KSR2 is an essential regulator of AMP kinase, energy expenditure, and insulin sensitivity - PubMed (original) (raw)

doi: 10.1016/j.cmet.2009.09.010.

Paul T Pfluger, Michele K Dougherty, Jeffery L Stock, Matthew Boehm, Oleg Chaika, Mario R Fernandez, Kurt Fisher, Robert L Kortum, Eun-Gyoung Hong, John Y Jun, Hwi Jin Ko, Aimee Schreiner, Deanna J Volle, Tina Treece, Amy L Swift, Mike Winer, Denise Chen, Min Wu, Lisa R Leon, Andrey S Shaw, John McNeish, Jason K Kim, Deborah K Morrison, Matthias H Tschöp, Robert E Lewis

Affiliations

KSR2 is an essential regulator of AMP kinase, energy expenditure, and insulin sensitivity

Diane L Costanzo-Garvey et al. Cell Metab. 2009 Nov.

Abstract

Kinase suppressors of Ras 1 and 2 (KSR1 and KSR2) function as molecular scaffolds to potently regulate the MAP kinases ERK1/2 and affect multiple cell fates. Here we show that KSR2 interacts with and modulates the activity of AMPK. KSR2 regulates AMPK-dependent glucose uptake and fatty acid oxidation in mouse embryonic fibroblasts and glycolysis in a neuronal cell line. Disruption of KSR2 in vivo impairs AMPK-regulated processes affecting fatty acid oxidation and thermogenesis to cause obesity. Despite their increased adiposity, ksr2(-/-) mice are hypophagic and hyperactive but expend less energy than wild-type mice. In addition, hyperinsulinemic-euglycemic clamp studies reveal that ksr2(-/-) mice are profoundly insulin resistant. The expression of genes mediating oxidative phosphorylation is also downregulated in the adipose tissue of ksr2(-/-) mice. These data demonstrate that ksr2(-/-) mice are highly efficient in conserving energy, revealing a novel role for KSR2 in AMPK-mediated regulation of energy metabolism.

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Figures

Figure 1

Figure 1. KSR1 and KSR2 interact with AMPK

(A) AMPK-related peptides detected from endogenous proteins co-precipitating with KSR2. KSR2-associated proteins were isolated and peptide fragments were detected by mass spectrometry. (B) Full length (FL) and truncated Pyo-tagged KSR1 and KSR2 constructs were expressed in COS-7 cells. (C) FLAG-tagged KSR1 or KSR2 expressed in 293T cells with the Myc-tagged AMPK α subunit. Cells were left untreated or treated with oligomycin. (D) Truncated FLAG-tagged KSR1 constructs expressed in 293T cells with the Myc-tagged AMPK α subunit. (E) Deletion constructs of FLAG-tagged KSR1 and the Myc-tagged AMPK α subunit expressed in 293T cells. (F) Truncated Pyo-tagged KSR2 constructs were expressed in COS-7 cells. (G) Pyo-tagged full length KSR1, KSR2 or KSR2 with a deletion of amino acids 327-392 expressed in COS-7 cells (left panel). FLAG-tagged full length KSR2 or KSR2 with a deletion of the CA3 region (ΔCA3) expressed with the Myc-tagged AMPK α subunit (right panel). (B-G) Whole cell lysates and anti-Pyo or anti-FLAG immunoprecipitates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with the indicated antibodies.

Figure 2

Figure 2. KSR1 expression regulates metabolism in mouse embryo fibroblasts

(A) ERK activation in WT MEFs and _ksr2_-/- MEFs expressing the indicated constructs were treated with 25 ng/ml of PDGF for the indicated times and phosphoERK was detected in situ. Representative expression of each construct is shown. Results are the mean ± S.D of triplicate determinations. (B) Glucose uptake in _ksr2_-/- MEFs and _ksr2_-/- MEFS expressing KSR2, KSR2.ΔCA3, or KSR2.Δ327-392. (C) FAO in _ksr2_-/- MEFs expressing the indicated constructs. (D) Glycolysis in NG108-15 cells expressing a non-targeting shRNA, or an shRNA against KSR2 with or without a vector encoding constitutively active AMPK (AMPK-CA). n = 8 for each condition. (E) NG108-15 cells expressing a non-targeting shRNA, or an shRNA against KSR2 were treated with 0-10 mM AICAR for 30 min at 37°C. (B-D) *, P<0.05; **, P<0.01; ***, P<0.001.

Figure 3

Figure 3. Targeted disruption of ksr2 causes obesity

(A) Expression of ksr2 (left panel) and ksr1 (right panel) mRNA in white adipose tissue (WAT), brown adipose tissue (BAT), liver, quadriceps (QUAD) and gastrocnemius (GASTROC) muscles, and whole brain. The expression of ksr2 and ksr1 was normalized relative to the expression of two control genes, GusB and Tbp, in each tissue. (B) Null (-/-), heterozygous (+/-) and WT (+/+) mice at day E18.5 (left), eight days (middle) and 24 weeks (right) of age. (C) Body weights of null (squares, n=8), and WT (diamonds, n=6) mice from three to 36 weeks of age. (D) Body weight and body composition of 12-week old WT (light bars, n=6 male and 7 female) and _ksr2_-/- (dark bars, n=5 of each sex) mice. (E) Wet weight of visceral (VISC), inguinal (ING), subcutaneous (SUB) and brown (BAT) adipose depots in WT (light bars, n=4) and _ksr2_-/- mice (dark bars, n=5). (F) Hematoxylin and eosin staining of histological sections from subcutaneous adipose tissue are shown (upper). Adipocyte cross-sectional area (lower) in WT (light bars) and _ksr2_-/- mice (dark bars). (G) WT and _ksr2_-/- brain lysates were immunoprecipitated with antibodies to the AMPK α subunit or a non-immune antibody. Immunoprecipitates and representative lysates were probed on western blot for expression of KSR2 and AMPKα. (H) Blood glucose levels at the indicated times in 6-7 month old WT (circles) and _ksr2_-/- mice (diamonds) following intraperitoneal injection of 0.25 g/kg AICAR. Basal blood glucose levels (mg/dl) in fed mice were 139.7 ± 8 (KSR2-/- female), 120.7 ± 12 (WT female), 152.8 ± 6 (KSR2-/- male), and 151.8 ± 23 (WT male). n=8 male, 4 female for each genotype. (I) Western blot analysis of total and phosphoThr172 AMPK α subunit (upper) and total and phosphoSer79 ACC (lower) from white adipose tissue of WT and _ksr2_-/- mice 15 min after injection with 0.25 g/kg AICAR. Graphs show the relative phosphorylation of each protein from five independent experiments. (J) Phosphorylation of AMPK Thr172 and ACC Ser79 in explants of subcutaneous adipose tissue incubated in DMEM at 37°C for 16 h with and without 1 mM AICAR. (D, E, H, I) *, p<0.05; **, P<0.01; ***, p<0.001 by single factor ANOVA or (I) unpaired, 2-tailed t-test.

Figure 4

Figure 4. _ksr2_-/- mice are hypothermic and hypophagic

(A) Hematoxylin and eosin staining of brown adipose tissue from WT (left) and _ksr2_-/- (right) mice. (B) Rectal temperature in 5-6 month old male and female WT (light bars, n=8 for each sex) and _ksr2_-/- mice (dark bars, n=6 males, n=11 females) during light (1 pm) and dark (9 pm) cycles (left panel). (C) UCP1 mRNA levels in BAT (left panel) in 9-10-month female WT (light bars, n=11) and _ksr2_-/- mice (dark bars, n=5). UCP1 protein levels (right panel) in 8-month WT and _ksr2_-/- female mice. (D) Average daily food intake in 6 WT males (age 11.5 ± 1.0 weeks), 7 WT females (age 10.6 ± 1.1 weeks), 5 _ksr2_-/- males (age 11.4 ± 0.6 week) and 5 _ksr2_-/- females (age 10.5 ± 1.0 week). (E) Serum leptin concentrations in 5-6 month WT and _ksr2_-/- mice. (F) Twenty four hour food intake following control PBS or 5mg/kg leptin injections in WT and _ksr2_-/- mice. n=5 for each genotype. (G) Neuropeptide mRNA expression in 8-9 month female WT and _ksr2_-/- mice. n=7 for each genotype. (B, D) *, P<0.05; **, P<0.01; ***, P<0.001

Figure 5

Figure 5. _ksr2_-/- mice are more active, but expend less energy than WT mice

(A) Oxygen consumption (left), carbon dioxide production (middle), and respiratory quotient (right) in WT and _ksr2_-/- mice. (B) Cumulative locomotor activity in WT and _ksr2_-/- mice. (C) Spontaneous (middle) and cumulative (right) energy expenditure in male and female WT and _ksr2_-/- mice. (D) Oxygen consumption (upper panel) and core body temperature of female WT and _ksr2_-/- mice at 32°C. (A-C) 6 WT males (age 11.5 ± 1.0 weeks), 7 WT females (age 10.6 ± 1.1 weeks), 5 _ksr2_-/- males (age 11.4 ± 0.6 week) and 5 _ksr2_-/- females (age 10.5 ± 1.0 weeks). (D) 4 WT (age 30.7 ± .02 weeks, and 3 _ksr2_-/- (32.6 ± .07 weeks) female mice. (A-C) *, P<0.05; **, P<0.01; ***, P<0.001, unpaired, 2-tailed t-test or (C) 2-Way ANOVA with Bonferroni’s post hoc test.

Figure 6

Figure 6. Lipid, glucose and insulin homeostasis is disrupted in _ksr2_-/- mice

(A) Lipolysis (left), serum concentrations of non-esterified free fatty acids (middle) and triglycerides (right) in 5-6 month WT (n=5, left; n=4 middle; n=7, right) and _ksr2_-/- mice (n=6, left; n=3 middle; n=4, right). (B) Fasting serum insulin in 5-6 month old WT (light bars, n=8) and _ksr2_-/- mice (dark bars, n=8). (C) Glucose infusion rate, (D) hepatic glucose production and hepatic insulin action, (E) glucose turnover, glycolysis, and glycogen/lipid synthesis in WT (n=11) and _ksr2_-/- mice (n=8). (F) Tissue glycogen from liver (left) and gastrocnemius (right) in 5-6 month old WT and _ksr2_-/- mice. (G) Glucose uptake in skeletal muscle, white adipose tissue, and brown adipose tissue, in WT (n=11) and _ksr2_-/- mice (n=8). (H) Ex vivo glucose uptake in isolated EDL muscle from WT and _ksr2_-/- mice treated with 100 nM insulin (left panel, n = 6 WT, 3 _ksr2_-/-), or 2 mM AICAR (right panel n = 5 WT, 5 _ksr2_-/-). (A-H) *, P<0.05, **, P<0.01, ***, P<0.001.

Figure 7

Figure 7. A model of KSR2-mediated regulation of metabolism

Arrows indicate activation. Lines with perpendicular bars attached indicate inhibition. Dashed lines denote decreased function or expression. Arrowheads denote corresponding changes in the amount or activity of the indicated molecule. See text for details.

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