forkhead box protein o1  (Proteintech)

Journal: Nutrients

Article Title: EGCG and Taurine Synergistically Ameliorate Lipid Metabolism Disorder by Modulating Gut Microbiota and PPARα/FAS Signaling Pathway

doi: 10.3390/nu17162595

Figure Lengend Snippet: Effects of EGCG and taurine on the expression of proteins related to fatty acid synthesis and catabolism. ( A ) Fox-o1 and PPARα protein bands; ( B ) FAS and LPL protein bands. ( C ) Relative expression level of PPARα; ( D ) relative expression level of FAS; ( E ) relative expression level of Fox-o1; ( F ) relative expression level of LPL; NC: normal diet control, MC: high-fat emulsion established model control, PC: simvastatin treatment positive control, EC: EGCG treatment, TT: taurine treatment, ETC: combined EGCG and taurine treatment. Data are means ± SEM (n = 2). a–e: Different letters in the same column indicate significant differences in the numerical values ( p < 0.05).

Article Snippet: Antibodies against fatty acid synthase (FAS), peroxisome proliferator-activated receptor alpha (PPARα), forkhead box protein o1 (Fox-o1), and lipoprotein lipase (LPL) were purchased from Proteintech Biotechnology Co., Ltd. (Wuhan, China).

Techniques: Expressing, Control, Emulsion, Positive Control

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Hepatic pannexin 1 (PANX1) overexpression attenuated hyperglycemia and alleviated fatty liver in db/db mice. a Oral glucose tolerance test (OGTT) exhibited no significant difference between the two sets of mice before adenoviral injection. The left panel displayed the OGTT curves, while the right panel presented the data for the area under the curve (AUC) ( n = 8). b Pyruvate tolerance test (PTT) curves on day 5 post adenoviral injection were presented in the left panel, and AUC data were displayed in the right panel ( n = 8). c The left panel displayed the OGTT curves on day 10 post adenoviral injection, while the right panel presented the data for the AUC ( n = 8). d Hepatic PANX1 overexpression reduced fasting blood glucose level of db/db mice on day 10 after adenoviral injection ( n = 8). Body weight ( e ) and liver weight ( f ) on day 11 post adenoviral injection ( n = 8). Morphological observation ( g ) and oil red O staining analysis ( h ) of livers from Ad-GFP and Ad-PANX1-injected db/db mice ( n = 5). Scale bar = 50 μm. Liver ( i , n = 8) and serum ( j , n = 7) triglyceride (TG) content of Ad-GFP and Ad-PANX1-injected db/db mice. k Serum adenosine triphosphate (ATP) content was increased after PANX1 overexpression in the liver of db/db mice. Serum ATP content was detected immediately after sacrifice ( n = 8). l Comparative mRNA levels of gluconeogenic genes in db/db mice livers ( n = 8). m The left panel displayed representative gel images of gluconeogenic proteins in db/db mouse livers, while the right panel presented quantitative data ( n = 7). * P < 0.05 or ** P < 0.01. Ad-GFP Ad-GFP-injected db/db mice, Ad-PANX1 Ad-PANX1-injected db/db mice, Akt protein kinase B, FOXO1 forkhead box protein O1, PEPCK phosphoenolpyruvate carboxykinase, G-6-Pase glucose-6-phosphatase

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Over Expression, Injection, Staining

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Hepatic pannexin 1 (PANX1) overexpression reduced hyperglycemia and attenuated fatty liver in high-fat diet (HFD)-fed mice. a The left panel displayed the pyruvate tolerance test (PTT) curves on day 5 following adenoviral injection in HFD-fed mice, and the right panel presented the corresponding area under the curve (AUC) data ( n = 7). b The left panel displayed the oral glucose tolerance test (OGTT) curves on day 10 post adenoviral injection in HFD-fed mice, and the right panel presented the corresponding AUC data. n = 8 (HFD + Ad-GFP) and 9 (HFD + Ad-PANX1). c PANX1 overexpression reduced fasting blood glucose level in HFD-fed mice on day 10 post adenoviral injection, from left to right, n = 9, 9, 8, and 9, respectively. d Liver overexpression of PANX1 increased global insulin sensitivity in HFD-fed mice diet. Another set of HFD-fed mice was used for the insulin tolerance test (ITT). n = 6 (HFD + Ad-GFP) and 8 (HFD + Ad-PANX1). The left panel displayed the ITT curves on day 7 post adenoviral injection in HFD-fed mice, and the right panel presented the corresponding AUC data. e PANX1 overexpression decreased global fat deposition in HFD-fed mice. The left panel displayed representative magnetic resonance imaging (MRI) images of whole-body fat, while the right panel presented quantitative data, n = 4 (Ad-GFP) and 5 (Ad-PANX1). Scale bar = 5 cm. f Hepatic PANX1 overexpression reduced hepatic lipid deposition in HFD-fed mice. The left panel displayed representative MRI images of liver fat, while the right panel presented quantitative data, n = 4 (Ad-GFP) and 5 (Ad-PANX1). Scale bar = 5 cm. Body weight ( g , n = 7) and liver weight ( h , from left to right, n = 7 and 8) on day 11 post adenoviral injection in HFD mice. Morphological observations ( i ) and oil red O staining analyses ( j ) of livers from Ad-GFP- and Ad-PANX1-injected HFD mice ( n = 5). Scale bar = 50 μm. Hepatic ( k , from left to right, n = 15 and 18) and serum ( l , n = 13) triglyceride (TG) content in HFD mice after PANX1 overexpression. m Overexpression of hepatic PANX1 led to an increase in serum adenosine triphosphate (ATP) content in HFD-fed mice, n = 13 (Ad-GFP) and n = 15 (Ad-PANX1). n Comparative mRNA levels of gluconeogenic genes in HFD-fed mice livers, from left to right, n = 7, 7, 6, 7, 7 and 7, respectively. o PANX1 overexpression reduced gluconeogenic proteins in HFD-fed mouse livers. The left panel showed representative gel images and the right panel presented quantitative data ( n = 7). * P < 0.05 or ** P < 0.01. HFD + Ad-GFP/Ad-GFP Ad-GFP-injected HFD-fed mice, HFD + Ad-PANX1/Ad-PANX1 Ad-PANX1-injected HFD-fed mice, Akt protein kinase B, FOXO1 forkhead box protein O1, PEPCK phosphoenolpyruvate carboxykinase, G-6-Pase glucose-6-phosphatase

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Over Expression, Injection, Magnetic Resonance Imaging, Staining

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Hepatic pannexin 1 (PANX1) restoration rescued the dysregulated glucolipid metabolism in PANX1-deficient mice. a Fasting blood glucose levels of wild-type (WT) and PANX1-deficient mice. The mice were fed on normal chow, from left to right, n = 8, 14, and 14 (WT), n = 11, 16, and 15 (PANX1 −/− ), respectively. b The left panel displayed the glucose tolerance test (OGTT) curves of 12-week-old WT and PANX1-deficient mice, and the right panel presented the area under the curve (AUC) data, n = 14 (WT) and 16 (PANX1 −/− ). c Body fat was increased in PANX1-deficient mice at the age of 15 weeks old. The left panel displayed the representative magnetic resonance imaging (MRI) images of whole-body fat, and the right panel presented the quantitative data ( n = 5). Scale bar = 5 cm. d Hepatic fat content was increased in PANX1-deficient mice at the age of 15 weeks old. The left panel displayed the representative MRI images of liver fat, and the right panel presented the quantitative data ( n = 5). Scale bar = 5 cm. e Schematic diagram of the grouping mode. 16 PANX1 −/− mice were randomly divided into two groups at the age of 16 weeks, and then injected with Ad-GFP and Ad-PANX1, respectively. f The left panel displayed the pyruvate tolerance test (PTT) curves on day 4 post adenoviral injection, and the right panel presented AUC data, n = 10 (WT + Ad-GFP), 8 (PANX1 −/− + Ad-GFP) and 8 (PANX1 −/− + Ad-PANX1). g The left panel displayed OGTT curves on day 8 post adenoviral injection, and the right panel presented the AUC data, n = 10 (WT + Ad-GFP), 8 (PANX1 −/− + Ad-GFP), and 8 (PANX1 −/− + Ad-PANX1). Body ( h , from left to right, n = 8, 8 and 7) and liver weight ( i, from left to right, n = 10, 8 and 7) on day 9 post adenoviral injection in WT and PANX1-deficient mice. j Morphological observations (left panel) and oil red O staining analyses (right panel) of adenoviral-injected-WT and PANX1-deficient mice ( n = 5). Scale bar = 50 μm. Hepatic ( k , from left to right, n = 10, 8 and 8) and serum ( l , from left to right, n = 9, 8 and 8) triglyceride (TG) levels of adenoviral-injected-WT and PANX1-deficient mice. m Hepatic PANX1 restoration rescued the decreased serum adenosine triphosphate (ATP) content in PANX1-deficient mice, n = 7 (WT + Ad-GFP), 7 (PANX1 −/− + Ad-GFP) and 6 (PANX1 −/− + Ad-PANX1). n Relative mRNA levels of gluconeogenic genes in livers of adenoviral-injected-WT and PANX1-deficient mice ( n = 8). o Hepatic PANX1 restoration reduced gluconeogenic proteins in PANX1-deficient mouse livers. The left panel displayed representative gel images, and the right panel presented quantitative data, n = 6 (WT + Ad-GFP) and 6 (PANX1 −/− + Ad-GFP), from left to right, n = 6, 6, 6, 5, and 6 (PANX1 −/− + Ad-PANX1), respectively. * P < 0.05 or ** P < 0.01. WT wild-type, PANX −/− PANX1-deficient mice, WT + Ad-GFP Ad-GFP-injected wild-type mice, PANX −/− + Ad-GFP Ad-GFP-injected PANX1-deficient mice, PANX −/− + Ad-PANX1 Ad-PANX1-injected PANX1-deficient mice, Akt protein kinase B, FOXO1 forkhead box protein O1, PEPCK phosphoenolpyruvate carboxykinase, G-6-Pase glucose-6-phosphatase

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Magnetic Resonance Imaging, Injection, Staining

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Pannexin 1 (PANX1)-mediated ATP release inhibited gluconeogenesis and reduced lipid deposition by activating P2 receptors in hepatocytes. a Representative immunofluorescent staining images of forkhead box protein O1 (FOXO1) in mouse hepatocytes infected with Ad-PANX1 or Ad-GFP ( n = 3). Scale bar = 25 μm. b Relative mRNA levels of gluconeogenic genes in mouse hepatocytes infected with Ad-GFP or Ad-PANX1 ( n = 5). c The left panel displayed representative gel images of gluconeogenic proteins in mouse hepatocytes infected with Ad-GFP or Ad-PANX1, with quantitative data presented in the right panel, n = 5 (Ad-GFP), from left to right, n = 5, 4, 5, 4 and 5 (Ad-PANX1), respectively. Treatment with PANX1 inhibitor PBN or P2 receptor inhibitor suramin abolished PANX1-induced FOXO1 nuclear exclusion ( d ) and inhibition of glucose production ( e ) in mouse hepatocytes ( n = 3). Scale bar = 25 μm. f Treatment with PBN or suramin abolished PANX1-induced activation of protein kinase B (Akt) in mouse hepatocytes. The left panel displayed the representative gel images, and the right panel presented the quantitative data ( n = 3). g Relative mRNA levels of key lipid metabolic genes in PANX1-overexpressed mouse hepatocytes ( n = 3). h Relative mRNA levels of key lipid metabolic genes in livers of Ad-PANX1-injected HFD mice, n = 7 (HFD + Ad-GFP), from left to right, n = 8, 7, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 9, 8, 9, 9, 9, 9, 9 and 9 (HFD + Ad-PANX1), respectively. i PANX1 overexpression reduced the mRNA level of FAS in db/db ( n = 8) and HFD mouse livers ( n = 7). j PANX1 overexpression decreased the fatty acid synthase (FAS) protein level in livers of db/db and HFD mice. The left panel displayed representative gel images, and the right panel presented the quantitative data. ( n = 7). k The FAS mRNA level was elevated in the livers of PANX1 -knockdown mice ( n = 7) and PANX1-deficient mice ( n = 8). l The FAS protein was elevated in the livers of hepatic PANX1 -knockdown mice and PANX1-deficient mice. The left panel presented the representative gel images, and the right panel displayed the quantitative data ( n = 7). m Treatment with PBN or suramin reversed PANX1-induced inhibition of FAS level in mouse hepatocytes. The left panel presented the representative gel images, and the right panel displayed the quantitative data ( n = 3). Treatment with PBN or suramin reversed PANX1-induced decrease in lipid accumulation ( n ) and triglyceride (TG) levels ( o ) in mouse hepatocytes ( n = 3). Scale bar = 25 μm. * P < 0.05 or. ** P < 0.01. Con control, PBN probenecid, S suramin, DAPI 4,6-diamino-2-phenyl indole, Ad-GFP Ad-GFP-infected mouse hepatocytes, Ad-PANX1 Ad-PANX1-infected mouse hepatocytes, PEPCK phosphoenolpyruvate carboxykinase, G-6-Pase glucose-6-phosphatase, SREBP1 sterol regulatory element-binding protein 1, ACC acetyl-coA carboxylase, SCD1 stearoyl-coA desaturase 1, PPARγ peroxisome proliferator-activated receptor γ, CHREBP carbohydrate response element binding protein, LXR liver x receptor, ACOX1 acyl-CoA oxidase 1, CPT1α carnitine palmitoyltransferase 1α, PPARα peroxisome proliferator-activated receptor α, SCAD short-chain acyl-coA dehydrogenase, MCAD medium-chain acyl-coA dehydrogenase, LCAD long-chain acyl-coA dehydrogenase, apoB apolipoprotein b, MTP microsomal triglyceride transfer protein, CD36 cluster of differentiation 36, FATP1 fatty acid transport protein 1, FATP2 fatty acid transport protein 2, FATP5 fatty acid transport protein 5, FABP1 fatty acid binding protein 1, FFAR1 free fatty acids receptor 1

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Staining, Infection, Inhibition, Activation Assay, Injection, Over Expression, Knockdown, Control, Binding Assay

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Pannexin 1 (PANX1) inhibited c-Jun N-terminal kinase-activator protein-1-fatty acid synthase (JNK-AP1-FAS) pathway to ameliorate hepatic lipid deposition. a PANX1 overexpression elevated calmodulin (CaM) protein level in mouse hepatocytes. The left panel showed the representative gel images, while the right panel displayed quantitative data ( n = 3). b Representative silver staining gel image of Co-IP products in Ad-CaM-treated mouse hepatocytes. The bands indicated by the arrows were pooled and analyzed by mass spectrometry (MS). Overexpression of CaM ( c ) or PANX1 ( d and e ) stimulated CaM-JNK interaction in mouse hepatocytes ( n = 3). f Representative immunofluorescent staining images of CaM and JNK in Ad-GFP- or Ad-PANX1-overexpressed mouse hepatocytes ( n = 3). Scale bar = 25 μm. g Representative immunofluorescent staining images of CaM and JNK in liver sections of HFD-fed mice injected with Ad-GFP or Ad-PANX1 ( n = 3). Scale bar = 25 μm. h and i CaM overexpression decreased the phosphorylations of JNK and AP1 in mouse hepatocytes. Panel ( h ) presented the representative gel images, and panel ( i ) displayed the quantitative data ( n = 3). PANX1 overexpression decreased JNK and AP1 phosphorylations in mouse hepatocytes ( j, from left to right, n = 3, 3, 5, and 4, respectively) and HFD-fed mouse livers ( k , n = 7). The left panel displayed the representative gel images, and the right panel presented the quantitative data. l Representative silver staining gel image of DNA-pulldown products in mouse hepatocytes. The bands indicated by the arrows were subjected to MS analysis. m Schematic diagram of nuclear proteins identified by MS analysis. V-maf musculoaponeurotic fibrosarcoma oncogene homolog K (MafK) overexpression inhibited the transcriptional activity of PANX1 gene promoter in HepG2 ( n ) and HEK 293 T ( o ) cells ( n = 3). p MafK overexpression reduced the mRNA level of PANX1 in mouse hepatocytes ( n = 3). q Overexpression of MafK decreased PANX1 protein level in mouse hepatocytes. The left panel showed representative gel images and the right panel displayed quantitative data ( n = 3). r siRNA silencing of MafK elevated PANX1 protein level in mouse hepatocytes. The left panel showed representative gel images and the right panel presented quantitative data ( n = 3). s The mRNA level of MafK was decreased in the livers of db/db mice, n = 8 ( db/m ) and 7 ( db/db ). t The protein level of MafK was reduced in livers of db/db mice. The upper panel showed representative gel images and the lower panel displayed quantitative data ( n = 3). * P < 0.05 or ** P < 0.01. Ad-GFP Ad-GFP-infected mouse hepatocytes, Ad-PANX1 Ad-PANX1-infected mouse hepatocytes, Ad-CaM Ad-CaM-treated mouse hepatocytes, FAS fatty acid synthase, Tfdp1 transcription factor dp 1, FoxO1 forkhead box protein O1, Stat1 signal transducer and activator of transcription 1, Nrf1 nuclear respiratory factor 1, Hnf1α hepatocyte nuclear factor 1α, Btf3 basic transcription factor 3, p-JNK phosphorylated c-Jun N-terminal kinase, p-AP1 phosphorylated activator protein-1, DAPI 4,6-diamino-2-phenyl indole, IP immunoprecipitation, pcDNA3.1 vector plasmid as control, pGFP GFP plasmid transfection, pMafK MafK plasmid transfection, si-NC control siRNA transfection, si-MafK MafK siRNA transfection

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Over Expression, Silver Staining, Co-Immunoprecipitation Assay, Mass Spectrometry, Staining, Injection, Activity Assay, Infection, Immunoprecipitation, Plasmid Preparation, Control, Transfection

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Family with sequence similarity 3 member A (FAM3A)-mediated ATP release was dependent on pannexin 1 (PANX1). a Overexpression of FAM3A elevated PANX1 mRNA level in mouse hepatocytes, from left to right, n = 3, 3, 3, 3, 3, 3, 6, and 6, respectively. b Overexpression of FAM3A upregulated PANX1 protein level in mouse hepatocytes. The upper panel showed representative gel images, and the lower panel displayed the quantitative data ( n = 6). c Representative immunofluorescent staining images of PANX1 in FAM3A-overexpressed mouse hepatocytes ( n = 3). Scale bar = 25 μm. Heat shock factor 1 (HSF1) overexpression enhanced the transcriptional activity of the PANX1 gene promoter in HepG2 ( d ) and HEK 293 T ( e ) cells ( n = 6). f Overexpression of HSF1 upregulated PANX1 protein level in mouse hepatocytes. The left panel showed representative gel images and the right panel displayed quantitative data ( n = 4). g Representative immunofluorescent staining images of HSF1 in FAM3A-overexpressed mouse hepatocytes ( n = 3). Scale bar = 25 μm. h Treatment with HSF1 inhibitor abolished FAM3A-induced increase in PANX1 protein level in mouse hepatocytes. The left panel showed representative gel images and the right panel displayed quantitative data ( n = 5). i Pannexin 1 inhibitor PBN treatment blocked FAM3A-induced ATP release in mouse hepatocytes ( n = 5). PBN treatment reversed FAM3A-promoted FOXO1 nuclear exclusion ( j ) and glucose production suppression ( k ) in mouse hepatocytes ( n = 3). Scale bar = 25 μm. PBN treatment reversed FAM3A-induced reduction in lipid accumulation ( l , n = 3) and triglyceride (TG) levels ( m , n = 5) in mouse hepatocytes. Scale bar = 25 μm. PBN treatment reversed FAM3A-induced reduction in lipid deposition ( n , n = 3) and TG content ( o , n = 6) in HepG2 cells. Scale bar = 25 μm. p PBN treatment blocked FAM3A-induced Akt and FOXO1 phosphorylations in mouse hepatocytes. The left panel showed representative gel images and the right panel presented quantitative data ( n = 3). * P < 0.05 or. ** P < 0.01. KRI HSF1 inhibitor KRIBB11, CXs connexins, Ad-GFP Ad-GFP-infected mouse hepatocytes, Ad-FAM3A Ad-FAM3A-infected mouse hepatocytes, Akt protein kinase B, FOXO1 forkhead box protein O1

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Sequencing, Over Expression, Staining, Activity Assay, Infection

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Hepatic family with sequence similarity 3 member A (FAM3A) overexpression failed to rescue the metabolic disorders of pannexin 1 (PANX1)-deficient mice. a Schematic diagram of the grouping mode. PANX1 −/− mice were randomly divided into two groups at the age of 14 weeks, and then injected with AAV-GFP or AAV-FAM3A, respectively. The mice were fed on normal chow. b The left panel showed oral glucose tolerance test (OGTT) curves on week 12 post AAV injection wild-type (WT) and PANX1-deficient mice, and the right panel displayed an area under the curve (AUC) data ( n = 9). c The left panel showed pyruvate tolerance test (PTT) curves on week 13 post-AAV injection in WT and PANX1-deficient mice, and the right panel presented AUC data, n = 10 (WT + AAV-GFP), 9 (PANX1 −/− + AAV-GFP) and 9 (PANX1 −/− + AAV-FAM3A). d The left panel displayed insulin tolerance test (ITT) curves on week 14 post-AAV injection in WT and PANX1-deficient mice, and the right panel presented AUC data ( n = 9). The upper panel showed representative magnetic resonance imaging (MRI) images of whole-body ( e ) and hepatic ( f ) fat in AAV-injected WT and PANX1-deficient mice, and the lower panel displayed quantitative data ( n = 4). Scale bar = 5 cm. Body ( g , from left to right, n = 10, 9 and 9) and liver weight ( h , n = 9) on the sacrificed day. i Morphological observations (left panel) and oil red O staining analyses (right panel) of AAV-WT and PANX1-deficient mice ( n = 5). Scale bar = 50 μm. Liver ( j ) and serum ( k ) triglyceride (TG) content of AAV-injected WT and PANX1-deficient mice ( n = 8). l Hepatic FAM3A overexpression failed to rescue the decreased serum adenosine triphosphate (ATP) content in PANX1-deficient mice ( n = 8). m FAM3A overexpression elevated ATP level in livers of PANX1-deficient mouse, n = 4 (WT + AAV-GFP), 5 (PANX1 −/− + AAV-GFP) and 5 (PANX1 −/− + AAV-FAM3A). n Relative mRNA levels of metabolic genes in livers of mice, from left to right, n = 7, 8, 8 and 7 (WT + AAV-GFP), n = 8 (PANX1 −/− + AAV-GFP) and 8 (PANX1 −/− + AAV-FAM3A). o The left panel showed representative gel images of metabolic proteins in mouse livers, and the right panel presented quantitative data ( n = 3). * P < 0.05 or ** P < 0.01. WT + AAV-GFP AAV-GFP-injected wild-type mice, PANX −/− + AAV-GFP AAV-GFP-injected PANX1-deficient mice, PANX −/− + AAV-FAM3A AAV-FAM3A-injected PANX1-deficient mice, FAS fatty acid synthase, Akt protein kinase B, FOXO1 forkhead box protein O1, PEPCK phosphoenolpyruvate carboxykinase, G-6-Pase glucose-6-phosphatase

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Sequencing, Over Expression, Injection, Magnetic Resonance Imaging, Staining

Journal: Military Medical Research

Article Title: PANX1-mediated ATP release confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis

doi: 10.1186/s40779-024-00543-6

Figure Lengend Snippet: Family with sequence similarity 3 member A (FAM3A) overexpression had no effect on the expressions of metabolic genes in pannexin 1 (PANX1)-deficient hepatocytes. a FAM3A overexpression had little effect on the extracellular adenosine triphosphate (ATP) content in PANX1-deficient hepatocytes, from left to right, n = 4, 4, 3, and 4, respectively. b FAM3A overexpression enhanced intracellular ATP content in PANX1-deficient hepatocytes ( n = 3). c Representative immunofluorescent staining images of forkhead box protein O1 (FOXO1) in PANX1-deficient hepatocytes with FAM3A overexpression ( n = 3). Scale bar = 25 μm. d FAM3A overexpression had little effect on suppressing glucose production in PANX1-deficient hepatocytes ( n = 3). e and f FAM3A overexpression failed to reduce lipid accumulation in PANX1-deficient hepatocytes. Panel ( e ) displayed representative lipid confocal images ( n = 3), and panel ( f ) presented quantitative triglyceride (TG) analysis ( n = 4). Scale bar = 25 μm. g and h FAM3A overexpression failed to activate the protein kinase B (Akt) pathway in PANX1-deficient mouse hepatocytes. Panel ( g ) presented representative gel images, and panel ( h ) displayed quantitative data. ( n = 3). * P < 0.05 or ** P < 0.01. i Proposed a model of PANX1-mediated ATP release that confers FAM3A’s suppression effects on hepatic gluconeogenesis and lipogenesis. PANX1-mediated ATP release activates purinergic P2 receptors in hepatocytes. On one hand, the activated Akt inhibits FOXO1 from repressing the expressions of PEPCK and G-6-Pase, thereby suppressing gluconeogenesis. On the other hand, activated CaM interacted with JNK to inhibit its activity, thereby repressing AP1 activity and FAS expression to reduce lipid deposition. Under obese conditions, FFAs upregulate PANX1 expression by inhibiting the expressions of the transcription factors MafK. Upregulation of hepatic PANX1 expression is an important compensatory mechanism for maintaining glucose and lipid homeostasis under obese conditions. FAM3A activates HSF1 to induce HSF1 expression, which in return confers its effects on ATP release induction and gluconeogenesis and lipogenesis repression in hepatocytes. Activation of the insulin-independent FAM3A-HSF1-PANX1-ATP-P2 receptor signaling pathway is effective in treating metabolic disorders. Akt protein kinase B, ATP adenosine triphosphate, CaM calmodulin, CX connexin, FAM3A family with sequence similarity 3 member A, FAS fatty acid synthase, FOXO1 forkhead box protein O1, G-6-Pase glucose-6-phosphatase, HSF1 heat shock factor 1, MafK v-maf musculoaponeurotic fibrosarcoma oncogene homolog K, P2R P2 receptor, PANX1 pannexin 1, p-AP1 phosphorylated activator protein-1, PI3K phosphoinositide 3-kinase, p-JNK phosphorylated c-Jun N-terminal kinase, FFAs free fatty acids, WT wild-type

Article Snippet: Anti-p-Akt (Ser473, #4060S), Akt (#9272S), forkhead box protein O1 (FOXO1, #2880S), p-FOXO1 (Ser256, #9461S), phosphorylated c-Jun N-terminal kinase (p-JNK, Thr183/Thr185, #4668S) and heat shock factor 1 (HSF1, #4356S) antibodies were obtained from Cell Signaling Technology (USA).

Techniques: Sequencing, Over Expression, Staining, Activity Assay, Expressing, Activation Assay