Background/Aims: Impaired adipogenesis may be the underlying cause in the development of obesity and type II diabetes. Mechanistically, the family of Homeobox transcription factors is implicated in the regulation of adipocyte fate. Hoxa5 is highly expressed in adipocytes, and its mRNA expression is decreased during differentiation. However, the function of Hoxa5 in adipose tissue has been poorly understood. The aim of this study is to unveil the role of Hoxa5 on adipocyte differentiation and its underlying mechanisms. Methods: Quantitative real-time PCR (qPCR) and western blot were performed to determine Hoxa5 expression in primary adipocytes and in adipose tissues from mice. Lipid accumulation was evaluated by bodipy staining. Dual luciferase assay was applied to explore the transcription factor of Hoxa5 and the transcriptional target gene modulated by Hoxa5. All measurements were performed at least for three times at least. Results: A significant reduction of Hoxa5 expression was observed in adipose tissue of High Fat Diet (HFD) induced obesity mice. We determined Hoxa5 increased adipocytes differentiation and mitochondrial biogenesis in adipocytes in vitro. CEBPβ was determined a transcription factor of Hoxa5 and inhibited methylation level of Hoxa5 by combining on the promoter of Hoxa5. Importantly, we found Fabp4, a known positive regulator of adipocytes differentiation, was transcriptional activation by Hoxa5. In addition, Hoxa5 promotes adipocytes differentiation by inhibiting PKA/HSL pathway. Conclusion: Our study demonstrated the promoting role of Hoxa5 in adipocytes differentiation and therefore bringing a new therapeutic mean to the treatment of obesity and type II diabetes.

Alpha melanocyte stimulating hormone (α-MSH) and Forkhead box C2 protein (Foxc2) enhance lipolysis in multiple tissues. However, their relationship in adipose fatty acid oxidation (FAO) remains unclear. Here, we demonstrated that α-MSH and Foxc2 increased palmitate oxidation to CO₂ in white (WAT) and brown adipose tissue (BAT). C/EBPβ expression was reduced by α-MSH and Foxc2. FFA level was elevated by α-MSH and pc-Foxc2 treatment along with increased FAO in white and brown adipocytes. The expression of FAO key enzymes, medium-chain acyl-CoA dehydrogenase (MCAD) and long-chain acyl-CoA dehydrogenase (LCAD) were increased in α-MSH and pc-Foxc2 group. Combination of α-MSH and Foxc2 treatment synergistically promoted FAO through increasing the activity of CPT-1 and phosphorylation of ACC. We found C/EBPβ bind to MC5R and Foxc2 promoter regions and inhibited FAO. cAMP level was increased by α-MSH and Foxc2 individually treated or combined treatment. Furthermore, cAMP/PKA pathway-specific inhibitor (H89) blocked the FAO, despite in α-MSH and Foxc2 both added group. While forskolin, the cAMP agonist, promoted FAO and enhanced the effect of α-MSH and Foxc2. Collectively, α-MSH and Foxc2 mutual promote FAO in WAT and BAT via cAMP/PKA signal pathway. And C/EBPβ as a transcription suppressor inhibits α-MSH and Foxc2 expression and FAO.

All possible diastereomeric C9-hydroxymethyl-, hydroxyethyl-, and hydroxypropyl-substituted 5-phenylmorphans were synthesized to explore the three-dimensional space around the C9 substituent in our search for potent MOR partial agonists. These compounds were designed to lessen the lipophilicity observed with their C9-alkenyl substituted relatives. Many of the 12 diastereomers that were obtained were found to have nanomolar or subnanomolar potency in the forskolin-induced cAMP accumulation assay. Almost all these potent compounds were fully efficacious, and three of those chosen for in vivo evaluation, 15, 21, and 36, were all extremely G-protein biased; none of the three compounds recruited beta-arrestin2. Only one of the 12 diastereomers, 21 (3-((1S,5R,9R)-9-(2-hydroxyethyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), was a MOR partial agonist with good, but not full, efficacy (E_max = 85%) and subnanomolar potency (EC₅₀ = 0.91 nM) in the cAMP assay. It did not have any KOR agonist activity. This compound was unlike morphine in that it had a limited ventilatory effect in vivo. The activity of 21 could be related to one or more of three well-known theories that attempt to predict a dissociation of the desired analgesia from the undesirable opioid-like side-effects associated with clinically used opioids. In accordance with the theories, 21 was a potent MOR partial agonist, it was highly G-protein biased and did not attract beta-arrestin2, and it was found to have both MOR and DOR agonist activity. All the other diastereomers that were synthesized were either much less potent than 21 or had either too little or too much efficacy for our purposes. It was also noted that a C9-methoxymethyl compound with 1R,5S,9R stereochemistry (41) was more potent than the comparable C9-hydroxymethyl compound 11 (EC₅₀ = 0.65 nM for 41 vs. 2.05 nM for 11). Both 41 and 11 were fully efficacious.

Four sets of diastereomeric C9-alkenyl 5-phenylmorphans, varying in the length of the C9-alkenyl chain, were designed to examine the effect of these spatially distinct ligands on opioid receptors. Functional activity was obtained by forskolin-induced cAMP accumulation assays and several compounds were examined in the [³⁵S]GTPgS assay and in an assay for respiratory depression. In each of the four sets, similarities and differences were observed dependent on the length of their C9-alkenyl chain and, most importantly, their stereochemistry. Three MOR antagonists were found to be as or more potent than naltrexone and, unlike naltrexone, none had MOR, KOR, or DOR agonist activity. Several potent MOR full agonists were obtained, and, of particular interest partial agonists were found that exhibited less respiratory depression than that caused by morphine. The effect of stereochemistry and the length of the C9-alkenyl chain was also explored using molecular modeling. The MOR antagonists were found to interact with the inactive (4DKL) MOR crystal structures and agonists were found to interact with the active (6DDF) MOR crystal structures. The comparison of their binding modes at the mouse MOR was used to gain insight into the structural basis for their stereochemically induced pharmacological differences.

Kappa opioid receptor (KOR) agonists have well-established antinociceptive effects. However, many KOR agonists have negative side effects, which limit their therapeutic potential. Some researchers have suggested that the development of biased agonists that preferentially stimulate KOR G-protein pathways over β-arrestin pathways may yield drugs with fewer adverse side effects. This was investigated in the current study. We describe the synthesis and characterization of three U50,488 analogues, 1, 2, and 3. We evaluated the acute and chronic antinociceptive effects of these compounds in mice using the warm-water tail flick assay and in a paclitaxel-induced neuropathic pain model. Side effects were investigated using open-field, passive wire hang, rotarod, elevated zero maze, conditioned place aversion, and whole-body plethysmography, with some tests being conducted in KOR or β-arrestin2 knock out mice. All compounds were highly potent, full agonists of the KOR, with varying signaling biases in vitro. In the warm-water tail withdrawal assay, these agonists were ~10 times more potent than U50,488, but not more efficacious. All KOR agonists reversed paclitaxel-induced neuropathic pain, without tolerance. Compound 3 showed no significant side effects on any test. Signaling bias did not correlate with the antinociceptive or side effects of any compounds and knockout of β-arrestin2 had no effect on U50,488-induced sedation or motor incoordination. These findings highlight the therapeutic potential of 3, with its lack of side effects typically associated with KOR agonists, and also suggest that G-protein signaling bias is a poor predictor of KOR agonist-induced side effects.

The 5-(3-hydroxy)phenylmorphan structural class of compounds are unlike the classical morphinans, 4,5-epoxymorphinans, and 6,7-benzomorphans, in that they have an equatorially oriented aromatic ring rather than the axial orientation of that ring found in the classical opioids. This modified and simplified opioid-like structure has been shown to retain antinociceptive activity, depending on its stereochemistry and substituents, and some of them have been found to be much more potent than morphine. A simple C9-hydroxy-5-(3-hydroxy)phenylmorphan enantiomer was found to be about 500 times more potent than morphine in vivo. We have previously examined C9-alkenyl and hydroxyalkyl substituents in the N-phenethyl-5-(3-hydroxy)phenylmorphan class of compounds. Comparable C9-alkyl (methyl through butyl) substituents, with their sets of diastereomers, have not been explored. All these compounds have now been synthesized to determine the effect chain-length and stereochemistry at the C9 position in the molecule might have on their interaction with opioid receptors. We now report the synthesis and in vitro activity of 16 compounds, the C9-methyl, ethyl, propyl, and butyl diastereomers, using the inhibition of forskolin-induced cAMP accumulation assay. Several potent (sub-nanomolar and nanomolar) MOR compounds were found to be selective agonists with varying efficacy. Of greatest interest, a selective MOR antagonist was discovered; it did not display any DOR or KOR agonist activity in vitro, was three times more potent than naltrexone, and was found to antagonize the EC90 of fentanyl at MOR to a greater extent than naltrexone.