Volume 22, Issue 1, Pages 163-174 (January 2018) Neuronal PAS Domain Protein 4 Suppression of Oxygen Sensing Optimizes Metabolism during Excitation of Neuroendocrine Cells  Paul V. Sabatini, Thilo Speckmann, Cuilan Nian, Maria M. Glavas, Chi Kin Wong, Ji Soo Yoon, Tatsuya Kin, A.M. James Shapiro, William T. Gibson, C. Bruce Verchere, Francis C. Lynn  Cell Reports  Volume 22, Issue 1, Pages 163-174 (January 2018) DOI: 10.1016/j.celrep.2017.12.033 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 NPAS4 Inhibits the Hypoxic Response in Pancreatic β Cells (A–E) Transduction of MIN6 cells with an adenovirus driving Npas4 expression (Ad-Npas4) significantly blunted the induction of the HIF1α target genes Ldha (A), Slc16a3 (B), Pdk1 (C), and Vegfa (D) following 6 hr in 1% O2, compared to cells infected with control virus (Ad-GFP) (n = 3). Co-immunoprecipitation experiments confirmed significantly reduced HIF1α binding to ARNT in Ad-Npas4-transduced MIN6 cells following 4 hr in 1% O2 (E), compared to Ad-GFP-infected cells (n = 4). IP, immunoprecipitation. (F–H) Transduction of dispersed C57BL/6 mouse islets with Ad-Npas4 inhibited the induction of HIF1α targets Slc16a3 (F), Ldha (G), and Sox9 (H) following 24 hr in 1% O2 (n = 5). (I and J) Ad-Npas4-transduced human islets exhibited reduced induction of HIF1α target genes LDHA (I) and SOX9 (J) following 24 hr in 1% O2 (n = 5). (K and L) The expression of Ldha (K) and Slc16a3 (L) were significantly increased in islets from Npas4flox/flox; Pdx1CreER+ (PN4 KO) mice, following 2.5 hr of exposure to 25 mM glucose and the PHD enzyme inhibitor JNJ-42041935 (100 μM) (n = 4). (M and N) 9-week-old PN4 KO islets have significantly impaired glucose-stimulated oxygen consumption and reduced maximal mitochondrial respiratory capacity. Area under the curve (N) was calculated from curves shown in (M) (n = 8). OCR, oxygen consumption rate; AUC, area under the curve; 2.8G/25G = 2.8 mM/25 mM glucose; Olm, oligomycin; Ant/Rot, antimycin A/rotenone. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, by Student’s t test or Mann-Whitney U test as appropriate. All data are presented as mean ± SEM. See also Figure S1. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Pdx1CreER-Mediated Deletion of Npas4 (PN4 KO) Results in Fasting Hyperglycemia, Glucose Intolerance, and β Cell Dedifferentiation (A) Weight gain was similar between control and PN4 KO mice on both chow (CHW) and HFD diets following tamoxifen administration, except for a mildly increased weight gain of HFD-fed PN4 KO mice early during HFD feeding (see inset; n = 15). (B) HFD-fed PN4 KO mice had significantly increased cumulative incidence of fasting hyperglycemia, defined by two consecutive glucose levels of 10 mM or higher following a 12-hr overnight fast, compared to diet-fed controls (n = 15). (C and D) Following 25 weeks of HFD feeding, HFD-fed PN4 KO mice were glucose intolerant in response to a 1 g/kg oral glucose bolus. Area under the curve (D) was calculated from curves shown in (C) (n = 19). (E) During high glucose in vitro insulin secretion, islets from HFD PN4 KO mice secreted significantly less insulin than diet-fed controls (n = 4). (F and G) No significant difference in β cell (F) or α cell (G) numbers were observed in PN4 KO mice compared to the diet-matched controls (n = 3). (H and I) Islets from PN4 KO have higher Sox9 mRNA (H) and protein (I) expression after 25 weeks on HFD (n = 4). (J) Increased numbers of non-insulin immunoreactive (INS−), β cell-lineage (GFP+) cells in both CHW- and HFD-fed PN4 KO islets (n = 3). (K) Increased numbers of glucagon immunoreactive (GCG+), β cell-lineage (GFP+) cells in both CHW- and HFD-fed PN4 KO islets (n = 3). (L) Representative images from HFD controls and CHW- and HFD-fed PN4 KO islets used for lineage-tracing GFP+ β cells, costained with insulin and glucagon (n = 3). Red/orange, IN4 KO; black/gray, control; solid lines, high-fat diet; dotted lines, control diet. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, by Student’s t test, Mann-Whitney U test, or one-way ANOVA with Dunnett’s post-test as appropriate. Scale bars, 25 μM. All data are presented as mean ± SEM. See also Figures S2 and S3. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 HFD-Fed PN4 KO Mice Demonstrate Alterations in Food Intake and Locomotor Activity (A) HFD PN4 KO mice displayed elevated fat mass compared to HFD-fed control mice (n = 6). (B) Light-phase (ZT0–11) food intake was significantly increased compared to diet-fed controls (n = 6). (C and D) Increased light-phase food intake was driven by increased meal size (D) as opposed to increased eating frequency (C; n = 6). (E–G) No alterations in water intake (E), frequency of drinking (F), or drink volume (G) were observed (n = 6). (H) Dark-phase (ZT12–23) locomotor activity was significantly reduced in HFD-fed PN4 KO mice (n = 6). Red, PN4 KO; black, littermate control. ∗p < 0.05; ∗∗p < 0.01 by Student’s t test or Mann-Whitney U test as appropriate. All data are presented as mean ± SEM. See also Figure S2. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 PN4 KO Mice Display an Elevated Response to Gluconeogenic Substrates and Impairments in Insulin Sensitivity (A and B) PN4 KO mice have elevated blood glucose levels during a 2 g/kg pyruvate tolerance test following 7 weeks of HFD feeding (A and B). Area under the curve (B) was calculated from curves shown in (A) (n = 6). (C and D) PN4 KO mice display increased blood glucose levels in response to a 2 g/kg glycerol tolerance test following 11 weeks of HFD feeding. Area under the curve (D) was calculated from curves shown in (C) (n = 12). (E and F) PN4 KO mice respond normally to a 0.75 U/kg insulin tolerance test following 7 weeks of HFD feeding. Area over the curve (F) was calculated from curves shown in (E) (n = 7). (G and H) PN4 KO mice have decreased response during a 0.75 U/kg insulin tolerance test at 11 weeks of HFD feeding. Area over the curve (G) was calculated from curves shown in (H) (n = 8). (I and J) Fasting plasma insulin (I ) and glucagon (J) levels are unaltered after 7 and 5 weeks of diet feeding, respectively, in both CHW- and HFD-fed PN4 KO mice (n = 4). Red, PN4 KO; black, control; solid lines, high-fat diet; dotted lines, control diet. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 by Student’s t test or Mann Whitney U test as appropriate. All data are presented as mean ± SEM. See also Figure S2. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 IN4 KO Mice Exhibit Later Onset of Glucose Intolerance than PN4 KO Mice (A) No alterations in body mass composition were observed in HFD-fed Npas4flox/flox; Ins1WT/Cre+ (IN4 KO) mice (n = 16). (B) Fasting glycemia was not elevated in HFD-fed IN4 KO mice (n = 16). (C–F) HFD-fed IN4 KO mice show no differences in body mass composition (C), food (D), or water (E) intake or activity (F) at 7 weeks of HFD feeding (n = 4). (G) Pyruvate tolerance was not different in IN4 KO mice compared with littermate controls following 7 weeks of HFD (n = 5). (H) Glycerol tolerance was not different in IN4 KO mice compared with littermate controls following 11 weeks of HFD (n = 6). (I) Insulin tolerance was not different in IN4 KO mice compared with littermate controls following 7 weeks of HFD (n = 6). (J and K) Following 17 weeks of HFD feeding, HFD-fed IN4 KO mice were glucose intolerant during a 2 g/kg oral glucose tolerance test. Area under the curve (K) was calculated from curves shownin (J) (n = 14). Red/orange, IN4 KO; black/gray, control; solid lines, high-fat diet; dotted lines, control diet. ∗p < 0.05, by Student’s t test. All data are presented as mean ± SEM. See also Figures S4, S5, and S6. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions

Figure 6 Depolarization-Mediated Induction of NPAS4 Is Blunted by Hypoxia and T2D (A and B) HIF1α expression was elevated in human islets following 4 hr of incubation with 5% O2 and 25 mM glucose, when compared to HIF1α expression at 2.8 mM glucose. HIF1α expression levels were calculated densitometrical (B) from western blots such as shown in (A) (n = 3). (C) Preincubation (24 hr) with the PHD enzyme inhibitor JNJ-42 significantly reduces glucose-stimulated Npas4 induction in mouse islets (n = 5). (D) High-glucose (25 mM)-mediated Npas4 induction in mouse islets was significantly reduced following 2 hr of stimulation in 1% O2 compared to 20% O2 (n = 6). (E) Human islets incubated for 2 hr at high glucose (25 mM) and low oxygen (1% O2) showed reduced NPAS4 protein levels compared to expression at control oxygen levels (20% O2). Representative of n = 3. (F) Mouse islets preincubated in 10% O2 for 24 hr prior to 2 hr of stimulation in 20% O2 and either low (2.8 mM) or high (25 mM) glucose had significantly elevated basal and decreased stimulatory Npas4 gene expression, respectively (n = 4). (G) Glucose-stimulated NPAS4 induction is lost in islets from those with T2D (n = 4). ∗p < 0.05; ∗p < 0.01, by Student’s t test, Mann-Whitney U test, or ANOVA followed by Tukey post-test as appropriate. All data are presented as mean ± SEM. Cell Reports 2018 22, 163-174DOI: (10.1016/j.celrep.2017.12.033) Copyright © 2017 The Author(s) Terms and Conditions