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AMPK is a kinase, which is allosterically activated by AMP and inhibited by ATP and is, therefore, generally activated in a low intracellular energy state. It is also directly activated by endocrine signals of the fasted state, including ghrelin, and is inhibited by endocrine signals of the fed state, including leptin, insulin and GLP1 [181]. For a graphic representation of intracellular energy sensing see Figure 1.2.

In fasting, ghrelin binds to its membrane-bound receptor, growth-hormone stimulating receptor (GhSR), induces phosphorylation and, with that, activation of AMPK, which in turn decreases acetyl-CoA carboxylase (ACC)

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and fatty acid synthase (FAS) activity in the VMN of the hypothalamus [182]. Subsequently, the concentration of malonyl-CoA is decreased [182], which reduces glycolysis and an increase in mitochondrial ß-oxidation by induction of carnitine palmitoyl-transferase (CPT1) expression [182, 183]. How exactly this AMPK pathway induces increased expression of NPY/AgRP and reduces POMC expression is not clear but it is likely to involve

induction of transcription factors in response to alterations in intracellular lipid metabolism. Ghrelin induces expression of the brain-specific

homeobox transcription factor (BSX) through induction of CPT1 [184]. Both expression of NPY and AgRP are induced by this transcription factor, in combination with cAMP-responsive element binding protein (CREB) and forkhead box O1 (FOXO1), respectively [185].

Leptin, on the other hand, decreases phosphorylation of AMPK in the fed state, with opposite effects on intracellular lipid metabolism, therefore increasing intracellular malonyl-CoA, inhibiting CPT1 and inhibiting BSX- dependent expression of NPY and AgRP. Furthermore, it inhibits FOXO1 and, in this way, inhibits AgRP production [186]. POMC expression is repressed by FOXO1, an effect that is alleviated through signal transducer and activator of transcription 3 (STAT3) [186], which is induced by insulin and leptin [187]. NPY suppression by leptin acts through a STAT3-

independent mechanism. At least part of the NPY suppression is signalled through a MAPK-dependent inhibition of voltage-dependent calcium

channels and, with that, inhibition of firing rates of NPY neurons of the ARC [188]. FOXO1 is further deactivated by Akt, which is induced by insulin and leptin through the phosphoinositide-3-kinase (PI3K) pathway [189, 190]. However, there appears to be another, AMPK-dependent pathway to induce NPY expression, which has not been widely described [191].

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Figure 1.2: Graphic overview of intracellular energy signalling. In the fasted state, ghrelin induces AMPK activity whilst it is inactive in the fed state through insulin, leptin and ATP derived from glucose. Sensors including AMPK, mTOR and STAT3 induce or inhibit cell growth,

intracellular metabolism and transcription of neurotransmitters NPY, AgRP and POMC. Signalling of insulin and leptin receptors is reduced through mTOR activity and through PTP1B and SOCS3, which are induced through inflammation and as a product of STAT3-induced gene transcription. ACC, acetyl-CoA carboxylase; AGRP, agouti-related peptide; AMPK, AMP- activated kinase; BSX, brain-specific homeobox transcription factor; CPT1, carnitine palmitoyl-transferase 1; FAS, fatty acid synthase; FOXO1,

forkhead box O1; GHSR, growth-hormone secretagogue receptor; IR, insulin receptor; IRS, insulin receptor substrate; JAK, janus kinase; MAPK, MCOA, malonyl-CoA; MTOR, mammalian target of rapamycin; NF B,

nuclear factor B; NPY, neuropeptide Y; OBR, leptin receptor; PDK1, pyruvate dehydrogenase kinase 1; PGC1 , PPAR coactivator 1 ; PI3K, POMC, pro-opiomelanocortin; phosphoinositide-3-kinase; PPAR,

peroxisome proliferator-activated receptor ; PTP1B, protein tyrosine phosphatase non-receptor type 1; SHP2, protein tyrosine phosphatase non-receptor type 11; SOCS3, suppressor of cytokine signalling 3; SREBP1, sterol regulatory element-binding protein 1; STAT3, signal transducer and activator of transcription 3; TG, triglyceride; TNF, tumour necrosis factor; TSC1/2, tuberous sclerosis 1 and 2.

This figure is based on the papers as referenced throughout this section and especially reviews [182, 184, 185, 192].

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Induction of PI3K through insulin and leptin leads to the activation mTOR [193], which is, in complex with regulatory-associated protein of mTOR (RAPTOR, mTOR complex 1), another crucial part of cellular energy sensing. MTOR promotes protein synthesis and cell growth by inducing messenger ribonucleic acid (mRNA) translation [194] and inhibiting

autophagy [195]. Importantly, mTOR also induces transcription of specific target genes by activition of transcription factors sterol regulatory

element-binding protein (SREBP) [196, 197], peroxisome proliferator- activated receptor (PPAR ) [198] and peroxisome proliferator-activated

receptor coactivator 1- (PGC1 ) [199], promoting triglyceride storage

and mitochondrial fatty acid oxidation. It directly suppresses

phosphorylation of insulin receptor substrate 1 (IRS1) and is, therefore, involved in insulin sensitivity in a negative feedback loop [200]. During fasting, mTOR is suppressed through a decrease in insulin and leptin signalling, a decrease in intracellular amino acid [201] and ATP

concentration signalled through AMPK [202], therefore improving insulin sensitivity, whilst the opposite occurs in overfeeding. In the hypothalamus, mTOR activation itself induces satiety [203], although it is unclear what downstream events are responsible for this effect.

Peripheral and central insulin and leptin resistance associated with obesity are also mediated through other mechanisms. Crucial proteins involved in this process are SOCS3 and PTP1B. In a negative feedback mechanism, activation of STAT3 through leptin induces expression of SOCS3 [204], which chronically inhibits leptin signalling through its receptor when

circulating leptin concentrations are elevated in obesity. SOCS3 diminishes phosphorylation and thus activation of janus kinase (JAK)2, protein

tyrosine phosphatase non-receptor type 11 (SHP2) and STAT3 through the leptin receptor [205, 206] whilst PTP1B blocks both the insulin and the

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leptin receptor [207], JAK2 and STAT3 [208]. Expression of both SOCS3 and PTP1B is induced by TNF through nuclear factor B (NF B), suggesting a mechanism through which inflammation induces insulin resistance [209]. On the other hand, muscle-specific insulin sensitivity is improved through exercise through intracellular signalling involving PI3K [210, 211], AMPK [212], Akt [210], resulting in an increase in ß-oxidation of fatty acids [213, 214].