Cooperation between subcellular NAD pools in the maintenance of NAD-dependent processes

Nicotinamide adenine dinucleotide (NAD) is needed as a cofactor in several processes throughout the cell, and its availability is decisive for the activity of many NAD-dependent enzymes. Owing to the membrane barriers in the compartmentalized eukaryotic cell, NAD cannot move freely. Understanding how subcellular NAD pools are established and maintained, as well as how they communicate with each other, is therefore crucial to understanding how NAD dependent processes are regulated in the eukaryotic cell. In this study we aimed at determining the role of the hitherto uncharacterized peroxisomal NAD pool. Moreover, we addressed experimentally the question as to how subcellular NAD pools communicate and cooperate upon altered NAD homeostasis. To do so we studied the consequences of excessive NAD+ consumption in the peroxisomes, cytosol or mitochondria using cell lines constitutively expressing the catalytic domain of PARP1 (PARP1cd) targeted to these organelles. The activity of the constructs was confirmed by PAR detection and a 30-40% decline in cellular NAD+ content. The excessive NAD+ consumption in peroxisomes did not severely affect the cellular phenotype, and peroxisomal processes were largely maintained. In line with this, upregulated expression of solute carrier SLC25A17 in these cells suggested that import of NAD+ into peroxisomes was increased to compensate for the excessive consumption. Interestingly, acetylation state of sirtuin targets in various compartments appeared unaltered. Thus, increased import of NAD+ into peroxisomes seemingly occurred without affecting NAD+-dependent deacetylation in other organelles. This is likely facilitated by the observed adjustment of the expression of NAD biosynthetic enzymes, NMNATs, and sirtuins in these cells. Likewise, excessive NAD+ consumption in the cytosol, and to a lesser extent, mitochondria, was also accompanied by altered expression of NMNATs and sirtuins combined with maintained acetylation state. This indicated that increased local NAD biosynthesis together with altered flux through sirtuins can maintain NAD+-dependent deacetylation despite significantly lowered cellular NAD+ content. In sum, our findings suggest that the maintenance of the peroxisomal pool might be more important than previously assumed. Moreover, we propose the existence of an interorganellar compensatory response capable of shifting the distribution of NAD within the cells upon compartment specific increase in NAD+ consumption. Maintenance of NAD+-dependent signaling in the affected or donating organelle(s) is proposed to occur via altered expression of local NAD producers and consumers

Impact of long-term fasting on the stomach-hypothalamus appetite regulating genes in Atlantic salmon postsmolts

Atlantic salmon will experience periods of fasting during its lifecycle. In nature, prolonged fasting periods occur owing to seasonal fluctuations in available feeds, migration or in relation to reproduction. In a culture setting, salmon is fasted mainly as part of planned operational handling prior to vaccination, delousing, transfer etc., and where fasting may last up to nine days. The mechanisms regulating the appetite during long-term fasting may vary among fish species. Here, we studied the impact of long-term fasting on neuro-endocrine regulation of appetite through the stomach-hypothalamic axis in Atlantic salmon post smolts (1.2 kg, ∼46 cm), reared in two experimental conditions (Fed and Fasted; triplicated tanks), and sampled after 4 weeks and 6 weeks of fasting. Fasted fish showed lower condition factor and hepatosomatic index at both sampling points compared to Fed group. In qPCR analysis, hypothalamic relative mRNA expression of agouti-related protein 1 (agrp1) was upregulated in fasted group at both sampling points. Among neuropeptide Y (npy) paralogs, only npya1 at 4 weeks was upregulated by fasting. As for cocaine- and amphetamine-regulated transcripts (cart), cart2a was elevated at 4 weeks, and cart2b at both 4 and 6 weeks in fasted group, while cart3a and cart4 showed no response to fasting. The pro-opiomelanocortin (pomc) a1, a2 and melanocortin-4 receptor (mc4r) a2 increased only after 6 weeks of fasting, while mc4rb1 did not respond to fasting. In stomach, 6 weeks of fasting resulted in a decrease of ghrelin1 (ghrl1), while expression of mboat4 was unaffected. The elevated levels of hypothalamic agrp1 and npya1 in fasted group support orexigenic roles for these neuropeptides. In addition, upregulation of cart2a, cart2b, pomca1 and pomca2 indicate that these play vital roles in appetite regulation and that fasting may halt and/or counteract hunger signals (agrp1 and npya1) to save energy from foraging search activities during catabolic conditions. Another possibility is that these neuropeptides play a role in fasting-induced stress. Based on the drop in mRNA expression of ghrl under catabolic conditions, we hypothesize that Ghrl might return as hunger signal once feed becomes available. We also propose that agrp1 is a potential appetite biomarker gene under feed deprived conditions.publishedVersio

Brain Distribution of 10 cart Transcripts and Their Response to 4 Days of Fasting in Atlantic Salmon (Salmo salar L.)

Cocaine- and amphetamine-regulated transcript (CART) has been known to be involved in feeding and energy balance in mammals, acting as an anorexigenic neuropeptide in hypothalamus. In Atlantic salmon, little is known about Cart brain localization and its function. In this study, in silico analysis revealed the existence of 10 cart paralogs, here named cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b, and 4. The Atlantic salmon Cart sequences shared from 19 to 50% of identity with the human homolog and between 25 and 90% of sequence identity among paralogs, except for Cart4 which only shared 18–23% of identity. We further explored cart mRNA expressions in 8 brain regions (Olfactory Bulb-OB, Telencephalon-TEL, Midbrain-MB, Cerebellum-CE, Hypothalamus-HYP, Saccus vasculosus-SV, Pituitary-PT, and Brain Stem-BS) of Atlantic salmon smolt under 4 days of fasting and continuous fed conditions. The cart paralogs analyzed were widely distributed among the brain regions and OB, TEL, HYP, MB, and BS seemed to be the major sites of expression. The expression of cart1a and 1b showed quite similar pattern in MB, HYP, and BS. The expression of cart2a had the highest in MB followed by HYP and TEL. The cart3a transcript was widely distributed in rostrocaudal regions of brain except in OB and SV whereas cart3b was predominantly expressed in BS followed by MB. Expression of cart4 was high in HYP followed by TEL. With regards to effect of feeding status the Atlantic salmon cart2b, which is the most abundant among the paralogs, was upregulated after 4 days of fasting in OB, MB, and HYP compared to fed group. This may suggest an unexpected, but possible orexigenic role of cart2b in Atlantic salmon or a fasting induced stress effect. No other significant effect was observed. Collectively, the differential expressions of the cart paralogs in different brain regions suggest that they may have roles in regional integration of appetite signals and are possibly involved in regulating other brain functions in Atlantic salmon. The fact that salmon has 10 cart paralogs, while mammalians only one, opens interesting perspectives for comparative research on evolutionary adaptations of gene function in the control of appetite and energy homeostasis.publishedVersio

Anorectic role of high dietary leucine in farmed Atlantic salmon (Salmo salar L.): Effects on feed intake, growth, amino acid transporters and appetite-control neuropeptides.

Leucine has been identified to modulate feed intake and energy homeostasis in fish as in other vertebrates. Under allostatic conditions, energy expenditure may change, and adjustments to the processes that govern the energy homeostatic system may be necessary. We investigated the responsiveness of appetite-related neuropeptides involved in feed intake regulation in Atlantic salmon (Salmo salar) reared with high (35 g/kg leucine) or control (27.3 g/kg leucine) leucine-supplemented diets and/or under chronic stressor conditions (chasing) for eight weeks. We also analysed the response of amino acid transporters potentially involved in uptake of branched-chain amino acids (BCAA), including leucine, into areas of the brain where nutrient sensors may signal locally or to other areas involved in appetite control. At the end of the experiment, all fish were subjected to a novel-acute stressor (confinement). Our results show that fish fed with high leucine diet had a lower feed intake, growth, and hepatosomatic index (HSI) when compared to fish fed control leucine diet. In addition, increased mRNA expression of amino acid solute carrier (slc) genes in the diencephalon, and genes related to appetite control, such as proopiomelanocortin a1 (pomca1), in both the diencephalon and telencephalon, imply their involvement in leucine anorectic effect. Stress, as high leucine, reduced feed intake, growth and HSI of fish fed control or high leucine diet and antagonized the high leucine effect on the slc genes mRNA expression. An increase of neuropeptide y a1 (npya1) was observed both due to high dietary leucine and/or stress treatment which may represent a compensatory regulatory mechanism with the aim to reverse the decrease in feed intake. In summary, our results confirm an anorectic role of high dietary leucine via the activation of amino acid sensing mechanisms in the brain. Further, corticotropin-releasing hormone 1 b1 (crh1b1) and npya1 showed to play a role in the regulation of appetite in Atlantic salmon under stress conditions and/or high leucine levels.publishedVersio

Regional Expression of npy mRNA Paralogs in the Brain of Atlantic Salmon (Salmo salar, L.) and Response to Fasting

Neuropeptide Y (NPY) is known as a potent orexigenic signal in vertebrates, but its role in Atlantic salmon has not yet been fully established. In this study, we identified three npy paralogs, named npya1, npya2, and npyb, in the Atlantic salmon genome. In silico analysis revealed that these genes are well conserved across the vertebrate’s lineage and the mature peptide sequences shared at least 77% of identity with the human homolog. We analyzed mRNA expression of npy paralogs in eight brain regions of Atlantic salmon post-smolt, and the effect of 4 days of fasting on the npy expression level. Results show that npya1 was the most abundant paralog, and was predominantly expressed in the telencephalon, followed by the midbrain and olfactory bulb. npya2 mRNA was highly abundant in hypothalamus and midbrain, while npyb was found to be highest expressed in the telencephalon, with low mRNA expression levels detected in all the other brain regions. 4 days of fasting resulted in a significant (p < 0.05) decrease of npya1 mRNA expression in the olfactory bulb, increased npya2 mRNA expression in the midbrain and decreased npyb mRNA expression in the pituitary. In the hypothalamus, the vertebrate appetite center, expression of the npy paralogs was not significantly affected by feeding status. However, we observed a trend of increased npya2 mRNA expression (p = 0.099) following 4 days of fasting. Altogether, our findings provide a solid basis for further research on appetite and energy metabolism in Atlantic salmon