POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels
- University of Texas Southwestern Medical Center, United States
- Howard Community College, United States
- UT Health San Antonio, United States
Figures
Figure 1 with 2 supplements
LEPR-expressing POMC neurons are required for normal liver insulin sensitivity in adult mice.
(A) Fed and fasting (16 hr) glucose one week before, and every week for four weeks after, PomcCreERt2::Leprflox/flox and littermate controls were injected with the last dose of tamoxifen (n = 12). (B) Fed and fasting (48 hr) insulin four weeks after tamoxifen was given (n = 4–6). (C) Fed and fasting (48 hr) glucagon four weeks after tamoxifen was given (n = 4–6). (D) Glucose excursion during an insulin tolerance test (ITT) only one week following the last injection of tamoxifen (n = 5–6). (E) Area under the curve for the ITT shown in B (n = 5–6). (F) Glucose infusion rate (GIR) needed to maintain euglycemia (119.3 ± 3.9 vs 122.0 ± 8.2 mg/dl) during an hyperinsulinemic-euglycemic clamp performed only one week following the last injection of tamoxifen (n = 6). (G) Glucose disposal (Rd) during the same hyperinsulinemic-euglycemic clamp (n = 6). (H) Basal and clamped hepatic glucose production (HPG) (n = 6). (I) Basal and clamped lipolysis rate as assessed by measuring free fatty acid (FFA) levels (n = 6). The data are expressed as the mean ± SEM. ***p<0.001, **p<0.01 and *p<0.05 versus littermate controls.
Figure 1—figure supplement 1
Validation of the PomcCreERt2 mice.
(A) PomcCreERt2 mice were crossed with Ai14(RCL-tdT)-D mice, injected with tamoxifen using the described paradigm. Using immunohistochemistry, tdTomato expression (red) was colocalized with β-endorphin (marker of POMC neurons) expression (green) 2 weeks later. (B) The inducible Cre model does not recombine in extra-hypothalamic areas, except for a few β-endorphin + cells located in the nucleus tractus solitarius in the HB and the pituitary. (C) Leptin-induced Stat3 activation in the arcuate nucleus of the hypothalamus. Mice were injected with tamoxifen using the described paradigm and leptin (5 mg/kg, i.p.) was administrated in 16h-fasted animals. Animals were decapitated 45 min later and pStat3 was evaluated by immunohistochemistry. A representative image of n = 3 animals per group is shown. CB, cerebellum, CTX, cortex; DR, dorsal raphe; HB, hindbrain; HIP, hippocampus; OB, olfactory bulb; PT, pituitary; SC, spinal cord; STR, striatum.
Figure 1—figure supplement 2
Glucagon stimulation test.
PomcCreERt2::Leprflox/flox and littermate control mice (n = 8) were treated with tamoxifen as described. 4 weeks after treatment, they were fasted for one hour and glucagon (120 µg/kg, i.p.) was administrated. Blood glucose was then monitored every 10 min for one hour. The data are expressed as the mean ± SEM.
Figure 2
LEPR-expressing POMC neurons are dispensable for the regulation of energy balance in adult mice.
(A) Body weight before, and up to four weeks after, PomcCreERt2::Leprflox/flox and littermate controls were injected with tamoxifen (n = 12). (B) Fat mass and C) Lean mass as assessed by nuclear magnetic resonance (NMR) four weeks following tamoxifen administration (n = 12). (D) Daily food intake, (E) Oxygen consumption (VO2), (F) Respiratory exchange ratio (RER), and G) locomotor activity in CaloSys Calorimetry System cages four weeks after the administration of tamoxifen (n = 5). Summary graphs showing average data for light (ZT0-ZT12) and dark (ZT12-ZD24) cycles are presented under each diurnal graph. The data are expressed as the mean ± SEM.
Figure 3
Deletion of LEPRs in adult POMC neurons impairs fasting-induced expression of orexigenic neuropeptides in the mediobasal hypothalamus.
(A) Pomc, (B) Agrp and (C) Npy mRNA expression in mediobasal hypothalamus of fed and fasted (48 hr) PomcCreERt2::Leprflox/flox and littermate control mice four weeks after tamoxifen was given (n = 8–14). The data are expressed as the mean ± SEM. ***p<0.001 versus littermate controls.
Figure 4
Deletion of LEPRs in adult POMC neurons impairs postprandial glycemia.
(A) Food intake and (B) Blood glucose up to six hours after food access was restored to 48 hr fasted PomcCreERt2::Leprflox/flox and littermate control mice, four weeks after tamoxifen was given (n = 8). The data are expressed as the mean ± SEM. **p<0.01 and *p<0.05 versus littermate controls.
Figure 5
LEPR-expressing POMC neurons are required for the fasting-induced fall in leptin levels, independent of changes in fat.
(A) Weight loss, and (B) fat-mass loss after a 48 hr fast in mice with constitutive (prenatal) deletion of LEPRs in POMC neurons and littermate controls (n = 7–10). (C) Plasma leptin levels, and D) visceral adipose tissue Lep mRNA expression in fed or fasted (48 hr) mice with constitutive deletion of LEPRs in POMC neurons and littermate controls (n = 7–14). (E) Weight loss, and (F) fat-mass loss after a 48 hr fast in PomcCreERt2::Leprflox/flox and littermate control mice four weeks after tamoxifen was given (n = 12). (G) Plasma leptin levels, and (H) visceral adipose tissue Lep mRNA expression in fed or fasted (48 hr) in PomcCreERt2::Leprflox/flox and littermate control mice four weeks after tamoxifen was given (n = 6–13). The data are expressed as the mean ± SEM. ***p<0.001, **p<0.01 and *p<0.05 versus littermate controls.
Figure 6
Deletion of LEPRs in adult POMC neurons impairs visceral adipose tissue expression of Adra2a with fasting.
(A) Expression of the nine adrenergic receptors in fed of fasted (24 hr – 48 hr) PomcCreERt2::Leprflox/flox and littermate control mice four weeks after tamoxifen was given (n = 8–14). (B) Comparison of the expression of Adra2a and (C) Adrb3 in epidydimal (eWAT) versus inguinal (iWAT) adipose tissue in an independent cohort of fed of fasted (48 hr) PomcCreERt2::Leprflox/flox and littermate control mice four weeks after tamoxifen was given (n = 5–6). The data are expressed as the mean ± SEM. ***p<0.001 and *p<0.05 versus littermate controls.
Figure 7
Pharmacological activation of ADRA2 stimulates leptin production.
(A) Visceral adipose tissue Lep mRNA expression one hour following an intraperitoneal (1 mg/kg) injection of the ADRA2 agonist clonidine (n = 10–12). (B) Plasma leptin levels up to two hours following the administration of clonidine in an independent cohort (n = 4–7). (C) Leptin release from epidydimal (eWAT) and (D) inguinal (iWAT) adipose tissue explants from fed and fasted (48 hr) PomcCreERt2::Leprflox/flox and littermate control mice following the addition of clonidine (1 µM) (n = 6). This experiment was performed four weeks after tamoxifen was given. The data are expressed as the mean ± SEM. ***p<0.001, **p<0.01, and *p<0.05 versus littermate controls.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Strain (Tg(Pomc-cre)1Lowl) | PomcCre mouse | PMID: 17556551 | RRID:IMSR_JAX:010714 |
| Strain (Tg(Pomc-cre/ERT2)#Jke) | PomcCreERt2 mouse | PMID: 24177424 | RRID:MGI:5569339 |
| Strain (Leprtm1.1Chua) | Leprflox/flox mouse | PMID: 15389315 | RRID:MGI:3511747 |
| Strain (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze) | Ai14(RCL-tdT)-D mouse | PMID: 20023653 | RRID:IMSR_JAX:007914 |
| Antibody (AB_331586) | phospho-Stat3 antibody | Tyr705, Cell Signaling Technology Cat# 9131, | RRID:AB_331586 |
| Antibody (AB_2314007) | β-endorphin antibody | Phoenix Pharmaceuticals Cat# H-022–33 | RRID:AB_2314007 |
| Antibody (AB_639922) | tdTomato antibody | Santa Cruz Biotechnology Cat# sc-33354, | RRID:AB_639922 |
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- https://doi.org/10.7554/eLife.33710.011
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POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels
eLife 7:e33710.
https://doi.org/10.7554/eLife.33710
