Liver-specific disruption of PPARgamma in leptin-deficient mice improves fatty liver but aggravates diabetic phenotypes

Abstract

To elucidate the function of PPARgamma in leptin-deficient mouse (ob/ob) liver, a PPARgamma liver-null mouse on an ob/ob background, ob/ob-PPARgamma(fl/fl)AlbCre(+), was produced using a floxed PPARgamma allele, PPARgamma(fl/fl), and Cre recombinase under control of the albumin promoter (AlbCre). The liver of ob/ob-PPARgamma(fl/fl)AlbCre(+) mice had a deletion of exon 2 and a corresponding loss of full-length PPARgamma mRNA and protein. The PPARgamma-deficient liver in ob/ob mice was smaller and had a dramatically decreased triglyceride (TG) content compared with equivalent mice lacking the AlbCre transgene (ob/ob-PPARgamma(fl/fl)AlbCre(-)). Messenger RNA levels of the hepatic lipogenic genes, fatty acid synthase, acetyl-CoA carboxylase, and stearoyl-CoA desaturase-1, were reduced in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice, and the levels of serum TG and FFA in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice were significantly higher than in the control ob/ob-PPARgamma(fl/fl)AlbCre(-) mice. Rosiglitazone treatment exacerbated the fatty liver in ob/ob-PPARgamma(fl/fl)AlbCre(-) mice compared with livers from nonobese Cre(-) mice; there was no effect of rosiglitazone in ob/ob-PPARgamma(fl/fl)AlbCre(+) mice. The deficiency of hepatic PPARgamma further aggravated the severity of diabetes in ob/ob mice due to decreased insulin sensitivity in muscle and fat. These data indicate that hepatic PPARgamma plays a critical role in the regulation of TG content and in the homeostasis of blood glucose and insulin resistance in steatotic diabetic mice.

Figure 1

Gene targeting and conditional deletion of exon 2 of the PPARγ gene. (a) Restriction maps of the wild-type allele, targeting vector, targeted allele, floxed allele, and null allele. The indicated 3′ probe was used to assess recombination events by Southern blot analysis. Open boxes represent exons and are numbered as indicated. PGK neomycin (PGK Neo) and thymidine kinase (TK) are positive and negative selection cassettes, respectively. Restriction sites: B, BamHI; E, EcoRI; S, SacI. (b) Southern blot analysis of BamHI-digested genomic DNA isolated from brain (B), liver (L), colon (C), spleen (S), kidney (K), white adipose (W), and tail (T) in ob/ob-PPARγ(fl/fl)AlbCre⁺ or ob/ob-PPARγ(fl/fl)AlbCre^– mice. Fragments hybridizing with 3′ probe from the wild-type, floxed, and deleted alleles migrate at approximately 14, 10, and 8 kb, respectively. (c) RNase protection analysis of PPARγ mRNA in OB/OB- or ob/ob-PPARγ(fl/fl)AlbCre mouse livers. Total RNA from three separate mouse livers in each genotype were hybridized with riboprobes for β-actin and PPARγ. The products were then separated on a 5.0% polyacrylamide gel. The size of the protected mRNA fragments for PPARγ and β-actin is as follows; wild-type PPARγ, 195 nt; null PPARγ, 165 nt; and β-actin; 250 nt.

Figure 2

Effect of hepatic PPARγ deficiency in ob/ob and OB/OB mice. (a–f) Nontreatment mice. (a) Liver of littermates. Livers of 8-week-old mice were used. (b–e) Histology of livers from OB/OB-PPARγ(fl/fl)AlbCre^– (b) and ob/ob-PPARγ(fl/fl)AlbCre^– (d) or OB/OB-PPARγ(fl/fl)AlbCre⁺ (c) and ob/ob-PPARγ(fl/fl)AlbCre⁺ mice (e). H&E staining was performed for liver sections (original magnification, ×100) from each genotyped mouse. (f) Total cholesterol and TG content in OB/OB-PPARγ(fl/fl)AlbCre^– (n = 5: 2 males and 3 females), OB/OB-PPARγ(fl/fl)AlbCre⁺ (n = 4: 2 males and 2 females), ob/ob-PPARγ(fl/fl)AlbCre^– (n = 6: 3 males and 3 females), and ob/ob-PPARγ(fl/fl)AlbCre⁺ (n = 15: 8 males and 7 females) mice. (g–m) Rosiglitazone-treated mice. (g) Rosiglitazone-treated livers. (h–k) H&E staining of livers from rosiglitazone-treated OB/OB- PPARγ(fl/fl)AlbCre^– (h) and ob/ob-PPARγ(fl/fl)AlbCre^– (j) or OB/OB- PPARγ(fl/fl)AlbCre⁺ (i) and ob/ob-PPARγ(fl/fl)AlbCre⁺ mice (k). (l) Body and liver weight in rosiglitazone-treated and control mice. For control groups: OB/OB-PPARγ(fl/fl)AlbCre^–, n = 4 (2 males and 2 females); OB/OB-PPARγ(fl/fl)AlbCre⁺ (n = 3, males); ob/ob-PPARγ(fl/fl)AlbCre^–, n = 7 (3 males and 4 females); and ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 5 (3 males and 2 females). Rosiglitazone groups: OB/OB-PPARγ(fl/fl)AlbCre^–, n = 5 (2 males and 3 females); OB/OB-PPARγ(fl/fl)AlbCre⁺, n = 4 (3 males and 1 female); ob/ob-PPARγ(fl/fl)AlbCre^–, n = 10 (6 males and 4 females); and ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 9 (5 males and 4 females). (m) Total cholesterol and TG content in rosiglitazone-treated mice. The mouse number for each genotype was described in l. Data are mean ± SE. *P < 0.001, **P < 0.01 compared with Cre^– mice. Rosi, rosiglitazone.

Figure 3

Northern blot analysis to assess the effect of PPARγ deficiency on hepatic gene expression in untreated and rosiglitazone-treated ob/ob mice. Total RNA was isolated from nonfasting male mice and 20 μg was subjected to electrophoresis on a 1.2% agarose gel, transferred to a nylon membrane, and hybridized with the indicated ³²P-labeled cDNA probes. (a) Northern blot of untreated ob/ob mice liver. (b) Northern blots of rosiglitazone-treated ob/ob mice liver. Quantitation of the bands was performed using the PhosphorImager from Molecular Dynamics and are expressed as the fold change, after correction for GAPDH levels, relative to OB/OB-PPARγ(fl/fl)AlbCre^– mice. Values are averages obtained from two animals.

Figure 4

Effect of PPARγ deficiency on the catabolism of serum TG in the ob/ob mouse. Lipoproteins were separated from 60 μl of pooled mouse plasma samples (n = 6 for each genotype) by FPLC. The concentration of TG (a) and cholesterol (b) in each eluted fraction is indicated on the y axis. Inset: immunoblot analysis of apoB, apoE, apoA-I, and apoA-II contained within the VLDL (V), LDL (L), and HDL (H) top fractions from each mouse genotype. (c) Measurement of serum TG after gavage with oil. The clearance rate of exogenous TG was measured in mice fasted for 4 hours as described in Methods. (d and e) Measurement of plasma lipase activities (d) and VLDL export rates (e). In d, for ob/ob-PPARγ(fl/fl)AlbCre^– mice, n = 7; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 10. In e, for ob/ob-PPARγ(fl/fl)AlbCre^–, n = 7; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 6. Each assay was performed as described in Methods. LPL, lipoprotein lipase; HL, hepatic lipase. (f) Northern blotting for hepatic lipase. The Northern blot was performed as described in the legend to Figure 3. All data are mean ± SE.*P < 0.01, Cre^– vs. Cre⁺ mice.

Figure 5

Effect of PPARγ deficiency on glucose homeostasis in the ob/ob mouse. (a) Blood glucose concentrations were measured in 10-week-old mice after no fasting and after 6 hours or 24 hours of fasting. For OB/OB-PPARγ(fl/fl)AlbCre^– mice, n = 15; OB/OB-PPARγ(fl/fl)AlbCre⁺, n = 16; ob/ob-PPARγ(fl/fl)AlbCre^–, n = 16; and ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 11. (b and c) Glucose tolerance test. 10-week-old (b) and 6-week-old (c) mice were injected with glucose (2 mg/g). For 10-week-old mice, OB/OB-PPARγ(fl/fl)AlbCre^– mice, n = 8; OB/OB-PPARγ(fl/fl)AlbCre⁺, n = 9; ob/ob-PPARγ(fl/fl)AlbCre^–, n = 19; and ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 15. For 6-week-old mice, ob/ob-PPARγ(fl/fl)AlbCre^–, n = 9; and ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 8. (d and e) Effect of rosiglitazone on glucose and insulin levels. Both of these measurements were performed using the same samples. For 20-week-old mice, ob/ob-PPARγ(fl/fl)AlbCre^–, n = 13; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 8. For 7-week-old mice, ob/ob-PPARγ(fl/fl)AlbCre^–, n = 11; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 6. (f–i) Hyperinsulinemic-euglycemic clamp. Measurements of (f) whole-body glucose uptake (g) suppression of basal endogenous glucose production (EGP) (h) white adipose glucose uptake, and (i) muscle glucose uptake. Male 5-week-old mice were used in this experiment. For ob/ob-PPARγ(fl/fl)AlbCre^– mice, n = 4; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 6. All data are mean ± SE. *P < 0.05, Cre ^– vs. Cre⁺ mice. (j–o) Effect of rosiglitazone on glucose levels (j and l), glucose tolerance (k), insulin levels (m), FFA (n), and TG (o) in ob/ob mice. The legend to Figure 2l describes the conditions for rosiglitazone treatment in the glucose analysis studies (j). For rosiglitazone-treated mice: ob/ob-PPARγ(fl/fl)AlbCre^–, n = 11; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 4. For control mice: ob/ob-PPARγ(fl/fl)AlbCre^–, n = 10; ob/ob-PPARγ(fl/fl)AlbCre⁺, n = 4. All data are mean ± SE. *P < 0.05, **P < 0.01, ***P < 0.001, rosiglitazone-treated vs. control Cre^– mice. ^†P < 0.05, ^††P < 0.01, ^†††P < 0.001, rosiglitazone-treated vs. control Cre⁺ mice. Rosi, rosiglitazone; Cont, control.