liver, providing insight into the molecular mechanisms underlying repression of hepatic steatosis. CONCLUSIONS: This study provides in vivo nonclinical proof-of-principle for the metabolic benefit of liver-specific inhibition of STK25 in the context of obesity and warrants future investigations to address the therapeutic potential of GalNAc-Stk25 ASO in the prevention and treatment of NAFLD.
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Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Enfermedad del Hígado Graso no Alcohólico/terapia , Oligonucleótidos Antisentido/farmacología , Proteínas Serina-Treonina Quinasas/metabolismo , Acetil-CoA Carboxilasa/metabolismo , Acetilglucosamina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Dieta Alta en Grasa , Regulación de la Expresión Génica/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/genética , Lipogénesis/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Obesos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Enfermedad del Hígado Graso no Alcohólico/sangre , Tamaño de los Órganos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Whole-body energy homeostasis at over-nutrition critically depends on how well adipose tissue remodels in response to excess calories. We recently identified serine/threonine protein kinase (STK)25 as a critical regulator of ectopic lipid storage in non-adipose tissue and systemic insulin resistance in the context of nutritional stress. Here, we investigated the role of STK25 in regulation of adipose tissue dysfunction in mice challenged with a high-fat diet. We found that overexpression of STK25 in high-fat-fed mice resulted in impaired mitochondrial function and aggravated hypertrophy, inflammatory infiltration and fibrosis in adipose depots. Reciprocally, Stk25-knockout mice displayed improved mitochondrial function and were protected against diet-induced excessive fat storage, meta-inflammation and fibrosis in brown and white adipose tissues. Furthermore, in rodent HIB-1B cell line, STK25 depletion resulted in enhanced mitochondrial activity and consequently, reduced lipid droplet size, demonstrating an autonomous action for STK25 within adipocytes. In summary, we provide the first evidence for a key function of STK25 in controlling the metabolic balance of lipid utilization vs lipid storage in brown and white adipose depots, suggesting that repression of STK25 activity offers a potential strategy for establishing healthier adipose tissue in the context of chronic exposure to dietary lipids.