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POMC neurons expressing leptin receptors coordinate metabolic responses to fasting via suppression of leptin levels

  1. Alexandre Caron
  2. Heather M Dungan Lemko
  3. Carlos M Castorena
  4. Teppei Fujikawa
  5. Syann Lee
  6. Caleb C Lord
  7. Newaz Ahmed
  8. Charlotte E Lee
  9. William L Holland
  10. Chen Liu
  11. Joel K Elmquist  Is a corresponding author
  1. University of Texas Southwestern Medical Center, United States
  2. Howard Community College, United States
  3. UT Health San Antonio, United States
Research Article
Cite this article as: eLife 2018;7:e33710 doi: 10.7554/eLife.33710
7 figures, 1 table and 1 additional file

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.

https://doi.org/10.7554/eLife.33710.002
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.

https://doi.org/10.7554/eLife.33710.003
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.

https://doi.org/10.7554/eLife.33710.004
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.

https://doi.org/10.7554/eLife.33710.005
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.

https://doi.org/10.7554/eLife.33710.006
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.

https://doi.org/10.7554/eLife.33710.007
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.

https://doi.org/10.7554/eLife.33710.008
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.

https://doi.org/10.7554/eLife.33710.009
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.

https://doi.org/10.7554/eLife.33710.010

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiers
Strain (Tg(Pomc-cre)1Lowl)PomcCre mousePMID: 17556551RRID:IMSR_JAX:010714
Strain (Tg(Pomc-cre/ERT2)#Jke)PomcCreERt2 mousePMID: 24177424RRID:MGI:5569339
Strain (Leprtm1.1Chua)Leprflox/flox mousePMID: 15389315RRID:MGI:3511747
Strain (Gt(ROSA)26Sortm14(CAG-tdTomato)Hze)Ai14(RCL-tdT)-D mousePMID: 20023653RRID:IMSR_JAX:007914
Antibody (AB_331586)phospho-Stat3 antibodyTyr705, Cell Signaling Technology Cat# 9131,RRID:AB_331586
Antibody (AB_2314007)β-endorphin antibodyPhoenix Pharmaceuticals Cat# H-022–33RRID:AB_2314007
Antibody (AB_639922)tdTomato antibodySanta Cruz Biotechnology Cat# sc-33354,RRID:AB_639922

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