Effects of AgRP and TTP2515 on α-MSH-induced cAMP production.

<p>(<b>A)</b> α-MSH-induced cAMP production in HEK293 cells overexpressing human MC4R (green diamond); inhibition of α-MSH(3 nM)-induced cAMP production by increasing amounts of AgRP (pink diamond) (<b>B)</b> α-MSH-induced cAMP production in HEK293 cells overexpressing human MC4-R in the presence of 20 nM AgRP, 3 nM α-MSH and increasing concentrations of TTP2515 (red diamond). (C+, cAMP production in the presence of α-MSH (3 nM) and AgRP (20 nM) and in the absence of TTP2515. C-, cAMP production in the presence of α-MSH (3 nM) no AgRP or TTP2515).</p

Effects of TTP2515 on metabolic and calorimetry parameters in AgRP KO and WT mice on a 45% high fat diet.

<p>(<b>A–F</b>) AgRP KO and WT mice received TTP2515 (30 mg/kg) and AgRP KO mice received water twice daily via oral gavage; all groups were on a 45% fat diet. (<b>A</b>) During days 1–4, there was an overall suppressive effect of TTP2515 treatment on food intake (p<0.05). Food intake was lower on days 2–4 in the WT TTP2515 and KO TTP2515 groups compared to the KO water group (p<0.05) (<b>B</b>) WT TTP2515 mice gained significantly less fat mass during treatment vs. the KO water group, however, this was not true for the KO TTP2515 group (p = 0.12). (<b>C</b>) Overall, there was a significant suppressive effect of treatment on cumulative body weight change (p = 0.01); body weight gain was more frequently significantly lower in the WT TTP2515 vs. the KO water group, rather than in the KO TTP2515 vs. the KO water group (<b>D,E</b>) Total and free T4 levels were elevated in both TTP2515 treatment groups compared to the KO water group. (<b>F–H</b>) AgRP KO mice were administered either TTP2515 (30 mg/kg days 1–7; 50 mg/kg days 7–10) or water twice daily. (<b>F,G</b>) On day 3 during the dark cycle, oxygen consumption and activity were significantly lower in TTP2515–treated mice. (<b>H</b>) RQ was not different between groups during the light or dark cycle at the 50 mg/kg dose (showing day 10, representative graph of this timeperiod). +p<0.05 KO Water vs KO TTP2515; *p<0.05 KO Water vs WT TTP2515; ^#p<0.05 vs water.</p

<b>Table 3.</b> VO2, EE and activity during the dark cycle in calorimetry experiments.

<p>In all VO2 and EE measurements above, adjustment for activity rendered VO2 and EE either significantly higher in the TTP2515 group or equivalent between groups. W, Watts; Values are mean ± SE.</p>**<p>p<0.01,</p>*<p>p<0.05 vs water.</p

<b>Table 1.</b> Effects of TTP2515 on hormone levels and liver weight in rats receiving icv AgRP infusion.

<p>All measurements reported at sacrifice in <i>ad lib</i> fed rats. Values are mean ± SE.</p>*<p>p<0.05 all groups;</p>†<p>p<0.05 vs AgRP+Water.</p

Effects of TTP2515 on refeeding and T4 after fasting in AgRP KO and WT mice.

<p>(<b>A–F</b>) AgRP KO and WT mice were fasted for 24 h and received either water or TTP2515 at 10, 25, or 50 mg/kg in three different experiments. (<b>A,B</b>) At the 10 mg/kg dose, no differences in food intake or body weight gain were observed. (<b>C,D</b>) At the 25 mg/kg dose, there was an overall significant attenuation in food intake (p<0.05) and weight gain (p<0.01) in the WT TTP2515 vs. the WT water group. Post-hoc analysis revealed that this was significant at the 20 h for food intake and at 9 and 20 h for weight gain. No differences in food intake or weight gain were observed between the KO TTP2515 vs KO water group. (<b>E</b>) At the 50 mg/kg dose, there was an overall significant attenuation in food intake in both the WT TTP2515 (p<0.01) and KO TTP2515 (p<0.05) groups vs. their water controls. Final cumulative food intake at the 20 h timepoint was significantly less in both TTP2515 groups vs. their water groups. (<b>F</b>) At the 50 mg/kg dose, final cumulative weight gain at 20 h was significantly less only in the WT TTP2515 vs WT water group. (<b>G</b>) A separate group of mice was fasted and blood samples were obtained before and after treatment. No difference in total T4 levels were observed before or after treatment. ***p<0.0001, **p<0.01, *p<0.05 vs respective water control.</p

Effects of TTP2515 in WT or AgRP KO mice acutely switched to a very high-fat diet.

<p>(<b>A–F</b>) Mice were switched from breeder chow to a 60% fat diet and started treatment with TTP2515 (30 mg/kg) or water twice daily. (<b>A–C</b>) Cumulative food intake, cumulative weight gain and fat mass were all significantly lower in TTP2515 treated mice. (<b>D</b>) Total T4 levels were significantly higher in TTP2515 treated mice. (<b>E</b>) Leptin levels tended to be lower in TTP2515 treated mice (p = 0.12). (<b>F</b>) Insulin levels were similar between groups. (<b>G–J</b>) In a separate experiment, AgRP KO mice on a 10% fat chow diet were switched to a 60% fat diet and started treatment with TTP2515 (5, 15, or 30 mg/kg) or water twice daily. (<b>G,I</b>) Cumulative caloric intake and cumulative weight gain were dose-dependently lower in TTP2515 treated mice compared to the water group. (<b>H</b>) Fat mass was lower in TTP2515 treated mice at both the 15 and 30 mg/kg doses vs. water. The 5 mg/kg group tended (p = 0.05) to have lower fat mass than the water group. (<b>J</b>) After 5 days of treatment, total T4 levels were equivalent between groups, however fasting and continued treatment revealed an increase in total T4 levels. *p<0.05, **p<0.01 vs water; <i>a</i>p<0.05 vs water, <i>b</i>p<0.05 vs 5 mg/kg, <i>c</i>p<0.05 vs 15 mg/kg.</p

Effects of TTP2515 in DIO and leptin-deficient mice.

<p>All measurements reported at sacrifice in <i>ad lib</i> fed mice unless otherwise indicated.</p><p>Values are mean ± SE.</p>*<p>p<0.05 vs water.</p

Effects of TTP2515 on metabolic and calorimetry parameters in leptin-deficient mice.

<p>Leptin-deficient mice received either TTP2515 at increasing doses (5–50 mg/kg) or water twice daily via oral gavage. (<b>A</b>) Food intake tended to be lower in TTP2515 treated mice during the 15 mg/kg treatment period and was significantly lower during the 30 and 50 mg/kg treatment periods. (<b>B</b>) Fat mass was significantly decreased after 24 days of TTP2515 treatment, while lean mass was not different between groups. (<b>C</b>) Body weight was significantly lower in TTP2515-treated mice on the 50 mg/kg dose. (<b>D</b>) Immediately upon starting the 50 mg/kg dose, mean respiratory quotient was significantly decreased in TTP2515 treated mice during the light cycle. (<b>E</b>) Total T4 levels at sacrifice were unchanged between groups. +p = 0.06, *p<0.05, **p<0.01 vs water.</p

Effects of TTP2515 on metabolic and calorimetry parameters in DIO mice.

<p>(<b>A–E</b>) DIO mice (16 weeks on 45% fat diet) received TTP2515 (30 mg/kg) or water twice daily via oral gavage. (<b>A,B</b>) TTP2515 treatment significantly decreased food intake and body weight gain. (<b>C</b>) Percent fat mass was significantly lower in TTP2515 treated mice at the end of the study. (<b>D</b>) TTP2515 treated mice had elevated total T4 levels. (<b>E</b>) Total T3 levels were similar between groups. (<b>F–H</b>) In a separate experiment DIO mice (45% fat diet for 15 weeks) received TTP2515 (30 mg/kg) or water twice daily via oral gavage. (<b>F,G</b>) Mean oxygen consumption and total activity were significantly decreased during the dark cycle of day 2 in TTP2515 treated mice. (<b>H</b>) Mean respiratory quotient was significantly decreased during the first half of the light cycle of day 3 in TTP2515 treated mice. **p<0.0001, *p<0.05 vs. water.</p

The <i>Pomc</i> neuronal enhancers nPE1 and nPE2 Share a common <i>cis</i>-regulatory code.

<p>(A) Evolutionary tree of placental mammalian lineages. The relative lengths of DNA sequences in kilobases (kb) separating nPE1 from nPE2 and nPE2 from exon 1 are illustrated by purple and green bars, respectively. (B) Alignment between an almost palindromic sequence carrying canonical homeodomain binding sites (HDBS) present in nPE1core and a remarkably similar sequence present within nPE2 region 1. (C) Scheme of nPE1core and regions 1 and 3 of nPE2 showing the relative positions of conserved HDBS. nPE1core carries a pair of inverted HDBS similar to another present in nPE2 region 1 (green boxes, full sequences depicted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1B</a>). Another pair of HDBS is present in region 3 of nPE2 (purple boxes). Canonical HDBS of the NKX subfamily are shown (blue boxes). Red letters indicate the mutated nucleotides. Grey boxes denote the critical enhancer regions determined previously in transgenic mice. (D) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE1core. The control nPE1core<i>Pomc</i>-EGFP carries the wild-type (wt) enhancer sequence whereas nPE1core(mut)<i>Pomc</i>-EGFP (mut) carries six nucleotide substitutions covering all HDBS (red letters in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1C</a>). Coronal brain sections showing EGFP expression in hypothalamic arcuate nucleus of nPE1core<i>Pomc</i>-EGFP (Left) but not in nPE1core(mut)<i>Pomc</i>-EGFP transgenic founder newborn mice (Right). (E) Two nearly identical transgenes were constructed to study the importance of the HDBS present in nPE2. The control transgene nPE2<i>Pomc</i>-EGFP carries the wild-type (wt) enhancer whereas nPE2(mut)<i>Pomc</i>-EGFP (mut) carries twelve nucleotide substitutions covering all HDBS (red letters in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004935#pgen.1004935.g001" target="_blank">Fig. 1C</a>). EGFP is expressed in the arcuate nucleus of founder transgenic mice carrying nPE2<i>Pomc</i>-EGFP (Left) but not nPE2(mut)<i>Pomc</i>-EGFP (Right). 3V, third ventricle.</p