Appetite Enhancement and Weight Gain by Peripheral Administration of TrkB Agonists in Non-Human Primates

Loss of function mutations in the receptor tyrosine kinase TrkB pathway resulted in hyperphagia and morbid obesity in human and rodents. Conversely, peripheral or central stimulation of TrkB by its natural ligands BDNF or NT4 reduced body weight and food intake in mice, supporting the idea that TrkB is a key anorexigenic signal downstream of the melanocortin-4 receptor (Mc4r) system. Here we show that in non-human primates TrkB agonists were anorexigenic when applied centrally, but surprisingly orexigenic, leading to gain in appetite, body weight, fat deposits and serum leptin levels, when given peripherally. The orexigenic and pro-obesity effects of peripherally administered TrkB agonists appear to be dose dependent, not associated with fluid retention nor with evidence of receptor down regulation. Our findings revealed that TrkB signaling exerts dual control on energy homeostasis in the primates that could be targeted for the treatment of either wasting disorders or obesity

6-OHDA-treated monkeys have decreased PGP9.5-ir in cardiac nerve bundles and fibers.

<p>(<b>A–D</b>) Microphotographs of PGP9.5-positive nerve bundles (<b>A,B</b>) and fibers (<b>C,D</b>) in control and 6-OHDA-treated animals. (<b>E</b>) Global PGP9.5-ir cardiac nerve bundle was reduced for both AAT (t(8) = 3.7, <i>P</i> = 0.006) and OD quantification (t(8) = 4.21, <i>P</i> = 0.003) in 6-OHDA compared to control animals. (<b>F</b>) PGP9.5-ir nerve bundle AAT was also decreased between groups in the lateral wall of the left ventricle. Within the 6-OHDA group, a significant effect of wall (ANOVA: F(3,16) = 4.632, <i>P</i> = 0.016) with the greatest reduction in the lateral compared to the septal wall (^a<i>P</i> = 0.022) was found. (<b>G</b>) Quantification of PGP9.5-ir fiber area density was significantly reduced in 6-OHDA-treated monkeys compared to controls (t(8) = 3.026, <i>P</i> = 0.016). (<b>H</b>) TH to PGP9.5 positive fibers ratio was significantly decreased in the anterior (t(8) = 4.335, <i>P</i> = 0.002) and inferior walls (t(8) = 4.43, <i>P</i> = 0.002) in 6-OHDA animals compared to controls. Within the 6-OHDA group there was a significant reduction in ratio between walls (ANOVA; F(3,16) = 3.299, <i>P</i> = 0.047) with significant loss in the inferior compared to septal wall (^b<i>P</i> = 0.044). Scale bar  = 25 µm. <i>*P</i><0.05, **<i>P</i><0.01, ^a<i>P</i> = 0.02, ^b<i>P</i> = 0.044. AAT, area above threshold; OD, optical density; PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p

GAP43 immunofluorescence increased in 6-OHDA monkeys and had minimal colabeling with TH.

<p>Cardiac sections of control (<b>A–D</b>) and 6-OHDA-treated (<b>F–I</b>) animals were labeled for GAP43 (red) and TH (green) immunofluorescence and counterstained with DAPI (blue nuclei). Arrows indicate GAP43 expression in nerve bundles. Arrowheads indicate colocalization of GAP43-ir and TH-ir. Inset (<b>d</b>) shows a positive control for GAP43 labeling in caudorostral diencephalon of rhesus embryonic day 38, run in parallel to these stainings. (<b>E</b>) Averaged scores demonstrate an increase of GAP43-ir in 6-OHDA monkeys. (t(8) = 9.073, <i>P</i><0.001). (<b>J</b>) Global GAP43 scores negatively correlate with AAT of TH-ir in cardiac nerve bundles. ***<i>P</i><0.001. AAT, area above threshold; GAP43, growth associated protein 43; TH, tyrosine hydroxylase.</p

6-OHDA-treated monkeys have reduced TH-ir and AADC-ir in the adrenal medulla.

<p>(<b>A-F</b>) Microphotographs of adrenal medulla sections stained for H&E (<b>A,B</b>), TH (<b>C,D</b>) and AADC (<b>E,F</b>) from control and 6-OHDA monkeys. Squares in A-F corresponds to respective insets a-f showing a higher magnification image of the selected area. (<b>G</b>) OD (TH: t(8) = 3.455, <i>P</i> = 0.009; AADC: t(8) = 2.192, <i>P</i> = 0.06) and (<b>H</b>) AAT (TH: t(5.791) = 4.437, <i>P</i> = 0.005; AADC: t(8) = 3.180, <i>P</i> = 0.013) quantifications of immunostainings showed significant reductions in 6-OHDA monkeys. Scale bar  = 50 µm; inset scale bar  = 50 µm. <i>*P</i><0.05, **<i>P</i><0.01. AADC, aromatic l-amino acid; AAT, area above threshold; OD, optical density; TH, tyrosine hydroxylase.</p

PGP9.5 and TH immunofluorescense demonstrated colabeling and decreased expression in cardiac nerve bundles of 6-OHDA monkeys.

<p>Microphotographs of immunofluorescence staining of PGP9.5 (red; <b>A,E</b>), TH (green; <b>B,F</b>) and counterstained with DAPI (blue nuclei; <b>C,G</b>) in controls (A–D) and 6-OHDA-treated animals (E–H). Colocalization of PGP9.5 and TH expression is identified as yellow. PGP9.5, protein gene product 9.5; TH, tyrosine hydroxylase.</p