In the hypothalamus, insulin takes on many roles involved in energy homoeostasis. Therefore, the aim of this study was to examine hypothalamic insulin expression during the initial phase of the ...metabolic response to fasting. Hypothalamic insulin content was assessed by both radioimmunoassay and Western blot. The relative expression of insulin mRNA was examined by qPCR. Immunofluorescence and immunohistochemistry were used to determine the distribution of insulin immunopositivity in the hypothalamus. After 6‐h fasting, both glucose and insulin levels were decreased in serum but not in the cerebrospinal fluid. Our study showed for the first time that, while the concentration of circulating glucose and insulin decreased, both insulin mRNA expression and insulin content in the hypothalamic parenchyma were increased after short‐term fasting. Increased insulin immunopositivity was detected specifically in the neurons of the hypothalamic periventricular nucleus and in the ependymal cells of fasting animals. These novel findings point to the complexity of mechanisms regulating insulin expression in the CNS in general and in the hypothalamus in particular.
Short‐term fasting increased the expression of insulin mRNA and protein insulin content in the rat hypothalamus. Increased insulin immunopositivity was detected in NeuN‐positive cells of periventricular nucleus and in ependymal cells around the third ventricle.
Purpose
Our previous study showed that 6-h fasting increased insulin expression in the hypothalamus of male rats. We, therefore, wanted to examine if this phenomenon occurs in female rats and whether ...it depended on the estrus cycle phase.
Methods
Female rats in proestrus or diestrus were either exposed to 6-h fasting or had
ad libitum
access to food. The serum, cerebrospinal fluid, and hypothalamic insulin levels were determined using radioimmunoassay. The hypothalamic insulin mRNA expression was measured by RT-qPCR, while the hypothalamic insulin distribution was assessed immunohistochemically.
Results
Albeit the short-term fasting lowered circulating insulin, both hypothalamic insulin mRNA expression and hypothalamic insulin content remained unaltered. As for the hypothalamic insulin distribution, strong insulin immunopositivity was noted primarily in ependymal cells lining the upper part of the third ventricle and some neurons mainly located within the periventricular nucleus. The pattern of insulin distribution was similar between the controls and the females exposed to fasting regardless of the estrous cycle phase.
Conclusion
The findings of this study indicate that the control of insulin expression in the hypothalamus differs from that in the pancreatic beta cells during short-term fasting. Furthermore, they also imply that the regulation of insulin expression in the female hypothalamus is different from males but independent of the estrus cycle phase.
Gladovanje predstavlja stanje tokom kojeg nema unosa hrane, pa se stoga potrebe za energijom zadovoljavaju razgradnjom endogenih energetskih depoa. U zavisnosti od dužine trajanja gladovanje može ...narušiti energetsku homeostazu. Hipotalamus kao mesto integracije signala o energetskom statusu organizma ima veoma važnu ulogu u regulaciji energetske homeostaze. Prethodne studije su pokazale da tokom kratkotrajnog gladovanja dolazi do smanjenja koncentracije insulina u krvi, dok je u hipotalamusu detektovana povećana količina insulina. Ovo neočekivano saznanje otvorilo je mnogobrojna pitanja, stoga je predmet ove doktorske disertacije bio ispitivanje uticaja šestočasovnog gladovanja na ekspresiju insulina u hipotalamusu i ispitivanje adaptivnog značaja ovog fenomena.U ekperimentima su korišćeni mužjaci pacova Wistar soja. Jedna grupa životinja imala je neometan pristup hrani (ad libitum),a drugoj je hrana uklonjena u trajanju od šest sati. Analizirana je genska i proteinska ekspresija insulina u hipotalamusu. Pokazano je da nakon šest sati gladovanja dolazi do povećanja količine insulina, kao i povećanja ekspresije iRNK za insulin. Proinsulin i insulin su detektovani u neuronima periventrikularnog jedra hipotalamusa i u ependimskim ćelijama koje okružuju treću moždanu komoru. Koncentracija glukoze i insulina u krvi bila je smanjena nakon kratkotrajnog gladovanja dok je u cerebrospinalnoj tečnosti ostala nepromenjena. Nakon kratkotrajnog gladovanja zabeležena je povećana količina aktivnog insulinskog receptora u periventrikularnom jedru. Pored toga, u membranskoj frakciji proteina izolovanih iz hipotalamusa zabeleženo je povećano prisustvo GLUT1 (55kDa), izoforme karakteristične za endotelne ćelije, povećanje neuronskih GLUT2 i GLUT3, dok je količina astrocitne GLUT1 (45 kDa) izoforme i GLUT4 ostala nepromenjena. Nakon šestočasovnog gladovanja nije zabeležena koekspresija aktivnog insulinskog receptora i glukoznih transportera. Količina glikogena u hipotalamusu ostala je nepromenjena. Ukupna i fosforilisana forma proteina uključenih u PI3K-AKT/PKB signalni put (IRS1, pIRS1-Tyr612, IRS2, pIRS2-Ser731, mTOR i pmTOR-Ser2448) ostala je nepromenjena nakon šestočasovnog gladovanja. Nasuprot tome, iako je ukupna količina ERK1/2 bila smanjena, detektovano je povećano prisustvo aktivne forme ovih enzima.Na osnovu dobijenih rezultata može se zaključiti da tokom kratkotrajnog gladovanja dolazi do de novo sinteze insulina u periventrikularnom jedru hipotalamusa. Takođe, rezultati ukazuju da se u početnoj fazi gladovanja procesi preuzimanja glukoze od strane nervnih ćelija kao i sinteza glikogena u hipotalamusu odvijaju nezavisno od dejstva insulina. Na osnovu promena detektovanih u količini glukoznih transportera rezultati sugerišu da se u kratkotrajnom gladovanju neuronima daje prioritet u snadbevanju glukozom odnosu na astrocite. Nakon kratkotrajnog gladovanja iako je detektovana aktivacija insulinskog receptora, ne dolazi do aktivacije PI3K-AKT/PKB signalnog puta, ali je zabeležena aktivacija ERK1/2. Dodatnom analizom signalnih puteva nishodno od ERK1/2 ostaje da se otkrije koja je uloga aktivacije ERK1/2 i da li de novosintetisani insulin tokom kratkotrajnog gladovanja doprinosi njihovoj aktivaciji.
