Hypothalamic UDP Increases in Obesity and Promotes Feeding via P2Y6-Dependent Activation of AgRP Neurons

Activation of orexigenic AgRP-expressing neurons in the arcuate nucleus of the hypothalamus potently promotes feeding, thus defining new regulators of AgRP neuron activity could uncover potential novel targets for obesity treatment. Here, we demonstrate that AgRP neurons express the purinergic receptor 6 (P2Y6), which is activated by uridine-diphosphate (UDP). In vivo, UDP induces ERK phosphorylation and cFos expression in AgRP neurons and promotes action potential firing of these neurons in brain slice recordings. Consequently, central application of UDP promotes feeding, and this response is abrogated upon pharmacologic or genetic inhibition of P2Y6 as well as upon pharmacogenetic inhibition of AgRP neuron activity. In obese animals, hypothalamic UDP content is elevated as a consequence of increased circulating uridine concentrations. Collectively, these experiments reveal a potential regulatory pathway in obesity, where peripheral uridine increases hypothalamic UDP concentrations, which in turn can promote feeding via PY6-dependent activation of AgRP neurons

Hexosamine Pathway Metabolites Enhance Protein Quality Control and Prolong Life

Aging entails a progressive decline in protein homeostasis, which often leads to age-related diseases. The endoplasmic reticulum (ER) is the site of protein synthesis and maturation for secreted and membrane proteins. Correct folding of ER proteins requires covalent attachment of N-linked glycan oligosaccharides. Here, we report that increased synthesis of N-glycan precursors in the hexosamine pathway improves ER protein homeostasis and extends lifespan in C. elegans. Addition of the N-glycan precursor N-acetylglucosamine to the growth medium slows aging in wild-type animals and alleviates pathology of distinct neurotoxic disease models. Our data suggest that reduced aggregation of metastable proteins and lifespan extension depend on enhanced ER-associated protein degradation, proteasomal activity, and autophagy. Evidently, hexosamine pathway activation or N-acetylglucosamine supplementation induces distinct protein quality control mechanisms, which may allow therapeutic intervention against age-related and proteotoxic diseases

Lowered Insulin Signalling Ameliorates Age-Related Sleep Fragmentation in <i>Drosophila</i>

<div><p>Sleep fragmentation, particularly reduced and interrupted night sleep, impairs the quality of life of older people. Strikingly similar declines in sleep quality are seen during ageing in laboratory animals, including the fruit fly <i>Drosophila</i>. We investigated whether reduced activity of the nutrient- and stress-sensing insulin/insulin-like growth factor (IIS)/TOR signalling network, which ameliorates ageing in diverse organisms, could rescue the sleep fragmentation of ageing <i>Drosophila</i>. Lowered IIS/TOR network activity improved sleep quality, with increased night sleep and day activity and reduced sleep fragmentation. Reduced TOR activity, even when started for the first time late in life, improved sleep quality. The effects of reduced IIS/TOR network activity on day and night phenotypes were mediated through distinct mechanisms: Day activity was induced by adipokinetic hormone, dFOXO, and enhanced octopaminergic signalling. In contrast, night sleep duration and consolidation were dependent on reduced S6K and dopaminergic signalling. Our findings highlight the importance of different IIS/TOR components as potential therapeutic targets for pharmacological treatment of age-related sleep fragmentation in humans.</p></div

Dnmt3a2/Dnmt3L Overexpression in the Dopaminergic System of Mice Increases Exercise Behavior through Signaling Changes in the Hypothalamus

Dnmt3a2, ade novoDNA methyltransferase, is induced by neuronal activity and participates in long-term memory formation with the increased expression of synaptic plasticity genes. We wanted to determine if Dnmt3a2 with its partner Dnmt3L may influence motor behavior via the dopaminergic system. To this end, we generated a mouse line, Dnmt3a2/3L(Dat/wt), with dopamine transporter (DAT) promotor driven Dnmt3a2/3L overexpression. The mice were studied with behavioral paradigms (e.g., cylinder test, open field, and treadmill), brain slice patch clamp recordings, ex vivo metabolite analysis, and in vivo positron emission tomography (PET) using the dopaminergic tracer 6-[F-18]FMT. The results showed that spontaneous activity and exercise performance were enhanced in Dnmt3a2/3L(Dat/wt)mice compared to Dnmt3a2/3L(wt/wt)controls. Dopaminergic substantia nigra pars compacta neurons of Dnmt3a2/3L(Dat/wt)animals displayed a higher fire frequency and excitability. However, dopamine concentration was not increased in the striatum, and dopamine metabolite concentration was even significantly decreased. Striatal 6-[F-18]FMT uptake, reflecting aromatic L-amino acid decarboxylase activity, was the same in Dnmt3a2/3L(Dat/wt)mice and controls. [F-18]FDG PET showed that hypothalamic metabolic activity was tightly linked to motor behavior in Dnmt3a2/3L(Dat/wt)mice. Furthermore, dopamine biosynthesis and motor-related metabolic activity were correlated in the hypothalamus. Our findings suggest that Dnmt3a2/3L, when overexpressed in dopaminergic neurons, modulates motor performance via activation of the nigrostriatal pathway. This does not involve increased dopamine synthesis

Reduced IIS causes day hyperactivity through increased octopaminergic signalling.

<p>(A) Two days feeding with mianserin hydrochloride (0.2 mg/ml) reverted the day activity phenotype of <i>dilp2-3,5</i> mutants (age 10 d), but not night activity, sleep, sleep bouts, and sleep bouts length (<i>w^Dah n = </i>17/24, <i>dilp2-3,5 n</i> = 17/27 +/− mianserin). GLM was used to determine significance of treatment by genotype interactions in sleep and activity behaviours on treatment with mianserin in controls and IIS mutants. Significant differences were seen in day activity (<i>p</i> = 0.0031), in day sleep (<i>p</i> = 0.0148), and day bout number (<i>p</i> = 0.002), but not in night behaviours (activity <i>p</i> = 0.31, sleep <i>p</i> = 0.49, bout number <i>p</i> = 0.72, night bout length <i>p</i> = 0.15). (B) Average activity count data (30 min bins) under 12∶12 h LD. (A) Kruskal Wallis test with Dunn's multiple comparison of selected pairs. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m.</p

Rapamycin rescued age-related night sleep fragmentation in an S6K-dependent manner.

<p>(A) Rapamycin treatment (9 d) did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, age 10 d, <i>n</i> = 21/20 control/rapamycin). (B) Average activity count data (30 min bins) under 12∶12 h LD. (C) Acute rapamycin treatment of 45-d-old flies for 3 d did not significantly affect day/night activity or wakefulness, but significantly increased night sleep duration, reduced night sleep fragmentation, and increased the length of night sleep periods (<i>w^Dah</i>, <i>n</i> = 64/64). Rapamycin-mediated night sleep, bout number, and bout length were independent of (D) 4E-BP (<i>n</i> = 19/19) and (E) reduced autophagy (a = <i>da-Gal4/UAS-ATG5-RNAi</i>, <i>n</i> = 20/17 or genetic controls c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21 and c2 = <i>UAS-ATG5-RNAi/+</i>, <i>n</i> = 23/19). Flies with reduced autophagy responded to rapamycin as controls in sleep (<i>p</i> = 0.81), bout number (<i>p</i> = 0.82), and night bout length (<i>p</i> = 0.42) (GLM). (F) Ubiquitous expression of constitutively active S6K blocked the rescue of night sleep fragmentation by rapamycin (c1 = <i>da-Gal4/+</i>, <i>n = </i>20/21, and c2 = <i>UAS-S6K^STDETE/+</i>, <i>n = </i>20/18, <i>S6K = da-Gal4/UAS-S6K^STDETE</i>, <i>n = </i>20/17). Flies expressing a constitutively active form of S6K significantly differed from controls in the response to rapamycin (sleep <i>p</i> = 0.01, bout number <i>p</i> = 0.03, night bout length <i>p</i> = <0.0001, GLM). Kruskal Wallis test with Dunn's multiple comparisons of selected pairs. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. Day behaviours of (D–F) are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s006" target="_blank">Figure S6B–D</a>.</p

Dopamine receptor mutants do not respond to rapamycin treatment.

<p>(A) <i>DopR1</i> mutants had similar activity and sleep features as rapamycin-fed flies. Behaviour of <i>DopR1</i> mutants was not affected by rapamycin feeding (age 10 d, <i>n</i> = 64 for all genotypes). Flies were fed with rapamycin for 9 d. (B) <i>dilp2-3,5</i>, <i>DopR1</i> mutants had similar activity and sleep features as <i>dilp2-3,5</i> mutants (<i>n = </i>64 for all genotypes). (C) QRT-PCR analysis of dopamine receptor (<i>DopR1</i>) expression normalized to <i>Rpl32</i> expression and controls in head extracts of <i>dilp2-3,5</i> mutants (age 10 d, <i>n</i> = 9) and <i>da-Gal4/UAS/INR^DN</i> flies and <i>da-Gal4/INR^DN;dfoxo</i> mutants (age 10 d, <i>n</i> = 3). (D) Mass spectrometry measurement of dopamine levels in head extracts of female flies (age 10 d, <i>n = </i>3). (E) QRT-PCR analysis of <i>DAT</i> expression, normalized to <i>Rpl32</i> expression (<i>n</i> = 9). (F) Behaviour of IIS mutants after short-term exposure (2 d) to the tyrosine hydroxylase inhibitor 3IY (5 mg/ml) (age 35 d, <i>n</i> = 32 for all genotypes). IIS mutants differed from controls in the nighttime activity and sleep response to 3IY treatment, but not in bout or bout length (activity <i>p</i> = 0.044, sleep <i>p</i> = 0.014, bout number <i>p</i> = 0.276, night bout length <i>p</i> = 0.463, GLM). (G) Behaviour of IIS mutants after short-term (12 h) exposure to METH (1 mg/ml) (age 25 d, <i>n</i> = 48 for all genotypes). IIS mutants differed from controls in daytime behaviours after METH treatment (activity <i>p</i> = 0.005, sleep <i>p</i> = <0.0001, bout number <i>p</i> = 0.0002, bout length <i>p</i> = 0.031, GLM) along with nighttime bouts (<i>p = </i><0.0001) and bout length (<i>p = </i><0.0001), whereas nighttime activity and sleep did not differ (activity <i>p = </i>0.796, sleep <i>p = </i>0.352, GLM). (A, B, G) Kruskal Wallis test with Dunn's multiple comparison of selected pairs. (F) Individual comparisons by Mann–Whitney U test. (C–E) Two-tailed <i>t</i> test. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. (C, E, F) QRT-PCR analysis normalized to <i>RNApolII</i> expression shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s005" target="_blank">Figure S5D</a>.</p

Day hyperactivity of IIS mutants is dependent on the <i>AkhR</i>.

<p>(A) Loss of <i>AkhR</i> abrogated the day activity phenotype of <i>dilp2-3,5</i> mutants (age 15 d, <i>w^Dah n</i> = 18, <i>dilp2-3,5 n</i> = 15, <i>AkhR n</i> = 18, <i>AkhR dilp-3,5 n</i> = 17). GLM was used to determine significance of genotype by genotype interactions in sleep and activity behaviours on loss of <i>AkhR</i> in controls and <i>dilp2-3,5</i> mutants. Significant differences were seen in day activity (<i>p</i> = 0.0057) and day bout number (<i>p</i> = 0.044) but not in day sleep (<i>p</i> = 0.14) or night behaviours (activity <i>p</i> = 0.09, sleep <i>p</i> = 0.63, bout number <i>p</i> = 0.22, night bout length <i>p</i> = 0.067). Corresponding nighttime behaviours are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s004" target="_blank">Figure S4A</a>. (B) Two-day tolbutamide (1.35 mg/ml) treatment increased day activity of <i>w^Dah</i> flies. Lack of <i>dfoxo</i>, <i>AkhR</i>, or <i>dilp2-3,5</i> blocked the tolbutamide effect on day activity (nighttime behaviour shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s004" target="_blank">Figure S4B</a>) (age 15 d, <i>w^Dah n</i> = 51/47, <i>AkhR n</i> = 30/34, <i>dfoxo^Δ94 n</i> = 36/34, <i>dilp2-3,5 n</i> = 22/18, +/− tolbutamide). Analysis of genotype by treatment interactions (GLM) in sleep and activity behaviours on tolbutamide treatment in <i>IIS</i>, <i>AkhR</i>, and <i>dfoxo</i> mutants compared to controls showed day activity (<i>p</i> = 0.049), day sleep (<i>p</i> = <0.0001), and bout number (<i>p</i> = 0.008) were significantly different. However, no differences were seen in night behaviours (activity <i>p</i> = 0.58, sleep <i>p</i> = 0.89, bout number <i>p</i> = 0.28). (C) Mass spectrometry measurement of octopamine levels in head extracts (age 10 d, <i>w^Dah n</i> = 7, <i>dilp2-3,5 n</i> = 6, <i>AkhR n</i> = 6, <i>AkhR,dilp2-3,5 n</i> = 6). (A and B) Kruskal Wallis test with Dunn's multiple comparison test (selected pairs). (C) Mann–Whitney test. (C) One-way ANOVA with Bonferroni's multiple comparison test. ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m.</p

<i>dfoxo</i> affected daytime activity and sleep phenotypes of <i>INR^DN</i> flies.

<p>Loss of <i>dfoxo</i> in <i>da-Gal4/UAS-INR^DN</i> flies but not in wild-type flies (age 20 d) decreased day activity but had no effect on night activity, had no significant effect on wakefulness (average activity per waking minute), increased day sleep duration but had no effect on night sleep duration, and reverted the low sleep bout phenotype of <i>da-Gal4/UAS-INR^DN</i> flies by day but not at night and increased night sleep bout duration. <i>dfoxo</i> indicates the <i>dfoxo^Δ94</i>allele (<i>n = </i>35 for all genotypes). Kruskal Wallis test with Dunn's multiple comparison test (selected pairs). ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m. Independent experiments verifying activity and sleep phenotypes of <i>INR^DN;dfoxo</i> double mutants are shown in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001824#pbio.1001824.s003" target="_blank">Figure S3</a>.</p

Reduced IIS affected activity and sleep and ameliorated age-related sleep fragmentation.

<p>(A) Locomotor activity over 9 d of <i>w^Dah</i> control and <i>dilp2-3,5</i> mutant flies under 12∶12 h LD and constant darkness 12∶12 h DD (<i>n = </i>12, age 20 d). Mean free running period (τ) in DD ± s.e.m. (B) Average activity count data (30 min bins) under 12∶12 h LD conditions (25 d <i>w^Dah n = </i>48, <i>dilp2-3,5 n = </i>31). (C) <i>dilp2-3,5</i> mutants were more active during the day and less active during the night compared to controls. (D) There was no significant difference in wakefulness (average activity per waking minute). (E) <i>dilp2-3,5</i> mutants slept more at night and less during the day than controls. (F) Minutes of sleep per 30 min (25 d <i>w^Dah n = </i>48, <i>dilp2-3,5 n = </i>31). (G) Day and night sleep of <i>dilp2-3,5</i> mutants were interrupted by fewer waking periods compared to controls. (H) <i>dilp2-3,5</i> flies had longer sleep bouts during the night. (I) Longer sleep bouts were more prevalent in <i>dilp2-3,5</i> mutants (age 25 d). (J) <i>w^Dah</i> control flies, but not <i>dilp2-3,5</i> mutants, show a significant age-related increase in night sleep bouts (age 10 d, 25 d, 45 d, 55 d, and 65 d). (B–F) <i>w^Dah</i> , <i>n</i> = 31, 43, 46, 26, and 31 for ages 10 d, 25 d, 45 d, 55 d, and 65 d, respectively; <i>dilp2-3,5</i>, <i>n</i> = 31, 31, 32, 29, and 43 for ages 10 d, 25 d, 45 d, 55 d, and 65 d, respectively. Kruskal Wallis test with Dunn's multiple comparison (selected pairs). ***<i>p</i><0.001, **<i>p</i><0.01, and *<i>p</i><0.05. Error bars represent s.e.m.</p