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. 2010 Oct 12;107(41):17704-9.
doi: 10.1073/pnas.1012665107. Epub 2010 Sep 27.

Suppressor of MEK Null (SMEK)/protein Phosphatase 4 Catalytic Subunit (PP4C) Is a Key Regulator of Hepatic Gluconeogenesis

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Suppressor of MEK Null (SMEK)/protein Phosphatase 4 Catalytic Subunit (PP4C) Is a Key Regulator of Hepatic Gluconeogenesis

Young-Sil Yoon et al. Proc Natl Acad Sci U S A. .
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Abstract

Fasting promotes hepatic gluconeogenesis to maintain glucose homeostasis. The cAMP-response element binding protein (CREB)-regulated transcriptional coactivator 2 (CRTC2) is responsible for transcriptional activation of gluconeogenic genes and is critical for conveying the opposing hormonal signals of glucagon and insulin in the liver. Here, we show that suppressor of MEK null 1 (SMEK1) and SMEK2 [protein phosphatase 4 (PP4) regulatory subunits 3a and 3b, respectively] are directly involved in the regulation of hepatic glucose metabolism in mice. Expression of hepatic SMEK1/2 is up-regulated during fasting or in mouse models of insulin-resistant conditions in a Peroxisome Proliferator-Activated Receptor-gamma Coactivator 1α (PGC-1α)-dependent manner. Overexpression of SMEK promotes elevations in plasma glucose with increased hepatic gluconeogenic gene expression, whereas depletion of the SMEK proteins reduces hyperglycemia and enhances CRTC2 phosphorylation; the effect is blunted by S171A CRTC2, which is refractory to salt-inducible kinase (SIK)-dependent inhibition. Taken together, we would propose that mammalian SMEK/PP4C proteins are involved in the regulation of hepatic glucose metabolism through dephosphorylation of CRTC2.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
SMEK enhances hepatic gluconeogenesis in vivo. (A) Q-PCR analysis showing SMEK1, SMEK2, PEPCK, G6Pase, and PGC-1α mRNA levels in ad libitum-fed (fed), 24-h fasted (fasted), or 24-h fasted and 5-h refed (refed) mice (*P < 0.05 and **P < 0.01, t test; n = 8). (B) Q-PCR analysis showing SMEK1, SMEK2, PEPCK, and PGC-1α mRNA in WT and db/db mice under ad libitum conditions (**P < 0.01, t test; n = 5). (C) Effects of Ad-SMEK2 or Ad-GFP on 4-h fasting glucose levels (Upper) or insulin levels (Lower) in WT mice (*P < 0.05, t test; n = 4). (D) Effects of Ad-SMEK2 or Ad-GFP on in situ hepatic glucose production in WT mice following 24 h of fasting (**P < 0.01 and *P < 0.05, t test; n = 10). The area under the curve (AUC) during the perfusion period for each condition is indicated. (E) Western blot analysis showing effects of Ad-SMEK2 on CRTC2, FOXO1a, or PGC-1α. (F) Transfection analysis was performed to determine the effects of SMEK/PP4C expression on CRTC2- or FOXO1a-dependent activation of G6Pase promoter activity in HepG2 cells (**P < 0.01 and *P < 0.05, t test; n = 3). pcf, pcDNA-flag empty vector. Data in A, B, and F represent the mean ± SD, and data in C and D represent the mean ± SEM.
Fig. 2.
Fig. 2.
SMEKs are crucial components of the PP4 complex. (A) Interaction of SMEK and PP4C or PP4R2 in rat primary hepatocytes. Coimmunoprecipitation assay was performed with anti-PP4C antibody or IgG control and blotted with antibodies against SMEK1, SMEK2, PP4C, and PP4R2. IP, immunoprecipitation. (B) Coimmunoprecipitation assay was performed with anti-PP4C antibody as in Fig. 2A to confirm the in vivo interaction with SMEK1 in db/db mouse liver. (C) Western blot analysis using whole-cell lysates (whole), nuclear fractions (Nuc), and cytoplasmic fractions (Cyt) showing subcellular localization of SMEK, PP4C, and PP4R2 in livers from normal lean mice. α-Tubulin and heat shock protein 90 (HSP90) was used for a cytoplasmic marker, whereas CREB was used for a nuclear marker. Several serine/threonine kinases known to regulate CRTC2 phosphorylation (AMPKα1, AMPKα2, SIK1, and SIK2) were also shown. (D) Effects of SMEK/PP4C on CRTC2 phosphorylation in an in vitro phosphatase assay. GST and GST-CRCT2 161–181 (WT and S171A) fusion proteins were isolated from bacteria and phosphorylated in vitro with AMPK. V, W, and M represent GST vector only, GST-WT CRTC2 161–181 amino acids, and GST-S171A CRTC2 161–181 amino acids, respectively. pcf, pcDNA-flag empty vector. (E) Effects of SMEK on CRTC2 translocation in primary hepatocytes. Effects of glucagon (GLU, 50 nM for 15 min) or aminoimidazole carboxamide ribonucleotide (AICAR) (500 μM for 30 min) on the nuclear localization of CRTC2 was shown. Ratio of nuclear CRTC2/CREB is indicated below. CREB is used as a marker for nuclear localization. (F) Effects of SMEK on CRTC2 translocation in primary hepatocytes. Ad-red fluorescent protein (RFP)-CRTC2 was coinfected with either Ad-GFP or Ad-SMEK2 in mouse primary hepatocytes. Effect of forskolin (FSK, 10 μM for 15 min) on nuclear localization of CRTC2 was shown as a control. DAPI staining was performed to indicate the location of nuclei.
Fig. 3.
Fig. 3.
SMEK knockdown reduces blood glucose levels and increases CRTC2 phosphorylation. (A) Effects of Ad-SMEK RNAi or control [Ad-unspecific (US)] RNAi on 4-h fasting glucose levels (Upper) and 4-h fasting insulin levels (Lower) in WT mice (**P < 0.01 and *P < 0.05, t test; n = 10). (B) Immunoblot analysis showing effects of Ad-SMEK RNAi on CRTC2 phosphorylation status or PGC-1α levels in WT mice. (C) Effects of Ad-SMEK RNAi or Ad-US RNAi on gluconeogenic gene expression in WT mice (**P < 0.01 and *P < 0.05, t test; n = 10). (D) Effects of Ad-SMEK RNAi or Ad-US RNAi on liver glycogen contents in WT mice (n = 10). Six-week-old mice were fed a high-fat diet for 6 wk, and hyperinsulinemic-euglycemic clamp studies were performed (**P < 0.01 and *P < 0.05, t test; n = 13–14). Basal hepatic glucose output (basal HGO) (E), clamp hepatic glucose output (clamp HGO) (F), glucose disposal (G), and infusion rates (H) were determined. Data in A and DH represent the mean ± SEM, and data in C represent the mean ± SD.
Fig. 4.
Fig. 4.
SMEK regulates CRTC2-dependent hepatic glucose metabolism in vivo. (A) Effects of Ad-SMEK RNAi or Ad-US RNAi on 4-h fasting glucose levels (Upper) or insulin levels (Lower) in db/db mice (*P < 0.05, t test; n = 5). (B) Pyruvate challenge analysis showing effects of SMEK knockdown on hepatic gluconeogenesis in db/db mice (**P < 0.01 and *P < 0.05, t test; n = 7). (C) Western blot analysis showing effects of Ad-SMEK RNAi or Ad-US on CRTC2 phosphorylation status, hepatic SMEK1, SMEK2, and PGC-1α expression in db/db mice. HSP90, heat shock protein 90. (D) Effects of Ad-WT CRTC2 or Ad-S171A CRTC2 on 4-h fasting gluconeogenic gene expression in db/db mice infected with Ad-SMEK RNAi (**P < 0.01, t test; n = 5). (E) Effects of Ad-WT CRTC2 or Ad-S171A CRTC2 on 4-h fasting glucose levels in db/db mice infected with Ad-SMEK RNAi (**P < 0.01 and *P < 0.05, t test; n = 5). (F) Proposed model for the regulation of CRTC2 phosphorylation and hepatic glucose production by interplay of serine/threonine kinases and phosphatases. Data in A, B, and E represent the mean ± SEM, and data in D represent the mean ± SD.

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