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1.
FIGURE 5.

FIGURE 5. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

Rgs16 mRNA expression is induced by ChREBP in a glucose-dependent manner. A and B, ChREBP, GK, and glucose are required for Rgs16 mRNA expression in ChREBP KO primary hepatocytes. C–F, Rgs16 mRNA induction by glycerol is ChREBP-dependent but GK-independent. 8-Bromo-cyclic AMP stimulates glucose production but inhibits Rgs16 mRNA expression in wild type and ChREBP KO primary hepatocytes. G–J, glucagon stimulates glucose production but inhibits Rgs16 mRNA expression in wild type primary hepatocytes. Error bars, S.E.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
2.
FIGURE 6.

FIGURE 6. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

Liver-specific expression of FGF21 induces hepatic Rgs16. A, Western blot of RGS16 protein in liver from wild type and transgenic Fgf21 (TgF21) (26) littermates (30 μg/lane; RGS16 antiserum provided by C. Beadling). B, Rgs16, CPT-1, and PEPCK mRNA steady-state levels assayed by qPCR. Shown are relative levels of mRNA in the liver of fed and fasted TgF21 and fed wild type compared with fasted wild type levels for each gene. C, transcription rates of Rgs16 and GAPDH in liver from fed and fasted wild type and Fgf21 transgenic mice. Liver was collected at ZT16 for each assay.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
3.
FIGURE 3.

FIGURE 3. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

Dietary simple carbohydrates strongly induce Rgs16 mRNA and protein. A, in situ hybridization of Rgs16 mRNA in liver. a, fasted liver at ZT16; b, fed liver at ZT16; c, liver at ZT9, day 3 of restricted feeding (RF) with water ad libitum (Rgs16 is expressed throughout the liver, highest in periportal, declining toward pericentral hepatocytes); d, liver at ZT16, day 3 with 5% sucrose-water ad libitum (Rgs16 expression is uniformly distributed in the liver). Scale bar, 200 mm; 12 h light/dark. B and C, Rgs16 mRNA (B) and protein (C) are induced by dietary sucrose. Liver was collected at ZT16 from mice denied chow from ZT4 to ZT16 and provided either 5% sucrose-water, 0.45% saccharine, or water ad libitum and assayed for Rgs16 mRNA by qPCR and protein by Western blot (30 μg protein/lane). Recombinant, untagged RGS16 expressed in COS-7 cells served as a marker (rRgs16). Error bars, S.E.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
4.
FIGURE 2.

FIGURE 2. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

RGS16 inhibits fatty acid oxidation in liver. A and B, decreased plasma β-hydroxybutyrate (A) and decreased fatty acid oxidation rate (B) in transgenic Rgs16 (TgR16) liver mitochondria compared with WT littermates. Mice were fed ad libitum a high fat diet for 2 weeks with Dox (0.1 mg/ml) (n = 6/group) and then fasted overnight and sacrificed at ZT7. C and D, increased plasma β-hydroxybutyrate (C) and increased fatty acid oxidation rate (D) in Rgs16 KO liver compared with WT littermates. TCA, tricarboxylic acids. Mice were fasted overnight and sacrificed at ZT7 (n = 6/group). A–D, 3 mice/group; TgR16 or Rgs16 KO compared with WT in each condition; *, p < 0.05; **, p < 0.01. E, gene expression ratio in transgenic Rgs16 (TgR16) and Rgs16 KO mice compared with their respective littermates. Shown is qPCR analysis of genes in pathways for fatty acid synthesis (FA Syn), glucose metabolism (Glu), and fatty acid oxidation (FA Ox) (3 mice/group, mRNA pooled prior to qPCR). Error bars, S.E.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
5.
FIGURE 7.

FIGURE 7. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

RGS16 inhibits hepatic fatty acid oxidation in a ChREBP-dependent manner. During the early fasting phase (ZT0–ZT6; gray font and arrows), glucagon stimulates hepatic glucose production from glycogen (48). The Gs-coupled glucagon receptor stimulates cAMP production, which activates the transcription factor CRTC2 (46) but inhibits Rgs16 mRNA accumulation (Fig. 5). We propose that a novel Gi/Gq-coupled receptor promotes fatty acid oxidation during the late fasting phase (after ZT6) (12). Fatty acid (FA) metabolites may serve as ligands of PPARα, which stimulates Fgf21 expression (26, 45). FGF21 promotes fatty acid lipolysis and release from white adipose tissue (WAT) (26, 49). Fatty acid oxidation in liver produces ATP and NADH necessary to drive hepatic gluconeogenesis during prolonged fasting. Glucose metabolites, such as xylulose 5-phosphate (37, 50), presumably activate ChREBP-dependent Rgs16 transcription and mRNA accumulation during the late fasting phase (Figs. 3 and 4). RGS16 inhibits Gi/Gq-mediated fatty acid oxidation (Figs. 1 and 2), thus modulating fatty acid utilization during periods of energy deprivation.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
6.
FIGURE 1.

FIGURE 1. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

Transgenic Rgs16 (TgR16) mice develop fatty liver. Transgenic Rgs16 mRNA and protein expression in liver is doxycycline-dependent. A, the transgenes ApoE::TA and TetON::Rgs16 were co-injected to obtain transgenic RGS16 tagged at the C terminus with a triple repeat of the Myc epitope (white box following mRgs16). The human ApoE promoter drives the Tet activator protein (TAS2s-M2) expression in liver. TAS2s-M2 binds TREs to induce expression of transgenic Rgs16-myc3 (TgR16) only in the presence of doxycycline. B and C, in situ hybridization. Transgenic Rgs16-myc mRNA is expressed throughout the liver of fed TgR16 transgenic mice (doxycyclin, 0.1 mg/ml) (B) but not in TgR16 liver (C), without Dox. D, RGS16 transgenic protein GAP activity was determined by single turnover GTPase assay in liver extracts from either transgenic Rgs16 (TgR16) or wild type mice. GTP-bound myristoylated recombinant Gαi1 was the substrate; recombinant RGS4 was assayed for comparison with liver extracts and background GTP hydrolysis on Gαi1 (no addition). E, Rgs16 transgene expression is doxycyclin-dependent and liver-specific. Doxycycline in the drinking water (0.001–0.1 mg/ml) induced expression of Rgs16-myc (TgR16) mRNA in liver but not in the other tissues indicated (assayed by qPCR). Inset, Western blot of liver protein in TgR16 and wild type littermates detected by RGS16 antiserum. F, fatty liver in transgenic Rgs16 (TgR16). Liver sections are sections of TgR16 and wild type male mice (high fat diet, 9.5 weeks with Dox (0.1 mg/ml)). Liver sections were stained with oil red O and H&E. G, Dox-dependent increase in hepatic triglyceride content in TgR16 mice (n = 6/group). H, increased hepatic triglyceride content in Gα11−/− mice independent of Dox (0.1 mg/ml). G and H, statistical analysis. A one-way analysis of variance test followed by Tukey's multiple comparison test was used. a versus b, p < 0.01. Mice were maintained on a high fat diet for 10 days, with or without Dox (0.1 mg/ml). Mice were fasted overnight before liver was collected at ZT8 (n = 4/group). Error bars, S.E.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.
7.
FIGURE 4.

FIGURE 4. From: Regulator of G Protein Signaling (RGS16) Inhibits Hepatic Fatty Acid Oxidation in a Carbohydrate Response Element-binding Protein (ChREBP)-dependent Manner.

ChREBP is required for Rgs16 mRNA and protein expression in mice on a high simple carbohydrate diet and during prolonged fasting. A, ChREBP−/− (KO) and WT mice (n = 3/group) were maintained on normal chow and 5% glucose-water for 3 days ad libitum before removing chow ZT4–ZT16. Liver was collected at ZT16 for qPCR analysis of Rgs16 mRNA expression. Statistical analysis was as follows: one-way analysis of variance test followed by Tukey's multiple comparison test. a versus c, p < 0.01; a versus d, p < 0.01; b versus d, p < 0.01; all other pairwise comparisons were not significantly different. B, SREBP-1c−/− (KO) mice and WT mice were treated as in A, except mice were provided 5% sucrose-water (n = 4/group). There was no statistically significant difference in Rgs16 mRNA expression between WT and SREBP KO mice, with or without sucrose-water. C and D, Rgs16 and liver pyruvate kinase mRNA expression in liver slices is induced by glucose in a dose- and ChREBP-dependent manner. Male mice (129SV/EvxC57BL/6) were provided normal chow and water ad libitum overnight, liver slices were collected at ZT4 and cultured 30 h in hepatocyte media at the indicated glucose concentrations (10 mm lactate was substituted for “0” glucose). Rgs16 mRNA (C) and liver pyruvate kinase mRNA (D) expression in liver slices from wild type and ChREBP−/− littermates. Shown is qPCR analysis of Rgs16 and liver pyruvate kinase expression normalized to the level in the liver of fed wild type mice harvested at ZT16. E (top), nuclear run-on transcription analysis; autoradiogram of 32P-labeled nascent transcript hybridized to the indicated cDNAs on nylon membranes. GAPDH served as a constitutively expressed internal control. Fed or fasted WT and ChREBP KO male mice were provided either water or 15% glucose-water ad libitum during restricted feeding. E (bottom), quantitation of Rgs16 transcription rate, normalized to GAPDH in each sample. Rgs16 mRNA steady-state levels were assayed by qPCR. Rgs16 transcription rate and mRNA levels in fasted mice are defined as 1.00. Shown is liver isolated at ZT5 day 3 restricted feeding (− Food) or refed normal chow (0.2 g) for 1 h (+ Food). RF, restricted feeding (n = 4/group). F, liver protein from ChREBP KO and WT mice in A (5% glucose) or WT mice in B (5% sucrose). Liver was collected at ZT16 for Western analysis of RGS16 protein expression (30 μg/lane). Error bars, S.E.

Victor Pashkov, et al. J Biol Chem. 2011 April 29;286(17):15116-15125.

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