Leptin-dependent control of glucose balance and locomotor activity by POMC neurons

Abstract

Leptin plays a pivotal role in regulation of energy balance. Via unknown central pathways, leptin also affects peripheral glucose homeostasis and locomotor activity. We hypothesized that, specifically, pro-opiomelanocortin (POMC) neurons mediate those actions. To examine this possibility, we applied Cre-Lox technology to express leptin receptors (ObRb) exclusively in POMC neurons of the morbidly obese, profoundly diabetic, and severely hypoactive leptin receptor-deficient Lepr(db/db) mice. Here, we show that expression of ObRb only in POMC neurons leads to a marked decrease in energy intake and a modest reduction in body weight in Lepr(db/db) mice. Remarkably, blood glucose levels are entirely normalized. This normalization occurs independently of changes in food intake and body weight. In addition, physical activity is greatly increased despite profound obesity. Our results suggest that leptin signaling exclusively in POMC neurons is sufficient to stimulate locomotion and prevent diabetes in the severely hypoactive and hyperglycemic obese Lepr(db/db) mice.

Figure 1. Generation of Mice Expressing HA-tagged Leptin Receptors in POMC Neurons

A) Strategy for generation of HA-ObRb/Pomc-Cre mice. B) HA-ObRb expressing neurons of HA-ObRb/Pomc-Cre mice are located in the arcuate nucleus of the hypothalamus, as shown by HA immunohistochemistry (IHC). Right Images: High magnification of areas marked in the left two photomicrographs. C) HA-ObRb is expressed in POMC neurons of HA-ObRb/Pomc-Cre mice, as demonstrated by double-fluorescent IHC for HA (red) and β-Endorphin (green). High magnification images are shown on the right. Arc, arcuate nucleus; 3v, third ventricle; ME, median eminence. Scale bars, 50 µm.

Figure 2. HA-tagged Leptin Receptors in POMC Neurons are Functional in vivo

A–C) Representative photomicrographs of P-STAT3 IHC in brains from leptin-treated (i.p. 4mg/kg, 45 min) HA-ObRb/Lepr^db/db (A), HA-ObRb/Pomc-Cre/Lepr^db/db (B), and wild type (C) mice. Matched medial (top) and caudal (bottom) arcuate sections are shown. Arc, arcuate nucleus; DMH, dorsomedial nucleus; VMH, ventromedial nucleus; PMV, ventral premammillary nucleus. Scale bars, 200 µm. D) Leptin activates nuclear P-STAT3 in two POMC neurons of HA- ObRb/Pomc-Cre/Lepr^db/db mice as shown by confocal microscopy.

Figure 3. Expression of HA-ObRb in POMC Neurons of Lepr^db/db Mice Reduces Body Weight and Food Intake

A)-B) Body weight curves. C)-D) Cumulative food intake (7–9 wks). E)-F) Adipose mass (12 wks of age). A)-D) Data were collected from 11 female and 14 male Lepr^db/db mice, 11 female and 17 male HA-ObRb/Pomc-Cre/Lepr^db/db mice, and 5 female and 9 male wild type mice. Fat mass was measured by Echo-MRI in 5 female and 4 male Lepr^db/db mice, 5 female and 7 male HA-ObRb/Pomc-Cre/Lepr^db/db mice, and 8 female and 6 male wild type mice. All animals are littermates. *, p < 0.05; **, p < 0.01, ***, p< 0.001.

Figure 4. Expression of HA-ObRb in POMC Neurons of Lepr^db/db Mice Normalizes Circulating Glucose Levels and Increases Insulin Sensitivity

A) Fed glucose levels and C) body weight of 5 wks old female Lepr^db/db mice (n=7), HA-ObRb/Pomc-Cre/Lepr^db/db mice (n=8), and wild type mice (n=8). B) Fed glucose levels and D) body weight of 5 wks old male fed Lepr^db/db mice (n=11), HA-ObRb/Pomc-Cre/Lepr^db/db mice (n=12), and wild type mice (n=14). E) Glucose levels in food-restricted Lepr^db/db mice. Over a period of 3 weeks, 8 wks old female Lepr^db/db mice (n=4) were pair-fed to ad libitum fed HA- ObRb/Pomc-Cre/Lepr^db/db littermates (n=4). Wild type littermates (n=6) were fed ad libitum. F) Cumulative intake over the 3 wks.G) Fasted serum insulin levels of 12 wks old female Lepr^db/db mice (n=7), HA-ObRb/Pomc-Cre/Lepr^db/db mice (n=8), and wild type mice (N=8). H) Insulin tolerance tests in 13 weeks old female Lepr^db/db mice (n=7), HA-ObRb/Pomc-Cre/Lepr^db/db mice (n=8), and 4 wild type mice. (*, Lepr^db/db vs HA-ObRb/Pomc-Cre/Lepr^db/db). Glucose was administered to wild type mice after 30 min to prevent severe hypoglycemia caused by the high insulin dose (1.5 U/kg). Shown are changes from baseline. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant.

Figure 5. Increased Activity of Lepr^db/db Mice Expressing HA-ObRb in POMC Neurons

A) Examples of locomotor activity from one Lepr^db/db, one HA-ObRb/Pomc-Cre/Lepr^db/db and one wild-type mouse. Black bar depicts lights-off period. B) Average nocturnal, C) diurnal, and D) 24 hours locomotor activity in 13 weeks old male Lepr^db/db mice (n=4), HA-ObRb/Pomc- Cre/Lepr^db/db mice (n=6), and wild type mice (n=5).*, p < 0.05; NS, not significant.

Figure 6. Hypothalamic POMC mRNA and α-MSH Peptide Levels are Increased in Lepr^db/db Mice Expressing Leptin Receptors Exclusively in POMC Neurons

A) - C) Arcuate POMC, NPY, and AgRP mRNA from 18 weeks old control Lepr^db/db mice (n=7) and HA-ObRb/Pomc-Cre/Lepr^db/db mice (n=5). D) - F) Hypothalamic α-MSH, NPY, and AgRP neuropeptide levels as measured by EIA’s in 18 weeks Lepr^db/db mice (n=3), HA-ObRb/Pomc- Cre/Lepr^db/db mice (n=3), and wild type mice (n=3). Numbers are depicted as percent of Lepr^db/db levels. *, p< 0.05; **, p<0.01; NS, not significant. G) Representative images of hypothalamic α-MSH immuno-staining in 18 wks old HA-ObRb/Pomc-Cre/Lepr^db/db and Lepr^db/db mice. Scale bar, 200 µm.