Effects of hypothalamic neurodegeneration on energy balance

Abstract

Normal aging in humans and rodents is accompanied by a progressive increase in adiposity. To investigate the role of hypothalamic neuronal circuits in this process, we used a Cre-lox strategy to create mice with specific and progressive degeneration of hypothalamic neurons that express agouti-related protein (Agrp) or proopiomelanocortin (Pomc), neuropeptides that promote positive or negative energy balance, respectively, through their opposing effects on melanocortin receptor signaling. In previous studies, Pomc mutant mice became obese, but Agrp mutant mice were surprisingly normal, suggesting potential compensation by neuronal circuits or genetic redundancy. Here we find that Pomc-ablation mice develop obesity similar to that described for Pomc knockout mice, but also exhibit defects in compensatory hyperphagia similar to what occurs during normal aging. Agrp-ablation female mice exhibit reduced adiposity with normal compensatory hyperphagia, while animals ablated for both Pomc and Agrp neurons exhibit an additive interaction phenotype. These findings provide new insight into the roles of hypothalamic neurons in energy balance regulation, and provide a model for understanding defects in human energy balance associated with neurodegeneration and aging.

Figure 1. Pattern of R26R Activation in Mice Carrying the PomcCre or AgrpCre Transgene

Whole brains from Tg.PomcCre/+; R26R/+ or Tg.AgrCre/+; R26R/+ mice of the indicated age were fixed and stained with Xgal as described in [9]. Photographs show the ventral brain surface, and coronal sections (not shown) indicate that Xgal-stained neurons lie in the arcuate nucleus of the hypothalamus. As assessed by the extent of Xgal staining, Cre-induced recombination in Pomc neurons is complete by embryonic day 17.5; however, Cre-induced recombination in Agrp neurons is just beginning at 2 wk of age (arrows) and is not complete until 3–4 wk of age.

Figure 2. Immunostaining of α-MSH or Agrp in Control and Tfam Mutant Animals of Different Ages

Hypothalami from control (Tfam ^flox/⁺; Tg.PomcCre/+ or Tfam ^flox/^flox; +/+), Pomc-specific Tfam mutant (Tfam ^flox/^flox; Tg.PomcCre/+), and Agrp-specific Tfam mutant (Tfam ^flox/^flox; Tg.AgrpCre/+) animals were harvested at the indicated ages and immunostained for α-MSH or Agrp as indicated. IR, immmunoreactivity.

Figure 3. Effect of Pomc-Specific Tfam Deficiency on Pomc and Agrp Immunostaining

Each column of panels shows coronal sections from a 7-mo-old control (Tfam ^flox/⁺; Tg.PomcCre/+ or Tfam ^flox/^flox; +/+), Pomc-specific Tfam mutant (Tfam ^flox/^flox; Tg.PomcCre/+), and Agrp-specific Tfam mutant (Tfam ^flox/^flox; Tg.AgrpCre/+) on the left, middle, and right, respectively. Each section was stained for α-MSH or Agrp as indicated. The three lower panels show coronal sections stained to reveal cell nuclei (DAPI) and immunostained for GFAP, and indicate that ablation of Pomc or Agrp neurons does not grossly disturb tissue architecture, cell number, or stimulate astrocytosis. The sections shown are representative of three different animals that were examined. IR, immmunoreactivity.

Figure 4. Expression of the R26R Cre Reporter Gene in Pomc- or Agrp-Specific Tfam Mutant Animals

Animals carrying the PomcCre or AgrpCre transgene together with the lacZ reporter allele for Cre recombination Gt(Rosa)26Sor^tm1Sor (R26R) were intercrossed with Tfam ^flox/^flox animals. F2 progeny of the indicated genotype were sacrificed at 7 mo of age, and hypothalamic coronal sections stained for Xgal. In this situation, Xgal staining serves as an autonomous histologic marker, and loss of Xgal staining in Tfam mutant animals is therefore secondary to cell death. Sections are representative of three to four per genotype group that were examined. The panel on the lower right shows the number of Agrp-expressing neurons in the control and Agrp-Tfam mutants as determined by fluorescence in situ hybridization to Agrp mRNA. **, p ≤ 0.01. Error bars = standard error of the mean.

Figure 5. Effect of Pomc- and/or Agrp-Specific Tfam Mutations on Body Weight

(A–D) show longitudinal measurements of body weight in animals of the indicated genotype and sex. (E) and (F) show comparisons of single (Pomc-specific) and double (Pomc- and Agrp-specific) Tfam deficiency for 6- and 9-mo-old animals. *, p ≤ 0.05; **, p ≤ 0.01. Error bars = standard error of the mean.

Figure 6. Lean Body Composition of Animals with Pomc- or Agrp-Specific Tfam Deficiency

Lean body mass of animals of the indicated genotype was determined as described in Materials and Methods. For Pomc-specific Tfam mutant animals (A), numbers of animals were: control n = 10, mutant n = 4 (male); and control n = 6, mutant n = 4 (female). For Agrp-specific Tfam mutant animals (C), numbers of animals were control n = 4, mutant n = 4 (male); and control n = 6, mutant n = 8 (female). (B) and (D) show comparisons of control and mutant animals of the indicated genotype and sex for fat masses of 7- to 10-mo-old animals. For Pomc-specific Tfam mutant animals (B), numbers of animals were control n = 10, mutant n = 4 (male); and control n = 6, mutant n = 4 (female). For Agrp-specific Tfam mutant animals (D), numbers of animals were control n = 4, mutant n = 4 (male); and control n = 6, mutant n = 8 (female). *, p ≤ 0.05; **, p ≤ 0.01. Error bars = standard error of the mean.

Figure 7. Food Intake and Energy Expenditure in Animals with Pomc- or Agrp-Specific Tfam Deficiency

(A and B) Daily food intake was measured for 7 d as described in Materials and Methods. For (A), numbers of animals used (control and Pomc-Tfam mutant) were 6-mo-old male (n = 13, n = 7); 6-mo-old female (n = 10, n = 6); > 8-mo-old male (n = 10, n = 6); and > 8-mo-old female (n= 13, n = 6). (C, D) O₂ consumption was measured over a 24-h period as described in Materials and Methods; panels illustrate results from a single control and a single Pomc-Tfam mutant animal before and after (10–12 d) corticosterone replacement; these results are representative of four control and four mutant animals that were examined. (E) Daily food intake was significantly increased after corticosterone replacement in Pomc-Tfam mutant animals (n = 6) but not in control (n = 9) animals. (F) Measurement of total serum T4 levels in control (n = 7) and Pomc-Tfam mutant (n = 5) animals. *, p ≤ 0.05; **, p ≤ 0.01. Error bars = standard error of the mean.

Figure 8. Compensatory Refeeding and Neuropeptide mRNA levels in Pomc-Specific and Agrp-Specific Tfam Deficiency

(A–D) 24-h compensatory refeeding after a 48-h fast was measured as described in Materials and Methods; data are shown either as (A and B) the ratio of food consumed over 24 h (refeeding) to normal daily food intake (averaged over 7 h prior to food deprivation), or as (C and D) percentage of weight recovery after 24 h of refeeding. For the Pomc-Tfam experiment (A and C), number of animals used was n = 23 (control) and n = 13 (Pomc-Tfam mutant); for the Agrp-Tfam experiment (B and D), number of animals used was n = 21 (control) and n = 15 (Agrp-Tfam). (E) The same refeeding defects were observed in Pomc-Tfam mutants after corticosterone replacement (control, n = 9; Pomc-Tfam mutant, n = 6). (F) Expression of Pomc, Agrp, and Npy in Pomc-Tfam mutants. Mice were fasted for 48 h 10 d after implanting corticosterone pellets, and expression of Pomc, Agrp, and Npy in the hypothalamus was measured by semi-quantitative RT-PCR. Values are shown as relative levels compared to free-fed controls. Numbers of animals used were n = 5 (control fed); n = 5 (control fasted); n = 3 (mutant fed); and n = 3 (mutant fasted). *, p ≤ 0.05; **, p ≤ 0.01. Error bars = standard error of the mean.