Journal: Nature Communications

Article Title: Gut microbiota-bile acid crosstalk contributes to the rebound weight gain after calorie restriction in mice

doi: 10.1038/s41467-022-29589-7

Figure Lengend Snippet: a Relative abundance of Parabacteroides distasonis in the contents of cecum among three groups. b The (un)conjugated (non-)12OH BAs percentage in the serum. c The BAs composition profiles in the serum. p -values were adjusted by the FDR’s method. d The composition level of LCA and UDCA in the serum. p -values were adjusted by the FDR’s method. e Relative mRNA expression of Tgr5 and Gcg genes in the distal ileum, n = 6 per group. f Active GLP-1 level in serum. g Relative mRNA expression of thermogenesis-related genes in the BAT. h UCP1 protein expression in the BAT, n = 3 per group. n = 8 per group in the experiments. Differences were assessed by a two-tailed multiple T-test in the GraphPad software. Data were expressed as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM in the bar plots. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest). Source data are provided as a Source data file. 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); Takeda G protein-coupled receptor 5 ( Tgr5 ); glucagon-like peptide-1 (GLP-1); brown adipose tissue (BAT); uncoupling protein 1 ( Ucp1 ); peroxisome proliferator-activated receptor-γ coactivator-1α ( Pgc1α ); elongation of very long-chain fatty acids 3 ( Elovl3 ); elongation of very long-chain fatty acids 6 ( Elovl6 ).

Article Snippet: The mice were intraperitoneally injected with GLP-1 receptor agonist, exendin-4 (exenatide, 24 nmol/kg, Cas: 141758-74-9, MedChemExpress), or PBS control once daily. n = 5 per group.

Techniques: Expressing, Two Tailed Test, Software

Journal: Nature Communications

Article Title: Gut microbiota-bile acid crosstalk contributes to the rebound weight gain after calorie restriction in mice

doi: 10.1038/s41467-022-29589-7

Figure Lengend Snippet: a Body weight (* p < 0.05. Differences between the CR + HF + PD group and the CR + HF + Vehicle group are marked in light-gray asterisks, differences between the CR + HF + PD group and the CR + HF + HKPD group are marked in dark-gray asterisks.) and the weight gain during the 4-week HFD. b Average energy intake during the first week of changing to HFD. c Raw average energy expenditures per hour during the period of light and dark, n = 4 per group. d Fasting blood glucose at the end of the experiment. e Fat mass and lean mass of the mice by NMR miniSpec LF50, n = 4 per group. f PCoA plots of the cecal microbiome based on the Jaccard similarity. g Mean abundance of the phyla among the three groups. h The bar plots show the mean percentage of (non-)12OH and (un)conjugated BAs in the serum. i The BAs composition profiles in the cecal contents among three groups. j The composition of unconjugated non-12OH BAs (LCA and UDCA). p -values were adjusted by the FDR’s method. k Relative mRNA expression of Tgr5 and Gcg in the distal ileum. l Active GLP-1 level in serum. m Relative mRNA expression of Ucp1 and Pgc1α in BAT. n UCP1 protein expression in BAT. o The body weight of UKO mice. p The fasting blood glucose level of UKO mice. n = 5 in the CR + HF + PD, CR + HF + Vehicle, CR + HF + HKPD, and CR + HF + PD (UKO) groups. n = 4 in the CR + HF + Vehicle (UKO) group. All differences were assessed by the two-tailed multiple T-test in the GraphPad software. All box and whiskers plots showed the box (from the 25th to 75th percentiles), the median value (in the transverse line), and the whiskers (go down to the smallest value and up to the largest), data in bar plots were expressed as means \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\pm$$\end{document} ± SEM. Source data are provided as a Source data file. High-fat diet (HFD); principal coordinate analysis (PCoA); 12α-hydroxylated bile acids (12OH BAs); non-12α-hydroxylated bile acids (non-12OH BAs); T-conjugated (taurine-conjugated); cholic acid (CA); deoxycholic acid (DCA); chenodeoxycholic acid (CDCA); muricholic acid (MCA); lithocholic acid (LCA); ursodeoxycholic acid (UDCA); bile acid (BA); Takeda G protein-coupled receptor 5 ( Tgr5 ); glucagon-like peptide-1 (GLP-1); uncoupling protein 1 ( Ucp1 ); peroxisome proliferator-activated receptor-γ coactivator-1α ( Pgc1α ); brown adipose tissue (BAT).

Article Snippet: The mice were intraperitoneally injected with GLP-1 receptor agonist, exendin-4 (exenatide, 24 nmol/kg, Cas: 141758-74-9, MedChemExpress), or PBS control once daily. n = 5 per group.

Techniques: Expressing, Two Tailed Test, Software

Journal: bioRxiv

Article Title: An inter-organelle contact between endosomal GLP-1R, ER VAP-B, and the mitochondrial AKAP SPHKAP triggers PKA-dependent MIC19 phosphorylation and β-cell mitochondrial remodelling

doi: 10.1101/2024.04.28.591531

Figure Lengend Snippet: a , Confocal microscopy analysis of hGLP-1R localisation in Ex-4-stimulated INS-1 832/3 SNAP/FLAG-hGLP-1R cells co-expressing dominant negative dynamin (Dyn) 1 and 2 K44A; white arrow indicates hGLP-1R plasma membrane localisation with Dyn1+2 K44A co-expression; yellow arrow indicates endosomal hGLP-1R localisation in a non-Dyn1+2 K44A expressing cell; size bar, 5 μm. b , EGFP-VAP-B co-IP with hGLP-1R in Veh versus Ex-4-stimulated INS-1 832/3 SNAP/FLAG-hGLP-1R cells co-expressing control pcDNA3.1 versus Dyn1+2 K44A. c , Schematic of the hGLP-1R-nanoluciferase (NLuc) – Venus-VAP-B interaction nanoBRET assay. d , Veh-subtracted Ex-4- versus exendin-F1 (Ex-F1)-induced BRET kinetic responses (left) and corresponding area under the curve (right) measuring hGLP-1R-NLuc – Venus-VAP-B interactions in INS-1 832/3 cells; n =3, *p<0.05 by paired t-test. e , Confocal microscopy analysis of hGLP-1R localisation in mouse primary islets transduced with SNAP/FLAG-hGLP-1R adenoviruses following 5-minute stimulation with the indicated agonists [Ex-4, Ex-F1, exendin-D3 (Ex-D3), tirzepatide (Tirz) and semaglutide (Sema) used at 100 nM; orforglipron (Orf) and danuglipron (Dan) used at 5 μM]; size bars, 20 μm. f , g , EGFP-VAP-B co-IP with hGLP-1R in INS-1 832/3 SNAP/FLAG-hGLP-1R cells stimulated for 5 minutes with the indicated agonists. Representative blots ( f ) and quantification of EGFP-VAP-B levels per condition normalised to SNAP ( g ); n =3.

Article Snippet: Custom analogues were from WuXi AppTec, China, and danuglipron and orforglipron were purchased from MedChemExpress.

Techniques: Confocal Microscopy, Expressing, Dominant Negative Mutation, Clinical Proteomics, Membrane, Co-Immunoprecipitation Assay, Control, Transduction

Journal: bioRxiv

Article Title: A Brain Reward Circuit Inhibited By Next-Generation Weight Loss Drugs

doi: 10.1101/2024.12.12.628169

Figure Lengend Snippet: a , Structure, naming convention, molecular formula, molecular weight (MW), half-life in humans, and species binding specificity of glucagon-like peptide-1 (GLP-1), liraglutide, danuglipron, and orforglipron. Amino acid sequences of liraglutide shown with its substitution and additions to GLP-1 highlighted in green and purple, respectively. b , Standard diet (SD) consumption over 2 hours post-administration of saline (Sal), liraglutide (Lira), vehicle (Veh), or danuglipron (Dan) ( n = 11 per injection, one-way ANOVA with Bonferroni correction, *** P <0.001). c , Schematic of the serine (TCA or S) to tryptophan (TGG or W) CRISPR-mediated substitution in Glp1r S33W (bottom) or in mouse Glp1r (top). d,e , Sanger sequencing chromatograms of ( d ) WT mouse Glp1r and ( e ) Glp1r S33W sequence, confirming the Glp1r S33W substitution in mice. f-h , Glucose tolerance test (GTT) on GLP1RAs. Comparison of blood glucose levels on ( f ) liraglutide (Lira), ( g ) danuglipron (Dan), ( h ) orforglipron (Orfo) followed by dextrose (Dex) in WT and Glp1r S33W mice ( n = 5-9 per injection, two-way ANOVA with Bonferroni correction, ** P <0.01; *** P <0.001). i-n , SD intake 1, 2, and 4 hours post-treatment of ( i, j ) Lira ( n = 9-11), ( k, l ) Dan ( n = 15-16), and ( m, n ) Orfo ( n = 8-9) and vehicle controls in WT and Glp1r S33W mice. o-t , High fat diet (HFD) intake 1, 2, and 4 hours post-treatment of ( o, p ) Lira ( n = 8-10), ( q, r ) Dan ( n = 14-16), and ( s, t ) Orfo ( n = 8-9) and their vehicle controls in WT and Glp1r S33W mice (two-way ANOVA with Bonferroni correction, * P <0.05; ** P <0.01; *** P <0.001). u , GTT measuring blood glucose levels following oral administration of danuglipron (oDan) and dextrose in Glp1r S33W and WT mice ( n = 6 per genotype, two-way ANOVA with Bonferroni correction, ** P <0.01; *** P <0.001). v-y , HFD intake 1, 2, and 4 hours after oral gavage of ( v ) danuglipron or vehicle and ( x ) orforglipron or saline ( n = 8-9, two-way ANOVA with Bonferroni correction, ** P <0.01; *** P <0.001). Comparison of the 4th hour between intraperitoneal (IP) and oral routes of administration with ( w ) danuglipron or ( y ) orforglipron (two-way ANOVA with Bonferroni correction, ** P <0.01; *** P <0.001). z , 7-day weight changes of mice on chronic HFD treated with orforglipron or saline daily at ZT6 ( n = 7 per injection, paired t-test, *** P <0.001). Data are represented as means ± SEM. * P <0.05; ** P <0.01; *** P <0.001. See Supplementary Table 1 for statistical details for all figures and Extended Data figures.

Article Snippet: Orforglipron powder (MedChemExpress, catalog no. HY-112185) was dissolved to 10mg/mL in Dimethyl sulfoxide (DMSO) and further diluted in 0.9% NaCl sterile saline to 0.1mg/mL.

Techniques: Molecular Weight, Binding Assay, Saline, Injection, CRISPR, Sequencing, Comparison

Journal: bioRxiv

Article Title: A Brain Reward Circuit Inhibited By Next-Generation Weight Loss Drugs

doi: 10.1101/2024.12.12.628169

Figure Lengend Snippet: GLP1RA activation across targeted GLP1R-expressing brain regions . a-d , GLP1R protein expression validated by a Glp1r-Cre;tdTomato mouse line and neuronal cFos activation 2 hours after danuglipron or liraglutide or 6 hours after orforglipron injection in WT and Glp1r S33W mice in the ( a ) DMH, ( b ) NTS, ( c ) AP, or ( d ) CeA. Scale bars = 200 µm. e-h , Quantification of cFos in the ( e ) DMH, ( f ) NTS, ( g ) AP and ( h ) CeA ( n = 3-4 per genotype, two-way ANOVA with Bonferroni correction, * P <0.05; ** P <0.01; *** P <0.001). i , Ratio of NTS/AP cFos activation in Glp1r S33W mice after danuglipron, orforglipron, and liraglutide ( n = 3-4 per injection, Kruskal-Wallis test, * P <0.05). j,k , Neuronal cFos activation in the NTS and AP 2 hours after danuglipron was administered to Glp1r S33W mice via ( j ) IP injection or ( k ) oral gavage. l , Quantification of cFos expression in the NTS (left) or AP (right) following danuglipron IP or oral delivery in Glp1r S33W mice ( n = 3 per delivery route, Welch’s t-test, * P <0.05). Data are represented as means ± SEM. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Orforglipron powder (MedChemExpress, catalog no. HY-112185) was dissolved to 10mg/mL in Dimethyl sulfoxide (DMSO) and further diluted in 0.9% NaCl sterile saline to 0.1mg/mL.

Techniques: Activation Assay, Expressing, Injection

Journal: bioRxiv

Article Title: A Brain Reward Circuit Inhibited By Next-Generation Weight Loss Drugs

doi: 10.1101/2024.12.12.628169

Figure Lengend Snippet: a , Representative image of the VTA from Glp1r-Cre mice injected with AAV- DIO- mGFP-2A-Synaptophysin-mRuby in the CeA; scale bar = 100 µm. CeA GLP1R fibers (red; pseudo-colored mGFP), synaptic terminals (green; pseudo-colored mRuby), and tyrosine hydroxylase-positive neurons (Th; blue, marking dopaminergic neurons) are shown, with a magnified view (right; scale bar = 50 µm). b , Schematic of proposed neural circuit from NTS → CeA → VTA → NAc. Arrows indicate neuron activation, blunted ends indicate neuron inhibition. Gray arrow from NTS → VTA indicates a known connection from NTS GLP-1 to VTA vGAT neurons . c , Schematic of genetically-encoded dopamine sensor, AAV-dLight1.3b, injection and fiber optic implant in the NAc of Glp1r S33W mice. d,g , Averaged Z-score traces showing dopamine release in the NAc in response to HFD following administration of ( d ) vehicle or danuglipron and ( g ) saline or orforglipron in Glp1r S33W mice. Traces are aligned to food retrieval time (t = 0) and averaged across five food trials per mouse. e-i , Quantified ( e,h ) area under the curve (AUC) for Z-scores and ( f,i ) maximum fluorescence Z-scores within the food retrieval window ( n = 9 for danuglipron, n = 7 for orforglipron, paired t-test, * P <0.05). j , Schematic of AAV-dLight1.3b injection and fiber optic implant into the NAc and AAV-DIO-hGLP1R injection into the CeA of Glp1r-Cre mice. k,n , Averaged Z-score traces showing dopamine release in the NAc in response to HFD following administration of ( k ) vehicle or danuglipron and ( n ) saline or orforglipron in CeA-hGLP1R mice. Traces are aligned to food retrieval time (t = 0) and averaged across five food trials per mouse. l- p , Quantification of ( l,o ) AUC for Z-scores and ( m,p ) maximum fluorescence Z-scores within the food retrieval window ( n = 8 for danuglipron, n = 7 for orforglipron, paired t-test, * P <0.05). Data are represented as means ± SEM. * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: Orforglipron powder (MedChemExpress, catalog no. HY-112185) was dissolved to 10mg/mL in Dimethyl sulfoxide (DMSO) and further diluted in 0.9% NaCl sterile saline to 0.1mg/mL.

Techniques: Injection, Activation Assay, Inhibition, Saline, Fluorescence