The STING-IRF3 pathway is involved in lipotoxic injury of pancreatic ß cells in type 2 diabetes.

Lipotoxic injury of pancreatic ß cells is an important pathological feature in type 2 diabetes mellitus (T2DM). Stimulator of interferon genes (STING) can recognize its own DNA leaked into the cytoplasm from damaged mitochondria or nuclei of the host cell, thus activating its downstream factor interferon regulatory factor 3 (IRF3), causing inflammation and apoptosis. The STING-IRF3 signaling pathway is closely related to glycolipid metabolism, but its relationship with the lipotoxicity of pancreatic ß cells has rarely been reported. Here, we investigated the role of the STING-IRF3 signaling pathway in lipotoxicity-induced inflammation, apoptosis, and dysfunction of pancreatic ß cells. We examined the activation of STING and IRF3 in islets of db/db mice and identified the role of the STING-IRF3 signaling pathway in palmitic acid (PA)-induced lipotoxic injury of INS-1, a rat insulinoma cell line. STING and phosphorylated IRF3 including downstream interferon-ß were upregulated in islets of db/db mice and PA-induced INS-1 cells. Gene silencing of STING or IRF3 ameliorated PA-induced INS-1 cell inflammation and apoptosis, and reversed impaired insulin synthesis. Additionally, PA induced downregulation of the phosphoinositide 3-kinase-AKT signaling pathway, and impaired high glucose-stimulated insulin secretion was reversed after knockdown of STING or IRF3. Our results suggest that activation of the STING-IRF3 pathway triggers inflammation and apoptosis of pancreatic ß cells, leading to ß-cell damage and dysfunction. Hence, inhibition of this signaling pathway may represent a novel approach for ß-cell protection in T2DM.

Activation of the STING-IRF3 pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease.

BACKGROUND: Nonalcoholic fatty liver disease (NAFLD) is a common result of obesity and metabolic syndrome. Hepatocyte injury and metabolic disorders are hallmarks of NAFLD. Stimulator of interferon genes (STING) and its downstream factor interferon regulatory factor 3 (IRF3) trigger inflammatory reaction in response to the presence of cytosolic DNA. STING has recently been shown to play an important role in early alcoholic liver disease. However, little is known about the role of STING-IRF3 pathway in hepatocyte injury. Here, we aimed to examine the effect of STING-IRF3 pathway on hepatocyte metabolism, inflammation and apoptosis. METHODS: We examined the activation of the STING-IRF3 pathway, a high-fat diet (HFD)-induced obese mouse model, and determined the role of this pathway in a free fatty acid (FFA)-induced hepatocyte inflammatory response, injury, and dysfunction in L-O2 human liver cells. RESULTS: STING and IRF3 were upregulated in livers of HFD-fed mice and in FFA-induced L-O2 cells. Knocking down either STING or IRF3 led to a significant reduction in FFA-induced hepatic inflammation and apoptosis, as evidenced by modulation of the nuclear factor κB (NF-κB) signaling pathway, inflammatory cytokines, and apoptotic signaling. Additionally, STING/IRF3 knockdown enhanced glycogen storage and alleviated lipid accumulation, which were found to be associated with increased expression of hepatic enzymes in glycolysis and lipid catabolism, and attenuated expression of hepatic enzymes in gluconeogenesis and lipid synthesis. CONCLUSIONS: Our results suggest that the STING-IRF3 pathway promotes hepatocyte injury and dysfunction by inducing inflammation and apoptosis and by disturbing glucose and lipid metabolism. This pathway may be a novel therapeutic target for preventing NAFLD development and progression.

Effects of microiontophoretic application of cocaine, alone and with receptor antagonists, upon the neurons of the medial prefrontal cortex, nucleus accumbens and caudate nucleus of rats.

The spontaneous extracellular electrical activity of 102 neurons, within the caudate nucleus (CN), medial prefrontal cortex (MPC), nucleus accumbens (NAc) and a control site, the lateral thalamic nucleus (LT), was studied. Cocaine depressed spontaneous activity in the majority of the cells studied from all regions except the lateral thalamus. Desipramine, which has been used clinically for the treatment of withdrawal of cocaine, also depressed neuronal activity in the caudate nucleus. In addition, of the three receptor antagonists tested, sulpiride, methysergide and naloxone, only the dopamine antagonist (sulpiride) affected cocaine-induced neuronal responses. This study further emphasizes the emerging importance of midbrain dopaminergic systems in the pharmacological effects of this important drug of abuse.

Amyloid beta-protein fragment 31-35 forms ion channels in membrane patches excised from rat hippocampal neurons.

Inside-out membrane patches excised from rat hippocampal neurons were used to test if ion channels could be formed by fragment 31-35 of amyloid beta-protein. The results showed: (1) after application of fragment 31-35 of amyloid beta-protein (5 microM) to either the inner or outer side of the patches, spontaneous currents could be recorded from those patches that had previously been 'silent'; (2) the fragment 31-35-induced conductance was cation-selective with a permeability ratio of P(Cs)/P(Cl)=23; (3) different levels of conductance, ranging from 25 to 500 pS, could be recorded in different patches, and in some cases, different conductances and spontaneous transitions among them could be recorded in a single patch; and (4) application of ZnCl(2) (1 mM) to the inner side of the patches reversibly blocked the newly formed channel activity; a similar effect was observed after application of CdCl(2) (1 mM). These results show that fragment 31-35 of amyloid beta-protein can insert into membrane patches from both sides and form cation-selective, Zn(2+)- and Cd(2+)-sensitive ion channels. It is proposed that fragment 31-35 in amyloid beta-protein might be the shortest active sequence known to date to form ion channels across neuronal membranes.

Nociceptive c-fos expression in supraspinal areas in avoidance of descending suppression at the spinal relay station.

The number and distribution of Fos-like-immunoreactive neurons in different supraspinal brain areas induced by formalin injection into one hindpaw was estimated in rats with transected dorsal half of the spinal cord at the thoracic level in an attempt to avoid most of the descending modulatory actions. The results showed that: (i) after spinal lesion, the peripheral noxious inputs, going up mainly through the ventral spinal cord, elicited a more widespread and densely located Fos-like-immunoreactive neurons in subcortical areas, many of them showed no Fos expression when noxious stimulation was given in rats with intact spinal cord; (ii) at the same time, a small number of subcortical areas, such as the lateral ventroposterior thalamic nucleus and dorsal raphe nucleus, exhibited no significant increase of nociceptive Fos-like immunoreactive neurons after spinal lesion as compared to that with intact spinal cord; and (iii) there appeared a prominent expansion of cortical areas with densely located Fos-like-immunoreactive neurons in spinal-lesioned rats as compared with the limited labelled areas in the control group with intact spinal cord. These results indicate that: (i) in avoiding the spinally descending modulatory mechanisms, more widespread supraspinal and cortical neurons will be recruited and activated in response to the noxious stimulation; and (ii) the descending systems exert differential actions on the spinal targets which project nociceptive signals to different supraspinal regions. The implication of these facts is discussed.

Beta-amyloid peptide fragment 31-35 induces apoptosis in cultured cortical neurons.

A synthetic fragment 31-35 of beta-amyloid peptide was used in cultured cortical neurons to examine whether this smaller sequence could trigger apoptotic degeneration in vitro by using morphological, biochemical and flow-cytometric examinations. The results showed that: (i) neurons treated with fragment 31-35 of beta-amyloid peptide exhibited membrane blebbing, compaction of nuclear chromatin, nuclear shrinkage and nuclear fragmentation; (ii) a typical DNA ladder was revealed by agarose gel electrophoresis following fragment 31-35 of beta-amyloid peptide exposure; (iii) the internucleosome DNA fragmentation was also detected by flow-cytometric examination following fragment 31-35 of beta-amyloid peptide exposure; and (iv) the DNA fragmentation induced by fragment 31-35 of beta-amyloid peptide in the above two examinations could be blocked by co-treatment with aurintricarboxylic acid or actinomycin D. It is suggested that fragment 31-35 of the beta-amyloid peptide may be a shorter sequence of beta-amyloid peptide responsible for triggering an apoptotic process in cultured neurons.

Effects of descending inhibitory systems on the c-Fos expression in the rat spinal cord during formalin-induced noxious stimulation.

This study provides morphological evidence for the activation of the descending modulatory control by nociceptive afferent pathways. Fos-like immunoreactivity in the spinal dorsal horn is used as an indicator of efficacy of transmission of noxious inputs at this level. Wistar rats were anesthetized with nembutal and the spinal dorsolateral funiculus was transected unilaterally at the level of T11,12. Two days later, an equal volume (0.2 ml) of formalin (5% in saline) was injected into the plantar aspect of two hindpaws. After 1 h of injection, rats were deeply anesthetized and killed for the immunocytochemical examination of Fos-like protein product by using an immunocytochemical technique. The results show that the mean number of Fos protein-like immunoreactive neurons is significantly lower in the superficial laminae and in the neck of the dorsal horn on the side ipsilateral to the intact dorsolateral funiculus than that on the opposite side (i.e. 18.4 +/- 1.0 vs 30.0 +/- 1.3 and 33.9 +/- 0.2 vs 56.8 +/- 1.7, respectively). We conclude that the peripheral noxious inputs (which ascend via the ventral half of the spinal cord in this study) can activate the supraspinal descending inhibitory systems, which in turn suppress the synthesis of Fos-like protein in the related dorsal horn neurons.

Lateral hypothalamus: site involved in pain modulation.

The present study is an attempt to examine the neuronal circuitry of a supraspinal site engaged in pain modulation. Five physiological measures were postulated as the criteria for defining a central nervous system site engaged in the circuitry of pain modulation. The lateral hypothalamus met these five measures: (i) 81% of the lateral hypothalamus neurons (247/304) responded to noxious stimuli using a single cell recording procedure; (ii) stimulation of the periaqueductal gray-dorsal raphe area or the habenula modulated 98% and 87% of the lateral hypothalamus noxious-evoked activity; (iii) microiontophoretically applied morphine modulated 77% of the lateral hypothalamus noxious evoked activity; (iv) electrical stimulation of the lateral hypothalamus produced behavioral analgesia proportional to the stimulus intensity as assessed by the tail flick assay; and (v) morphine application into the lateral hypothalamus produced behavioral analgesia in a dose-response manner using the tail flick assay. In conclusion, the lateral hypothalamus can be considered one of the pain modulation sites.

Serotonergic, noradrenergic and galaninergic projections to the nucleus parafascicularis.

Immunocytochemical staining for serotonin (5-HT), tyrosine hydroxylase (TH) and galanin (GAL) was combined with horseradish peroxidase (HRP) retrograde tract-tracing technique to analyze the localizations of 5-HT-, catecholamine (CA)- and GAL-containing neurons in the brainstem which project to the nucleus parafascicularis (PF) in rats. It is demonstrated that most of the retrogradely HRP-labeled neurons (70%) in bilateral periaqueductal gray (PAG) and raphe nuclei are positively immunostained by antiserum to 5-HT, and that most of the retrogradely HRP-labeled neurons (over 80%) in bilateral locus coeruleus (LC) are positively immunostained by antisera to both TH and GAL. The possible functions of these PF-petal serotonergic, catecholaminergic (actually noradrenergic) and galaninergic projections are discussed.

Morphine and norepinephrine-induced antinociception at the spinal level is mediated by adenosine.

The purpose of this study was to examine whether adenosine or serotonin is involved in mediation of the antinociception produced by norepinephrine at the spinal cord level. Aminophylline, an adenosine receptor antagonist and naloxone given intrathecally (i.t.) were used to test the antinociception produced by i.t. norepinephrine, serotonin, morphine or the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA) by using the tail-flick assay in rats. It was observed that (1) aminophylline blocked the antinociception produced by norepinephrine, but exhibited no effect on the antinociception produced by serotonin, (2) aminophylline blocked the antinociception produced by morphine similarly to naloxone, (3) aminophylline blocked the antinociception produced by NECA and (4) naloxone failed to block the antinociception produced by NECA and serotonin. The results suggest that adenosine is involved in mediation of the norepinephrine-produced antinociception at the spinal level and that norepinephrine and adenosine may act in a sequential manner in norepinephrine-induced antinociception.

Neurokinin A, calcitonin gene-related peptide, and dynorphin A (1-8) in spinal dorsal horn contribute to descending inhibition evoked by nociceptive afferent pathways: an immunocytochemical study.

Immunocytochemical technique was used to compare the contents of neurokinin A (NKA), calcitonin gene-related peptide (CGRP), and dynorphin A (1-8) (DynA) on two sides of the lumbar dorsal horn of rats in which the unilateral thoracic dorsalateral funiculus (DLF) was transected while formalin (0.2 ml, 0.5%) was injected equally into two hindpaws. The results showed that all the NKA-like, CGRP-like, and DynA (1-8)-like immunoreactivities were significantly lower in the superficial laminae of the dorsal horn on the side ipsilateral to the lesioned DLF than that on the side with intact DLF. This implies that peripheral noxious inputs activate the supraspinal descending inhibitory systems which in turn modulate the transmission of noxious message at the spinal level by changing the release of related neuropeptides.

Changes of spinal substance P, calcitonin gene-related peptide, somatostatin, Met-enkephalin and neurotensin in rats in response to formalin-induced pain.

Changes of substance P (SP)-, calcitonin gene-related peptide (CGRP)-, somatostatin (SS)-, Met-enkephalin (Met-Enk)- and neurotensin (NT)- immunoreactive materials on two sides of the lumbar dorsal horn were inspected microscopically and quantified with a computer-assisted image processing system in rats with intact or totally transected spinal cord 2 h after injection of 0.2 ml of 0.5% formalin into the right hindpaw subcutaneously. The results showed that the SP-like immunoreactivity (SP-LI), CGRP-LI, SS-LI, Met-Enk-LI, and NT-LI were significantly higher in fibers and terminals in superficial laminae of the dorsal horn ipsilateral to the formalin injection in both of the experimental groups. It is supposed that the increased contents of these peptides reflect an increased biosynthesis, transport, and release of these peptides in primary afferents and spinal intrinsic neurons in response to the long-lasting inflow of noxious messages, and that these changes seem to be produced even in the condition when the supraspinal effects have been excluded.

Habenulopetal catecholaminergic projections in the rat brain: a combined Fluoro-Gold/catecholamine fluorescence study.

Habenulopetal catecholamine (CA)-containing neurons were determined using Fluoro-Gold retrograde tracing combined with a CA-fluorescence technique. A robust number of habenulopetal CA neurons were found mainly in the ipsilateral locus coeruleus (LC), while a considerable number of habenulopetal non-CA neurons were found in the LC, subcoeruleus, A5, A4 and A7 areas.

Effects of stimulation of superior colliculus on nociceptive unit discharges in parafascicular neurons and nocifensive reflex of hind limb in rat.

Effects of stimulation of the superior colliculus (SC) on the spontaneous and nociceptive discharges of parafascicular (PF) neurons were investigated in 43 urethane-anesthetized rats. Two groups of PF cells were sampled according to their responses to noxious stimuli: 59 of them were 'nociceptive-on' and 12 'nociceptive-off'. Following stimulation of the intermediate and deep layers of SC, the firing rate of nociceptive discharge of 'nociceptive-on' cells was inhibited significantly (-76 +/- 5%, P less than 0.01) in 75% cases tested, while the nociceptive response of 'nociceptive-off' cells was disinhibited markedly (79 +/- 9%, P less than 0.01) in 67% cases by the same stimulation. In 30 animals of this series the latency of hind limb withdrawal reflex elicited by noxious skin heating was compared before and after SC stimulation. In 24 cases in which the stimulating electrodes were positioned exactly in intermediate-deep layers of SC, SC stimulation lengthened the latency by 62 +/- 8% (P less than 0.01), while in 6 cases in which the electrodes drifted from these areas, the latency was not changed following the same stimulation.

Effects of intrathecal monoamine antagonists and naloxone on the descending inhibition of the spinal transmission of noxious input in rats: study with a new experimental model.

An electrophysiological model has been developed to explore the transmitters and their relationships in the descending control of spinal transmission of noxious inputs. Nociceptive discharges were recorded extracellularly in parafascicular (Pf) neurons, and the caudal stump of longitudinally isolated dorsal half of the lower thoracic spinal cord was stimulated to simulate the descending volleys coming from the supraspinal structures. Nociceptive discharges in 34 Pf cells were markedly suppressed (83.2 +/- 13.9%) by the preceding spinal stimulation. Phentolamine, methysergide and naloxone were separately administered with random sequence in each of 25 cells by an intrathecal route to observe if the descending inhibition could be blocked by these drugs. The results suggested that in the dorsal spinal cord there exist at least two neurochemically different descending inhibitory fiber systems which comprise either the long descending adrenergic or serotonergic fibers and, most of the adrenergic fibers are succeeded by a propriospinal opioidergic neuron while a few of the serotonergic fibers have such a succession.

Suppression of nociceptive responses in parafascicular neurons by stimulation of substantia nigra: an analysis of related inhibitory pathways.

A total of 166 neurons in parafascicular nucleus (PF) were studied, 85 from intact animals, 72 following dorsal spinal cord transection (D.Sp.C.X.), and 9 following complete transection of the spinal cord. Two patterns of nociceptive responses were identified following noxious stimulation and these responses were classified as 'nociceptive-on' and 'nociceptive-off' neurons, respectively. The effects of stimulating the substantia nigra (SNS) on the spontaneous and on the nociceptive evoked discharges were observed and compared in intact, D.Sp.C.X. and completely transected spinal cord rats. The results show that SNS significantly suppresses both the spontaneous and the nociceptive evoked discharges elicited by peroneal nerve stimulation. With an intact spinal cord, SNS suppressed both the spontaneous [-37 +/- 3.2% (P less than 0.05)] and the nociceptive evoked discharges [-52.8 +/- 2.8% (P less than 0.01)] of the 'nociceptive-on' cells respectively, while in the 'nociceptive-off' cells the same stimulation elicited an even more prominent suppression upon both discharges (-47.7 +/- 5.4%, P less than 0.01 and -64.9 +/- 5.0%, P less than 0.01), respectively. After D.Sp.C.X., the suppressive effects on the 'nociceptive-on' cells following SNS were diminished (-28.1 +/- 3.5% and -36.9 +/- 2.6%, respectively) but not abolished, while in the 'nociceptive-off' cells, the inhibitory effects on SNS were unchanged. In addition, the suppressive effects of SNS on the spontaneous activity of PF neurons in cases with completely cut spinal cords remains unchanged. These results suggest that SNS modulates the spontaneous and the noxious evoked responses of the PF neurons by way of supraspinal connections besides the previously described descending projecting pathways.

Interrelations of opioids with monoamines in descending inhibition of nociceptive transmission at the spinal level: an immunocytochemical study.

This study was designed to reexamine a previous proposal of whether the opioid-like substances (OLS) being acting mainly as an intrinsic spinal mediator in the descending inhibition of nociception of the bulbospinally projecting NE-ergic, and/or 5-HT-ergic terminals in the dorsal horn by using an immunocytochemical method. The effects of intrathecal (i.t.) phentolamine (Ph), cyproheptadine (Cyp), and naloxone (Nal), administered separately or coadministered by two of them, on the expression of Fos-like-immunoreactive (FLI) neurons were observed on both sides of the lumbar dorsal horn of rats, in which equal volumes of formalin were injected into two hindpaws and the ipsilateral dorsolateral funiculus (DLF) was transected at the thoracic level antecedently. The results showed: (1) when rats were pretreated with i.t. saline, the number of nociceptive FLI neurons was significantly lowered 44% (p<0.01) on the side of the lumbar dorsal horn with intact DLF compared to the opposite side with sectioned DLF; (2) when rats were separately pretreated with i.t. Ph, Cyp and Nal, the reduction of FLI neurons on the DLF-intact side were decreased by 27% (p<0.01), 21% (p<0.01), and 25% (p<0.01), respectively; (3) when rats were pretreated with combined i.t. Ph+Cyp, the reduction on the intact side was eliminated almost completely (4%); (4) when rats were pretreated with combined i.t. Ph+Nal, the reduction on the intact side was 21% (p<0.01); and (5) when rats were pretreated with i.t. Cyp+Nal, the reduction on the intact side was 9.1%. These results suggest that: (1) nearly all the suppressive action exerted by the DLF-descending fibers are produced by the release of either NE or 5-HT as neurotransmitters at the spinal level; (2) most of the opioid-like substances act as an intrinsic spinal mediator mainly for the descending NE-ergic, but in a lesser extent for the 5-HT-ergic terminals in the dorsal horn circuitry; and (3) some OLS-ergic interneurons may only be activated by local nociceptive input.

Locus coeruleus modulates thalamic nociceptive responses via adrenoceptors.

This study investigated the parafascicular (PF) neuronal nociceptive responses and their modulation following electrical stimulation of the locus coeruleus (LC) and intrathecal (i.t.) or intracerebroventricular (i.c.v.) administration of two alpha-adrenoceptor antagonists, the alpha2-antagonist, yohimbine, and the alpha1-antagonist, prazosin. The main results were as follows: (1) the nociceptive evoked discharges in PF neurons were suppressed by preceding stimulation of LC; (2) the suppressive effect of LC stimulation on PF neurons was replaced by a facilitatory effect following pretreatment of i.t. yohimbine in 14 units tested, while i.t. prazosin failed to alter the LC-induced suppression, even when the prazosin dose was doubled; (3) i.c.v. pretreatment with prazosin strengthened the suppressive effect of LC stimulation on PF neurons; (4) i.c.v. norepinephrine (NE) administration induced, in PF neurons, a biphasic response to noxious stimulation; an early, brief (about 10 min) inhibitory effect followed by a late, long-lasting facilitatory effect; and (5) i.c.v. pretreatment of yohimbine or prazosin prevented the inhibitory or facilitatory responses released by NE, respectively. These results provide evidence that: (1) the LC-descending projections exhibit a suppressive effect on nociceptive transmission at the spinal level through alpha2-receptors; and (2) the LC-ascending projections exhibit dual effects, facilitatory and inhibitory, at the medial thalamus (PF) level through alpha1- and alpha2-receptors, respectively.

Morphological evidence for the activation of descending modulatory control by nociceptive afferent pathways: an immunocytochemical study.

Immunocytochemical technique was used to compare the content of substance P (SP), Met-enkephalin (Met-Enk) and neurotensin (NT) on two sides of the lumbar dorsal horn of rats in which the unilateral dorsolateral funiculus was transected while formalin (0.2 ml, 5%) was injected equally into two hindpaws. The results showed that the SP-like immunoreactivity (SP-LI) and Met-Enk-LI were significantly higher and the NT-LI was significantly lower in the superficial laminae of dorsal horn on the side ipsilateral to the intact DLF than that on the opposite side, implying that peripheral noxious inputs can activate the supraspinal descending inhibitory systems which in turn modulate the transmission of noxious message at the spinal level by changing the activities of related peptidergic neurons.

Norepinephrine modulates single hypothalamic arcuate neurons via alpha(1)and beta adrenergic receptors.

The effects of norepinephrine (NE) on the electrophysiological activities of single hypothalamic arcuate neurons were studied using extracellular recording of 385 neurons from 169 brain slices in rats. The results showed that: (1) of 236 neurons selected randomly and tested with NE application, 137 (58.0%) were excited, 67 (28.4%) were inhibited, and 32 (13.6%) failed to respond; (2) substitution of low Ca(2+)-high Mg(2+) artificial cerebrospinal fluid (ACSF) for normal ACSF abolished the NE-induced inhibitory effect but failed to abolish the excitatory effect; (3) both the NE-induced excitatory and inhibitory effects were antagonized partly by phentolamine, prazosin, and propranolol but not by yohimbine; (4) naloxone and glibenclamide, a blocker of adenosine triphosphate-sensitive (K(ATP)) channels, blocked the NE-induced inhibitory effect; and (5) neurons that were inhibited by NE were also inhibited by morphine and cromakalim, an agonist of K(ATP) channels, and moreover, the morphine-induced inhibitory effect could be blocked by glibenclamide, while the cromakalim-induced inhibitory effect was not blocked by naloxone. These results imply that: (a) NE excites arcuate neurons through a mechanism that is insensitive to lowering the extracellular Ca(2+) suggesting a direct postsynaptic response through alpha(1)- and beta-adrenergic receptors, while NE inhibits cells through at least an inhibitory interneuron in arcuate and so is dependent on a Ca(2+)-sensitive presynaptic release mechanism; and (b) the inhibitory interneuron may be opioidergic, being excited first through alpha(1)- and beta-adrenergic receptors, after which the released opioids inhibit the neurons being recorded with an involvement of activation of K(ATP) channels. This possibility needs to be substantiated in much more detail.