Early-life adversity increases morphine tolerance and persistent inflammatory hypersensitivity through upregulation of δ opioid receptors in mice.

ABSTRACT: Exposure to severely stressful events during childhood is associated with poor health outcomes in later life, including chronic pain and substance use disorder. However, the mediators and mechanisms are unclear. We investigated the impact of a well-characterized mouse model of early-life adversity, fragmented maternal care (FC) between postnatal day 2 and 9, on nociception, inflammatory hypersensitivity, and responses to morphine. Male and female mice exposed to FC exhibited prolonged basal thermal withdrawal latencies and decreased mechanical sensitivity. In addition, morphine had reduced potency in mice exposed to FC and their development of tolerance to morphine was accelerated. Quantitative PCR analysis in several brain regions and the spinal cords of juvenile and adult mice revealed an impact of FC on the expression of genes encoding opioid peptide precursors and their receptors. These changes included enhanced abundance of δ opioid receptor transcript in the spinal cord. Acute inflammatory hypersensitivity (induced by hind paw administration of complete Freund's adjuvant) was unaffected by exposure to FC. However, after an initial recovery of mechanical hypersensitivity, there was a reappearance in mice exposed to FC by day 15, which was not seen in control mice. Changes in nociception, morphine responses, and hypersensitivity associated with FC were apparent in males and females but were absent from mice lacking δ receptors or ß-arrestin2. These findings suggest that exposure to early-life adversity in mice enhances δ receptor expression leading to decreased basal sensitivity to noxious stimuli coupled with accelerated morphine tolerance and enhanced vulnerability to persistent inflammatory hypersensitivity.

Src Kinase Inhibition Attenuates Morphine Tolerance without Affecting Reinforcement or Psychomotor Stimulation.

BACKGROUND: Prolonged opioid administration leads to tolerance characterized by reduced analgesic potency. Pain management is additionally compromised by the hedonic effects of opioids, the cause of their misuse. The multifunctional protein ß-arrestin2 regulates the hedonic effects of morphine and participates in tolerance. These actions might reflect µ opioid receptor up-regulation through reduced endocytosis. ß-Arrestin2 also recruits kinases to µ receptors. We explored the role of Src kinase in morphine analgesic tolerance, locomotor stimulation, and reinforcement in C57BL/6 mice. METHODS: Analgesic (tail withdrawal latency; percentage of maximum possible effect, n = 8 to 16), locomotor (distance traveled, n = 7 to 8), and reinforcing (conditioned place preference, n = 7 to 8) effects of morphine were compared in wild-type, µ, µ, and ß-arrestin2 mice. The influence of c-Src inhibitors dasatinib (n = 8) and PP2 (n = 12) was examined. RESULTS: Analgesia in morphine-treated wild-type mice exhibited tolerance, declining by day 10 to a median of 62% maximum possible effect (interquartile range, 29 to 92%). Tolerance was absent from mice receiving dasatinib. Tolerance was enhanced in µ mice (34% maximum possible effect; interquartile range, 5 to 52% on day 5); dasatinib attenuated tolerance (100% maximum possible effect; interquartile range, 68 to 100%), as did PP2 (91% maximum possible effect; interquartile range, 78 to 100%). By contrast, c-Src inhibition affected neither morphine-evoked locomotor stimulation nor reinforcement. Remarkably, dasatinib not only attenuated tolerance but also reversed established tolerance in µ mice. CONCLUSIONS: The ability of c-Src inhibitors to inhibit tolerance, thereby restoring analgesia, without altering the hedonic effect of morphine, makes c-Src inhibitors promising candidates as adjuncts to opioid analgesics.

Integrin-Linked Kinase in Muscle Is Necessary for the Development of Insulin Resistance in Diet-Induced Obese Mice.

Diet-induced muscle insulin resistance is associated with expansion of extracellular matrix (ECM) components, such as collagens, and the expression of collagen-binding integrin, α2ß1. Integrins transduce signals from ECM via their cytoplasmic domains, which bind to intracellular integrin-binding proteins. The integrin-linked kinase (ILK)-PINCH-parvin (IPP) complex interacts with the cytoplasmic domain of ß-integrin subunits and is critical for integrin signaling. In this study we defined the role of ILK, a key component of the IPP complex, in diet-induced muscle insulin resistance. Wild-type (ILK(lox/lox)) and muscle-specific ILK-deficient (ILK(lox/lox)HSAcre) mice were fed chow or a high-fat (HF) diet for 16 weeks. Body weight was not different between ILK(lox/lox) and ILK(lox/lox)HSAcre mice. However, HF-fed ILK(lox/lox)HSAcre mice had improved muscle insulin sensitivity relative to HF-fed ILK(lox/lox) mice, as shown by increased rates of glucose infusion, glucose disappearance, and muscle glucose uptake during a hyperinsulinemic-euglycemic clamp. Improved muscle insulin action in the HF-fed ILK(lox/lox)HSAcre mice was associated with increased insulin-stimulated phosphorylation of Akt and increased muscle capillarization. These results suggest that ILK expression in muscle is a critical component of diet-induced insulin resistance, which possibly acts by impairing insulin signaling and insulin perfusion through capillaries.

Mice Lacking beta2-Integrin Function Remain Glucose Tolerant in Spite of Insulin Resistance, Neutrophil Infiltration and Inflammation.

Beta2-integrins are important in leukocyte trafficking and function, and are regulated through the binding of cytoplasmic proteins, such as kindlin-3, to their intracellular domain. Here, we investigate the involvement of beta2-integrins in the regulation of metabolic disease using mice where the kindlin-3 binding site in the beta2-integrin cytoplasmic tail has been mutated (TTT/AAA-beta2-integrin knock-in (KI) mice), leading to expressed but dysfunctional beta2-integrins and significant neutrophilia in vivo. Beta2-integrin KI mice fed on a high fat diet showed normal weight gain, and normal accumulation of macrophages and lymphocytes in white adipose tissue (WAT) and liver, but increased neutrophil numbers especially in WAT. In addition, beta2-integrin KI mice fed on a high fat diet showed significantly increased peripheral insulin resistance in response to high-fat feeding. However, this was associated with improved glucose disposal following glucose load. Interestingly, beta2-integrin KI neutrophils produced more elastase in vitro, in response to stimulation. Beta2-integrin KI mice displayed variability of tissue inflammatory status, with liver and WAT exhibiting little or no difference in inflammation compared to high fat fed controls, whereas skeletal muscle demonstrated a raised inflammatory profile in association with higher elastase levels and diminished signalling through the IRS1-PKB pathway. In conclusion, although expression of dysfunctional beta2-integrins increased neutrophil production and infiltration into tissue, skeletal muscle was the most affected tissue exhibiting evidence of higher neutrophil activity and insulin resistance. Thus, beta2-integrins modulate glucose homeostasis during high fat feeding predominantly through actions on skeletal muscle to affect metabolic phenotype in vivo.