ABA induces the increase of the [cAMP]_i in hNCI-H716 cells.

<p>(A) hNCI-H716 cells were incubated for the indicated time in the absence or presence of 200 μM ABA (squares), or of 10 mM glutamine (rhombi); [cAMP]_i was then measured on cell extracts. Data are mean±SD of at least 3 different experiments; *, p<0.05 compared with untreated cells; #, p<0.05 compared to glutamine-treated cells (for the same time). (B) hNCI-H716 cells were transfected with an empty plasmid (control) or with a LANCL2-containing plasmid (LANCL2). After 48 h from transfection, cells were lysed and a Western blot analysis was performed using an anti-LANCL2 monoclonal antibody [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0140588#pone.0140588.ref019" target="_blank">19</a>]; a representative blot is shown, confirming LANCL2 overexpression after transfection. LANCL2 expression was normalized on vinculin levels. (C) After 48 h from transfection, cells were stimulated for 2.5 min in the absence or presence of 200 μM ABA. [cAMP]_i was measured on cell extracts and data, expressed as fold increase over values in unstimulated cells, are expressed as mean±SD of at least 3 different experiments; basal cAMP values were not significantly different upon transfection. *, p<0.05 compared to control. (D) After 48 h from transfection, cells were incubated for 2 h in the absence or presence of 200 μM ABA. GLP-1 levels in the culture media were then estimated with an ELISA kit. Data, expressed as fold increase over values in unstimulated cells, are expressed as mean±SD of at least 3 different experiments. *, p<0.05 compared to untreated cells.</p

ABA induces GLP-1 release and transcription in hNCI-H716 cells.

<p>(A) hNCI-H716 cells were incubated for 2 h in the absence or presence of ABA (at the indicated concentrations), or of 200 mM glucose or 10 mM glutamine (gln). In some experiments, cells were pre-incubated for 10 min in the absence or presence of 20 μM 2′,3′-Dideoxyadenosine, a specific adenylyl cyclase inhibitor (grey bar) or of 1 μM of a cell permeable PKA inhibitor (protein kinase A inhibitor 14–22 amide, myristoylated, black bar), prior to stimulation with 200 μM ABA. GLP-1 levels in the culture media were then estimated with an ELISA kit. Data, expressed as fold increase over values in untreated cells, are expressed as mean±SD of at least 3 different experiments. *, p<0.05 compared to untreated cells. (B) hNCI-H716 cells were incubated for 2 h in the absence or presence of 200 μM ABA and qPCR was performed with specific primers for GLP-1 and glucagon; *, p<0.05 compared to expression in untreated cells.</p

Effect of oral ABA on plasma GLP-1, insulin and glucose levels in rats.

<p>ABA (50 mg/Kg, black squares) or vehicle alone (open squares) were orally administered to rats pre-treated with Sitagliptin (6 animals per experimental group) and blood samples were collected at 0, 20, 40 and 60 min to evaluate plasma GLP-1 (A), insulin (C) and glucose (E). The AUC corresponding to the curves of GLP-1 (B), insulin (D) and glycemia (F) were calculated. Inset to panel A: blood samples were collected from the portal vein of rats not pre-treated with Sitagliptin, 10 min after ABA or vehicle administration and GLP-1 levels were evaluated (n = 5 rats per group). *, p<0.05 and **, p<0.01 compared with the corresponding value in vehicle-treated animals; #, p<0.05 and ##, p<0.01 compared with time zero.</p

Discovery of Novel and Selective SIRT6 Inhibitors

SIRT6 is an NAD⁺-dependent deacetylase with a role in the transcriptional control of metabolism and aging but also in genome stability and inflammation. Broad therapeutic applications are foreseen for SIRT6 inhibitors, including uses in diabetes, immune-mediated disorders, and cancer. Here we report on the identification of the first selective SIRT6 inhibitors by in silico screening. The most promising leads show micromolar IC₅₀s, have significant selectivity for SIRT6 versus SIRT1 and SIRT2, and are active in cells, as shown by increased acetylation at SIRT6 target lysines on histone 3, reduced TNF-α secretion, GLUT-1 upregulation, and increased glucose uptake. Taken together, these results show the value of these compounds as starting leads for the development of new SIRT6-targeting therapeutic agents

ABAp increases after an oral glucose load in healthy subjects, but not in T2D patients.

<p>After overnight fasting, a pre-test blood sample was taken from 7 healthy subjects and from 9 T2D patients, all of whom subsequently underwent a standard OGTT. The values of plasma ABA (A), glucose (B) and insulin (C) shown are the mean ± SD from the healthy controls (black rhombi) and from the T2D subjects (grey squares). * p<0.05 relative to time zero values.</p

Fasting ABAp in NGT subjects and T2D patients.

<p>Fasting ABAp was determined by HPLC-MS in 21 male T2D patients (squares) and in 27 sex-, age- and BMI-matched NGT subjects (rhombi). Results are ordered by increasing value. The circled areas indicate the possible existence of two sub-groups within the T2D patients, one with higher-than-normal ABAp levels and one with ABAp values similar to those of the NGT group. Inset: a box-and-whisker plot drawn from the same data sets. * p = 0.013</p

Pre-partum impairment and post-partum restoration of the ABAp increase after oral glucose load in GDM subjects.

<p>The values of plasma ABA (A), glucose (B) and insulin (C) shown are the mean ± SD from seven NGT (black rhombi) and from nine GDM subjects (grey squares), who underwent a standard OGTT at the 24^th-28^th week (pre-partum) and again 2–3 months after childbirth (post-partum). Post-partum restoration of the ABAp increase during OGTT in the GDM subjects was accompanied by restoration of a normal glycemic profile. * p<0.05 compared to time zero values; ^§ p<0.05 compared to NGT.</p

Higher fasting ABAp in T2D patients compared to NGT controls.

<p>Higher fasting ABAp in T2D patients compared to NGT controls.</p

Diminished increase of ABAp after oral glucose load in GDM and reversal to normal after childbirth.

<p>Diminished increase of ABAp after oral glucose load in GDM and reversal to normal after childbirth.</p