DHHC7 knockout in liver promotes obesity development and increases the plasma cholesterol under high fat diet feeding.

A. Real-time PCR analysis the expression of DHHC3 and DHHC7 in liver from CON and D7LKO mice to show that DHHC7 expression is diminished in D7LKO mouse liver. B. Western blot of total liver extracts from D7LKO and CON mice with anti-DHHC7 (i), anti-Hsp90 (ii), respectively. Panel iii shows the expression DHHC7 in mouse heart. C Densitometric analysis of protein expression in B, the level of indicated protein was set to 1 in control. D. The growth curve of D7LKO mice and control mice. The mice (group of 6) were feed with High fat diet as indicated time and body weight were measured each week. E. Food intake of Control and D7LKO mice. F. The oxygen consumption of Control and D7LKO mice. G, H and I. The fat mass of Brown, Inguinal and epididymal fat of control and D7LKO mice (panel i); The right of panel i represents quantitative analysis of left panel i; The panel ii showed the H&E staining of each individual fat to show the increased adipocyte size in D7LKO mice. Scal bar is 25μM. J and K. The plasma glucose and triglycerides of Control and D7LKO mice. L, M and N. The plasma total cholesterol, high density lipoprotein and low density lipoprotein cholesterol of Control and D7LKO mice. In all plotting panels, the bar graphs represent Means+ S.D., n=3-6. ** p<0.01. ***p<0.001.

Inactivation of DHHC7 in the liver promotes the Prg4 expression and Overexpression DHHC7 in liver promotes the development of obesity under HFD feeeding.

A. The Volcan plot of liver tissue proteomics of HFD male mice. The position of Prg4 is indicated by an arrow. B. Western blot of total liver extracts from Control and D7LKO mouse liver with anti-Prg4 (i), DHHC7 (ii) and HSP90(iii), respectively. The Right: Densitometric analysis of protein expression in the left, the level of indicated protein was set to 1 in control. C. Western blot of total liver extracts from the mice, which were administrated with empty and Prg4 adenoviral recombinant vector, with anti-Prg4 and anti-GFP antibodies, respectively. Right: Densitometric analysis of Prg4 expression in left, the level of indicated protein was set to 1 in GFP. The levels of DHHC7 were normalized to that of GFP encoding internally from adenoviral vectors. D. The growth curve of the mice (group of 6) administrated with either empty recombinant adenoviral vector or that encoding Prg4 under HFD feeding. The insert: the image of mice administrated with either empty recombinant adenoviral vector (GFP) or that encoding Prg4 (Prg4) under HFD feeding. E. F and G. The mass of Brown, inguinal and epididymal fat of the mice administrated with either empty recombinant adenoviral vector (GFP) or that encoding Prg4 (Prg4) after 12-week HFD feeding. H and I. The plasma level of triglycerides and total cholesterols in the mice administrated with either empty recombinant adenoviral vector (GFP) or that encoding Prg4 (Prg4) at 12 week HFD. Feeding. In all the plotting panels, the bar graphs represent Means+ S.D., n=3-6. ** p<0.01. ***p<0.001.

Inactivation of DHHC7 in the liver enhances PKA-CREB pathway.

Western blot of total liver extract from either control or D7LKO mice with anti-phospho-PKA substrate (i), DHHC7 (ii) and HSP90 (iii) antibodies, respective. The Molecular Weight is marked at left in panel i. B. Western blot of total liver extract from either control or D7LKO mice with anti-phospho-CREB at Ser133 (i), CREB (ii), Prg4 (iii), DHHC7 (iv) and HSP90 (v) antibodies, respectively. The right: the densitometric analysis of the expression of each protein in right. The level of each protein in control was set as 1. C. TPC assay of Gαi in the liver protein from either control (con) or D7LKO. Panel i. palmitoylated. Panel ii, iii and iv show the level of total Gαi、 DHHC7 and HSP90 in total liver extracts. Left: Densitometric analysis of relative levels of palmitoylated Gai and DHHC7 in that the total level for each protein in control mouse liver was set as 1. In all the plotting panels, the bar graphs represent Means+ S.D., n=3. * p<0.05. ** p<0.01. ***p<0.001.

Prg4 suppresses adipocyte PKA signaling and HSL phosphorylation

A. 1) Western blot of total brown fat tissue lysate from control or D7LKO mice with anti-phospho-HSL at Ser563 (i), HSL (ii), Prg4 (iiii) and FASN antibodies, respectively. 2). Densitometric analysis of the level of indicated proteins in 1). The levels of each protein in control mice were set as 1. B and C. The same as A except Inguinal and Epididymal fat, respectively. D. Western blot of total brown fat extracts from control or D7LKO mice with anti-phospho-PKA substrate antibodies to show that inactivation of DHHC7 in the liver inhibited PKA substrate phosphorylation in adipocytes. E. The treatment of adipocytes with Prg4 suppresses HSL phosphorylation at 563. The differentiated Brown, Biege and 3T3-L1 adipocytes were incubated with the culture medium prepared from 293T cells expressing with or without Prg4 (see Material and Methods). 4 hours later, the total cell lysates were prepared for Western blot with anti-phospho-HSL at Ser563 (i). HSL (ii) and Prg4 (iii), respectively. 2) Densitometric analysis of indicated protein in 1). The level of indicated protein was set to 1 in control cells. In all plotting the panels, the bar graphs represent Means+ S.D., n=3. * p<0.05. ** p<0.01. ***p<0.001.

Prg4 interacts with GPR146 and this interaction is required for Prg4 to suppress HSL phosphorylation at S563.

A. Co-immunoprecipitation assay of Flag tagged GPR146 and Prg4 co-expressed in HEK293T cells with anti-M2(Flag) affinity gel. Anti-M2 immunoprecipitates were probed with anti-Prg4 antibodies (i). Panel ii and iii showed the input levels of Prg4 and Flag-GPR146, respectively. B. Schematic representation of Prg4 structure and its two mutants. The two major domains of Prg4: SMB and HEM were indicated. C. Co-immunoprecipitation assay of the interaction between GPR146 with different forms of Prg4 to show that SMB domain of Prg4 mediates the interaction between Prg4 and GPR146. The experiments were performed essentially as A, except two Prg4 mutants were also used. ΔSMBPrg4: SMB domain was deleted. ΔHEMPrg4: HEM domain was deleted. D. The interaction between Prg4 and GPR146 is required for Prg4 to suppress HSL phosphorylation at Ser563. The experiments were carried out similar to Fig. 4E except ΔSMBPrg4 and ΔHEMPrg4 were also used. E The work model of Prg4 in liver and adipose tissue. In plotting panels, the bar graphs represent Means+ S.D., n=3. * p<0.05. ** p<0.010. ***p<0.001.