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8, 12, 1183–1189 (2007)
doi:10.1038/sj.embor.7401086
Figures and Tables
Tripeptidyl peptidase II promotes fat formation in a conserved fashion
Renée M McKay, James P McKay, Jae Myoung Suh, Leon Avery & Jonathan M Graff
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Figure 1
Analysis of Caenorhabditis elegans TPPII and cholecystokinin signalling. (A) Alignment of worm (ce) and human (h) TPPII. (B) TPPII double-stranded RNA was injected into N2 (wild-type) worms. F1 progeny were examined with DIC microscopy, which showed that TPPII-RNAi worms were paler, indicating decreased fat stores. The TPPII-RNAi and control worms were stained with Nile Red, a lipid-specific dye the emission intensity of which correlates with fat content, and photographed under fluorescence microscopy. Worms are at the same developmental stage. (C) CKR-1 GFP transgenic worms were examined and the fluorescence pattern indicated that CKR-1 is expressed in a subset of neurons (arrow). (D) Analysis of feeding behaviour (pumps/min) following CKR-1 (R-1), CKR-2 (R-2), CKR-1+CKR-2 (R-1+R-2) and TPPII RNAi in an rrf-3, neural RNAi sensitized, background. The pumping rate of eat-2 mutant worms was measured as a positive control (Cont) for altered feeding behaviour. n=10, three replicates. (E) qPCR analysis of CKR-1 expression in CKR-1-RNAi (left panel) and CKR-2 expression in CKR-2-RNAi (right panel) worms and control (Cont). (F) A TPPIIGFP transgene was expressed in the intestinal fat-storing cells (bracket) and a few head neurons (arrow). Error bars indicate s.d. CKR, cholecystokinin receptor; DIC, differential interference contrast; qPCR, quantitative PCR; RNAi, RNA interference; TPP, tripeptidyl peptidase.
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Figure 2
TPPII expression in mammalian fat. (A) qPCR analysis of TPPII expression in the brain, skeletal muscle and adipose depots of mice. (B) qPCR analysis of TPPII expression in GWAT and MWAT collected from control and ob/ob littermate mice. (C) qPCR analysis of TPPII expression during 3T3-L1 adipogenesis. Error bars indicate s.d. BAT, brown adipose tissue; GWAT, gonadal white adipose tissue; MWAT, mesenteric white adipose tissue; qPCR, quantitative PCR; TPP, tripeptidyl peptidase.
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Figure 3
TPPII is necessary and sufficient for mammalian adipogenesis. (A,B) 3T3-L1 cells expressing control RNAi, TPPII RNAi1 or TPPII RNAi2 were analysed by (A) real-time PCR and (B) western blots for TPPII expression. (C) 3T3-L1 cells expressing control RNAi (Cont), TPPII RNAi1 or TPPII RNAi2 were adipogenically induced and then stained with the fat-specific dye Oil Red O (red) to visualize lipid content. (D) Lipid content of cells in (C) was quantified. (E) Molecular analysis of 3T3-L1 cells in (C,D). C/EBP and PPAR are adipogenic transcription factors; adipsin, aP2 and lipoprotein lipase (LPL) are expressed in differentiated adipocytes. Pref1 and Gata2 are preadipocyte markers. (F) Western blot analysis showing that TPPII virus increases TPPII protein levels in NIH3T3 cells. (G) NIH3T3s infected with GFP or TPPII virus were adipogenically induced. Lipid accumulation was visualized with Oil Red O staining. (H) Alignment of human (h) and mouse (m) TPPII, highlighting the marked structural conservation in the unusual carboxy-terminal extension. Diagram of the TPPII-Asp44Ala (D44A) and TPPII- N catalytic mutants forms of TPPII and the C-terminal (Cterm) and protease domain (PD) constructs. Arrows indicate increased adipogenesis. (I) NIH3T3 cells were infected with viruses expressing GFP, TPPII or two catalytic TPPII mutants, TPPII-Asp44Ala or TPPII-N, and adipogenically induced. Lipid accumulation was assessed with Oil Red O staining. (J) Fat content of cells shown in (I) was quantified. Error bars indicate s.d.; *P<0.05. C/EBP, CCAAT/enhancer-binding protein; GFP, green fluorescent protein; NC, no change; PPAR, peroxisome proliferator-activated receptor; TPP, tripeptidyl peptidase.
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Figure 4
Tpp2 heterozygous mutant mice are lean. (A) Schema of the wild-type (WT) Tpp2 locus and the mutant (Mut) Tpp2 allele. Splice acceptor (SA) and the lacZ-neomycin selectable marker ( -geo) cassette were inserted and disrupt gene expression from the third exon (E3). (B) Western blot showing reduction of TPPII protein in GWAT from Tpp2 heterozygous mouse compared with wild-type control. (C) Genomic DNA was extracted from pups of multiple Tpp2 heterozygous (het) intercrosses and then genotyped for the presence of the wild-type and mutant Tpp2 alleles. The data indicate that Tpp2 homozygous (homo) mutant mice die in utero. (D) Average body weight (left panel) and average body fat percentage (right panel) of 16-week-old Tpp2 heterozygous mice (n=15) and control (Cont) littermates (n=14). (E) Photograph of representative gonadal WAT of control and Tpp2 heterozygous littermates. (F) Average weights of IWAT, GWAT and MWAT from Tpp2 heterozygous mice (n=5) and wild-type littermate controls (n=7). (G) Average weights of liver, heart, spleen and kidney isolated from the mice in (F). (H) Histological sections of control and Tpp2 heterozygote GWAT and IWAT. (I,J) Plasma of control (n=5) and Tpp2 heterozygous (n=5) mice were analysed for insulin (I) and leptin (J) levels. (K) Food intake of wild-type (n=6) and Tpp2 heterozygous mice (n=5) was measured daily for 1 week, averaged and then plotted. Error bars indicate s.d.; *P<0.05; **P<0.01; NS, not significant by t-test. GWAT, gonadal white adipose tissue; IWAT, inguinal white adipose tissue; MWAT, mesenteric white adipose tissue; TPP, tripeptidyl peptidase.
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