Hypothalamic JNK1-hepatic fatty acid synthase axis mediates a metabolic rewiring that prevents hepatic steatosis in male mice treated with olanzapine via intraperitoneal: Additional effects of PTP1B inhibition

Abstract

Olanzapine (OLA), a widely used second-generation antipsychotic (SGA), causes weight gain and metabolic alterations when administered orally to patients. Recently, we demonstrated that, contrarily to the oral treatment which induces weight gain, OLA administered via intraperitoneal (i.p.) in male mice resulted in body weight loss. This protection was due to an increase in energy expenditure (EE) through a mechanism involving the modulation of hypothalamic AMPK activation by higher OLA levels reaching this brain region compared to those of the oral treatment. Since clinical studies have shown hepatic steatosis upon chronic treatment with OLA, herein we further investigated the role of the hypothalamus-liver interactome upon OLA administration in wild-type (WT) and protein tyrosine phosphatase 1B knockout (PTP1B-KO) mice, a preclinical model protected against metabolic syndrome. WT and PTP1B-KO male mice were fed an OLA-supplemented diet or treated via i.p. Mechanistically, we found that OLA i.p. treatment induces mild oxidative stress and inflammation in the hypothalamus in a JNK1-independent and dependent manner, respectively, without features of cell dead. Hypothalamic JNK activation up-regulated lipogenic gene expression in the liver though the vagus nerve. This effect concurred with an unexpected metabolic rewiring in the liver in which ATP depletion resulted in increased AMPK/ACC phosphorylation. This starvation-like signature prevented steatosis. By contrast, intrahepatic lipid accumulation was observed in WT mice treated orally with OLA; this effect being absent in PTP1B-KO mice. We also demonstrated an additional benefit of PTP1B inhibition against hypothalamic JNK activation, oxidative stress and inflammation induced by chronic OLA i.p. treatment, thereby preventing hepatic lipogenesis. The protection conferred by PTP1B deficiency against hepatic steatosis in the oral OLA treatment or against oxidative stress and neuroinflammation in the i.p. treatment strongly suggests that targeting PTP1B might be also a therapeutic strategy to prevent metabolic comorbidities in patients under OLA treatment in a personalized manner.

Keywords: Hypothalamus; Inter-organ crosstalk; Liver; Metabolic side-effects; Olanzapine; PTP1B.

Graphical abstract

Fig. 1

OLA dietary treatment promoted intrahepatic lipid accumulation in WT, but not in PTP1B–KO male mice. A. Representative images of liver sections with H&E (20x, scale bars-100 μm) (WT Chow diet: n = 3; WT OLA sup. diet: n = 3; PTP1B–KO Chow diet: n = 3; PTP1B–KO OLA sup. diet: n = 3) and ORO staining (WT Chow diet: n = 4; WT OLA sup. diet: n = 4; PTP1B–KO Chow diet: n = 4; PTP1B–KO OLA sup. diet: n = 4) and intrahepatic TGs (WT Chow diet: n = 9; WT OLA sup. diet: n = 9; PTP1B–KO Chow diet: n = 6; PTP1B–KO OLA sup. diet: n = 7, groups were compared using Student's t-test). B. Representative Western blot images of hepatic FAS with densitometric quantification normalized for Vinculin protein levels (WT Chow diet: n = 12; WT OLA sup. diet: n = 12; PTP1B–KO Chow diet: n = 12; PTP1B–KO OLA sup diet: n = 12). C. Quantification of hepatic lipid fluxes (WT Chow diet: n = 9; WT OLA sup. diet: n = 9; PTP1B–KO Chow diet: n = 6; PTP1B–KO OLA sup. diet: n = 7). D. RNAseq data: heatmap of hepatic gene expression comparing WT or PTP1B–KO mice fed an OLA supplemented diet versus each genotype-matched control group (fed a chow diet). The log₂(fold change) for each gene is presented. E. Principal categories of steatosis-related gene families regulated in WT mice fed an OLA supplemented diet in comparison to WT mice fed a chow diet. F. WT male mice received a daily oral gavage of OLA (10 mg/kg) or VEH during 8 weeks. Representative Western blot images of FAS protein levels in liver and densitometric quantification normalized for Vinculin protein levels (WT VEH: n = 6; WT OLA: n = 6, groups were compared using Student's t-test). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05.

Fig. 2

OLA i.p. administration prevented intrahepatic lipid accumulation in WT and PTP1B–KO male mice despite of FAS upregulation found in WT mice. WT and PTP1B–KO male mice received a daily i.p. injection of OLA (10 mg/kg) during 8 weeks. A. Representative images of liver sections with H&E (20x, scale bars-100 μm) (WT VEH: n = 3; WT OLA: n = 3; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3) and ORO staining (WT VEH: n = 3; WT OLA: n = 3; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3) and intrahepatic TGs (WT VEH: n = 4; WT OLA: n = 5; PTP1B–KO VEH: n = 8; PTP1B–KO OLA: n = 9, groups were compared using Student's t-test). B. Representative Western blot images of hepatic FAS and densitometric quantification normalized for Vinculin protein levels (WT VEH: n = 5; WT OLA: n = 5; PTP1B–KO VEH: n = 7; PTP1B–KO OLA: n = 6). C.Fasn, Insig1, Insig2 and Srebf1 mRNA levels using Tbp and Actb as housekeeping genes (WT VEH: n = 5; WT OLA: n = 5; PTP1B–KO VEH: n = 7; PTP1B–KO OLA: n = 8). D. ATP levels in livers of male mice from both genotypes (WT VEH: n = 15; WT OLA: n = 18; PTP1B–KO VEH: n = 10; PTP1B–KO OLA: n = 14). E. Representative Western blot images of hepatic ACC and AMPK phosphorylation and densitometric quantification normalized for Vinculin protein levels (WT VEH: n = 4–5; WT OLA: n = 5; PTP1B–KO VEH: n = 6–7; PTP1B–KO OLA: n = 5–6). F. Representative Western blots of FAS and ACC and AMPK phosphorylation in primary hepatocytes from OLA-treated mice and densitometric quantification normalized for Vinculin protein levels (WT VEH: n = 3–4; WT OLA: n = 3–4; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3). G. Cell viability of mouse primary hepatocytes treated in vitro with OLA for 48 h (12.5 μM) using the crystal violet method (DMSO: n = 5; OLA: n = 5, groups were compared using Student's t-test) (left panel). ORO staining (middle panel, 5x, scale bars-2 mm) and respective zoom and the corresponding quantification in mouse primary hepatocytes treated OLA for 48 h (12.5 μM) (DMSO: n = 5; OLA: n = 5, groups were compared using Student's t-test) (left panel). H. Representative Western blot images of FAS and pACC protein levels of mouse primary hepatocytes treated as detailed in G. Densitometric quantification normalized for Vinculin protein levels (n = 4). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Hypothalamic JNK phosphorylation paralleled with increased hepatic FAS levels in WT male mice treated with OLA via i.p. A. Representative Western blot images of hypothalamic JNK phosphorylation and total JNK and densitometric quantification normalized for total JNK and Vinculin protein levels, respectively (WT VEH: n = 4; WT OLA: n = 7; PTP1B–KO VEH: n = 6; PTP1B–KO OLA: n = 7). B. Representative Western blot images of JNK phosphorylation and total JNK in GT1-7 hypothalamic neurons (12.5 μM, 5 min, DMSO: n = 3; OLA: n = 3 independent experiments, groups were compared using Student's t-test), microglia (12.5 μM, 60 min, DMSO: n = 5; OLA: n = 5 independent experiments, groups were compared using Student's t-test) and astrocytes (12.5 μM, 60 min, DMSO: n = 3; OLA: n = 3 independent experiments, groups were compared using Student's t-test) treated with OLA. Densitometric quantification of phospho-JNK and total JNK normalized for total JNK and Vinculin protein levels, respectively. C. Experimental design (left panel). Representative Western blot images of hypothalamic JNK phosphorylation and total JNK in WT male mice 30 min after receiving an intrahypothalamic injection with OLA (15 nmol) (middle panel). Densitometric quantification of phospho-JNK and total JNK normalized for total JNK and Vinculin protein levels, respectively (right panel) (DMSO: n = 7; OLA: n = 9, groups were compared using Student's t-test). D. Representative Western blot images of hepatic FAS in WT male mice 48 h after receiving an intrahypothalamic injection with OLA (15 nmol) and densitometric quantification normalized for Vinculin levels (WT DMSO: n = 7; WT OLA: n = 10, groups were compared using Student's t-test). E. Representative Western blot images of hepatic pACC and pAMPK in WT male mice 30 min, 8 and 48 h after receiving an intrahypothalamic injection with OLA (15 nmol) and densitometric quantification normalized for Vinculin levels (30 min: WT DMSO: n = 7; WT OLA: n = 8; 8 h: WT DMSO: n = 3; WT OLA: n = 3; 48 h: WT DMSO: n = 5; WT OLA: n = 7, groups were compared using Student's t-test). F. WT male mice were vagotomized 7 days prior to OLA (15 nmol) intrahypothalamic injection and after 48 h livers were collected. Representative Western blot images of hepatic FAS and densitometric quantification normalized for Vinculin levels (Sham: DMSO: n = 3; OLA: n = 4; Vagotomized: DMSO: n = 6, OLA: n = 6). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 4

JNK1 deletion prevented OLA-induced FAS upregulation in the liver. A. Experimental design of the intrahypothalamic injection of adeno-associated viruses encoding Cre recombinase in C57BL/6J male JNK2-KO/JNK1^flox-flox mice 4 days prior to OLA i.p. daily injections for 8 weeks after which hypothalamus and liver were collected. Representative Western blot images of hypothalamic total JNK and Vinculin protein levels. B. Representative Western blot images of hepatic FAS in mice treated as in A, and densitometric quantification normalized for Vinculin levels (AAV-GFP OLA: n = 8; AAV-Cre OLA: n = 7, groups were compared using Student's t-test). C. Body weight variance at the beginning (week 0) and end (week 8) of the OLA i.p. treatment (AAV-GFP: n = 8; AAV-Cre: n = 8) (left panel). Body weight variance at the end of the OLA i.p. treatment (AAV-GFP: n = 8; AAV-Cre: n = 8, groups were compared using Student's t-test) (right panel). D. Thermographic pictures and quantification of BAT (left panel) and tail (right panel) maximal temperature of male mice via i.p. with OLA (AAV-GFP: n = 4; AAV-Cre: n = 4, groups were compared using Student's t-test). E. Representative Western blot of BAT UCP-1 and densitometric quantification normalized for Vinculin levels in mice treated with OLA via i.p. (AAV-GFP: n = 13; AAV-Cre: n = 12, groups were compared using Student's t-test). F. Representative Western blot of hypothalamic phospho-JNK protein levels in male mice injected AMPKα1-CA or GFP adenoviruses 5 days prior to OLA or DMSO intrahypothalamic injection for 30 min before sampling and respective densitometric quantification normalized for Vinculin levels (AMPKα1-CA VEH: n = 4; AMPKα1-CA OLA: n = 5, groups were compared using Student's t-test). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 5

OLA i.p. administration decreases antioxidant metabolites and increases lipid peroxidation in the hypothalamus of WT male mice. A. Metabolomic analysis of the hypothalamus showing vitamins and related molecules, amino acids, neurotransmitters and intermediates, nucleosides and nucleotides, bioenergetics substrates and intermediates of metabolic pathways (WT VEH: n = 6; WT OLA: n = 6; PTP1B–KO VEH: n = 6; PTP1B–KO OLA: n = 6, groups were compared using Student's t-test). B. Lipidomic analysis of the hypothalamus from mice receiving OLA via i.p. (WT VEH: n = 6; WT OLA: n = 6; PTP1B–KO VEH: n = 6; PTP1B–KO OLA: n = 6, groups were compared using Student's t-test). C. Representative images of hypothalamic sections analyzed by immunohistochemistry against neuroketals and 3-nitrotyrosine in WT and PTP1B–KO male mice i.p. injected with OLA or VEH (20x, scale bars-100 μm) (WT VEH: n = 3; WT OLA: n = 3; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3). D. Representative LDI-MS imaging (one sample per group) of peroxidized lipid species corresponding to (left to right)1. PA (22:2; O), C₂₅H₄₅O₉P, [M+H–H2O]⁺; 2. PA (21:1; O2), C₂₄H₄₅O₁₀PNa, [M+Na]⁺; 3. PA (27:2; O), C₃₀H₅₅O₉PNa, [M+Na]⁺; 4. PE (24:2; O3); C₂₉H₅₄NO₁₁P, [M+H]⁺; 5. PA (27:2; O), C₃₀H₅₅O₉PK, [M+K]⁺; 6. PA (27:2; O), C₃₀H₅₅O₉PK, [M+K]⁺; 7. PA (27:2; O2), C₃₀H₅₅O₉PK, [M+K]⁺; 8. PA (27:2; O2), C₃₀H₅₅O₁₀PK , [M+K]⁺; 9. PC (23:3; O2), C₃₁H₅₆NO₁₀PNa, [M+Na]⁺; 10. PE (26:3; O2), C₃₁H₅₆NO₁₀PNa, [M+Na]⁺; 11. PC (25:1; O), C₃₃H₆₄NO₉PNa, [M+Na]⁺; 12. PE (30:3; O2), C₃₅H₆₄NO₁₀P, [M+H–H2O]⁺; 13. PC (25:3; O2), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 14. PE (28:3; O2), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 15. PC (25:3; O3), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 16. PS (27:2; O), C₃₃H₆₀NO₁₁PNa , [M+Na]⁺; 17. PC (25:1; O2), C₃₃H₆₄NO₁₀PK, [M+K]⁺; 18. PC (25:3; O3), C₃₃H₆₀NO₁₁PK, [M+K]⁺; 19. PS (27:2; O), C₃₃H₆₀NO₁₁PK, [M+K]⁺; 20. PE (30:3; O3), C₃₅H₆₄NO₁₁PNa, [M+Na]⁺; 21. PG (30:3; O2), C₃₆H₆₅O₁₂PNa, [M+Na]⁺; 22. PG (30:3; O2), C₃₆H₆₅O₁₂PK, [M+K]⁺ (more information in Supplementary Table 4). Intensity of each ion (arbitrary units) is color-coded (WT VEH: n = 3; WT OLA: n = 3; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3). E. Representative confocal images of MitoTracker Red CM-H₂Xros staining in GT1-7 hypothalamic neurons stimulated with OLA (12.5 μM) during a short time-period (4 min) with or without pre-treatment with a selective PTP1B inhibitor (iPTP1B, 20 μM, 2 h) and quantification (DMSO: n = 16; OLA: n = 15; iPTP1B: DMSO: n = 8; OLA: n = 7 independent experiments). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

Effects of OLA i.p. administration in astrocyte and microglia activation in the hypothalamus: protective effect of PTP1B inhibition.A. T2 maps of the brain from WT and PTP1B–KO mice treated with OLA via i.p. (WT VEH: n = 5; WT OLA: n = 4; PTP1B–KO VEH: n = 4; PTP1B–KO OLA: n = 4). B. NMR images of the brain from mice receiving an intrahypothalamic OLA injection at 48 h post-injection. Inflamed area is indicated by a white square (WT VEH: n = 5; WT OLA: n = 5). C. Representative confocal images of Iba1 and GFAP immunofluorescence (20x (upper panel), scale bars-100 μm and 63x (lower panel), scale bars-50 μm) of OLA-treated mice via i.p. (WT VEH: n = 4; WT OLA: n = 5; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3; left panels) and quantification of GFAP⁺ cells (WT VEH: n = 4; WT OLA: n = 5; PTP1B–KO VEH: n = 3; PTP1B–KO OLA: n = 3, right panel). D. Representative Western blot images of IκBα in microglia and astrocytes pre-treated with a selective PTP1B inhibitor (iPTP1B, 20 μM, 2 h) prior to OLA addition (12.5 μM, 30 min) and their respective densitometric quantifications normalized for Vinculin levels (Microglia: DMSO: n = 9; OLA: n = 9; iPTP1B: DMSO: n = 6; OLA: n = 6; Astrocytes: DMSO: n = 11; OLA: n = 11; iPTP1B: DMSO: n = 6; OLA: n = 6 independent experiments). Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001.

Fig. 7

JNK1/2 deletion in the hypothalamus of mice treated with OLA via i.p. prevented hypothalamic inflammation, but not oxidative stress.A. Representative images of neuroketals and 3-nitrotyrosine immunohistochemistry of hypothalamic sections of C57BL/6J JNK2-KO/JNK1^flox-flox mice injected AAV-GFP or AAV-Cre in the hypothalamus prior to the treatment with OLA via i.p. (20x, scale bars-100 μm) (AAV-GFP OLA: n = 5; AAV-Cre OLA: n = 5, groups were compared using Student's t-test). B. Representative LDI-MS imaging (one sample per group) of peroxidized lipid species corresponding to (left to right)1. PA (22:2; O), C₂₅H₄₅O₉P, [M+H–H2O]⁺; 2. PA (21:1; O2), C₂₄H₄₅O₁₀PNa, [M+Na]⁺; 3. PA (27:2; O), C₃₀H₅₅O₉PNa, [M+Na]⁺; 4. PE (24:2; O3); C₂₉H₅₄NO₁₁P, [M+H]⁺; 5. PA (27:2; O), C₃₀H₅₅O₉PK, [M+K]⁺; 6. PA (27:2; O), C₃₀H₅₅O₉PK, [M+K]⁺; 7. PA (27:2; O2), C₃₀H₅₅O₉PK, [M+K]⁺; 8. PA (27:2; O2), C₃₀H₅₅O₁₀PK, [M+K]⁺; 9. PC (23:3; O2), C₃₁H₅₆NO₁₀PNa, [M+Na]⁺; 10. PE (26:3; O2), C₃₁H₅₆NO₁₀PNa, [M+Na]⁺; 11. PC (25:1; O), C₃₃H₆₄NO₉PNa, [M+Na]⁺; 12. PE (30:3; O2), C₃₅H₆₄NO₁₀P, [M+H–H2O]⁺; 13. PC (25:3; O2), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 14. PE (28:3; O2), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 15. PC (25:3; O3), C₃₃H₆₀NO₁₀PNa, [M+Na]⁺; 16. PS (27:2; O), C₃₃H₆₀NO₁₁PNa, [M+Na]⁺; 17. PC (25:1; O2), C₃₃H₆₄NO₁₀PK, [M+K]⁺; 18. PC (25:3; O3), C₃₃H₆₀NO₁₁PK, [M+K]⁺; 19. PS (27:2; O), C₃₃H₆₀NO₁₁PK, [M+K]⁺; 20. PE (30:3; O3), C₃₅H₆₄NO₁₁PNa, [M+Na]⁺; 21. PG (30:3; O2), C₃₆H₆₅O₁₂PNa, [M+Na]⁺; 22. PG (30:3; O2), C₃₆H₆₅O₁₂PK [M+K]⁺ (more information in Supplementary Table 4).), in hypothalamic sections of JNK2-KO/JNK1^flox-flox mice injected AAV-GFP and AAV-Cre in the hypothalamus prior to the treatment with OLA via i.p. Intensity of each ion (arbitrary units) is color-coded (AAV-GFP OLA: n = 3; AAV-Cre OLA: n = 3). C. Representative confocal images from MitoTracker Red CM-H₂Xros staining of GT1-7 hypothalamic neurons pre-treated with SP600125 (20 μM, 2 h) or NAC (10 mM, 2 h) prior to OLA (12.5 μM) stimulation during a short time-period (4 min) and quantification (SP600125: DMSO: n = 8; OLA: n = 8; NAC: DMSO: n = 7; OLA: n = 7). D. Representative Western blot images of phosphorylated and total JNK in GT1-7 hypothalamic neurons cell line pre-treated with NAC (10 mM, 2 h) prior to OLA addition (12.5 μM, 5 min). Densitometric quantification of phospho-JNK and total JNK normalized for total JNK and Vinculin protein levels, respectively (DMSO: n = 5; OLA: n = 5; NAC: DMSO: n = 5; OLA: n = 5 independent experiments). E. Representative confocal images of Iba1 and GFAP immunofluorescence (20x, scale bars-100 μm and 63x, scale bars-50 μm) in hypothalamic sections of JNK2-KO/JNK1^flox-flox mice injected AAV-GFP and AAV-Cre in the hypothalamus prior to the treatment with OLA via i.p. (AAV-GFP OLA: n = 5; AAV-Cre OLA: n = 4) and quantification of GFAP⁺ cells (right panels, AAV-GFP OLA: n = 5; AAV-Cre OLA: n = 4, groups were compared using Student's t-test). F. Representative Western blot images of IκBα in microglia (left panels, n = 6 independent experiments, groups were compared using Student's t-test) and astrocytes (right panels, n = 6 independent experiments, groups were compared using Student's t-test) pre-treated with SP600125 (20 μM, 2 h) prior to OLA (12.5 μM, 60 min) stimulation and their respective densitometric quantifications normalized for Vinculin levels. Results are expressed in fold of change versus DMSO without SP600125 condition. Each point/bar corresponds to mean ± SEM; comparisons between groups: *p < 0.05; **p < 0.01; ***p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)