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. 2010 Mar;120(3):720-34.
doi: 10.1172/JCI39620. Epub 2010 Feb 15.

PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice

Ryoichi Banno  1 , Derek Zimmer, Bart C De Jonghe, Marybless Atienza, Kimberly Rak, Wentian Yang, Kendra K Bence
Affiliations

PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice

Ryoichi Banno et al. J Clin Invest. 2010 Mar.

Abstract

Protein tyrosine phosphatase 1B (PTP1B) and SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) have been shown in mice to regulate metabolism via the central nervous system, but the specific neurons mediating these effects are unknown. Here, we have shown that proopiomelanocortin (POMC) neuron-specific deficiency in PTP1B or SHP2 in mice results in reciprocal effects on weight gain, adiposity, and energy balance induced by high-fat diet. Mice with POMC neuron-specific deletion of the gene encoding PTP1B (referred to herein as POMC-Ptp1b-/- mice) had reduced adiposity, improved leptin sensitivity, and increased energy expenditure compared with wild-type mice, whereas mice with POMC neuron-specific deletion of the gene encoding SHP2 (referred to herein as POMC-Shp2-/- mice) had elevated adiposity, decreased leptin sensitivity, and reduced energy expenditure. POMC-Ptp1b-/- mice showed substantially improved glucose homeostasis on a high-fat diet, and hyperinsulinemic-euglycemic clamp studies revealed that insulin sensitivity in these mice was improved on a standard chow diet in the absence of any weight difference. In contrast, POMC-Shp2-/- mice displayed impaired glucose tolerance only secondary to their increased weight gain. Interestingly, hypothalamic Pomc mRNA and alpha-melanocyte-stimulating hormone (alphaMSH) peptide levels were markedly reduced in POMC-Shp2-/- mice. These studies implicate PTP1B and SHP2 as important components of POMC neuron regulation of energy balance and point to what we believe to be a novel role for SHP2 in the normal function of the melanocortin system.

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Figures

Figure 2
Figure 2. POMC-Ptp1b–/– mice are resistant to HFD-induced obesity.
(A) Body weights of male POMC-Ptp1b–/– (n = 24) and -Ptp1b+/– mice (n = 27) and Ptp1b+/+ controls (n = 31) on HFD. (B) Body weights of female POMC-Ptp1b–/– (n = 20) and -Ptp1b+/– mice (n = 21) and Ptp1b+/+ controls (n = 24) on HFD. (C) Body weights of male POMC-Ptp1b–/– (n = 11) and -Ptp1b+/– mice (n = 10) and Ptp1b+/+ controls (n = 10) on a chow diet. (D) Body weights of female POMC-Ptp1b–/– (n = 8) and -Ptp1b+/– mice (n = 6) and Ptp1b+/+ controls (n = 8) on a chow diet. (E) Male POMC-Ptp1b–/– (KO) mice have decreased epididymal fat pad weight on HFD compared with Ptp1b+/+ (WT) controls. (F) Male POMC-Ptp1b–/– mice have decreased fat mass on HFD as determined by DEXA. (G) Lean mass of male POMC-Ptp1b–/– and Ptp1b+/+ control mice on HFD as determined by DEXA; for E–G, n = 8/genotype, 17–18 weeks of age. (H) Male POMC-Ptp1b–/– mice have decreased length compared with Ptp1b+/+ controls on HFD (n = 10/group). All values are mean ± SEM. *P < 0.05 for POMC-Ptp1b–/– versus Ptp1b+/+ controls by ANOVA (A and B) or 2-tailed Student’s t test (E–H).
Figure 1
Figure 1. Generation of mice with POMC neuron deficiency of PTP1B or SHP2.
(A) Deletion efficiency of POMC-Cre as assessed by immunohistochemistry for GFP. Ptp1b+/+;Z/EG control hypothalamus (left) compared with POMC-Ptp1b–/–;Z/EG hypothalamus (right). (B and C) Immunohistochemistry for αMSH was performed in colchicine-injected Ptp1b+/+ or Shp2+/+ controls compared with POMC-Ptp1b–/– or -Shp2–/– mice. 3V, third ventricle. Scale bars: 50 μm. (D) Quantification of POMC cell number and cell size in hypothalamic sections of Ptp1b+/+ and Shp2+/+ (WT) compared with POMC-Ptp1b–/– and POMC-Shp2–/– (KO) mice; adult (3–8 months old) mice were analyzed, n = 3 mice per genotype, 2–4 sections per mouse. (E) Detection of deletion of Ptp1b or Shp2 alleles (Δ) in POMC-Ptp1b–/– and -Shp2–/– mice compared with Ptp1b+/+ and Shp2+/+ controls. DNA was extracted from different tissues (P, pituitary; H, hypothalamus; B, whole brain; L, liver; K, kidney; F, fat; M, skeletal muscle; and hindbrain), and deletion of the floxed allele was detected by PCR. Recombination was detected only in pituitary, hypothalamus, whole brain, and hindbrain of POMC-Ptp1b–/– and -Shp2–/– mice. A PCR reaction with IL-2 was used as an internal control. (F) PTP1B protein levels in Ptp1b+/+ controls compared with POMC-Ptp1b–/– mice (top panels) and SHP2 protein levels in Shp2+/+ controls compared with POMC-Shp2–/– mice (bottom panels) as determined by immunoblotting. SHP2 and ERK2 protein levels, respectively, are shown as a loading control. Hypo, hypothalamus.
Figure 3
Figure 3. POMC-Ptp1b–/– mice have increased energy expenditure.
(A) Cumulative food intake for 3 days on HFD at the age of 5–6 weeks (n = 8/group). (B) POMC-Ptp1b–/– mice have decreased feed efficiency on HFD at the age of 6 weeks compared with Ptp1b+/+ controls (n = 8/group). FI, food intake. (C) POMC-Ptp1b–/– mice have increased energy expenditure as indicated by increased VO2 particularly during the dark cycle on HFD at the age of 17 weeks compared with Ptp1b+/+ controls (n = 8/group); no significant differences between genotypes were found in RQ values. (D) Locomotor activity of mice on HFD at the age of 17 weeks measured during the calorimetry experiment (n = 8/group). (E) POMC-Ptp1b–/– mice have increased body temperature on HFD at the age of 10 weeks compared with Ptp1b+/+ controls (n = 11/group). All data were collected from male mice; WT, Ptp1b+/+, KO, POMC-Ptp1b–/– mice. All values are mean ± SEM. *P < 0.05 POMC-Ptp1b–/– versus Ptp1b+/+ controls by 2-tailed Student’s t test, #P = 0.06 by 2-tailed Student’s t test.
Figure 4
Figure 4. Leptin sensitivity is improved in POMC-Ptp1b–/– mice.
(A) Leptin sensitivity is enhanced in POMC-Ptp1b–/– mice compared with Ptp1b+/+ controls. Male mice (8–10 weeks of age on a chow diet) were injected with leptin (0.5 μg/g body weight every 12 hours) or saline i.p. during the indicated period. POMC-Ptp1b–/– mice show suppression of food intake and body weight in response to leptin, whereas Ptp1b+/+ controls do not respond to this low dose of leptin (n = 8/group leptin; n = 4/group saline). Food intake and body weight were measured for 2 days prior to the start of injections and normalized to 100% for day 0 values. (B) Twelve-week-old male Ptp1b+/+ (WT) and POMC-Ptp1b–/– (KO) mice on chow diet were fasted overnight and injected with leptin (3 μg/g BW i.p.). Shown are equivalent ARC sections stained for ACTH (green) and pStat3 (red). Scale bar: 100 μm for left, middle, and right panels; 50 μm for higher-magnification images. (C) Cells double-labeled for pStat3 and ACTH were counted bilaterally in the ARC as described in Methods. Data are presented as the percentage of ACTH cells that were also pStat3-positive. All values are mean ± SEM. *P < 0.05, **P < 0.01, #P < 0.06, POMC-Ptp1b–/– versus Ptp1b+/+ controls by 2-tailed Student’s t test.
Figure 5
Figure 5. POMC-Shp2–/– mice are sensitive to HFD-induced obesity.
(A) Body weights of male POMC-Shp2–/– mice (n = 8) versus Shp2+/+ controls (n = 11) on HFD. (B) Body weights of female POMC-Shp2–/– mice (n = 7) versus Shp2+/+ controls (n = 6) on HFD. (C) Body weights of male POMC-Shp2–/– mice (n = 15) versus Shp2+/+ controls (n = 21) on a chow diet. (D) Body weights of female POMC-Shp2–/– mice (n = 17) versus Shp2+/+ controls (n = 14) on a chow diet. (E) Male POMC-Shp2–/– (KO) mice have increased epididymal fat pad weight compared with Shp2+/+ (WT) mice on HFD. (F) Male POMC-Shp2–/– mice have increased fat mass on HFD as determined by DEXA of abdomen. (G) Lean mass of males on HFD as determined by DEXA. For E–G, n = 8/genotype, 17–18 weeks of age. (H) Male POMC-Shp2–/– mice have increased length compared with Shp2+/+ controls (n = 10/genotype). All values are mean ± SEM. *P < 0.05, POMC-Shp2–/– versus Shp2+/+ controls by 2-tailed Student’s t test.
Figure 6
Figure 6. POMC-Shp2–/– mice have decreased energy expenditure.
(A) Cumulative food intake for 3 days on HFD at the age of 11 weeks (n = 7/group). (B) POMC-Shp2–/– mice have increased feed efficiency on HFD compared with Shp2+/+ controls (11 weeks old; n = 7/group). (C) POMC-Shp2–/– mice have decreased energy expenditure on HFD compared with Shp2+/+ controls as indicated by decreased VO2 during both the light and dark cycles; POMC-Shp2–/– mice show decreased RQ during the dark cycle compared with Shp2+/+ controls (14 weeks old; n = 8/group). (D) Locomotor activity of POMC-Shp2–/– mice is decreased on HFD (14 weeks old; n = 8/group). All data were collected from male mice; WT, Shp2+/+; KO, POMC-Shp2–/– mice. All values are mean ± SEM. *P < 0.05, POMC-Shp2–/– versus Shp2+/+ controls by 2-tailed Student’s t test.
Figure 7
Figure 7. Leptin sensitivity is impaired in POMC-Shp2–/– mice.
(A) Weight-matched male mice (8–10 weeks of age on chow diet) were selected for this experiment from a large cohort of mice to control for body weight differences. Mice were injected with leptin every 12 hours (2 μg/g BW/injection) or saline i.p. during the indicated period. Shp2+/+ controls show a greater suppression of food intake and body weight than POMC-Shp2–/– mice (n = 6/group leptin; n = 3–5 saline controls). Food intake and body weight were measured for 2 days prior to the start of injections and normalized to 100% for day 0 values. All values are mean ± SEM. *P < 0.05, POMC-Shp2–/– versus Shp2+/+ controls by 2-tailed Student’s t test. (B) Consistent with impaired leptin sensitivity, hypothalamic expression of Pomc mRNA is reduced in both the fed and fasted states in POMC-Shp2–/– mice (KO) versus Shp2+/+ controls (WT). Hypothalamic expression of both Agrp mRNA (C) and Npy mRNA (D) is similar in POMC-Shp2–/– mice and Shp2+/+ controls. All mice were males in either the fed state or 24-hour fasted state and on HFD for 14–16 weeks; n = 6–8 per genotype. All values are mean ± SEM. *P < 0.05, fed versus fasted within genotype; #P < 0.05, WT fed versus KO fed; †P < 0.05, WT fasted versus KO fasted, by 2-tailed Student’s t test.
Figure 8
Figure 8. αMSH peptide levels in the PVN of POMC-Ptp1b–/– and -Shp2–/– mice.
(A) αMSH staining of hypothalamic projections to the PVN of 5- to 6-week-old female POMC-Ptp1b–/– and -Shp2–/– (KO) mice compared with their respective Ptp1b+/+ and Shp2+/+ (WT) controls on a chow diet; scale bar: 200 μm. (B) Quantification of αMSH staining in PVN of 5- to 6-week-old female POMC-Ptp1b–/– and -Shp2–/– mice on a chow diet (n = 3–7 mice per genotype, 3–4 PVN-containing sections per animal; data are expressed as percentage relative to WT controls, mean ± SEM. (C) αMSH peptide content in whole hypothalamus of 18-week-old male POMC-Ptp1b–/– and -Shp2–/– mice on HFD compared with WT controls. Data are expressed as pmol αMSH per μg protein, mean ± SEM; **P < 0.001, WT versus KO by 2-tailed Student’s t test.
Figure 9
Figure 9. Insulin sensitivity and glucose homeostasis are improved in POMC-Ptp1b–/– mice.
(A) GTT for POMC-Ptp1b–/– mice and Ptp1b+/+ control mice on HFD (8 weeks old). (B) ITT for POMC-Ptp1b–/– mice and Ptp1b+/+ control mice on HFD (16 weeks old). (C) GTT for POMC-Ptp1b–/– mice and Ptp1b+/+ control mice on chow diet (16 weeks old). (D) ITT for POMC-Ptp1b–/– mice and Ptp1b+/+ controls on a chow diet (17 weeks old). Male mice (n = 8/genotype) were used for GTT and ITT experiments. Hyperinsulinemic-euglycemic clamp analysis of POMC-Ptp1b–/– mice on a chow diet reveals improved insulin sensitivity. Body weights (E), epididymal fat pad weights (F), and basal blood glucose (G) of male POMC-Ptp1b–/– mice (KO) and Ptp1b+/+ (WT) control mice on a chow diet were similar prior to clamp. (H) POMC-Ptp1b–/– mice have increased insulin sensitivity as shown by a greater GIR compared with Ptp1b+/+ controls. (I) POMC-Ptp1b–/– mice have a greater insulin-induced suppression of HGP during the clamp compared with controls (WT clamp vs. KO clamp). (J) POMC-Ptp1b–/– mice have increased Rd compared with Ptp1b+/+ controls. Male mice were 8 weeks of age on a chow diet (n = 8/group). All values are mean ± SEM. *P < 0.05, **P < 0.01 Ptp1b+/+ versus POMC-Ptp1b–/– by 2-tailed Student’s t test; #P < 0.05, Ptp1b+/+ versus POMC-Ptp1b–/– by AUC.
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