Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus.

Insulin acts in the brain to suppress feeding, whereas neuropeptide Y (NPY) has the opposite effect. Since fasting lowers plasma insulin levels and increases hypothalamic synthesis of NPY, we proposed that insulin may inhibit hypothalamic NPY gene expression. To test this hypothesis, we used RIA and in situ hybridization histochemistry to determine if centrally administered insulin could reduce levels of both NPY and its messenger RNA (mRNA) in discreet hypothalamic regions during fasting. Three groups of Long-Evans rats were entered into a 72-h study protocol. One group was fed ad libitum during this period, while the others were fasted. Fed rats received intracerebroventricular (icv) injections of saline vehicle at 12-h intervals, whereas fasted groups received icv vehicle alone or with insulin (4 mU/12 h). In vehicle-only treated rats, fasting significantly increased expression of preproNPY mRNA in the arcuate nucleus to 179 +/- 20% of fed controls. Administration of icv insulin during fasting abolished this increase (99 +/- 14% of fed controls; P less than 0.05 vs. fasted, vehicle-treated rats). Central insulin administration during fasting also reduced immunoreactive NPY concentrations in samples punched from the paraventricular nucleus (PVN) (875 +/- 122 pg/punch) to levels below vehicle-only treated rats (1396 +/- 435 pg/punch; P less than 0.05), similar to free-feeding control values (814 +/- 170 pg/punch). By comparison, neither fasting nor central insulin administration altered NPY levels in four other hypothalamic regions (supraoptic, ventromedial, dorsomedial, and arcuate nuclei). Continuous icv insulin infusion at a lower dose (2 mU/day) produced a similar result during a shorter period (48 h) of food deprivation in Wistar rats. In this study, central insulin infusion also inhibited the fasting-related increase in arcuate preproNPY mRNA levels and did not affect plasma glucose or insulin levels. This suggests that insulin acts locally to inhibit hypothalamic NPY mRNA expression. We conclude that the increase of levels of NPY in the PVN and preproNPY mRNA in the arcuate nucleus during fasting are inhibited by icv insulin. Fasting, therefore, increases NPY biosynthesis along an arcuate nucleus-PVN pathway in the hypothalamus via a mechanism dependent on low insulin levels.

ObjectiveTo investigate whether antidrug antibodies and/or drug non‐trough levels predict the long‐term treatment response in a large cohort of patients with rheumatoid arthritis (RA) treated with adalimumab or etanercept and to identify factors influencing antidrug antibody and drug levels to optimize future treatment decisions.MethodsA total of 331 patients from an observational prospective cohort were selected (160 patients treated with adalimumab and 171 treated with etanercept). Antidrug antibody levels were measured by radioimmunoassay, and drug levels were measured by enzyme‐linked immunosorbent assay in 835 serial serum samples obtained 3, 6, and 12 months after initiation of therapy. The association between antidrug antibodies and drug non‐trough levels and the treatment response (change in the Disease Activity Score in 28 joints) was evaluated.ResultsAmong patients who completed 12 months of followup, antidrug antibodies were detected in 24.8% of those receiving adalimumab (31 of 125) and in none of those receiving etanercept. At 3 months, antidrug antibody formation and low adalimumab levels were significant predictors of no response according to the European League Against Rheumatism (EULAR) criteria at 12 months (area under the receiver operating characteristic curve 0.71 [95% confidence interval (95% CI) 0.57, 0.85]). Antidrug antibody–positive patients received lower median dosages of methotrexate compared with antidrug antibody–negative patients (15 mg/week versus 20 mg/week; P = 0.01) and had a longer disease duration (14.0 versus 7.7 years; P = 0.03). The adalimumab level was the best predictor of change in the DAS28 at 12 months, after adjustment for confounders (regression coefficient 0.060 [95% CI 0.015, 0.10], P = 0.009). Etanercept levels were associated with the EULAR response at 12 months (regression coefficient 0.088 [95% CI 0.019, 0.16], P = 0.012); however, this difference was not significant after adjustment. A body mass index of ≥30 kg/m2 and poor adherence were associated with lower drug levels.ConclusionPharmacologic testing in anti–tumor necrosis factor–treated patients is clinically useful even in the absence of trough levels. At 3 months, antidrug antibodies and low adalimumab levels are significant predictors of no response according to the EULAR criteria at 12 months.