RESUMO
Metabolic reprogramming is fundamental to biological homeostasis, enabling cells to adjust metabolic routes after sensing altered availability of fuels and growth factors. ULK1 and ULK2 represent key integrators that relay metabolic stress signals to the autophagy machinery. Here, we demonstrate that, during deprivation of amino acid and growth factors, ULK1/2 directly phosphorylate key glycolytic enzymes including hexokinase (HK), phosphofructokinase 1 (PFK1), enolase 1 (ENO1), and the gluconeogenic enzyme fructose-1,6-bisphosphatase (FBP1). Phosphorylation of these enzymes leads to enhanced HK activity to sustain glucose uptake but reduced activity of FBP1 to block the gluconeogenic route and reduced activity of PFK1 and ENO1 to moderate drop of glucose-6-phosphate and to repartition more carbon flux to pentose phosphate pathway (PPP), maintaining cellular energy and redox homeostasis at cellular and organismal levels. These results identify ULK1/2 as a bifurcate-signaling node that sustains glucose metabolic fluxes besides initiation of autophagy in response to nutritional deprivation.
Assuntos
Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia , Glucose/metabolismo , Glicólise , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Via de Pentose Fosfato , Proteínas Serina-Treonina Quinases/metabolismo , Estresse Fisiológico , Aminoácidos/deficiência , Aminoácidos/metabolismo , Animais , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/genética , Biomarcadores Tumorais/metabolismo , Morte Celular , Proteínas de Ligação a DNA/metabolismo , Feminino , Frutose-Bifosfatase/metabolismo , Genótipo , Células HCT116 , Hexoquinase/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Células MCF-7 , Masculino , Camundongos Knockout , Fenótipo , Fosfofrutoquinase-1/metabolismo , Fosfopiruvato Hidratase/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas Supressoras de Tumor/metabolismoRESUMO
BACKGROUND AND AIMS: NAFLD represents an increasing health problem in association with obesity and diabetes with no effective pharmacotherapies. Growing evidence suggests that several FGFs play important roles in diverse aspects of liver pathophysiology. Here, we report a previously unappreciated role of FGF4 in the liver. APPROACH AND RESULTS: Expression of hepatic FGF4 is inversely associated with NAFLD pathological grades in both human patients and mouse models. Loss of hepatic Fgf4 aggravates hepatic steatosis and liver damage resulted from an obesogenic high-fat diet. By contrast, pharmacological administration of recombinant FGF4 mitigates hepatic steatosis, inflammation, liver damage, and fibrogenic markers in mouse livers induced to develop NAFLD and NASH under dietary challenges. Such beneficial effects of FGF4 are mediated predominantly by activating hepatic FGF receptor (FGFR) 4, which activates a downstream Ca2+ -Ca2+ /calmodulin-dependent protein kinase kinase beta-dependent AMP-activated protein kinase (AMPK)-Caspase 6 signal axis, leading to enhanced fatty acid oxidation, reduced hepatocellular apoptosis, and mitigation of liver damage. CONCLUSIONS: Our study identifies FGF4 as a stress-responsive regulator of liver pathophysiology that acts through an FGFR4-AMPK-Caspase 6 signal pathway, shedding light on strategies for treating NAFLD and associated liver pathologies.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Caspase 6/metabolismo , Caspase 6/farmacologia , Dieta Hiperlipídica/efeitos adversos , Ácidos Graxos/metabolismo , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fator 4 de Crescimento de Fibroblastos/farmacologia , Fator 4 de Crescimento de Fibroblastos/uso terapêutico , Humanos , Fígado/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/uso terapêuticoRESUMO
unc-51-like autophagy activating kinase 1 and 2 (Ulk1/2) regulate autophagy initiation under various stress conditions. However, the physiological functions of these Ser/Thr kinases are not well characterized. Here, we show that mice with liver-specific double knockout (LDKO) of Ulk1 and Ulk2 (Ulk1/2 LDKO) are viable, but exhibit overt hepatomegaly phenotype. Surprisingly, Ulk1/2 LDKO mice display normal autophagic activity in hepatocytes upon overnight fasting, but are strongly resistant to acetaminophen (APAP)-induced liver injury. Further studies revealed that Ulk1/2 are also dispensable for APAP-induced autophagy process, but are essential for the maximum activation of c-Jun N-terminal kinase (JNK) signaling both in vivo and in isolated primary hepatocytes during APAP treatment. Mechanistically, APAP-induced inhibition of mechanistic target of rapamycin complex 1 releases Ulk1 from an inactive state. Activated Ulk1 then directly phosphorylates and increases the kinase activity of mitogen-activated protein kinase kinase 4 and 7 (MKK4/7), the upstream kinases and activator of JNK, and mediates APAP-induced liver injury. Ulk1-dependent phosphorylation of MKK7 was further confirmed by a context-dependent phosphorylation antibody. Moreover, activation of JNK and APAP-induced cell death was markedly attenuated in Mkk4/7 double knockdown hepatocytes reconstituted with an Ulk1-unphosphorylatable mutant of MKK7 compared to those in cells rescued with wild-type MKK7. CONCLUSION: Together, these findings reveal an important role of Ulk1/2 for APAP-induced JNK activation and liver injury, and understanding of this regulatory mechanism may offer us new strategies for prevention and treatment of human APAP hepatotoxicity. (Hepatology 2018;67:2397-2413).
Assuntos
Acetaminofen/efeitos adversos , Analgésicos não Narcóticos/efeitos adversos , Antipiréticos/efeitos adversos , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/deficiência , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/etiologia , Fígado/enzimologia , Proteínas Serina-Treonina Quinases/deficiência , Animais , Masculino , CamundongosRESUMO
Inflammation plays a central role in the etiology of diabetic nephropathy, a global health issue. We observed a significant reduction in the renal expression of fibroblast growth factor 1, a known mitogen and insulin sensitizer, in patients with diabetic nephropathy and in mouse models implying that fibroblast growth factor 1 possesses beneficial anti-inflammatory and renoprotective activities in vivo. To test this possibility, we investigated the effects of chronic intraperitoneal administration of fibroblast growth factor 1 into both the streptozotocin-induced type 1 diabetes and db/db type 2 diabetes models. Indeed, recombinant fibroblast growth factor 1 significantly suppressed renal inflammation (i.e., cytokines, macrophage infiltration), glomerular and tubular damage, and renal dysfunction in both type 1 and type 2 diabetes mice. Fibroblast growth factor 1 was able to correct the elevated blood glucose levels in type 2 but not in type 1 diabetic mice, suggesting that the anti-inflammatory effect of fibroblast growth factor 1 was independent of its glucose-lowering activity. The mechanistic study demonstrated that fibroblast growth factor 1-mediated inhibition of the renal inflammation in vivo was accompanied by attenuation of the nuclear factor κB and c-Jun N-terminal kinase signaling pathways, further validated in vitro using cultured glomerular mesangial cells and podocytes. Thus, fibroblast growth factor 1 holds great promise for developing new treatments for diabetic nephropathy through countering inflammatory signaling cascades in injured renal tissue.
Assuntos
Anti-Inflamatórios/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Nefropatias Diabéticas/prevenção & controle , Fator 1 de Crescimento de Fibroblastos/farmacologia , Rim/efeitos dos fármacos , Animais , Glicemia/efeitos dos fármacos , Glicemia/metabolismo , Linhagem Celular , Citocinas/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/complicações , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/complicações , Nefropatias Diabéticas/sangue , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Fator 1 de Crescimento de Fibroblastos/sangue , Humanos , Mediadores da Inflamação/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Rim/metabolismo , Rim/patologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , NF-kappa B/metabolismo , Ratos Wistar , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacosRESUMO
Production of protein therapeutics often involves in vitro refolding from bacterial inclusion bodies and subsequent PEGylation to improve protein stability and plasma half-life. Here, we devised a novel strategy for one-step production of site-specific mono-PEGylated proteins with good bioactivity and improved biostability by integrating PEGylation and protein refolding (IPPR). Using lysozyme and recombinant human fibroblast growth factor 21 (rhFGF21) as model proteins, we showed that both PEGylation and refolding of denatured proteins have been simultaneously accomplished by IPPR with high efficiency of refolding yield and bioconjugation. PEGylated rhFGF21 by IPPR has a similar capacity as the native rhFGF21 to stimulate glucose uptake in 3T3-L1 cells, but exhibits prolonged blood glucose and triglyceride lowering activity levels in the ob/ob diabetic mouse model. Hence, IPPR will significantly facilitate the generation of protein therapeutics.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Muramidase/química , Polietilenoglicóis/química , Redobramento de Proteína , Células 3T3-L1 , Animais , Células Cultivadas , Modelos Animais de Doenças , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Camundongos Obesos , Muramidase/metabolismo , Polietilenoglicóis/metabolismo , Polietilenoglicóis/farmacocinética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Using fusion tags, expression of recombinant human fibroblast growth factor 18 (rhFGF18) in mammalian cells and Escherichia coli has been extensively used for fundamental research and clinical applications, including chondrogenesis and osteogenesis, hair growth, and neuroprotection. However, high-level rhFGF18 expression is difficult and the products are often not homogeneous. Furthermore, fusion-tagged protein has higher immunogenicity and lower bioactivity, and the removal of the fused tag is expensive. To overcome the limitations of fusion-tagged expression of protein and to prepare soluble highly bioactive rhFGF18, we have developed a rapid and efficient expression strategy. Optimized hFGF18 gene was amplified by polymerase chain reaction and cloned into pET22b and pET3c vectors, then transformed into E. coli strains Origima (DE3) and BL21 (DE3)PlysS. The best combination of plasmid and host strain was selected, and only Origima (DE3)/pET3c-rhFGF18 was screened for high-level expressed rhFGF18. Under optimal conditions in a 30-L fermentor, the average bacterial yield and expression level of rhFGF18 of three batches were more than 652 g and 30 % respectively, after treatment with 1 mM isopropyl-thio-ß-galactopyranoside for 10 h at 25 °C. The target protein was purified by CM Sepharose FF and heparin affinity chromatography. The purity of rhFGF18 was shown by HPLC to be higher than 95 %, and the yield was 155 mg/L. In vitro MTT assays demonstrated that the purified rhFGF18 could stimulate significant proliferation of NIH3T3 cells, and animal experiments showed that rhFGF18 could effectively regulate hair growth. In conclusion, this may be a better method of producing rhFGF18 to meet the increasing demand in its pharmacological application.
Assuntos
Proliferação de Células/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/metabolismo , Folículo Piloso/efeitos dos fármacos , Animais , Cromatografia de Afinidade , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Escherichia coli/genética , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Cabelo/efeitos dos fármacos , Cabelo/crescimento & desenvolvimento , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Plasmídeos , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNARESUMO
BACKGROUND & AIMS: Non-alcoholic fatty liver disease (NAFLD) has emerged as a major liver disease increasingly in association with non-alcoholic steatohepatitis (NASH), cirrhosis and hepatocellular carcinoma (HCC). However, there are currently no approved therapies for treating NAFLD and NASH. Fibroblast growth factor 4 (FGF4) has recently been shown as a promising drug candidate for several metabolic diseases. METHODS: Mice fed a high-fat diet with high fructose/glucose drinking water (HF/HFG, Western-like diet) for 21 weeks were intraperitoneally injected with non-mitogenic recombinant FGF4â³NT (rFGF4â³NT, 1.0 mg/kg body weight) every other day for 8 weeks. Primary mouse hepatocytes cultured in medium containing high glucose/palmitic acid (HG/PA) or TNFα/cyclohexane (TNFα/CHX) were treated with 1.0 µg/ml rFGF4â³NT. Changes in parameters for histopathology, lipid metabolism, inflammation, hepatocellular apoptosis and fibrosis were determined. The Caspase6 activity and AMPK pathway were assessed. RESULTS: Administration of rFGF4â³NT significantly attenuated the Western-like diet-induced hepatic steatosis, inflammation, liver injury and fibrosis in mice. rFGF4â³NT treatment reduced fatty acid-induced lipid accumulation and lipotoxicity-induced hepatocyte apoptosis, which were associated with inhibition of Caspase6 cleavage and activation. Inhibition of AMP-activated protein kinase (AMPK) by Compound C or deficiency of Ampk abrogated rFGF4â³NT-induced hepatoprotection in primary hepatocytes and in mice with NASH. CONCLUSION: rFGF4â³NT exerts significant protective effects on NASH via an AMPK-dependent signaling pathway. Our study indicates that FGF4 analogs may have therapeutic potential for the Western-like diet induced NASH.
Assuntos
Carcinoma Hepatocelular , Água Potável , Neoplasias Hepáticas , Hepatopatia Gordurosa não Alcoólica , Proteínas Quinases Ativadas por AMP , Animais , Cicloexanos/efeitos adversos , Água Potável/efeitos adversos , Ácidos Graxos , Fator 4 de Crescimento de Fibroblastos/efeitos adversos , Frutose/efeitos adversos , Glucose/efeitos adversos , Inflamação , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/patologia , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/patologia , Ácido Palmítico/farmacologia , Fator de Necrose Tumoral alfa/efeitos adversosRESUMO
Several members of the FGF family have been identified as potential regulators of glucose homeostasis. We previously reported that a low threshold of FGF-induced FGF receptor 1c (FGFR1c) dimerization and activity is sufficient to evoke a glucose lowering activity. We therefore reasoned that ligand identity may not matter, and that besides paracrine FGF1 and endocrine FGF21, other cognate paracrine FGFs of FGFR1c might possess such activity. Indeed, via a side-by-side testing of multiple cognate FGFs of FGFR1c in diabetic mice we identified the paracrine FGF4 as a potent anti-hyperglycemic FGF. Importantly, we found that like FGF1, the paracrine FGF4 is also more efficacious than endocrine FGF21 in lowering blood glucose. We show that paracrine FGF4 and FGF1 exert their superior glycemic control by targeting skeletal muscle, which expresses copious FGFR1c but lacks ß-klotho (KLB), an obligatory FGF21 co-receptor. Mechanistically, both FGF4 and FGF1 upregulate GLUT4 cell surface abundance in skeletal muscle in an AMPKα-dependent but insulin-independent manner. Chronic treatment with rFGF4 improves insulin resistance and suppresses adipose macrophage infiltration and inflammation. Notably, unlike FGF1 (a pan-FGFR ligand), FGF4, which has more restricted FGFR1c binding specificity, has no apparent effect on food intake. The potent anti-hyperglycemic and anti-inflammatory properties of FGF4 testify to its promising potential for use in the treatment of T2D and related metabolic disorders.
Assuntos
Fator 4 de Crescimento de Fibroblastos/farmacologia , Hipoglicemiantes/farmacologia , Músculo Esquelético/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Diabetes Mellitus Tipo 2/tratamento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Fator 4 de Crescimento de Fibroblastos/administração & dosagem , Fator 4 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/administração & dosagem , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Glucose/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/metabolismo , Inflamação , Resistência à Insulina , Ligantes , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Músculo Esquelético/metabolismo , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Comunicação Parácrina , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.
Assuntos
Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/inervação , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sistema Nervoso Simpático/fisiologia , Termogênese , Proteína Desacopladora 1/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Metabolismo Energético , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Proteínas de Transferência de Fosfolipídeos/deficiência , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação , Transdução de Sinais , Proteína Desacopladora 1/deficiência , Proteína Desacopladora 1/genéticaRESUMO
Increased lipogenesis has been linked to an increased cancer risk and poor prognosis; however, the underlying mechanisms remain obscure. Here we show that phosphatidic acid phosphatase (PAP) lipin-1, which generates diglyceride precursors necessary for the synthesis of glycerolipids, interacts with and is a direct substrate of the Src proto-oncogenic tyrosine kinase. Obesity-associated microenvironmental factors and other Src-activating growth factors, including the epidermal growth factor, activate Src and promote Src-mediated lipin-1 phosphorylation on Tyr398, Tyr413 and Tyr795 residues. The tyrosine phosphorylation of lipin-1 markedly increases its PAP activity, accelerating the synthesis of glycerophospholipids and triglyceride. Alteration of the three tyrosine residues to phenylalanine (3YF-lipin-1) disables lipin-1 from mediating Src-enhanced glycerolipid synthesis, cell proliferation and xenograft growth. Re-expression of 3YF-lipin-1 in PyVT;Lpin1-/- mice fails to promote progression and metastasis of mammary tumours. Human breast tumours exhibit increased p-Tyr-lipin-1 levels compared to the adjacent tissues. Importantly, statistical analyses show that levels of p-Tyr-lipin-1 correlate with tumour sizes, lymph node metastasis, time to recurrence and survival of the patients. These results illustrate a direct lipogenesis-promoting role of the pro-oncogenic Src, providing a mechanistic link between obesity-associated mitogenic signaling and breast cancer malignancy.
Assuntos
Neoplasias da Mama/patologia , Proteína Tirosina Quinase CSK/genética , Fosfatidato Fosfatase/metabolismo , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/mortalidade , Proteína Tirosina Quinase CSK/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Lipogênese/fisiologia , Masculino , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos Mutantes , Camundongos Transgênicos , Fosfatidato Fosfatase/genética , Fosforilação , Proto-Oncogene Mas , Tirosina/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Insulin resistance is associated with a greatly increased risk of type 2 diabetes. Administration of fibroblast growth factor-1 (FGF-1) resulted in a marked improvement in insulin sensitivity. However, the underlying molecular mechanism whereby FGF-1 represses insulin resistance remains largely unknown. Here, we sought to delineate the role of FGF-1 in insulin resistance with respect to its anti-inflammatory capability. In this study, we found that FGF-1 had positive effects on glucose intolerance, hepatic lipid accumulation, and insulin resistance, while it markedly repressed cytokine secretion (TNF-α and IL-6) in serum and reduced liver inflammation in diet-induced obesity (DIO) mice. Further, FGF-1 treatment significantly represses TNF-α-induced insulin resistance in vitro and in vivo. These results indicate that FGF-1 likely ameliorates insulin resistance via a mechanism that is independent of its glucose-lowering activity. Subsequent experiments demonstrated that FGF-1 ameliorated insulin resistance, and inflammation was accompanied by decreased c-Jun N-terminal kinase (JNK) signaling. In addition, it is likely that FGF-1 impedes JNK phosphorylation via blocking the transforming growth factor-ß activated kinase 1 (TAK1) and TAK1 binding protein 1 (TAB1) interaction. These findings reveal that FGF-1 regulates insulin sensitivity and may represent an attractive therapeutic target for preventing the development of insulin resistance.
RESUMO
Fibroblast growth factor-20 (FGF20) is a paracrine member of the FGF family that is preferentially expressed in the substantia nigra pars compacta (SNpc). Previous studies have demonstrated that FGF20 enhances the survival of dopaminergic neurons suggesting the potential use of FGF20 to treat Parkinson's disease (PD). However, the reduced solubility of the bacterial recombinant human FGF20 (rhFGF20) and the absence of efficient strategies to transport rhFGF20 across the blood-brain barrier (BBB) have halted its clinical application. In the present study, we have examined the efficiency of fuzing a small ubiquitin-related modifier (SUMO) to rhFGF20 to enhance its soluble expression and further investigated the efficacy of FUS-guided, rhFGF20-liposome transport across the BBB. We also examined the bioavailability and behavioral improvement in a 6-hydroxydopamine-lesioned rat model of PD following 2 weeks' FUS-liposomal combinatorial treatment. Our results showed that, in contrast with rhFGF20 or LIP-FGF20, the FUS-LIP-rhFGF20 treatment could significantly improve the apomorphine-induced rotations by protecting against the loss of dopaminergic neurons in the SNpc. Our Results suggest that our combinatorial method would help overcome key challenges that hinder the currently available methods for the use of rhFGF20 in PD treatment.
Assuntos
Fatores de Crescimento de Fibroblastos/administração & dosagem , Doença de Parkinson/diagnóstico por imagem , Doença de Parkinson/tratamento farmacológico , Proteolipídeos/administração & dosagem , Animais , Linhagem Celular Tumoral , Corpo Estriado/diagnóstico por imagem , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Fatores de Crescimento de Fibroblastos/metabolismo , Humanos , Lipossomos , Masculino , Camundongos , Células NIH 3T3 , Oxidopamina/toxicidade , Células PC12 , Doença de Parkinson/metabolismo , Proteolipídeos/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Resultado do Tratamento , Ultrassonografia/métodosRESUMO
Obesity is characterized by excessive fatty acid conversion to triacylglycerols (TAGs) in adipose tissues. However, how signaling networks sense fatty acids and connect to the stimulation of lipid synthesis remains elusive. Here, we show that homozygous knock-in mice carrying a point mutation at the Ser86 phosphorylation site of acetyltransferase Tip60 (Tip60 SA/SA ) display remarkably reduced body fat mass, and Tip60 SA/SA females fail to nurture pups to adulthood due to severely reduced milk TAGs. Mechanistically, fatty acids stimulate Tip60-dependent acetylation and endoplasmic reticulum translocation of phosphatidic acid phosphatase lipin 1 to generate diacylglycerol for TAG synthesis, which is repressed by deacetylase Sirt1. Inhibition of Tip60 activity strongly blocks fatty acid-induced TAG synthesis while Sirt1 suppression leads to increased adiposity. Genetic analysis of loss-of-function mutants in Saccharomyces cerevisiae reveals a requirement of ESA1, yeast ortholog of Tip60, in TAG accumulation. These findings uncover a conserved mechanism linking fatty acid sensing to fat synthesis.
Assuntos
Retículo Endoplasmático/enzimologia , Lisina Acetiltransferase 5/metabolismo , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Transativadores/metabolismo , Triglicerídeos/biossíntese , Acetilação , Animais , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Ácidos Graxos/metabolismo , Feminino , Histona Acetiltransferases/genética , Histona Acetiltransferases/metabolismo , Cinética , Lisina Acetiltransferase 5/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , Fosfatidato Fosfatase/genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Transativadores/genética , Triglicerídeos/químicaRESUMO
The efficiency of intestinal absorption of dietary fat constitutes a primary determinant accounting for individual vulnerability to obesity. However, how fat absorption is controlled and contributes to obesity remains unclear. Here, we show that inhibition of endoplasmic-reticulum-associated degradation (ERAD) increases the abundance of triacylglycerol synthesis enzymes and fat absorption in small intestine. The C2-domain protein AIDA acts as an essential factor for the E3-ligase HRD1 of ERAD to downregulate rate-limiting acyltransferases GPAT3, MOGAT2, and DGAT2. Aida-/- mice, when grown in a thermal-neutral condition or fed high-fat diet, display increased intestinal fatty acid re-esterification, circulating and tissue triacylglycerol, accompanied with severely increased adiposity without enhancement of adipogenesis. Intestine-specific knockout of Aida largely phenocopies its whole-body knockout, strongly indicating that increased intestinal TAG synthesis is a primary impetus to obesity. The AIDA-mediated ERAD system may thus represent an anti-thrifty mechanism impinging on the enzymes for intestinal fat absorption and systemic fat storage.
Assuntos
Gorduras na Dieta/metabolismo , Degradação Associada com o Retículo Endoplasmático , Absorção Intestinal , Intestino Delgado/enzimologia , Obesidade/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Triglicerídeos/biossíntese , Animais , Esterificação , Camundongos Endogâmicos C57BL , Proteínas de Transferência de Fosfolipídeos/genéticaRESUMO
The recent discovery of metabolic roles for fibroblast growth factor 1 (FGF1) in glucose homeostasis has expanded the functions of this classically known mitogen. To dissect the molecular basis for this functional pleiotropy, we engineered an FGF1 partial agonist carrying triple mutations (FGF1ΔHBS) that diminished its ability to induce heparan sulfate (HS)-assisted FGF receptor (FGFR) dimerization and activation. FGF1ΔHBS exhibited a severely reduced proliferative potential, while preserving the full metabolic activity of wild-type FGF1 in vitro and in vivo. Hence, suboptimal FGFR activation by a weak FGF1-FGFR dimer is sufficient to evoke a metabolic response, whereas full FGFR activation by stable and sustained dimerization is required to elicit a mitogenic response. In addition to providing a physical basis for the diverse activities of FGF1, our findings will impact ongoing drug discoveries targeting FGF1 and related FGFs for the treatment of a variety of human diseases.
Assuntos
Fator 1 de Crescimento de Fibroblastos/química , Hepatócitos/efeitos dos fármacos , Mitógenos/química , Receptores de Fatores de Crescimento de Fibroblastos/química , Células 3T3-L1 , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Fator 1 de Crescimento de Fibroblastos/genética , Fator 1 de Crescimento de Fibroblastos/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Expressão Gênica , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mitógenos/genética , Mitógenos/metabolismo , Mitógenos/farmacologia , Modelos Moleculares , Células NIH 3T3 , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Estabilidade Proteica , Ratos , Receptores de Fatores de Crescimento de Fibroblastos/genética , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
PEGylation is a widely applied approach to improve the pharmacokinetic and pharmacodynamic properties of protein therapeutics. The current solution-phase PEGylation protocols often suffer from poor yield of homogeneously PEGylated bioactive products and hence fall short of being commercially attractive. To improve upon these techniques, here we developed a novel, solid-phase PEGylation methodology using a hydrophobic interaction chromatography (HIC) resin. Two variations of the HIC-based PEGylation are described that are tailored towards conjugation of proteins with hydrophobicity index above (lysozyme) and below (fibroblast growth factor 1, FGF-1) that of the mPEG-butyraldehyde (mPEG) chain used. In the case of lysozyme, the protein was first immobilized on the HIC, and the HIC-bound protein was then conjugated by passing over the column. In the case of FGF-1, the mPEG solution was first immobilized on the HIC, and the FGF-1 solution was then passed through the column. Circular dichroism (CD) spectroscopy demonstrated HIC-based PEGylation almost retained the secondary structures of proteins. Bioactivity assay showed that the recovery of activity of HIC-based PEGylated rhFGF-1 (i.e. 92%) was higher than that of liquid-phase PEGylated rhFGF-1 (i.e. 61%), while HIC-based PEGylated lysozyme showed the same activity recovery (i.e. 7%) as the liquid-phase PEGylated form. For specific proteins, the HIC-based solid-phase PEGylation maybe offer a more promising alternative than the current PEGylation methods and is expected to have a major impact in the area of protein-based therapeutics.
Assuntos
Polietilenoglicóis/química , Cromatografia/métodos , Dicroísmo Circular , Fator 1 de Crescimento de Fibroblastos/análise , Humanos , Interações Hidrofóbicas e Hidrofílicas , Muramidase/análise , Estrutura Secundária de Proteína , Proteínas Recombinantes/análise , SoluçõesRESUMO
Fibroblast growth factor 21 (FGF21) is an endocrine-acting hormone that has the potential to treat metabolic diseases, such as type 2 diabetes and obesity. Development of FGF21 into a therapeutic has been hindered due to its low intrinsic bio-stability, propensity towards aggregation and its susceptibility to in vivo proteolytic degradation. In order to address these shortcomings, we've developed recombinant human FGF21 variants by strategically introducing cysteine residues via site-directed mutagenesis, and have also developed a solid-phase nickel affinity PEGylation strategy, whereby engineered, surface-exposed cysteine residues of immobilized proteins were used as a platform to efficiently and site-selectively conjugate with PEG-maleimide. The engineered PEGylated FGF21 conjugates retained its biological functions, as well as demonstrated an increase in half-life by over 211.3 min. By demonstrating the biological activity of the FGF21 analog as a prototype, we have also provided a "generalized" solid-phase approach to effectively increase serum half-life of protein therapeutics.