Molecular Metabolism ( IF 7.0 ) Pub Date : 2020-04-18 , DOI: 10.1016/j.molmet.2020.100985 Milagros Trotta 1 , Estefanía Pilar Bello 1 , Ramiro Alsina 1 , María Belén Tavella 2 , José Luis Ferrán 3 , Marcelo Rubinstein 4 , Viviana Florencia Bumaschny 5
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Objective
Hypothalamic arcuate proopiomelanocortin (Arc-POMC) neurons are involved in different physiological processes such as the regulation of energy balance, glucose homeostasis, and stress-induced analgesia. Since these neurons heterogeneously express different biological markers and project to many hypothalamic and extrahypothalamic areas, it is proposed that Arc-POMC neurons could be classified into different subpopulations having diverse physiological roles. The aim of the present study was to characterize the contribution of the subpopulation of Arc-POMC neurons cosecreting gamma-aminobutyric acid (GABA) neurotransmitter in the control of energy balance.
Methods
Arc-Pomc expression restricted to GABAergic-POMC neurons was achieved by crossing a reversible Pomc-deficient mouse line (arcPomc−) with a tamoxifen-inducible Gad2-CreER transgenic line. Pomc expression was rescued in the compound arcPomc−/−:Gad2-CreER female and male mice by tamoxifen treatment at postnatal days 25 (P25) or 60 (P60), and body weight, daily food intake, fasting glycemia, and fasting-induced hyperphagia were measured. POMC recovery was quantified by immunohistochemistry and semiquantitative RT-PCR. Neuropeptide Y (NPY) and GABAergic neurons were identified by in situ hybridization. Arc-POMC neurons projecting to the dorsomedial hypothalamic nucleus (DMH) were studied by stereotactic intracerebral injection of fluorescent retrobeads into the DMH.
Results
Tamoxifen treatment of arcPomc−/−:Gad2-CreER mice at P60 resulted in Pomc expression in ∼23–25% of Arc-POMC neurons and ∼15–23% of Pomc mRNA levels, compared to Gad2-CreER control mice. Pomc rescue in GABAergic-POMC neurons at P60 normalized food intake, glycemia, and fasting-induced hyperphagia, while significantly reducing body weight. Energy balance was also improved in arcPomc−/−:Gad2-CreER mice treated with tamoxifen at P25. Distribution analysis of rescued POMC immunoreactive fibers revealed that the DMH is a major target site of GABAergic-POMC neurons. Further, the expression of the orexigenic neuropeptide Y (NPY) in the DMH was increased in arcPomc−/− obese mice but was completely restored after Pomc rescue in arcPomc−/−:Gad2-CreER mice. Finally, we found that ∼75% of Arc-POMC neurons projecting to the DMH are GABAergic.
Conclusions
In the present study, we show that the expression of Pomc in the subpopulation of Arc-GABAergic-POMC neurons is sufficient to maintain normal food intake. In addition, we found that DMH-NPY expression is negatively correlated with Pomc expression in GABAergic-POMC neurons, suggesting that food intake may be regulated by an Arc-GABAergic-POMC → DMH-NPY pathway.
中文翻译：
下丘脑Pomc表达仅限于GABA能神经元，可抑制Npy过表达并恢复肥胖小鼠的食物摄入。
目的
下丘脑弓形proopiomelanocortin（Arc-POMC）神经元参与不同的生理过程，例如能量平衡，葡萄糖稳态和压力诱导的镇痛的调节。由于这些神经元异质表达不同的生物学标记并投射到许多下丘脑和下丘脑区域，因此提出将Arc-POMC神经元分为具有不同生理作用的不同亚群。本研究的目的是表征共同分泌γ-氨基丁酸（GABA）神经递质的Arc-POMC神经元亚群在控制能量平衡中的作用。
方法
弧形POMC限于GABA能-POMC神经元表达通过几根可逆实现的POMC缺陷型小鼠系（弧POMC -用他莫昔芬诱导型）GAD2 -CreER转基因系。在复合弧Pomc -/-：Gad2中拯救了pomc表达-在出生后第25天（P25）或第60天（P60）通过他莫昔芬治疗的雌性和雄性小鼠的Crere，并测量体重，每日食物摄入量，空腹血糖和空腹诱发的食欲亢进。通过免疫组织化学和半定量RT-PCR定量POMC的回收率。通过原位杂交鉴定神经肽Y（NPY）和GABA能神经元。通过立体定向脑内向DMH注射荧光后向珠，研究了投射到背丘下丘脑核（DMH）的Arc-POMC神经元。
结果
他莫昔芬治疗电弧POMC - / - ：GAD2在P60 -CreER小鼠导致POMC表达在电弧POMC神经元的~23-25％和~15-23％POMC mRNA水平相比，GAD2 -CreER对照小鼠。P60时对GABAergic -POMC神经元进行Pomc抢救可使食物摄入，血糖和空腹诱发的食欲亢进正常化，同时显着减轻了体重。电弧Pomc -/-的能量平衡也得到改善：Gad2-在P25时用他莫昔芬治疗的-CreER小鼠。拯救的POMC免疫反应性纤维的分布分析表明，DMH是GABAergic-POMC神经元的主要靶位。此外，在弧形Pomc -/-肥胖小鼠中，DMH中的致病性神经肽Y（NPY）的表达增加，但是在弧形Pomc -/-：Gad2- CreER小鼠中，在Pomc拯救后，其完全恢复。最后，我们发现投射到DMH的Arc-POMC神经元中约有75％是GABA能的。
结论
在本研究中，我们表明，Arc- GABAergic -POMC神经元亚群中Pomc的表达足以维持正常的食物摄入。此外，我们发现DMH-NPY表达与GABAergic -POMC神经元中的Pomc表达负相关，这表明食物摄取可能受Arc-GABAergic-POMC→DMH-NPY途径的调节。
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