Characterization of Sirtuin Inhibitors in Nematodes Expressing a Muscular Dystrophy Protein Reveals Muscle Cell and Behavioral Protection by Specific Sirtinol Analogues

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High Sugar-Induced Insulin Resistance in Drosophila Relies on the Lipocalin Neural Lazarillo

In multicellular organisms, insulin/IGF signaling (IIS) plays a central role in matching energy needs with uptake and storage, participating in functions as diverse as metabolic homeostasis, growth, reproduction and ageing. In mammals, this pleiotropy of action relies in part on a dichotomy of action of insulin, IGF-I and their respective membrane-bound receptors. In organisms with simpler IIS, this functional separation is questionable. In Drosophila IIS consists of several insulin-like peptides called Dilps, activating a unique membrane receptor and its downstream signaling cascade. During larval development, IIS is involved in metabolic homeostasis and growth. We have used feeding conditions (high sugar diet, HSD) that induce an important change in metabolic homeostasis to monitor possible effects on growth. Unexpectedly we observed that HSD-fed animals exhibited severe growth inhibition as a consequence of peripheral Dilp resistance. Dilp-resistant animals present several metabolic disorders similar to those observed in type II diabetes (T2D) patients. By exploring the molecular mechanisms involved in Drosophila Dilp resistance, we found a major role for the lipocalin Neural Lazarillo (NLaz), a target of JNK signaling. NLaz expression is strongly increased upon HSD and animals heterozygous for an NLaz null mutation are fully protected from HSD-induced Dilp resistance. NLaz is a secreted protein homologous to the Retinol-Binding Protein 4 involved in the onset of T2D in human and mice. These results indicate that insulin resistance shares common molecular mechanisms in flies and human and that Drosophila could emerge as a powerful genetic system to study some aspects of this complex syndrome

The cellular homeostasis of the gut: what the Drosophila model points out

The digestive tract is subjected to many aggressions throughout animal life. Since disruptions of gut physiology impact on animal fitness and survival, maintenance of gut integrity and functionality is essential for the individual. Over the last 40 years, research on rodents has aimed at understanding how cellular homeostasis of the digestive tract is maintained when challenged with disruptions. Following the discovery of stem cells in the digestive tract of Drosophila, a flurry of studies made an important contribution to our understanding of how the proliferation and the differentiation of these cells are controlled and participate in the renewal of the digestive tract. Insights into these mechanisms in Drosophila have revealed many similarities with mammalian intestinal stem cells. For instance, the highly conserved EGFR, JAK/STAT, Wingless/Wnt, Hedgehog, Integrins, BMP/TGFβ, Hippo and Insulin pathways all participate in adult intestinal cellular homeostasis. Here, we provide a literature review of recent advances in the field highlighting the adult Drosophila midgut as a convenient model for dissecting mechanisms involved in the maintenance of the cellular homeostasis of the digestive tract in conventionally reared conditions. In addition, we shed light on recently published data putting Drosophila forward as a genetic tool to decipher the mechanisms underlying intestinal diseases and intestinal tumour progression

Metabolic and growth defects induced by High Sugar Diet.

<p>(A) After 60′ of starvation, L3 larvae were transferred on high sucrose (20× in PBS), and the levels of circulating glucose and trehalose were monitored from 1′ to 60′ after transfer, revealing a modification of glucose levels, but not trehalose. Note that the starvation before transfer to 20× sucrose induces itself a slight increase in basal glycemia. (B) Glucose and (C) trehalose levels as measured in the hemolymph of wandering larvae fed from eclosion on 1× or 5× sucrose diet. (D) Total triacylglycerides (TAGs) and (E) circulating DAGs in larvae fed on 1× and 5× sucrose diet after eclosion. (F) Effect of 1× or 5× sucrose diet on the rate of transcription of the <i>ACC</i> gene in mid-L3 larvae. (G) Weight of adult males emerged after larvae were fed on 1× (ctrl) or 5× sucrose diet (High Sugar Diet, HSD). (H) Effect of control diet or HSD on the developmental timing, assessed at the time of white pupa formation. (I) Measurement of food intake of L3 larvae previously fed on ctrl diet or HSD, as measured by blue food ingestion. (J) Differential expression of the <i>PECK</i> gene on ctrl diet or HSD in mid-L3 larvae.</p

High Sugar Diet induces peripheral Dilp-resistance.

<p>(A) A two-fold increase in <i>DILP2</i> and <i>DILP5</i> transcription is observed in larval brain upon feeding a HSD (fold changes are presented, f.c.). (B) Dilp2 and Dilp5 immuno-staining of the insulin-producing cells (IPCs) in L3 larvae fed ctrl and HSD. (C) Quantification of fluorescence in IPCs (fold changes are presented, f.c.). (D) and (E) Insulin stimulation test of fat body explants from control or HSD-fed larvae. After a short incubation to human insulin (0,5 µM, 20 min) the amount of tGPH fluorescence was quantified as an evaluation of insulin sensitivity. In D, representative images of fat bodies after incubation. Cell membranes outlined with the tGPH marker are shown in inserts. (F) The dFoxo targets <i>Inr</i> and <i>4EBP</i> are upregulated in HSD conditions, indicative of a general reduction of IIS in HSD-fed larvae (mid-L3 larval samples, fold changes are presented, f.c.). (G) Circulating Dilp2-Flag in the hemolymph of larvae fed either control of HSD. Larvae express a Flag-tagged Dilp2 in the IPCs (<i>Dilp2-Gal4>Flag-Dilp2</i>) and circulating levels of Dilp2-Flag are quantified using an Elisa method (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036583#s4" target="_blank">Material and Methods</a>). (H) Forced expression of a bacterial sodium channel in the brain IPCs during larval development (<i>Dilp2-Gal4>NaChBac,</i> see (6)) promotes Dilps secretion and prevents HSD-induced hyperglycemia.</p

NLaz is required for High Sugar Diet-induced Dilp-resistance.

<p>(A) Changes in expression of <i>Puc</i>, <i>NLaz</i>, <i>Karl</i> and <i>GLaz</i> in HSD <i>vs</i> control conditions (fold changes are presented, f.c.). (B) Glycemia of control, heterozygous and homozygous <i>NLaz</i> mutant larvae fed either normal diet (light grey bars) or HSD (dark bars). (C) and (D) Fat body explants from <i>NLaz</i> mutant larvae fed either control or HSD were exposed to human insulin (0,5 µM, 20 min.). The amount of tGPH fluorescence was quantified as an evaluation of insulin sensitivity. (E) Glycemia of control larvae or larvae with a fat body-specific knock-down of <i>NLaz</i> (<i>NLaz-RNAi</i>), fed either normal diet (light grey bars) or HSD (dark bars).</p

Sirtuin inhibition protects from the polyalanine muscular dystrophy protein PABPN1

Oculopharyngeal muscular dystrophy (OPMD) is caused by polyalanine expansion in nuclear protein PABPN1 [poly(A) binding protein nuclear 1] and characterized by muscle degeneration. Druggable modifiers of proteotoxicity in degenerative diseases, notably the longevity modulators sirtuins, may constitute useful therapeutic targets. However, the modifiers of mutant PABPN1 are unknown. Here, we report that longevity and cell metabolism modifiers modulate mutant PABPN1 toxicity in the muscle cell. Using PABPN1 nematodes that show muscle cell degeneration and abnormal motility, we found that increased dosage of the sirtuin and deacetylase sir-2.1/SIRT1 exacerbated muscle pathology, an effect dependent on the transcription factor daf-16/FoxO and fuel sensor aak-2/AMPK (AMP-activated protein kinase), while null mutants of sir-2.1, daf-16 and aak-2 were protective. Consistently, the Sir2 inhibitor sirtinol was protective, whereas the Sir2 and AMPK activator resveratrol was detrimental. Furthermore, rescue by sirtinol was dependent on daf-16 and not aak-2, whereas aggravation by resveratrol was dependent on aak-2 and not daf-16. Finally, the survival of mammalian cells expressing mutant PABPN1 was promoted by sirtinol and decreased by resveratrol. Altogether, our data identify Sir2 and AMPK inhibition as therapeutic strategies for muscle protection in OPMD, extending the value of druggable proteins in cell maintenance networks to polyalanine diseases