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Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

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Science Signaling  21 Jul 2015:
Vol. 8, Issue 386, pp. ra73
DOI: 10.1126/scisignal.aaa4374
  • Fig. 1 Tamoxifen-induced GRK2 ablation prevents diet-induced obesity and reverses HFD-induced glucose intolerance.

    Mice were fed HFD for 8 weeks, injected with tamoxifen, and maintained for five more weeks on the HFD. (A) Body weight evolution (expressed in grams) along the 13 weeks of high-fat feeding. (B) Daily food intake: no significant differences were found between any pair of conditions. (C and D) Intraperitoneal GTTs were performed either at 8 weeks of HFD (before tamoxifen administration, dotted lines) or 5 weeks after tamoxifen injection, after a total of 13 weeks of HFD (solid lines) in control (Cre−/−GRK2fl/fl) mice (C, black) and in Cre+/−GRK2fl/fl mice (D, red). (E) Histogram showing the GTT area under the curve (AUC) of data in (C) and (D). a.u., arbitrary units. (F and G) Insulin tolerance tests (ITTs) were performed in mice described in (C) and (D), before and after tamoxifen treatment. (H) Histogram showing the ITT area under the curve of data in (F) and (G). (I and J) Serum concentrations of fasting glucose (I) and insulin (J) from HFD-fed control (Cre−/−GRK2fl/fl) and Tam-GRK2−/− mice. Results in (A) to (J) are means ± SEM of seven to eight mice per group. Statistical analysis was performed by two-way repeated-measures analysis of variance (ANOVA) followed by Bonferroni’s post hoc test (A to H) or by unpaired two-tailed t test (I and J). *P < 0.05; **P < 0.01; ***P < 0.005. Open symbols, dotted lines: Pre-Tam; filled symbols, solid lines: Post-Tam; black: Cre−/−GRK2fl/fl; red: Cre+/−GRK2fl/fl.

  • Fig. 2 GRK2 loss during a HFD enhances insulin signaling in insulin-sensitive tissues.

    (A to C) After the 13 weeks of HFD, control and Tam-GRK2−/− mice were treated with either vehicle or insulin (Ins), and tissue lysates from muscle (A), eWAT (B), and liver (C) were subjected to Western blot and probed with antibodies against GRK2, total and phosphorylated AKT (P-AKT) (Ser473), and GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Representative immunoblots and densitometric analysis of four mice per group are shown. Results are expressed as percentage of stimulation over vehicle-treated mice (basal). Results are means ± SEM of seven to eight mice per genotype, three to four mice per treatment. Statistical significance was analyzed by unpaired two-tailed t test (A and C) or by Mann-Whitney test (B). *P < 0.05.

  • Fig. 3 Tamoxifen-induced GRK2 depletion reduces epididymal adipose mass and adipocyte size and increases markers of lipolysis.

    (A) eWAT weight in control (Tam-GRK2+/+) and Tam-GRK2−/− mice at the end of the 13-week HFD. (B) Sections of epididymal fat pads from control and Tam-GRK2−/− mice stained with hematoxylin and eosin (magnification, ×10; scale bars, 50 μm). Representative photomicrographs are shown. Relative adipocyte size was calculated by determining individual adipocyte areas using image analysis software (ImageJ) in at least four different randomly chosen fields per mouse, in five mice per genotype. (C) eWAT lysates were immunoblotted for PPARγ, C/EBPα, and FAS using GAPDH as a loading control. Representative immunoblots and densitometric analysis are shown. n.s., not significant. (D) eWAT lysates were immunoblotted for HSL and subjected to real-time quantitative polymerase chain reaction (qPCR) to measure the expression of HSL (lipe). qPCR results were normalized against ppia and b2m mRNAs. Data are represented as means ± SEM of seven to eight mice per genotype for (B) and (C), and six mice per genotype for (D). Statistical significance was analyzed by unpaired two-tailed t test. *P < 0.05; **P < 0.01; ***P < 0.005. (E) Non-esterified fatty acid (NEFA) release from eWAT explants isolated from control or Tam-GRK2−/− mice (n = 4 to 6 per genotype) was measured in supernatants at the indicated times of isoproterenol stimulation. (F) Circulating NEFA serum concentrations in 13-week HFD-fed controls and Tam-GRK2−/− mice either fed or fasted for 16 hours. Data are represented as means ± SEM of four to five mice per group. Statistical significance was analyzed by two-way ANOVA followed by Bonferroni post hoc test (E) and by Kruskal-Wallis followed by Dunn’s multiple comparison test (F). *P < 0.05; **P < 0.01.

  • Fig. 4 Decreasing GRK2 abundance during a HFD prevents fat accumulation within BAT and augments the expression of fatty acid oxidation and thermogenic markers.

    (A) Sections of BAT from control (Tam-GRK2+/+) and Tam-GRK2−/− mice after 13 weeks of HFD were stained with hematoxylin and eosin (scale bars, 50 μm). Representative photomicrographs from five mice per genotype are shown. (B) BAT lysates were immunoblotted for GRK2, CPT1, UCP1, and GAPDH. Representative immunoblots and densitometric analysis are shown. (C) qPCR was used to measure the expression of HSL (lipe), CPT1 (cpt1b), UCP1 (ucp1), PGC1α (ppargc1a), and Dio2 (dio2) mRNAs. Results were normalized against ppia and b2m mRNAs. Data are means ± SEM of seven to eight mice per genotype for (B) and six mice per genotype for (C). Statistical significance was analyzed by unpaired two-tailed t test. *P < 0.05. (D) NEFA release from interscapular BAT explants isolated from control or Tam-GRK2−/− mice (n = 4 to 6 mice per genotype) was measured in supernatants at the indicated times after isoproterenol stimulation. Statistical significance was analyzed by two-way ANOVA followed by Bonferroni post hoc test. **P < 0.01. (E) Intraperitoneal GTTs were performed in mice treated or not with propranolol before the GTTs. Histogram shows the GTT area under the curve (AUC). Results are means ± SEM of four to six mice per group. Statistical analysis was performed by two-way repeated-measures ANOVA followed by Bonferroni’s post hoc test. *P < 0.05. Open symbols, dotted lines: propranolol-treated; filled symbols, solid lines: no treatment; black: Tam-GRK2+/+; red: Tam-GRK2−/−.

  • Fig. 5 Tamoxifen-induced GRK2 loss protects against the development of NAFLD and associated inflammation in the liver.

    (A) Liver sections from HFD-fed control (Cre−/−GRK2fl/fl) and Tam-GRK2−/− mice were stained with hematoxylin and eosin (scale bars, 50 μm). Images are representative of four to five mice per genotype. (B) Liver weight in control and Tam-GRK2−/− mice. (C) Representative immunoblots and densitometric analysis are shown for FAS, PPARγ, and GAPDH. (D) qPCR was used to measure the expression of mRNAs encoding PPARγ (pparg), FAS (fasn), and CPT1 (cpt1a). Results were normalized against ywhaz and gapdh mRNAs. Data in (B), (C), and (E) are means ± SEM of seven to eight mice per genotype. (E and F) Immunohistochemical analysis of liver sections stained with F4/80 antibody and counterstained with hematoxylin. Representative photomicrographs are shown, and characteristic macrophage arrangements known as hepatic crown-like structures (hCLS) are indicated by arrows. Scale bars, 50 μm (E). Positively stained area was quantified using ImageJ software in at least four different randomly chosen fields per mouse, in four to five mice per genotype (F). (G and H) qPCR was used to measure the hepatic expression of the F4/80-encoding mRNA (G) and the ratio between mRNAs encoding mannose receptor C type 1 (mrc1) and iNOS (nos2) as M2 and M1 markers, respectively (H). Results were normalized against ywhaz and gapdh mRNAs. Values are represented as fold change over control mice and are means ± SEM of six mice per genotype. Statistical significance was analyzed by unpaired two-tailed t test. *P < 0.05.

  • Fig. 6 GRK2 depletion during the course of a HFD can reverse an already established obese and insulin-resistant phenotype, potentially through its combined effects in different tissues.

    Besides its role as a GPCR kinase, GRK2 inhibits insulin signaling and may also control the GPCR-mediated transmodulation of the insulin pathway. A reduction in GRK2 abundance after obesity and insulin resistance have been established leads to the following: (i) an increased adrenergic-dependent lipolytic capacity in eWAT that provides BAT with FFAs (in turn, BAT displays an enhanced expression of oxidative and thermogenic markers, and consequently, both tissues may contribute to the concerted protection against excessive fat mass accretion); (ii) improved insulin signalling in muscle, which can underlie the enhanced insulin-induced glucose clearance; (iii) increased insulin signalling in liver associated with a decrease in triacylglyceride (TG) accumulation and inflammation within this tissue. All these processes may mediate the protective effects of lowering GRK2 abundance against excessive body weight gain and HFD-induced insulin resistance.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/8/386/ra73/DC1

    Fig. S1. Characterization of an obese and insulin-resistant phenotype after 8 weeks of HFD, before tamoxifen treatment, and quantification of tamoxifen-induced GRK2 depletion.

    Fig. S2. HFD-induced weight gain in control mice compared with SD-fed mice.

    Fig. S3. CreER expression or translocation to the nucleus does not affect body weight gain or insulin-induced phosphorylation of AKT in muscle, eWAT, or liver.

    Fig. S4. The abundance of components in or inhibitors of the insulin signaling pathway is not changed upon GRK2 loss in muscle or liver.

    Fig. S5. CreER expression or translocation to the nucleus does not affect lipid accumulation in the liver.

    Fig. S6. Decreased abundance of inflammation markers in the liver upon tamoxifen-induced GRK2 loss.

    Table S1. Primers used for RT-PCR analysis.

  • Supplementary Materials for:

    Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2

    Rocio Vila-Bedmar, Marta Cruces-Sande, Elisa Lucas, Hanneke L. D. M. Willemen, Cobi J. Heijnen, Annemieke Kavelaars, Federico Mayor Jr.,* Cristina Murga*

    *Corresponding author. E-mail: cmurga{at}cbm.csic.es (C.M.); fmayor{at}cbm.csic.es (F.M.)

    This PDF file includes:

    • Fig. S1. Characterization of an obese and insulin-resistant phenotype after 8 weeks of HFD, before tamoxifen treatment, and quantification of tamoxifen-induced GRK2 depletion.
    • Fig. S2. HFD-induced weight gain in control mice compared with SD-fed mice.
    • Fig. S3. CreER expression or translocation to the nucleus does not affect body weight gain or insulin-induced phosphorylation of AKT in muscle, eWAT, or liver.
    • Fig. S4. The abundance of components in or inhibitors of the insulin signaling pathway is not changed upon GRK2 loss in muscle or liver.
    • Fig. S5. CreER expression or translocation to the nucleus does not affect lipid accumulation in the liver.
    • Fig. S6. Decreased abundance of inflammation markers in the liver upon tamoxifen-induced GRK2 loss.
    • Table S1. Primers used for RT-PCR analysis.

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    Citation: R. Vila-Bedmar, M. Cruces-Sande, E. Lucas, H. L. D. M. Willemen, C. J. Heijnen, A. Kavelaars, F. Mayor Jr., C. Murga, Reversal of diet-induced obesity and insulin resistance by inducible genetic ablation of GRK2. Sci. Signal. 8, ra72 (2015).

    © 2015 American Association for the Advancement of Science

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