Journal: Metabolites

Article Title: Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice

doi: 10.3390/metabo11060387

Figure Lengend Snippet: In vitro characterization of leptin-expressing islet organoids. ( a ) Schematic illustration of vAd-RIP-leptin-OFF. The rat insulin-1 promoter (rIns-1) drives expression of the synthetic transcription factor tTA and the green fluorescent protein ZsGreen in pancreatic beta-cells. The TRE-tight promoter drives expression of mouse leptin (mLeptin) and the red fluorescent protein mCherry. The two expression cassettes are separated by a transcription blocker sequence (TB). Binding of tTA to the TRE-tight promoter induces in the absence of doxycycline the expression of leptin and mCherry, while addition of doxycycline turns-off the expression of the two proteins. IRES-elements in the cassettes ensures stoichiometric expression of the two proteins under the same promoter. ( b ) Representative maximum projection of a 3D-stack of leptin-expressing islet organoids, obtained by confocal imaging, cultured for 4 weeks in vitro (1st set of experiments), showing reflection, expression of ZsGreen, mCherry and their overlay. Scale bar: 50 µm. ( c ) Representative maximum projection of a 3D-stack of leptin-expressing islet organoids, obtained by confocal imaging, cultured for 4 weeks in vitro (2nd set of experiments), showing reflection, expression of ZsGreen, mCherry and their overlay. Scale bar: 50 µm.

Article Snippet: The mouse leptin cDNA was obtained from pCMV6.mouse leptin (#MC208876, OriGene, Rockville, MD, USA) and subcloned into pTRE-tight (Clontech, Takara, Mountain View, CA, USA) to create pTRE-tight.mLeptin.

Techniques: In Vitro, Expressing, Sequencing, Binding Assay, Imaging, Cell Culture

Journal: Metabolites

Article Title: Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice

doi: 10.3390/metabo11060387

Figure Lengend Snippet: In vivo characterization of leptin-expressing islet organoids from the first set of experiments. ( a ) Photograph of the eye containing the leptin-expressing islet organoid graft (red arrow) 9 weeks after transplantation. ( b ) Maximum projection of a 3D-stack of a leptin-expressing islet organoid graft (red arrow) obtained by confocal imaging 9 weeks after transplantation; ( ba ) reflection, ( bb ) mCherry fluorescence, ( bc ) ZsGreen fluorescence, ( bd ) overlay image; scale bar 500 µm. ( c ) Bodyweight of transplanted and control mice over the entire period of the experiment. ( d ) Area under the curve (AUC) for ipGTT of transplanted and control mice over the entire period of the experiment. ( e ) Fasting blood glucose of transplanted and control mice over the entire period of the experiment. ( f ) Plasma insulin levels of transplanted and control mice at the end of the experiment. ( g ) Plasma C-peptide levels of transplanted and control mice at the end of the experiment. ( d , e ) * p < 0.05. ( c – g ) n = 3.

Article Snippet: The mouse leptin cDNA was obtained from pCMV6.mouse leptin (#MC208876, OriGene, Rockville, MD, USA) and subcloned into pTRE-tight (Clontech, Takara, Mountain View, CA, USA) to create pTRE-tight.mLeptin.

Techniques: In Vivo, Expressing, Transplantation Assay, Imaging, Fluorescence, Control, Clinical Proteomics

Journal: Metabolites

Article Title: Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice

doi: 10.3390/metabo11060387

Figure Lengend Snippet: Additional information related to . ( a ) Timeline of the second set of experiments. ( b ) Individual traces from the ipGTTs summarized as AUC in d. Data are expressed as mean ± SEM. Data points that include saturated glucose measurements are labeled with #. * p < 0.05; ** p < 0.01; *** p < 0.001; n = 7 for transplanted and 5 for control group. ( c ) Plasma leptin levels of transplanted mice over the period of the experiment, n = 3–7.

Article Snippet: The mouse leptin cDNA was obtained from pCMV6.mouse leptin (#MC208876, OriGene, Rockville, MD, USA) and subcloned into pTRE-tight (Clontech, Takara, Mountain View, CA, USA) to create pTRE-tight.mLeptin.

Techniques: Labeling, Control, Clinical Proteomics

Journal: Metabolites

Article Title: Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice

doi: 10.3390/metabo11060387

Figure Lengend Snippet: In vivo characterization of leptin-expressing islet organoids from the second set of experiments. ( a ) Photograph of the eye containing the leptin-expressing islet organoid graft (red arrow) 6 weeks after transplantation. ( b ) Maximum projection of a 3D-stack of a leptin-expressing islet organoid graft (red arrow) obtained by confocal imaging 12 weeks after transplantation; ( ba ) reflection, ( bb ) mCherry fluorescence, ( bc ) ZsGreen fluorescence, ( bd ) overlay image; scale bar 300 µm. ( c ) Bodyweight, ( d ) Area under the curve (AUC) for ipGTT, ( e ) Fasting blood glucose, ( f ) Plasma insulin levels and ( g ) Plasma C-peptide levels of transplanted and control mice over the period of the experiment. ( c – g ) * p < 0.05, ** p < 0.01, *** p < 0.001; n = 7 for transplanted and 5 for control group.

Article Snippet: The mouse leptin cDNA was obtained from pCMV6.mouse leptin (#MC208876, OriGene, Rockville, MD, USA) and subcloned into pTRE-tight (Clontech, Takara, Mountain View, CA, USA) to create pTRE-tight.mLeptin.

Techniques: In Vivo, Expressing, Transplantation Assay, Imaging, Fluorescence, Clinical Proteomics, Control

Journal: Metabolites

Article Title: Ectopic Leptin Production by Intraocular Pancreatic Islet Organoids Ameliorates the Metabolic Phenotype of ob/ob Mice

doi: 10.3390/metabo11060387

Figure Lengend Snippet: Characterization of leptin-expressing islet organoid grafts from animals treated with doxycycline, removed at the end of the experiment. ( a ) Fluorescent microscopy image of a leptin-expressing islet organoid graft from transplanted non-doxycycline-treated animal; ( aa ) DAPI staining; ( ab ) ZsGreen fluorescence; ( ac ) mCherry fluorescence; ( ad ) overlay. ( b ) Fluorescent microscopy image of a leptin-expressing islet organoid graft from transplanted doxycycline-treated animal; ( ba ) DAPI staining, ( bb ) ZsGreen fluorescence, ( bc ) mCherry fluorescence, ( bd ) overlay. ( c ) Immunohistochemistry image of a leptin-expressing islet organoid graft from transplanted non-doxycycline treated animal; ( ca ) DAPI staining, ( cb ) C-peptide staining, ( cc ) leptin staining, ( cd ) overlay. ( d ) Immunohistochemistry image of a leptin-expressing islet organoid graft from transplanted doxycycline treated animal; ( da ) DAPI staining, ( db ) C-peptide staining, ( dc ) leptin staining, ( dd ) overlay. ( a – d ) All scale bars 50 µm.

Article Snippet: The mouse leptin cDNA was obtained from pCMV6.mouse leptin (#MC208876, OriGene, Rockville, MD, USA) and subcloned into pTRE-tight (Clontech, Takara, Mountain View, CA, USA) to create pTRE-tight.mLeptin.

Techniques: Expressing, Microscopy, Staining, Fluorescence, Immunohistochemistry