Nutritional energy stimulates NAD+ production to promote tankyrase-mediated PARsylation in insulinoma cells.

The poly-ADP-ribosylation (PARsylation) activity of tankyrase (TNKS) regulates diverse physiological processes including energy metabolism and wnt/β-catenin signaling. This TNKS activity uses NAD+ as a co-substrate to post-translationally modify various acceptor proteins including TNKS itself. PARsylation by TNKS often tags the acceptors for ubiquitination and proteasomal degradation. Whether this TNKS activity is regulated by physiological changes in NAD+ levels or, more broadly, in cellular energy charge has not been investigated. Because the NAD+ biosynthetic enzyme nicotinamide phosphoribosyltransferase (NAMPT) in vitro is robustly potentiated by ATP, we hypothesized that nutritional energy might stimulate cellular NAMPT to produce NAD+ and thereby augment TNKS catalysis. Using insulin-secreting cells as a model, we showed that glucose indeed stimulates the autoPARsylation of TNKS and consequently its turnover by the ubiquitin-proteasomal system. This glucose effect on TNKS is mediated primarily by NAD+ since it is mirrored by the NAD+ precursor nicotinamide mononucleotide (NMN), and is blunted by the NAMPT inhibitor FK866. The TNKS-destabilizing effect of glucose is shared by other metabolic fuels including pyruvate and amino acids. NAD+ flux analysis showed that glucose and nutrients, by increasing ATP, stimulate NAMPT-mediated NAD+ production to expand NAD+ stores. Collectively our data uncover a metabolic pathway whereby nutritional energy augments NAD+ production to drive the PARsylating activity of TNKS, leading to autoPARsylation-dependent degradation of the TNKS protein. The modulation of TNKS catalytic activity and protein abundance by cellular energy charge could potentially impose a nutritional control on the many processes that TNKS regulates through PARsylation. More broadly, the stimulation of NAD+ production by ATP suggests that nutritional energy may enhance the functions of other NAD+-driven enzymes including sirtuins

Diverse fuels promote TNKS autoPARsylation and turnover in INS-1 cells.

<p><b>A. Left panels:</b> Cells were pretreated with XAV939 (4 μM) or DMSO in the presence of 3 mM glucose for 30 min before glucose was raised to the indicated final concentration for 5 hr. Equal aliquots of the lysates were immunoblotted (upper panels). TNKS in the remaining lysates was precipitated by incubation with resins of GST-IRAP_aa78-109 (15 μg), which contained a hexapeptide sequence (IRAP_aa96-101) that bound to the ANK domain of TNKS [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122948#pone.0122948.ref049" target="_blank">49</a>]. The precipitates were immunoblotted sequentially for PAR and TNKS. Each lane corresponds to a 15-cm plate. The data shown are representative of 2 independent experiments. <b>Right panels:</b> Cells were treated for 7 hr with the indicated glucose concentration in combination with either XAV939 (4 μM) or DMSO. Equal aliquots of the lysates were immunoblotted (upper panels). PARsylated species in the remaining lysates were precipitated by incubation with resins of GST fused to the WWE domain of RNF146 (20 μg) and immunoblotted for TNKS (two exposures) and RNF146 (lower panels). Each lane corresponds to a 10-cm plate. The data shown are representative of 4 independent experiments. <b>B.</b> Cells were treated with 3 or 14 mM glucose for 30 min before MG-132 (10 μM) or vehicle (DMSO) was added for another 30 min. Equal aliquots of the lysates were immunoblotted (upper panels). Ubiquitinated species in the remaining lysates were precipitated by incubation with resins of GST fused to the ubiquitin-binding motifs of S5a (20 μg, lanes 1–4), using GST as negative control (20 μg, lane 5). The precipitates were sequentially immunoblotted with anti-TNKS antibodies to detect ubiquitinated TNKS, and with anti-ubiquitin antibodies for loading control. The bar graph shows densitometer analysis of ubiquitinated TNKS (mean ± SEM; n = 3). The data shown are representative of 2 independent experiments. <b>C.</b> Cells were treated with FK866 (0.2 μM) or DMSO in the presence of 3 or 14 mM glucose. <b>D</b>. Cells were treated with 3 mM glucose (lane 1), 1 mM NMN in combination with 3 mM glucose (lane 2), or 11 mM glucose (lane 3). <b>E.</b> Cells were treated with the indicated concentration of glucose along with a glucokinase activator (GKA, 10 μM RO-28-1675 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0122948#pone.0122948.ref050" target="_blank">50</a>]) or DMSO. <b>F.</b> Cells were treated with 3 mM glucose, either alone (lane 3) or in combination with alanyl-glutamine (10 mM in lane 1; 20 mM in lane 2) or pyruvate (10 mM, lane 4). For <b>C-F,</b> the treatment duration was 6–8 hr. Each lane represents 10% of whole-cell extracts from a well in 24-well plates. The immunoblots are representative of 2–3 experiments, each performed in 2–4 replicates. The bar graphs show densitometer quantification of TNKS abundance (mean ± SEM).</p

NAD⁺ analysis of INS-1 cells.

<p><b>A.</b> Confluent cultures of INS-1 cells were treated with the NAMPT inhibitor FK866 (0.2 μM) in the presence of 11 mM glucose for the indicated duration. NMN (200 μM) was added 90 min prior to the final time point. NAD⁺ content (mean ± S.E.M.) was plotted on a semilog scale as % of pretreatment level. <b>B.</b> Confluent INS-1 cultures were treated for 6 hr with the TNKS inhibitor XAV939 (4 μM), the PARP1 inhibitor PJ-34 (12 μM), or DMSO vehicle (0.1% v/v) in the presence of 11 mM glucose. NAD⁺ consumption (mean ± S.E.M.) during the treatment was determined as described in Materials and Methods.</p

Glucose and fatty acids increase NAD⁺ production in INS-1 cells.

<p><b>A.</b> Cells were treatment with 3 or 14 mM glucose in the presence of XAV939 (4 μM) or DMSO for 7 hr. ATP content was determined as described in Materials and Methods and normalized to protein content. <b>B.</b> Cells were grown to confluence in regular RPMI (11 mM glucose). For each 12-well plate, 2 wells served as untreated controls while the remainder were switched to fresh RPMI containing the indicated combination of glucose (3 or 14 mM), FK866 (0.2 μM), DMSO, and sodium azide (0.8 mM). Cells were lysed 7 hr later for NAD⁺ analysis. NAD⁺ contents (shown in the first 7 bars) were used to calculate the amount of NAD⁺ produced <i>vs</i>. consumed during the 7-hr treatment (shown in the last 6 bars) using the formula described in Materials and Methods. <b>C.</b> Cells were treated for 6 hr with 0, 0.2 or 0.3 mM fatty acids (a 2:1 mixture of linoleic acid and oleic acid) in the presence of 0.1 mM albumin and 3 mM glucose. NAD⁺ production during the treatment was determined as in <b>B.</b> Data shown (mean ± S.E.M.) are representative of 4 (<b>A</b>-<b>B</b>) or 2 (<b>C</b>) independent experiments.</p