L4 - Hypothalamus and the Control of Energy Balance

what did early studies on energy balance do and find?

• used rat lesion experiments to identify which parts of the hypothalamus regulates food intake

• focus was on the arcuate nucleus (ARCN) located ventrally in the tuberal hypothalamus

• lesions of the ARCN produced complicated phenotypes with tendency to overeat/obesity or starvation

• results demonstrated that tuberal hypothalamus specifically the ARCN plays a central role in controlling energy balance and homeostasis

what is the view of energy homeostasis?

• in the ARCN of the tuberal hypothalamus, distinct neuron populations are activated based on energy status

• some neurons are activated during fasting (low energy) states

• some neurons are activated during obese (high energy) states

what are the two major classes of neurons in the ARCN?

POMC and NPY

what do the POMC and NPY neurons express?

• leptin and insulin receptors

• leptin and insulin provide direct measures of the body’s energy and glucose/fat levels

• POMC and NPY are able to monitor the amount of leptin and insulin as they have leptin and insulin receptors

what happens when leptin binds POMC neurons?

activates POMC neurons

what happens when leptin binds NPY neurons?

inhibits NPY neurons

what do POMC and NPY neuron axons project to?

• project to Mc4r neuron

• when Mc4r neuron is stimulated or repressed it coordinates an array of activities by other centres

what does activation of NPY neurons lead to?

• leads the brain to coordinate an array of activities that stimulate food intake and reduce energy expenditure

• low energy → NPY activated → eat more, burn less

what does activation of POMC lead to?

• leads the brain to coordinate an array of activities that reduce food intake and increase energy expenditure

• high energy → POMC activated → eat less, burn more

how do neurons of the ARCN act?

antagonistically, resulting in an antagonistic balance

describe the molecular specification of ARCN neurons

• early tuberal progenitors in the hypothalamus are defined by expression of Nkx2.1

• TF such as Tbc3 and Rax are then activated, the tuberal progenitors will either become an ARC tuberal progenitor or ARC neuronal precursor

• addition TFs then determine whether the ARCN neuron will differentiate into a POMC neuron or NPY neuron

how can we study specification?

• aim is to understand the molecular pathways, by observing which TFs are expressed, in which cells and at what developmental time point

• gain of function, loss of function, genetic lineage tracing, conditional knockout or conditional gene activation - all used to determine whether a specific gene is required in a specific molecular pathway

why did researchers study Islet1?

• to test whether Islet1 is expressed in early ARCN neuronal progenitors

• whether Islet1 is required for differentiation of Pomc neurons

what have studies into Islet1 shown?

• that Islet1 is expressed in relatively late progenitor cells that are about to become POMC neurons

• that Islet1 expression occurs before POMC expression

• that Islet1 is important for proper development of POMC expressing ARCN neurons

how was Islet1 and POMC expression analysed experimentally?

• as POMC is a small neuropeptide it is difficult to detect

• antibodies against its precursor protein ACTH are used to visualise POMC expression

• mouse embryos were examined at multiple developmental stages using immunohistochemistry to detect Islet1 and ACTH

• in adults, a combination of immunohistochemistry and transgenic POMC-EGFP reporter line was used to determine whether POMC-expressing cells also express Islet1

what were the findings from the experimental analysis of Ilset1 and POMC expression?

• Islet1 is expressed before POMC

• in adult mice, POMC-EGFP cells consistently co-express Ilset1, indicating that once progenitor cells acquire a transcriptional profile, it is maintained into adulthood

how does Islet1 directly activate POMC?

• Islet1 directly activates POMC transcription by binding to specific homeodomain DNA motifs present within the neuronal POMC enhances nPE1 and nPE2

• when Islet1 binds to these enhancer regions, it activates transcription of the POMC gene, both nPE1 and nPE2 are required for normal POMC expression

what happens if the POMC enhancer sequences are mutated, and what does this mean?

• Islet1 can no longer recognise or bind the DNA

• results in loss of POMC transcription

• it means that Islet1 binding to nPE1 and nPE2 is necessary for activation of POMC expression

• Islet1 is necessary for POMC expression

how is the function role of nPE1 and nPE2 enhancers demonstrated?

• mutations are introduced into nPE1 and nPE2 enhancer elements to disrupt their ability to drive gene expression

• when the enhancer sequences are mutated, reporter expression in POMC neurons is lost

• this demonstrates that nPE1 and nPE2 are required enhancer elements for activating POMC gene expression in the hypothalamus

what type of experiment must be done to confirm whether Islet1 is required for POMC neurons?

a conditional knockout of Islet1

why does it matter when Islet1 is knocked out?

• Islet1 is required for early vascular development

• Islet1 must be knocked out conditionally at a later developmental stage after vascular development has occurred

• a constitutive KO can’t be used as it will lead to embryonic lethality, meaning no embryos survive long enough for POMC neurons to form

why is a conditional knockout used to investigate Islet1 function?

• this approach allows to investigate Islet1 function in hypothalamic neuron differentiation without disrupting essential early developmental processes

explain the conditional KO of Islet1 experiment

• Islet1 was conditionally KO and POMC expression was assessed using anti-ACTH antibody at two developmental stages

• in the Islet1 KO embryos - neither Islet1 or ACTH was detected at either stage, indicating a failure of POMC neuron differentiation

• in control embryos - both Islet1 and ACTH were expressed at both stages

what did the results of the Islet1 conditional KO experiment demonstrate?

• that Islet1 is essential for the differentiation of POMC-expressing neurons in the arcuate nucleus

why is the expression of both Islet1 and POMC analysed by immunohistochemistry?

• Islet1 immunohistochemistry confirms that the conditional KO successfully removes Islet1 protein

• ACTH staining shows whether POMC neurons differentiate

• loss of both Islet1 and ACTH demonstrates that failure of POMC differentiation is due to Islet1 deletion

How are behavioural consequences of Islet1/POMC loss examined, and what do they lead to?

• to determine functional role of POMC neurons, mice with a conditional KO of Islet1 were allowed to develop into adulthood and compared with control mice

• Islet1 KO mice fail to generate POMC neurons showing early onset obesity

• this phenotype reflects the normal role of POMC as a neurohormone, which activates neural circuits that reduce food intake and increase energy expenditure

• loss of POMC therefore disrupts energy homeostasis, leading to extensive weight gain

why do Islet1 KO mice exhibit early onset obsesity?

• as Islet1 is knocked out, POMC neurons fail to develop and cannot activate brain circuits that normally reduce food intake and increase energy expenditure, therefore leading to early onset obesity in the mice