LTKO mice maintain euglycemic and glucose tolerant on a high-fat diet.

<p>(A) Fasting glucose levels in 4.5-month male control and LTKO mice (n=8) after the treatment with a high-fat diet for 3.5 months. (B) Non-fasting blood glucose levels in 4-month male control and LTKO mice (n=8) after the treatment with the high-fat diet for 3 months. (C, D) Glucose tolerance tests and the AUC analysis in 4.5-month male control and LTKO mice (n=8) after the treatment with the high-fat diet for 3.5 months, respectively. (E, F) Expression of genes involved in glucose metabolism was analyzed in the liver of control and LTKO mice (n=4) treated with the high-fat diet for 5 months by real-time PCR. <i>Pck1</i>, phosphoenoylpyruvate carboxykinase 1; <i>G6pc</i>, glucose-6-phosphatase, catalytic; <i>Pdk2</i>, pyruvate dehydrogenase kinase 2; <i>Gck</i>, glucokinase; <i>Pklr</i>, pyruvate kinase, liver and red blood cell type. Data represent mean ± SEM. * indicates a significance with <i>P</i><0.05 in control vs. LTKO mice.</p

Body composition of LTKO mice fed a high-fat diet.

<p>(A, B) Body weight and length measurements of control and LTKO mice (n=6) after a high-fat diet (HFD) treatment for 5 months, respectively. (C, D) Body fat and bone mineral density (BMD) analyses of the above HFD treated mice by DEXA, respectively. Data represent mean ± SEM.</p

Sirt6 overexpression has no significant effect on glucose toerance in LTKO mice.

<p>(A) Sirt6 overexpression was assessed by Western blot analysis in liver lysates from control and LTKO mice injected with SIRT6 or GFP adenoviruses (n=6). (B) Glucose tolerance tests in 4-month-old control and LTKO mice injected with SIRT6 or GFP adenoviruses (n=6). (C) Expression of genes involved in glucose metabolism was analyzed in the livers of SIRT6 or GFP adenovirus infected control and LTKO mice (n=6) by real-time PCR. Data represent mean ± SEM. * indicates a significance with <i>P</i><0.05 between loxp-GFP and loxp-SIRT6 groups.</p

Insulin sensitivity in LTKO mice fed chow diet.

<p>(A) Insulin tolerance tests (ITT) in 3-month male control and LTKO mice (n=6) after 3-hour fasting and an intraperitoneal injection of 0.75 U human regular insulin (humulin R, Lilly) per kg body weight. (B) The data in Panel A were replotted as percentage of basal blood glucose as a function of injection time. (C) Plasma insulin levels in 4-month male control and LTKO mice (n=12) after an overnight 16-hour fasting. (D) Plasma insulin levels in 4-month male control and LTKO mice (n=6) under <i>ad libitum</i> conditions. Data represent mean ± SEM. * indicates a significance with <i>P</i><0.05 in control vs. LTKO mice.</p

Gck knockdown impairs glucose tolerance in both wild-type and LTKO mice.

<p>(A) Gck protein was analyzed in the livers of 3-month-old control and LTKO mice by Western blots. (B) Gck knockdown was assessed by Western blots in liver lysates from control and LTKO mice injected with shGck or shGFP adenoviruses. (C, D) Glucose tolerance tests and insulin tolerance tests in 6-month-old male control and LTKO mice injected with shGck or shGFP adenoviruses (n=5-6), respectively. Data represent mean ± SEM. *, <i>P</i><0.05 between LTKO-shGFP and LTKO-shGck groups; #, <i>P</i><0.05 between loxp-shGFP and loxp-shGck groups.</p

Tomtom analysis results for conserved motifs and experimental validation.

<p><b>(A-D)</b> Transcription factors predicted for 20 consensus sequences (as query motif) by Tomtom analysis. Selected set of DHS overlapped motif aligning with their TF’s PWM (top) and query motif (bottom) with binding specificity mentioned by p-values. <b>(E-F)</b> Validation of <i>FOXO3</i> and <i>SOX2</i> binding to predicted BMo location in <i>SESN3</i> upstream region by ChIP analysis. <b>(G)</b> Overexpression of <i>FOXO3</i> and <i>SOX2</i> activated the <i>SESN3</i> gene expression in human HepG2 hepatoma cells. <b>(H)</b> Knockout of <i>FOXO3</i> or <i>SOX2</i> using CRISPR/Cas9 approach downregulated the <i>SESN3</i> gene in human HEK293 cells. (* p<0.05).</p

Transcripts of Human <i>SESN3</i> gene reported in ENSEMBL database.

<p>Transcripts of Human <i>SESN3</i> gene reported in ENSEMBL database.</p

Block diagram showing occurrence of conserved motifs.

<p><b>(A)</b> Location of twenty motifs identified and their distribution in 5 kb upstream sequences across human-<i>SESN3</i> & its other primate/rodent orthologous species were shown in the block diagram. The combined best matches of a sequence to a group of motifs were shown by combined p value. Sequence strand specified as “+” (input sequence was read from left to right) and “-” (input sequence was read on its complementary strand from right to left) with respect to the occurrence of motifs. Coordinates of each motif across species is shown as a sequence scale (from left to right, in blue) below the diagram. DNase I hypersensitive region was shown in 5kb upstream region of <i>SESN3</i> in <b>(B)</b> human cell lines and <b>(C)</b> mouse liver (8 week adult and 14.5 days embryo) using ENCODE project, represented by UCSC browser visualization tool. An overlap of DHS signal was found and shown as dark band over respective motifs in block diagram. The two coordinates on x-axis represents the <i>5kb upstream regions</i> as base distance (in blue) and genic distance (with respect to gene start site, in red) of <i>SESN3</i> gene.</p

E4orf1 suppresses glucose output in the presence of gluconeogenic stimulators in HepG2.

<p>(A) Western blot showing Ad36 E4orf1 protein expression in HepG2 cells following transfection with V5-Ad36 E4orf1 plasmid DNA. (B) Glucose output by HepG2 cells following E4orf1 or null Vector transfection. Cells were stimulated with gluconeogenic cAMP (1 mM) (C) and Dexamethasone (500 nM) (D) in the absence or presence of insulin (I). Expressed as mean mM glucose per mg of protein, ± SD. E4orf1 significantly suppresses glucose output in the basal and insulin stimulated conditions (p = 0.0001 and 0.003, respectively).</p

E4orf1 transfection increases Ras abundance and suppresses Glut2 levels in HepG2.

<p>A) Densitometry for Ras and Glut2 expression normalized to GAPDH. E4orf1 transfected cells have (i.) Significantly greater Ras expression in basal and insulin stimulated condition (p = 0.001 and 0.004, respectively) and (ii.) Significantly lower Glut2 levels in the basal condition (p = 0.04). B) Western blots of Ras and Glut2 expression in HepG2.</p