Geneviève Roussy

Abstract The epidemic of type 2 diabetes mellitus (T2DM) is fueled by added fructose consumption. Here, we thus combined high-fat/high-fructose diet, with multiple low-dose injections of streptozotocin (HF/HF/Stz) to emulate the long-term complications of T2DM. HF/HF/Stz rats, monitored over 56 weeks, exhibited metabolic dysfunctions associated with the different stages of the T2DM disease progression in humans: an early prediabetic phase characterized by an hyperinsulinemic period with modest dysglycemia, followed by a late stage of T2DM with frank hyperglycemia, normalization of insulinemia, marked dyslipidemia, hepatic fibrosis and pancreatic β-cell failure. Histopathological analyses combined to [ 18 F]-FDG PET imaging further demonstrated the presence of several end-organ long-term complications, including reduction in myocardial glucose utilization, renal dysfunction as well as microvascular neuropathy and retinopathy. We also provide for the first time a comprehensive µ-PET whole brain imaging of the changes in glucose metabolic activity within discrete cerebral regions in HF/HF/Stz diabetic rats. Altogether, we developed and characterized a unique non-genetic preclinical model of T2DM adapted to the current diet and lifestyle that recapitulates the major metabolic features of the disease progression, from insulin resistance to pancreatic β-cell dysfunction, and closely mimicking the target-organ damage occurring in type 2 diabetic patients at advanced stages.

RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3–4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs. RNA interference (RNAi) is gaining acceptance as a potential therapeutic strategy against peripheral disease, and several clinical trials are already underway with 21-mer small-interfering RNA (siRNA) as the active pharmaceutical agent. However, for central affliction like pain, such innovating therapies are limited but nevertheless crucial to improve pain research and management. We demonstrate here the proof-of-concept of the use of 27-mer Dicer-substrate siRNA (DsiRNA) for silencing targets related to CNS disorders such as pain states. Indeed, low dose DsiRNA (0.005 mg/kg) was highly efficient in reducing the expression of the neurotensin receptor-2 (NTS2, a G-protein-coupled receptor (GPCR) involved in ascending nociception) in rat spinal cord through intrathecal (IT) administration formulated with the cationic lipid i-Fect. Along with specific decrease in NTS2 mRNA and protein, our results show a significant alteration in the analgesic effect of a selective-NTS2 agonist, reaching 93% inhibition up to 3–4 days after administration of DsiRNA. In order to ensure that these findings were not biased by unsuspected off-target effects (OTEs), we also demonstrated that treatment with a second NTS2-specific DsiRNA also reversed NTS2-induced antinociception, and that NTS2-specific 27-mer duplexes did not alter signaling through NTS1, a closely related receptor. Altogether, DsiRNAi represents a potent tool for dissecting nociceptive pathways and could further lead to a new class of central active drugs. IntroductionThe use of synthetic double-stranded RNA oligonucleotides to trigger RNA interference (RNAi) and specifically to reduce the expression of targeted genes is a standard research method that is routinely used in vitro. The ability to manipulate gene expression levels can help to elucidate the functions of gene products and facilitate the observation of subsequent phenotypic changes in a controlled experimental fashion.1Hannon GJ RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3487) Google Scholar,2Agrawal N Dasaradhi PV Mohmmed A Malhotra P Bhatnagar RK Mukherjee SK RNA interference: biology, mechanism, and applications.Microbiol Mol Biol Rev. 2003; 67: 657-685Crossref PubMed Scopus (762) Google Scholar RNAi is also gaining acceptance as a research tool in vivo, and over a hundred publications already report use of this method in live animals.3Whelan J First clinical data on RNAi.Drug Discov Today. 2005; 10: 1014-1015Crossref PubMed Scopus (63) Google Scholar,4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar In addition to elucidating gene function in vivo, it can be used for validating the potential significance of specific genes as targets for small-molecule drug development programs.5Sah DW Therapeutic potential of RNA interference for neurological disorders.Life Sci. 2006; 79: 1773-1780Crossref PubMed Scopus (75) Google Scholar Over 30 pharmaceutical and biotechnology companies are actively pursuing RNAi-based therapeutics, and several compounds are already in clinical trials. In spite of the rapid progress made in in vivo applications of RNAi, the field is still in its infancy; the methods in use are neither routine nor standardized. The use of small-interfering RNA (siRNA) in the central nervous system (CNS) may be more complex than its use in other organs such as the liver. In general, the greatest challenges to the in vivo use of RNAi relate to delivery (especially with and to the potential for off-target effects by in is of a for receptor Google are the products of double-stranded by the J A of RNA interference in PubMed Scopus Google Scholar synthetic RNA duplexes that are as experimental to trigger the by In several demonstrated that the use of synthetic are for can show than 21-mer J as potent RNAi 2005; PubMed Scopus Google MA RNAi and 2005; PubMed Scopus Google Scholar are by 21-mer in a MA is by of 2005; PubMed Scopus Google Scholar and the with this is to the of in silencing complex these are synthetic RNA duplexes in and are to as Dicer-substrate of the of the Dicer-substrate in the of in vivo are in P J MA and in and in vivo delivery of 2006; PubMed Scopus Google of gene expression by a and a 2006; PubMed Scopus Google Scholar in vivo demonstrated the use of formulated with cationic by to the use of by to In this we to specific the delivery of in vivo to peripheral organs and demonstrated in a of MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 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The ability to manipulate gene expression levels can help to elucidate the functions of gene products and facilitate the observation of subsequent phenotypic changes in a controlled experimental fashion.1Hannon GJ RNA interference.Nature. 2002; 418: 244-251Crossref PubMed Scopus (3487) Google Scholar,2Agrawal N Dasaradhi PV Mohmmed A Malhotra P Bhatnagar RK Mukherjee SK RNA interference: biology, mechanism, and applications.Microbiol Mol Biol Rev. 2003; 67: 657-685Crossref PubMed Scopus (762) Google Scholar RNAi is also gaining acceptance as a research tool in vivo, and over a hundred publications already report use of this method in live animals.3Whelan J First clinical data on RNAi.Drug Discov Today. 2005; 10: 1014-1015Crossref PubMed Scopus (63) Google Scholar,4Behlke MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google Scholar In addition to elucidating gene function in vivo, it can be used for validating the potential significance of specific genes as targets for small-molecule drug development programs.5Sah DW Therapeutic potential of RNA interference for neurological disorders.Life Sci. 2006; 79: 1773-1780Crossref PubMed Scopus (75) Google Scholar Over 30 pharmaceutical and biotechnology companies are actively pursuing RNAi-based therapeutics, and several compounds are already in clinical trials. In spite of the rapid progress made in in vivo applications of RNAi, the field is still in its infancy; the methods in use are neither routine nor standardized. The use of small-interfering RNA (siRNA) in the central nervous system (CNS) may be more complex than its use in other organs such as the liver. In general, the greatest challenges to the in vivo use of RNAi relate to delivery (especially with and to the potential for off-target effects by in is of a for receptor Google are the products of double-stranded by the J A of RNA interference in PubMed Scopus Google Scholar synthetic RNA duplexes that are as experimental to trigger the by In several demonstrated that the use of synthetic are for can show than 21-mer J as potent RNAi 2005; PubMed Scopus Google MA RNAi and 2005; PubMed Scopus Google Scholar are by 21-mer in a MA is by of 2005; PubMed Scopus Google Scholar and the with this is to the of in silencing complex these are synthetic RNA duplexes in and are to as Dicer-substrate of the of the Dicer-substrate in the of in vivo are in P J MA and in and in vivo delivery of 2006; PubMed Scopus Google of gene expression by a and a 2006; PubMed Scopus Google Scholar in vivo demonstrated the use of formulated with cationic by to the use of by to In this we to specific the delivery of in vivo to peripheral organs and demonstrated in a of MA Progress towards in vivo use of siRNAs.Mol Ther. 2006; 13: 644-670Abstract Full Text Full Text PDF PubMed Scopus (448) Google A for the of to RNA interference (RNAi) in 2006; PubMed Scopus Google not the and gene delivery to the the 2002; Full Text Full Text PDF PubMed Scopus Google Scholar silencing of gene targets in the CNS use of a intrathecal (IT) of siRNA to be are J PubMed Scopus Google of in PubMed Scopus Google Scholar cationic can be used in vivo to facilitate delivery of The cationic lipid was used by and efficient intrathecal delivery of RNA to the spinal cord and peripheral 2005; PubMed Scopus Google Scholar to siRNA through to in the spinal the use of siRNA by with to P SK P N A siRNA by 2006; PubMed Scopus Google Scholar and demonstrated the of of siRNA formulated in to the in rat in J of in PubMed Scopus Google this we demonstrate the of 27-mer DsiRNA in reducing the expression of a specific (GPCR) gene in rat spinal cord and of DsiRNA formulated in by a in the neurotensin receptor-2 mRNA and levels for 3–4 The in NTS2 in the changes in effects were the and our results the of DsiRNA in pain

Central administration of the neuropeptide neurotensin (NT) was shown to induce antinociceptive responses both spinally and supraspinally. Although NTS2 receptors play an important role in modulating the activity of spinal neurons, we have recently implicated NTS1 receptors in NT's analgesic effects in acute spinal pain paradigms. The current experiments were thus designed to examine the antinociceptive effects of intrathecal administration of NTS1 agonists in formalin-induced tonic pain in rats. We first established, using immunoblotting and immunohistochemical approaches, that NTS1 receptors were present in small- and medium-sized dorsal root ganglion cells and localized in the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal injection of NT (1-15 microg/kg) or NTS1 preferring agonists on the nocifensive response to intraplantar formalin. Both NTS1-agonists, PD149163 (10-120 microg/kg) and NT69L (1-100 microg/kg), dose-dependently attenuated the formalin-induced behaviors. Accordingly, NTS1 agonists markedly suppressed pain-evoked c-fos expression in the superficial, nucleus proprius and neck regions of the spinal dorsal horn. The concomitant administration of PD149163 with the NTS1 antagonist SR48692 (3 microg/kg) significantly reversed PD149163-induced antinociception, confirming the implication of NTS1 in tonic pain. In contrast, NT69L's analgesic effects were partly abolished by co-administration of SR48692, indicating that NT69L-induced effects may also be exerted through interaction with NTS2. These results demonstrate that NTS1 receptors play a key role in the mediation of the analgesic effects of NT in persistent pain and suggest that NTS1-selective agonists may represent a new line of analgesic compounds.

BACKGROUND: Central neurotensin (NT) administration results in a naloxone-insensitive antinociceptive response in animal models of acute and persistent pain. Both NTS1 and NTS2 receptors were shown to be required for different aspects of NT-induced analgesia. We recently demonstrated that NTS2 receptors were extensively associated with ascending nociceptive pathways, both at the level of the dorsal root ganglia and of the spinal dorsal horn. Then, we found that spinally administered NTS2-selective agonists induced dose-dependent antinociceptive responses in the acute tail-flick test. In the present study, we therefore investigated whether activation of spinal NTS2 receptors suppressed the persistent inflammatory pain symptoms observed after intraplantar injection of formalin. RESULTS: We first demonstrated that spinally administered NT and NT69L agonists, which bind to both NTS1 and NTS2 receptors, significantly reduced pain-evoked responses during the inflammatory phase of the formalin test. Accordingly, pretreatment with the NTS2-selective analogs JMV-431 and levocabastine was effective in inhibiting the aversive behaviors induced by formalin. With resolution at the single-cell level, we also found that activation of spinal NTS2 receptors reduced formalin-induced c-fos expression in dorsal horn neurons. However, our results also suggest that NTS2-selective agonists and NTS1/NTS2 mixed compounds differently modulated the early (21-39 min) and late (40-60 min) tonic phase 2 and recruited endogenous pain inhibitory mechanisms integrated at different levels of the central nervous system. Indeed, while non-selective drugs suppressed pain-related behaviors activity in both part of phase 2, intrathecal injection of NTS2-selective agonists was only efficient in reducing pain during the late phase 2. Furthermore, assessment of the stereotypic pain behaviors of lifting, shaking, licking and biting to formalin also revealed that unlike non-discriminative NTS1/NTS2 analogs reversing all nociceptive endpoint behaviors, pure NTS2 agonists specifically inhibited paw lifting, supporting a role of NTS2 in spinal modulation of persistent nociception. CONCLUSION: The present study provides the first demonstration that activation of NTS2 receptors produces analgesia in the persistent inflammatory pain model of formalin. The dichotomy between these two classes of compounds also indicates that both NTS1 and NTS2 receptors are involved in tonic pain inhibition and implies that these two NT receptors modulate the pain-induced behavioral responses by acting on distinct spinal and/or supraspinal neural circuits. In conclusion, development of NT agonists targeting both NTS1 and NTS2 receptors could be useful for chronic pain management.