1. Volume 6, Issue 1, Pages 77-86 (July 2000)
Decreased IRS-2 and Increased SREBP-1c Lead to Mixed Insulin Resistance and Sensitivity in Livers of Lipodystrophic and ob/ob Mice 
Iichiro Shimomura, Morihiro Matsuda, Robert E. Hammer, Yuriy Bashmakov, Michael S. Brown, Joseph L. Goldstein 
Molecular Cell 
Volume 6, Issue 1, Pages (July 2000)
DOI: /S (05)

2. Figure 1
Changes in mRNA and Protein Levels in Livers of Wild-Type, Transgenic aP2-SREBP-1c436, and ob/ob Mice Treated with Leptin
A microosmotic pump delivering PBS (−) or mouse recombinant leptin (+) was placed in the subcutaneous tissue. On day 12 after pump insertion, mice were killed and livers were removed. Mice in this experiment are the same as those previously used for measurements of leptin, glucose, and insulin in blood (Shimomura et al. 1999b).
(A) Aliquots (10 μg) of pooled total RNA from livers of four mice were subjected to electrophoresis and hybridized with indicated 32P-labeled probe.
(B) Northern blotting was carried out as described in (A). For immunoblots, pooled liver lysates (50 μg) were subjected to 8% SDS-PAGE, blotted onto nitrocellulose, and incubated with 1 μg/ml of rabbit anti-rat insulin receptor β subunit, anti-rat IRS-1, or anti-mouse IRS-2 followed by 0.25 μg/ml horseradish peroxidase–coupled donkey anti-rabbit IgG. Filters were exposed to Reflection NEF496 film for 2 s (insulin receptor) or 1 s (IRS-1 and IRS-2).
WT, wild type; Tg, transgenic aP2-SREBP-1c436 mice.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

3. Figure 2
Immunoblot Analysis of IRS-1, IRS-2, Akt, and Phospho-Akt in Livers of Wild-Type and Transgenic aP2-SREBP-1c436 Mice
Wild-type female mice and transgenic female littermates (4 months old) were used. PBS (0.2 ml) with or without 5 U of regular human insulin (Eli Lilly) was injected into the portal vein. The liver was excised 1 min after injection and extracted. Pooled liver lysates (50 μg) from four animals were subjected to 8% SDS-PAGE, transferred to nitrocellulose, and incubated with 1 μg/ml of rabbit anti-rat IRS-1, anti-mouse IRS-2, anti-mouse Akt, or anti-mouse phospho-Akt(Ser 473) followed by 0.25 μg/ml horseradish peroxidase–coupled donkey anti-rabbit IgG. Filters were exposed to film for 1 s (IRS-1 and IRS-2), 1 min (Akt), or 3 min (Phospho-Akt).
WT, wild type; Tg, transgenic aP2-SREBP-1c436 mice.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

4. Figure 3
Changes in Hepatic mRNAs in Mice that Were Fasted or Refed and in Streptozotocin-Treated Diabetic Rats Treated with or without Insulin
(A) C57BL/6J male mice 12 weeks of age were divided into three groups. Five mice were maintained on chow diet, five were fasted for 24 hr, and five were fasted for 24 hr and then refed the chow diet for 6 hr prior to killing. Pooled RNA (10 μg) from five mice was subjected to blot hybridization with indicated 32P-labeled cDNA probe. Plasma glucose levels (mean ± SEM) were 180 ± 13, 105 ± 6, and 176 ± 8 mg/dl for nonfasted, fasted, and refed groups, respectively. Insulin levels were 0.72 ± 0.15, 0.10 ± 0.02, and 2.0 ± 0.49 ng/ml, respectively. Leptin levels were 3.9 ± 0.2, 2.1 ± 0.3, and 4.7 ± 1.4 ng/ml, respectively.
(B) Rats were fasted for 24 hr, after which diabetes was induced by an intravenous injection of streptozotocin (STZ; 65 mg/kg body weight) in 50 mM sodium citrate (pH 4.5) (Shimomura et al. 1999c). Control rats were fasted and injected with 50 mM sodium citrate solution (pH 4.5). After injection, animals were fasted for a further 24 hr, after which plasma glucose levels were checked, and diabetes was confirmed (glucose > 250 mg/ml). Animals were then fed a chow diet for 12 hr, after which insulin (INS) was administered to the STZ + INS group. Insulin-treated animals received a combination of human regular insulin (3 U; Eli Lilly) intraperitoneally and human NPH insulin (4 U; Eli Lilly) subcutaneously, each given in 0.2 ml of PBS. Rats in control and STZ groups received 0.2 ml PBS intraperitoneally and subcutaneously. The animals were fasted for 6 hr and then anesthetized with halothane. Total RNA was isolated from livers and pooled, after which 10 μg aliquots were subjected to blot hybridization with indicated 32P-labeled cDNA probe. Plasma glucose levels were 165 ± 19, 505 ± 53, and 97 ± 5 mg/dl for control, STZ, and STZ + INS groups, respectively. Plasma leptin levels were 1.6 ± 0.6, 0.35 ± 0.10, and 8.8 ± 2.4 ng/ml, respectively.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

5. Figure 4
Effects of Insulin and Glucagon on Expression of Insulin Receptor, IRS-1, and IRS-2 mRNAs in Cultured Rat Hepatocytes
Hepatocytes were isolated from one fed rat as described in Experimental Procedures. Aliquots of 6 × 106 hepatocytes were plated in collagen I–coated dishes (100 mm diameter) in Hepatocyte Attachment Medium supplemented with 5% fetal calf serum and 100 nM triiodothyronine. After 4 hr at 37°C, the medium was replaced with medium 199 containing Earle's salts and 5 mM glucose supplemented with 1 nM insulin, 100 nM dexamethasone, and 100 nM triiodothyronine but no serum. The cells were then washed, and the experiment was initiated with addition of insulin and/or glucagon in medium 199.
(A) Time course of insulin action. After pretreatment for 16 hr at 37°C, cells were incubated in the absence or presence of 100 nM insulin for indicated time. For comparison, the lane corresponding to the 0 time point was photographed twice, and both photographs are shown (denoted by asterisks).
(B) Effect of insulin concentration. After pretreatment, cells were incubated for 12 hr with indicated concentration of insulin.
(C) Effect of glucagon on insulin action. After pretreatment, cells were incubated for 12 hr in absence or presence of 100 nM insulin with indicated concentration of glucagon.
(A–C) The cells were homogenized, and aliquots of total RNA (10 μg) were subjected to blot hybridization as described in Figure 1.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

6. Figure 5
Effects of Insulin and Glucagon on Expression of SREBP-1a and SREBP-1c mRNAs in Cultured Rat Hepatocytes as Measured by RNase Protection
Total RNA (10 μg) isolated from hepatocytes in Figure 4 was subjected to RNase protection assay. The data were normalized relative to the 18S RNA signal and plotted as fold change relative to the SREBP-1a mRNA level in the first lane of each blot.
(A) Time course of insulin action. For comparison, the lane corresponding to the 0 time point was photographed twice, and both photographs are shown (denoted by asterisks).
(B) Effect of insulin concentration.
(C) Effect of glucagon on insulin action.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

7. Figure 6
Effect of Insulin and Glucagon on Expression of Insulin Receptor, IRS-1, and IRS-2 Proteins in Cultured Rat Hepatocytes
Hepatocytes were isolated and pretreated as described in Figure 5. (A) Cells were incubated for 12 hr with the indicated concentration of insulin.
(B) Cells were incubated in the absence or presence of 100 nM insulin for the indicated time.
(C) Cells were incubated for 12 hr in the absence or presence of 100 nM insulin with indicated concentration of glucagon. After treatment, cells were lysed and lysates (50 μg) were subjected to 8% SDS-PAGE and blotted with antibodies as described in Figure 1B. Filters were exposed to film for 2 s (insulin receptor) or 1 s (IRS-1 and IRS-2).
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

8. Figure 7
Acute Effects of Insulin on Tyrosine Phosphorylation of IRS-1 and IRS-2 and on IRS-Associated PI 3-Kinase Activity in Cultured Rat Hepatocytes
Hepatocytes were pretreated as described in Figure 4. After the standard 16 hr pretreatment, cells were preincubated for 24 hr without or with 100 nM insulin as indicated, after which cells were treated acutely for 10 min with fresh medium with or without 100 nM insulin. The cells were then harvested and lysates prepared. Lysates (1 mg) were subjected to immunoprecipitation with antibodies against IRS-1 or IRS-2.
(A) One half of each immunoprecipitate was used for immunoblotting with anti-phosphotyrosine antibody. Filters were exposed to film at room temperature for 2 min.
(B) The other half of each immunoprecipitate was used to measure PI 3-kinase activity. Arbitrary units of activity are plotted as the fold change relative to the value in hepatocytes that received no insulin (first lane in each group). Inset shows a photograph of the thin layer chromatogram (exposed to film at room temperature for 12 hr) that was quantified.
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

9. Figure 8
Acute Effects of Insulin on Insulin Receptor, IRS-1, IRS-2, Akt, and Phospho-Akt Proteins in Cultured Rat Hepatocytes
Hepatocytes were pretreated as described in Figure 4. After the standard 16 hr pretreatment, the cells were preincubated for 24 hr without (lanes A and C–G) or with (lanes B and H–L) 100 nM insulin. The medium was removed, cells were washed with PBS, and dishes received fresh medium containing the indicated concentration of insulin. After incubation for zero time (lanes A and B) or 5 min (lanes C–L), cells were harvested, lysates were prepared, and lysates (50 μg) were subjected to 8% SDS-PAGE and blotted with antibodies as described in Figure 1B. Filters were exposed to film at room temperature for 1 s (IRS-1 and IRS-2), 1 min (Akt), or 3 min (Phospho-Akt).
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )

10. Figure 9
Model Illustrating How Hyperinsulinemia Secondary to Leptin Deficiency Creates a Vicious Cycle of Insulin Resistance and Sensitivity Coexisting in Livers of Lipodystrophic and ob/ob Mice
Molecular Cell 2000 6, 77-86DOI: ( /S (05) )