GIP increases human adipocyte LPL expression through CREB and TORC2-mediated trans-activation of the LPL gene

Abstract

GIP (glucose-dependent insulinotropic polypeptide) is a gastrointestinal hormone that regulates pancreatic islet function. Additionally, emerging evidence suggests an important physiological role for GIP in the regulation of adipocyte metabolism. In previous studies on the lipogenic effects of GIP, it was shown to increase adipocyte lipoprotein lipase (LPL) activity in both differentiated 3T3-L1 cells and human adipocytes through a pathway involving activation of protein kinase B (PKB)/Akt. In the current study, we examined the effects of GIP on LPL gene expression. GIP in the presence of insulin increased LPL gene expression in human adipocytes and LPL promoter activity in GIP receptor-expressing HEK-293 cells, and both effects were greatly reduced by the transcription inhibitor actinomycin D. Subsequent studies established that GIP increased phosphorylation of Serine 133 in cAMP-response element binding protein (CREB) and the nuclear localization of cAMP-responsive CREB coactivator 2 (TORC2) through a pathway involving phosphatidylinositol 3-kinase (PI3-K), PKB, and AMP-activated protein kinase (AMPK). However, in the presence of insulin, GIP failed to activate the cAMP/PKA pathway. Knockdown of CREB and TORC2 using RNA interference reduced LPL expression, supporting a functional regulatory role. GIP-induced phospho-CREB and TORC2 were shown to bind to a cAMP-response element (-II) site in the human LPL promoter and GIP increased protein-protein interactions of these two factors. The lipogenic effects of GIP in the presence of insulin are therefore at least partially mediated by upregulation of adipocyte LPL gene transcription through a pathway involving PI3-K/PKB/AMPK-dependent CREB/TORC2 activation.

Fig. 1.

GIP but not GLP-1 strongly increases LPL activity and expression in human adipocytes. Human adipocytes were serum starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for the indicated periods of time with GIP or GLP-1 (100 nM) in the presence of insulin (1 nM). A: Effect of GIP or GLP-1 on LPL activity. LPL activity was determined as described in Materials and Methods. B: Effect of GIP or GLP-1 on LPL protein expression. Human adipocytes were treated as described above and Western blot analyses were performed using antibodies against LPL and β-actin. C: The effect of GIP and GLP-1 on LPL mRNA expression. Human adipocytes were treated as described above and real-time RT-PCR performed to quantify LPL mRNA levels; shown as the fold difference versus control normalized to 18S rRNA expression levels. D: Effects of transcription inhibition on GIP-mediated LPL activity. Human adipocytes were treated for 24 h with GIP or GLP-1 (100 nM) in the presence of transcription inhibitor, actinomycin D (5 μg/ml). LPL activity was determined as described in Materials and Methods. E: Effects of transcription inhibition on GIP-mediated LPL expression. Human adipocytes were treated as described above and Western blot analyses were performed using antibodies against LPL and β-actin. F: The effect of GIP and GLP-1 on LPL promoter activity. GIPR-HEK-293 cells were transfected with LPL promoter reporter construct (chr8+: 19840052 - 19841249; -992 to +206 of LPL gene, 1 µg) and then treated for 24 h with insulin (1 nM) plus GIP or GLP-1 (100 nM). The reporter activities are shown as the relative luciferase activity normalized to protein concentration. All data represent three independent experiments, each carried out in duplicate and Western blots are representative of n = 3. Significance was tested using ANOVA with Newman-Keul's post hoc test where ** represents P < 0.05 versus Control and ## represents P < 0.05 versus GIP.

Fig. 2.

GIP modulates CREB/TORC2 activity in human adipocytes. Human adipocytes were serum starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP or GLP-1 (100 nM) in the presence of insulin (1 nM). A: Effects of GIP on phosphorylation of CREB and nuclear localization of TORC2. Nuclear extracts were isolated and Western blot analyses performed with antibodies against phosphorylated Ser133-CREB, CREB, TORC2, and Histone H3. Cytoplasmic extracts were immunoprecipitated (IP) with phospho-Serine/Threonine followed by immunoblotting (IB) for TORC2. Input represents one-tenth of total cytoplasmic extracts used in assay. B: Confocal microscopy. Human adipocytes were treated as described above. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 μm and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are representative of n = 3.

Fig. 3.

CREB/TORC2 are functionally involved in the regulation of LPL expression. A: Effects of different CREB constructs on GIP-responsiveness of the LPL promoter. GIPR-HEK-293 cells were cotransfected with an LPL promoter reporter construct (chr8+: 19840052 - 19841249; -992 to +206 of LPL gene, 2 µg), and various CREB constructs or control vector pCMV5 (1 µg). After transfection, cells were treated with insulin (1 nM) plus GIP (100 nM), and the reporter activities are shown as the relative luciferase activity normalized to protein concentration. All data represent three independent experiments, each carried out in triplicate. Significance was tested using ANOVA with Newman-Keuls post hoc test, where ** represents P < 0.05 versus untreated Vector Control, ## represents P < 0.05 versus respective untreated Control. B, C: RNAi-mediated suppression of CREB and TORC2 reduces GIP-stimulated LPL expression. B: Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL protein expression. Human adipocytes were transfected with a pool of three siRNAs for CREB and TORC2 and incubated for 72 h. At 48 h after transfection, cells were treated with insulin (1 nM) plus GIP (100 nM) for 24 h and Western blot analyses were performed as described in Materials and Methods, using antibody against phospho-CREB (Serine-133), CREB, TORC2, LPL, Histone H3, and β-actin. C. Effects of CREB and TORC2 siRNAs on GIP-stimulated LPL mRNA expression. Human adipocytes were treated as described above and real-time RT-PCR performed to quantify LPL mRNA levels; shown as the fold difference versus control normalized to 18S rRNA expression levels. Significance was tested using ANOVA with Newman-Keul's post hoc test where ** represents P < 0.05 versus Control siRNA, ## represents P < 0.05 versus GIP.

Fig. 4.

GIP regulates CREB/TORC2 through a pathway involving PI3-K/PKB/AMPK. Human adipocytes were serum starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). A: Effect of PI3-K inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PI3-K inhibitors, LY 294002 (40 μM) or wortmannin (400 nM). B: Effect of AMPK modulation on CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of AMPK inhibitor (Compound C, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine, 40 μM) or AMPK activator (5-(3-(4-(2-(4-Fluorophenyl)ethoxy)phenyl)propyl)furan-2-carboxylic acid, FPPF, 50 μM). AMPK inhibitor or activator was added to cells during 1 h preincubation as well as GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed. TORC2 and histone H3 blots are from nuclear extracts and phospho-AMPK Thr172 and AMPK blots are from cytoplasmic extracts. C: Effect of AMPK modulation on LPL activity. Human adipocytes were treated as described above and LPL activity was determined as described in Materials and Methods. D: Effect of GIP or GLP-1 on cAMP accumulation. Human adipocytes were treated as described above and incubated with GIP or GLP-1 (100 nM) in the presence of insulin (1 nM) and IBMX (0.5 mM). cAMP concentrations determined in the cell extracts. E: Effect of PKA inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated as described above and stimulated insulin (1 nM) plus GIP, GLP-1 (100 nM) in the presence or absence of H-89 or Rp-cAMPs. H-89 (10 μM) or Rp-cAMPs (200 μM) was added to cells during 1 h preincubation as well as during GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed with antibodies against phosphorylated Thr172-AMPK, AMPK, TORC2, and histone H3. F: Effect of PKB inhibition on GIP-mediated CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PKB inhibitor (Akt IV, 500 nM). Nuclear extracts were isolated from each sample and Western blot analyses were performed with antibodies against phosphorylated Ser133-CREB, CREB, and histone H3. Western blots are representative of n = 3. All data represent three independent experiments, each carried out in duplicate. Significance was tested using ANOVA with Newman-Keul's post hoc test where ** represents P < 0.05 versus Control.

Fig. 4.

GIP regulates CREB/TORC2 through a pathway involving PI3-K/PKB/AMPK. Human adipocytes were serum starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). A: Effect of PI3-K inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PI3-K inhibitors, LY 294002 (40 μM) or wortmannin (400 nM). B: Effect of AMPK modulation on CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of AMPK inhibitor (Compound C, 6-[4-(2-Piperidin-1-yl-ethoxy)-phenyl)]3-pyridin-4-yl-pyrrazolo[1,5-a]pyrimidine, 40 μM) or AMPK activator (5-(3-(4-(2-(4-Fluorophenyl)ethoxy)phenyl)propyl)furan-2-carboxylic acid, FPPF, 50 μM). AMPK inhibitor or activator was added to cells during 1 h preincubation as well as GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed. TORC2 and histone H3 blots are from nuclear extracts and phospho-AMPK Thr172 and AMPK blots are from cytoplasmic extracts. C: Effect of AMPK modulation on LPL activity. Human adipocytes were treated as described above and LPL activity was determined as described in Materials and Methods. D: Effect of GIP or GLP-1 on cAMP accumulation. Human adipocytes were treated as described above and incubated with GIP or GLP-1 (100 nM) in the presence of insulin (1 nM) and IBMX (0.5 mM). cAMP concentrations determined in the cell extracts. E: Effect of PKA inhibition on GIP-mediated decreases in phospho-AMPK Thr172 and nuclear CREB/TORC2 activity. Human adipocytes were treated as described above and stimulated insulin (1 nM) plus GIP, GLP-1 (100 nM) in the presence or absence of H-89 or Rp-cAMPs. H-89 (10 μM) or Rp-cAMPs (200 μM) was added to cells during 1 h preincubation as well as during GIP stimulation. Nuclear/cytoplasmic extracts were isolated from each sample and Western blot analyses were performed with antibodies against phosphorylated Thr172-AMPK, AMPK, TORC2, and histone H3. F: Effect of PKB inhibition on GIP-mediated CREB/TORC2 activity. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PKB inhibitor (Akt IV, 500 nM). Nuclear extracts were isolated from each sample and Western blot analyses were performed with antibodies against phosphorylated Ser133-CREB, CREB, and histone H3. Western blots are representative of n = 3. All data represent three independent experiments, each carried out in duplicate. Significance was tested using ANOVA with Newman-Keul's post hoc test where ** represents P < 0.05 versus Control.

Fig. 5.

Effects of PI3-K, AMPK, PKA, or PKB inhibition on GIP-mediated LPL mRNA and protein expression. Human adipocytes were serum starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). A: Effect of PI3-K, AMPK, PKA or PKB inhibition on GIP-mediated LPL transcription. Human adipocytes were treated for 24 h with insulin (1 nM) plus GIP (100 nM) in the presence or absence of PI3-K inhibitors (40 μM of LY 294002 or 400 nM of wortmannin), AMPK inhibitor (40 μM of Compound C) or AMPK activator (50 μM of FPPF), PKA inhibitors (10 μM of H-89 or 200 μM of Rp-cAMPs), or PKB inhibitor (500 nM of Akt IV). Real-time RT-PCR performed to quantify LPL mRNA levels; shown as the fold difference versus control normalized to 18S rRNA expression levels. B: Effect of PI3-K, AMPK, PKA, or PKB inhibition on GIP-mediated LPL expression. Human adipocytes were treated as described in A and Western blot analyses were performed using antibodies against LPL and β-actin. All data represent three independent experiments, each carried out in duplicate and Western blots are representative of n = 3. Significance was tested using ANOVA with Newman-Keul's post hoc test where ** represents P < 0.05 versus Control.

Fig. 6.

GIP increases protein-protein interaction between phospho-CREB and TORC2 in the nucleus. A: Coimmunoprecipitation. Human adipocytes were serum-starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM) and in the presence or absence of PI3-K inhibitors, LY 294002 (40 μM), wortmannin (400 nM) or AMPK activator (50 μM). Nuclear extracts were isolated from each sample and immunoprecipitated (IP) with phospho-CREB (Ser133) followed by immunoblotting (IB) for TORC2. Input represents one-tenth of total nuclear extract used in the coimmunoprecipitation assay. B: Confocal microscopy. Human adipocytes were treated as described above and fixed following stimulation with GIP (100 nM) in the presence or absence of PI3-K inhibitors or AMPK activator. Immunocytochemical staining was performed using antibodies against phospho-CREB (Ser133, green) or TORC2 (red) and nuclei were stained with DAPI (blue). The scale bar indicates 20 μm and all imaging data were analyzed using the Northern Eclipse program (ver.6). Shown are representative of n = 3.

Fig. 7.

Phospho-CREB/TORC2 binds CRE-II sequence of human LPL promoter. A: Identification of putative CRE sequences in the promoters of several adipocyte-specific genes. Potential CREs present in these promoters are indicated by the box-enclosed regions and consensus CRE sequences shown at the top of the figure. B, C: Representative gel of a ChIP assay for the binding of phospho-CREB (B) and TORC-2 (C) in the human LPL promoter. Human adipocytes were serum-starved in DMEM/Ham's F-12 medium (1:1, v/v) containing 0.1% BSA overnight and treated for 24 h with GIP (100 nM) in the presence of insulin (1 nM). Phospho-CREB and TORC2 were respectively immunoprecipitated from intact chromatin isolated from human adipocytes using anti-phospho-CREB (Ser 133) and anti-TORC2 antibody. Precipitated DNA fragments were analyzed by PCR using primers flanking the CRE-I and CRE-II sites in the LPL promoter. An isotype-matched IgG was used as negative control and 1% Input (PCR product of one-hundredth of the total isolated DNA used in the ChIP assay) as positive control, respectively. Shown are representative of n = 3.

Fig. 8.

Proposed pathway by which GIP increases LPL activity in adipocytes. GIP receptor interaction results in PI3-K activation, increased phosphorylation of Ser473 and Thr308 in PKB, and reduced phosphorylation of Ser428 in LKB1 and Thr172 in AMPK. An associated decrease in AMPK phosphorylation leads to dephosphorylation of TORC2, thus allowing increased translocation from the cytoplasm and into the nucleus. PKB also increases phospho-CREB Ser133 levels in the nucleus. TORC2 complexes with phospho-CREB Ser133 in the nucleus and binds to CRE-II of the human LPL promoter, thus turning on the transcriptional machinery for up-regulation of LPL.