Improved metabolic phenotype of hypothalamic PTP1B-deficiency is dependent upon the leptin receptor

Abstract

Protein tyrosine phosphatase 1B (PTP1B) is a known regulator of central metabolic signaling, and mice with whole brain-, leptin receptor (LepRb) expressing cell-, or proopiomelanocortin neuron-specific PTP1B-deficiency are lean, leptin hypersensitive, and display improved glucose homeostasis. However, whether the metabolic effects of central PTP1B-deficiency are due to action within the hypothalamus remains unclear. Moreover, whether or not these effects are exclusively due to enhanced leptin signaling is unknown. Here we report that mice with hypothalamic PTP1B-deficiency (Nkx2.1-PTP1B(-/-)) display decreased body weight and adiposity on high-fat diet with no associated improvements in glucose tolerance. Consistent with previous reports, we find that hypothalamic deletion of the LepRb in mice (Nkx2.1-LepRb(-/-)) results in extreme hyperphagia and obesity. Interestingly, deletion of hypothalamic PTP1B and LepRb (Nkx2.1-PTP1B(-/-):LepRb(-/-)) does not rescue the hyperphagia or obesity of Nkx2.1-LepRb(-/-) mice, suggesting that hypothalamic PTP1B contributes to the central control of energy balance through a leptin receptor-dependent pathway.

Keywords: BAT, Brown adipose tissue; CNTF, Ciliary neurotrophic factor; Cre, Cre recombinase; GTT, Glucose tolerance test; HFD, High-fat diet; HPA, hypothalamus–pituitary–adrenal; Hypothalamus; IL-6, Interleukin-6; ITT, Insulin tolerance test; JAK2, Janus kinase 2; LepRb, Leptin receptor long form; Leptin; Nkx2.1, NK2 homeobox 1 protein or thyroid transcription factor-1; Obesity; PI3K, Phosphatidylinositol 3-kinase; POMC, Proopiomelanocortin; PTP1B, Protein tyrosine phosphatase 1B; PTPs, Protein tyrosine phosphatases; Phosphatase; Prdm16, PR domain containing 16; SHP2, Src homology 2 domain-containing protein tyrosine phosphatase; STAT3, Signal transducer and activator of transcription 3; UCP1, Uncoupling protein 1; WAT, White adipose tissue; db/db, Leptin receptor-deficient mice; ob/ob, leptin-deficient mice.

Figure 1

Detection of PTP1B deletion in Nkx2.1-PTP1B deficient mouse models. (A) PTP1B protein levels in the hypothalamus and brain of Nkx2.1-PTP1B^–/– (KO) mice compared with PTP1B fl/fl controls (WT). SHP2 protein levels are shown as a loading control. (B) Detection of deletion of PTP1B or LepRb floxed alleles in PTP1B fl/fl:LepRb fl/fl, Nkx2.1-LepRb^–/–, and Nkx2.1-PTP1B^–/–:LepRb^–/– mice. DNA was isolated from different tissues [hypothalamus (Hypo), extrahypothalamic brain, pituitary (Pit), lung, hindlimb, perigonadal white adipose tissue (WAT), brown adipose (BAT), and liver], and deletion of floxed allele was detected by PCR.

Figure 2

Nkx2.1-PTP1B^–/– mice have reduced body weight and adiposity on HFD. (A) Body weights of male Nkx2.1-PTP1B^–/– (n=9), Nkx2.1-PTP1B^+/– (n=8), and control PTP1B fl/fl (n=14) mice on chow. (B) Body weights of male Nkx2.1-PTP1B^–/– (n=6), Nkx2.1-PTP1B^+/– (n=6), and control PTP1B fl/fl (n=17) mice on HFD. (C) Body weights of female Nkx2.1-PTP1B^–/– (n=10), Nkx2.1-PTP1B^+/– (n=8), and control PTP1B fl/fl (n=9) mice on chow. (D) Body weights of female Nkx2.1-PTP1B^–/– (n=15), Nkx2.1-PTP1B^+/– (n=11), and control PTP1B fl/fl (n=17) mice on HFD. (E) Epididymal fat pad weight for male Nkx2.1-PTP1B^–/– (n=9 on chow, 6 on HFD) and control PTP1B fl/fl (n=14 on chow, 9 on HFD) mice on chow or HFD. (F) Body length for male Nkx2.1-PTP1B^–/– (n=8 on chow, 6 on HFD) and control PTP1B fl/fl (n=14 on chow, 9 on HFD) mice on chow or HFD. (G) Fat mass as determined by NMR of male Nkx2.1-PTP1B^–/– (n=6) and control PTP1B fl/fl (n=9) mice on HFD. (H) Lean mass as determined by NMR of male Nkx2.1-PTP1B^–/– (n=6) and control PTP1B fl/fl (n=9) mice on HFD. All values are mean±SEM. Weight curves analyzed by two-way ANOVA with repeated measures: #p≤0.05. Fishers LSD post hoc pairwise comparisons between Nkx2.1-PTP1B^–/– and wildtype controls:*p<0.05. Body composition and body length data analyzed by two tailed Student′s t-test: *p<0.05.

Figure 3

Nkx2.1-PTP1B^–/–:LepRb^–/– mice show no difference in body weight compared to Nkx2.1-LepRb^–/– mice. (A) Body weights of male Nkx2.1-PTP1B^–/–:LepRb^–/– (n=8), Nkx2.1-LepRb^–/– (n=9), control PTP1B fl/fl:LepRb fl/fl (n=14) and control LepRb fl/fl (n=9) mice on chow. (B) Body weights of male Nkx2.1-PTP1B^–/–:LepRb^–/– (n=5), Nkx2.1-LepRb^–/– (n=7), control PTP1B fl/fl:LepRb fl/fl (n=11) and control LepRb fl/fl (n=7) mice on HFD. (C) Body weights of female Nkx2.1-PTP1B^–/–:LepRb^–/– (n=13), Nkx2.1-LepRb^–/– (n=13), control PTP1B fl/fl:LepRb fl/fl (n=15) and control LepRb fl/fl (n=15) mice on chow. (D) Body weights of female Nkx2.1-PTP1B^–/–:LepRb^–/– (n=6), Nkx2.1-LepRb^–/– (n=13), control PTP1B fl/fl:LepRb fl/fl (n=6) and control LepRb fl/fl (n=5) mice on HFD. All values are mean±SEM. Weight curves analyzed by two-way ANOVA with repeated measures: *p<0.05.

Figure 4

Male Nkx2.1-PTP1B^–/–:LepRb^–/– mice show no difference in body composition compared to Nkx2.1-LepRb^–/– mice. Fat mass (A) and lean mass (B) as determined by NMR of Nkx2.1-PTP1B^–/–:LepRb^–/– (n=8), Nkx2.1-LepRb^–/– (n=8), and control PTP1B fl/fl:LepRb fl/fl (n=3) mice on chow. (C) Body length of Nkx2.1-PTP1B^–/–:LepRb^–/– (n=8), Nkx2.1-LepRb^–/– (n=8), and control PTP1B fl/fl:LepRb fl/fl (n=3) mice on chow. Fat mass (D) and lean mass (E) as determined by NMR of Nkx2.1-PTP1B^–/–:LepRb^–/– (n=5) and Nkx2.1-LepRb^–/– (n=6) mice on HFD. (F) Body length of Nkx2.1-PTP1B^–/–:LepRb^–/– (n=5) and Nkx2.1-LepRb^–/– (n=6) mice on HFD. Body composition and body length on chow analyzed by one-way ANOVA followed by Student–Newman–Keuls pairwise comparison: *p<0.05 indicated group vs. control.

Figure 5

Nkx2.1-PTP1B^–/– mice have decreased food intake on HFD whereas Nkx2.1-PTP1B^–/–:LepRb^–/– mice show similar hyperphagia as Nkx2.1-LepRb^–/– mice. Average daily (A) and cumulative (B) food intake of male Nkx2.1-PTP1B^–/– (n=5) and control PTP1B fl/fl (n=6) on HFD. (C) 7 day feed efficiency of male Nkx2.1-PTP1B^–/– (n=5) and control PTP1B fl/fl (n=6) on HFD. (D) Core temperature of male Nkx2.1-PTP1B^–/– (n=5) and control PTP1B fl/fl (n=14) on HFD at 8 weeks of age. Average daily (E) and cumulative (F) food intake of male and female Nkx2.1-PTP1B^–/–:LepRb^–/– (n=9), Nkx2.1-LepRb^–/– (n=6), and control PTP1B fl/fl:LepRb fl/fl (n=6) mice on chow. (G) 4 day feed efficiency of male Nkx2.1-PTP1B^–/–:LepRb^–/– (n=4), Nkx2.1-LepRb^–/– (n=2), and control PTP1B fl/fl:LepRb fl/fl (n=3) mice on chow. (H) Core temperature of male Nkx2.1-PTP1B^–/–:LepRb^–/– (n=4), Nkx2.1-LepRb^–/– (n=2), control PTP1B fl/fl:LepRb fl/fl (n=8) mice on chow at 14 weeks of age. 24 hour food intake analyzed by two tailed Student′s t-test or one-way ANOVA followed by Student–Newman–Keuls pairwise comparison: *p<0.05 indicated group vs. control. Cumulative food intake analyzed by two-way ANOVA with repeated measures: *p<0.05.

Figure 6

Glucose tolerance is unchanged in Nkx2.1-PTP1B deficient models compared to controls. (A) GTT for male Nkx2.1-PTP1B^–/– (n=9) and control PTP1B fl/fl (n=8) mice on chow at 11 weeks of age. (B) Area under the curve (AUC) for blood glucose during GTT of male chow cohort. (C) GTT for male Nkx2.1-PTP1B^–/– (n=6) and control PTP1B fl/fl (n=9) mice on HFD at 17 weeks of age. (D) AUC for blood glucose during GTT of male HFD cohort. (E) GTT for female PTP1B^–/–:LepRb^–/– (n=5), Nkx2.1-LepRb^–/– (n=7), and control PTP1B fl/fl:LepRb fl/fl (n=6) on chow at 13 weeks of age. (F) AUC for blood glucose during GTT of female chow cohort. GTT analyzed by two-way ANOVA with repeated measures: *p<0.05.

Figure 7

Comparison of the anatomic specificity of various CNS PTP1B deficient mouse models. LepRb and POMC are highly expressed in the arcuate nucleus (ARC) of the hypothalamus and are also found in the nucleus of the solitary tract (NTS) of the medulla. Outside of the NTS and hypothalamic nuclei including the ventromedial hypothalamic nucleus (VMH), dorsomedial hypothalamic nucleus (DMH), lateral hypothalamic area (LHA), and ventral premamillary nucleus (PMV), LepRb-expressing neurons are distributed broadly throughout the brain, with known extrahypothalamic sites including the cortex, hippocampus (Hippo), subfornical organ (SFO), ventral tegmental area (VTA), and the parabrachial nucleus (PBN).