Fig 1.
A. 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. 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.
Fig 2.
Glucose and fatty acids increase NAD+ production in INS-1 cells.
A. 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. 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 vs. consumed during the 7-hr treatment (shown in the last 6 bars) using the formula described in Materials and Methods. C. 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. Data shown (mean ± S.E.M.) are representative of 4 (A-B) or 2 (C) independent experiments.
Fig 3.
Diverse fuels promote TNKS autoPARsylation and turnover in INS-1 cells.
A. Left panels: 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-IRAPaa78-109 (15 μg), which contained a hexapeptide sequence (IRAPaa96-101) that bound to the ANK domain of TNKS [49]. 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. Right panels: 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. 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. C. Cells were treated with FK866 (0.2 μM) or DMSO in the presence of 3 or 14 mM glucose. D. 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). E. Cells were treated with the indicated concentration of glucose along with a glucokinase activator (GKA, 10 μM RO-28-1675 [50]) or DMSO. F. 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 C-F, 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).
Fig 4.
Cell-type specificity of the glucose effects on TNKS.
A. Confluent cultures of mouse MIN6 insulinoma cells, human HEK293 renal epithelial cells, and mouse 3T3-L1 preadipocytes were treated in 24-well plates for 7 hr with glucokinase activator (10 μM GKA, lane 1) in 3 mM glucose, 3 mM glucose alone (lane 2), 14 mM glucose (lane 3), or XAV939 (4 μM, lane 4) in 3 mM glucose. Whole-cell extracts representing 10% of each well were immunoblotted for the indicated proteins. The immunoblots are representative of 2 independent experiments, each performed in 4 or 6 replicates per condition. The bar graphs indicate densitometer analysis of TNKS abundance (mean ± S.E.M). B. Confluent cultures of the indicated cell types were treated for 7 hr with 3 or 14 mM glucose as indicated by L (low) or H (high) in combination with XAV939 (4 μM) or DMSO. Lysates were incubated with GST as controls (lane 1 for MIN6; lane 5 for HEK293 and 3T3-L1) or with GST-WWE (15 μg, the remaining lanes) to precipitate PARsylated species as in Fig 3A. Lysates (upper panels) and the precipitates (lower panels) were immunoblotted for TNKS and PARP1. The precipitates were also Coomassie-stained for GST fusion proteins. Each lane represents a 15-cm plate (10 cm for MIN6 cells). PARsylated PARP1 was not detectable in 3T3-L1 cells. The data are representative of 2 or 3 experiments. C. Cells were treated with 3 (L) or 14 mM (H) glucose for 30 min before MG-132 (10 μM) or DMSO was added for another 30 min prior to harvesting for ubiquitination analysis as in Fig 3B. Each lane represents a 10-cm plate. The data are representative of 2 experiments, each performed in triplicate.