PMC

Reconstitution of the IRS-1 KO and IRS-1-IRS-3 double-KO cells with various IRS proteins or IRS-1-IRS-2 chimeras. IRS-1 KO cells (A) or IRS-1-IRS-3 double-KO cells (B) were reconstituted with (+) IRS-1, IRS-2, IRS-3, or IRS-4 as described in Materials and Methods. Differentiation was induced as described in the legend to Fig. . On day 6, the cells were fixed and stained with oil red O. (C) Schematic diagram showing the structures of IRS-1-IRS-2 chimeras. (D) On day 6 of differentiation, wild-type and IRS-1 KO cells and IRS-1 KO cells reconstituted with IRS-1, IRS-2, N1.C2, and N2.C1 were fixed and stained with oil red O.

Expression of total proteins and phosphoproteins of p42/44 MAPK (A) and p38 MAPK (B). Wild-type, IRS-1 KO, IRS-2 KO, IRS-3 KO, and IRS-4 KO cells and IRS-1 KO cells reconstituted with (+) IRS-1, IRS-4, or the N1.C2 chimera were differentiated, and protein lysates were prepared on days 0, 2, and 6 of differentiation. The lysates were separated by sodium dodecyl sulfate-0% polyacrylamide gel electrophoresis and analyzed by Western blotting using antibodies against p44/42 MAPK and p38 MAPK (left) or phospho-p44/42 MAPK and phospho-p38 MAPK (right).

Overall tyrosine phosphorylation of proteins decreases during differentiation. Wild-type, IRS-1 KO, IRS-2 KO, IRS-3 KO, and IRS-4 KO cells and IRS-1 KO cells reconstituted with (+) IRS-1, IRS-4, or the N1.C2 chimera were differentiated, and protein lysates were prepared on days 0, 2, and 6 of differentiation. The lysates were separated by sodium dodecyl sulfate-6% polyacrylamide gel electrophoresis and analyzed by Western blotting using antiphosphotyrosine antibody (4G10). Representative blots are shown. Numbers on the left are protein molecular weight markers.

Protein expression of adipocyte differentiation markers in wild-type, IRS-1 KO, IRS-2 KO, IRS-3 KO, and IRS-4 KO cells on days 0, 2, and 6 of differentiation. Protein lysates were prepared from the cells and analyzed for the adipogenic markers PPARγ, C/EBPα, FAS, GLUT 4, STAT 5, and UCP-1 by Western blot analysis as described in Materials and Methods. The results are representative of at least two independent experiments.

Protein expression of adipocyte differentiation markers in wild-type and IRS-1 KO cells and IRS-1 KO cells reconstituted with (+) IRS-1, IRS-4, or the N1.C2 chimera on days 0, 2, and 6 of differentiation. Protein lysates were prepared from the cells and analyzed for the adipogenic markers PPARγ, C/EBPα, FAS, GLUT 4, STAT 5, and UCP-1 by Western blot analysis as described in Materials and Methods. The results are representative of at least two independent experiments.

IRS-1 and IRS-3 play important roles in brown adipocyte differentiation. (A) Differentiation of wild-type and all four IRS KO brown adipocytes. (B) Comparison of differentiation of wild-type, IRS-1 KO, IRS-3 KO, and IRS-1-IRS-3 double-KO adipocytes. Brown adipose precursor cells isolated from newborn wild-type and different IRS KO mice were grown to confluence. Differentiation was induced as described in Materials and Methods. On day 6, the cells were fixed and stained with oil red O. The results are representative of at least four independent experiments.

Expression of Wnt 10a mRNA in wild-type and different IRS KO cells (A) and IRS-1 KO cells reconstituted with (+) various IRS proteins (B) at the preadipocyte stage. Quantitative RT-PCR analysis using the SYBR Green protocol was used to measure the expression levels of Wnt 10a in various cell lines as described in Materials and Methods. The data are from three independent experiments and are expressed as the mean plus standard error of the mean. Significance is determined relative to wild-type (A) or IRS-1 KO (B) cells by Student's t test. **, P < 0.01; ***, P < 0.0001.

mRNA expression of adipocyte differentiation markers in wild-type and IRS-1 KO cells and IRS-1 KO cells reconstituted with (+) IRS-1, IRS-4, or the N1.C2 chimera on days 0, 2, and 6 of differentiation. Total RNA was isolated from the cells, and 20 μg was subjected to Northern analysis with specific probes for the adipogenic markers PPARγ, C/EBPα, C/EBPδ, and FAS. The data are presented as arbitrary units after normalization to day 2 wild-type levels for each experiment. The results are means of three independent experiments. The average standard error of the mean was 21.4% at all data points.

mRNA expression of adipocyte differentiation markers in wild-type, IRS-1 KO, IRS-2 KO, IRS-3 KO, and IRS-4 KO cells on days 0, 2, and 6 of differentiation. Total RNA was isolated from the cells, and 20 μg of RNA was subjected to Northern analysis with specific probes for the adipogenic markers PPARγ, C/EBPα, C/EBPδ, and FAS. Expression of PGC-1 was measured by quantitative RT-PCR analysis using the SYBR Green protocol as described in Materials and Methods. The data are presented as arbitrary units after normalization to day 2 wild-type levels for each experiment. The results are means of three independent experiments. The average standard error of the mean was 20.3% at all data points.

Expression of IRS mRNAs and proteins in wild-type brown adipocytes during differentiation. (A) Quantitative real-time PCR by the ABI Taqman procedure was used to measure the transcript numbers for each IRS in total RNA extracted from wild-type cells on days 0, 3, and 6 of differentiation. Details of RNA preparation, primers, probes, and the method of quantification are described in Materials and Methods. The data are presented as relative numbers of transcripts per cell using calibration curves prepared for each IRS. (B) Protein expression of IRS-1 and IRS-2 in wild-type brown adipocyte differentiation. Specific antibodies against IRS-1 or IRS-2 were used in Western blot analysis as described in Materials and Methods. (C) Blow-up graph of panel A to better illustrate the expression levels of IRS-3 and IRS-4 mRNAs during differentiation. The results are means from two to four experiments.