Whole-organism screening for gluconeogenesis identifies activators of fasting metabolism

Abstract

Improving the control of energy homeostasis can lower cardiovascular risk in metabolically compromised individuals. To identify new regulators of whole-body energy control, we conducted a high-throughput screen in transgenic reporter zebrafish for small molecules that modulate the expression of the fasting-inducible gluconeogenic gene pck1. We show that this in vivo strategy identified several drugs that affect gluconeogenesis in humans as well as metabolically uncharacterized compounds. Most notably, we find that the translocator protein ligands PK 11195 and Ro5-4864 are glucose-lowering agents despite a strong inductive effect on pck1 expression. We show that these drugs are activators of a fasting-like energy state and, notably, that they protect high-fat diet-induced obese mice from hepatosteatosis and glucose intolerance, two pathological manifestations of metabolic dysregulation. Thus, using a whole-organism screening strategy, this study has identified new small-molecule activators of fasting metabolism.

Figure 1. Rapid pharmacological profiling of gluconeogenesis

(a) the pck1 promoter is activated during the larval ‘feeding to fasting’ transition. The arrow points to the remaining yolk at 4 dpf, which is consumed by 5 dpf. The arrowhead points to Tg(pck1:Venus) expression in the pronephros. Exposure levels during image capturing were kept constant. Black scale bars, 200μm; white scale bars, 40μM. (b) Time course showing glucose levels and pck1 promoter dynamics from 3 to 10 dpf in never fed larvae. pck1 promoter activation starts after 3 dpf and is incremental until 7 dpf. Glucose levels reach a plateau between 4.5 and 6 dpf, before the calorie deficit leads to a net depletion. (c) Visualization of pck1 promoter dynamics in the liver using Tg(pck1:Venus) reporter zebrafish. Zebrafish larvae were treated with isoprenaline (Iso) and metformin (Met) from 4 to 6 dpf. (d) Glucose levels after treatment with isoprenaline and metformin (n=5×10 zebrafish/condition). **P < 0.01; ***P < 0.001; two-tailed t-test. Data are represented as mean values ± s.e.m..

Figure 2. A small molecule screen identifies functionally conserved as well as unknown modulators of gluconeogenesis

(a) Differential pck1 promoter activation by small molecules tested during the primary screen. 2400 compounds from two bioactive small molecule libraries were screened in duplicates. Neg Con = Negative Control, 1% DMSO; Pos Con = Positive Control, 10μM isoprenaline; Bud = Budenoside; Czp = Clozapine; Dox = Doxepin; For = Formoterol; FX = Fluoxetine; PK = PK 11195. (b) The scatterplot shows representative hit compounds found in the primary screen. FDA approved drugs that are known to influence glucose homeostasis in humans including glucocorticoids, beta-adrenergic agonists, tricyclic antidepressants (TriCA) and Czp activate Tg(pck1:Luc2) and increase glucose levels. Glucose concentrations are unaffected by FX and decrease after PK treatment despite high Tg(pck1:Luc2) activity suggesting compensatory gluconeogenesis (10μM from 3 to 5 dpf, P-values see Table 1). Blue dashed line indicates glucose levels of control animals. Orange dashed line indicates 1-fold log₂ change of pck1 promoter activity.

Figure 3. TSPO ligands enhance a gluconeogenic fasting response

(a,b) Time course analyses of pck1 promoter activity (a) and glucose levels (b) during the ‘feeding to fasting’ transition in control vs. PK 11195 treated zebrafish. Glucose levels are reduced at all time points despite incrementally increasing Tg(pck1:Luc2) activity (control vs. 10μM PK 11195, 0.1% DMSO, n=5×10 larvae per condition and time point). (c) Structure of the TSPO ligands PK 11195, PKD1, Ro5-4864 and the benzodiazepine Clonazepam. (d) PK 11195, PKD1 and Ro5-4864 enhance pck1 promoter activity and act synergistically during co-treatment with isoprenaline. (e) In contrast, baseline and isoprenaline stimulated glucose levels are reduced by TSPO ligand treatment [10μM isoprenaline (Iso), 10μM PK 11195 (PK), 10μM PKD1, 5μM Ro5-4864 (Ro), 0.1% DMSO; 4 to 6 dpf; n=5×10 larvae/condition]. (f,g) Dose-response curves of Ro5-4864, PK 11195 and Clonazepam effect on pck1 promoter activity (f) and glucose levels (g) (stimulation with 10μM isoprenaline; 4 to 6 dpf; n = 4 × 10/condition and dilution). *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test. Data are represented as mean values ± s.e.m..

Figure 4. TSPO ligands induce a fasting-like energy state in the liver

(a) MA-scatterplot of a microarray comparison of liver tissue of PK 11195 and vehicle treated zebrafish. PK 11195 induces expression of a Ppar-α target gene set (PTS, blue). A subset of this gene cluster, the Ppar-α enrichment set (PES, orange), shows enriched expression in the top ranks of differentially regulated genes compared to all genes (Gene comparison set, GCS, grey) (control vs. 10μM PK 11195 from 4 to 6 dpf, n=2/condition of ~ 20 livers). Orange dashed line, median value of the average log₂ signal intensity; blue dashed line, 90th percentile of all genes. (b) The 21 members of the PES gene set are also enriched at the top of the list of differentially regulated genes in mouse liver after a long physiological fast as shown by a Kolmogorov-Smirnov (K-S) statistic for cumulative distribution probability: Rank sorting of differentially regulated genes in fasted vs. fed mouse livers (C57BL/6, Standard Diet, n=3 microarray comparisons, 24h fast vs. fed) shows a significant enrichment of 19 out of the 21 PES orthologues [p < 10⁻¹⁰ by K-S statistic for all 21 genes]. Every horizontal black line marks one of the displayed genes rank sorted from top to bottom; yellow, up-regulated genes; blue, down-regulated genes. (c) qPCR analysis of PES genes in zebrafish livers (control vs. 10μM PK 11195, 6 dpf, n=5/condition) and (d) mouse livers (fed vs. fast; n = 3/condition, and fast + vehicle vs. fast + 2×5mg/kg bw PK 11195; 8h fast; n=6/condition; SD; 11–12 weeks male C57BL/6). *p < 0.05, **p < 0.01; two-tailed t-test. Data are represented as mean values ± s.e.m..

Figure 5. PK 11195 improves hepatosteatosis and glucose tolerance in diet-induced obese mice

(a) Hypoglycemic effects of PK 11195 treatment in fasted mice (1mg/kg bw PK 11195 in saline, standard diet, n=6/condition, 24h fast). (b) Representative images of Hematoxylin-Eosin (HE) and Oil-Red O (ORO) stained livers after 5 weeks of vehicle or PK 11195 treatment in diet-induced obese mice (1mg/kg bw PK 11195, s.c. injections for five days per week, n=5/condition, HFD from 10 to 19 weeks, start of intervention at 14 weeks). ORO staining shows reduced lipid accumulation in livers of the PK 11195 treatment group. Scale bar, 100μm (c) Srebp-1a protein levels are reduced in the liver in response to PK 11195 treatment (See Supplementary Figure 9 for uncut gels). (d) At four weeks of treatment, mice were subjected to an i.p. glucose tolerance test after a 6h fast. Two additional injections of PK 11195 (2mg/kg bw) were administrated at 8 am and 1h prior to the glucose challenge at 2 pm. PK 11195 treated animals show a significantly improved glucose tolerance (20min, P=0.029; 40min, P=0.015; 60min, P=0.032; 120min, P=0.025). (e) mRNA transcript levels were quantified by qPCR for two pro-inflammatory markers, TNFα and Il-6, from livers of control and PK 11195 treated mice (from (b), n = 4/condition). *P < 0.05; **P < 0.01; ***P < 0.001; two-tailed t-test. All data are represented as mean values ± s.e.m..