Hypothalamic insulin signaling is essential to the maintenance of glucose and energy homeostasis. During pathological states, such as obesity and type 2 diabetes mellitus, insulin signaling is impaired. One key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. We used a hypothalamic, neuronal cell model, mHypoE-44, to understand how the highly prevalent nonesterified fatty acid, palmitate, affects neuronal insulin signaling. Through Western blot analysis, we discerned that prolonged exposure to palmitate impairs insulin activation, as assessed by phosphorylation of Akt. We investigated the role of endoplasmic reticulum (ER) stress, which is known to promote cellular insulin resistance and apoptosis in peripheral tissues. Palmitate treatment induced ER stress through a c-Jun N-terminal kinase (JNK)-dependent pathway because a selective JNK inhibitor blocked palmitate activation of the ER stress pathways eIF2 alpha and X-box binding protein-1. Interestingly, JNK inhibition did not prevent the palmitate-mediated cleaved caspase-3 increase, an apoptotic marker, or insulin signaling attenuation. However, pretreatment with the AMP kinase activator, aminoimidazole carboxamide ribonucleotide, blocked JNK phosphorylation and importantly prevented caspase-3 cleavage and restored insulin signaling during short-term exposure to palmitate. Thus, activation of AMP kinase prevents the deleterious effects of palmitate on hypothalamic neurons by inhibiting the onset of insulin resistance and apoptosis.

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Central insulin signaling is critical for the prevention of insulin resistance. Hyperinsulinemia contributes to insulin resistance, but it is not yet clear whether neurons are subject to cellular insulin resistance. We used an immortalized, hypothalamic, clonal cell line, mHypoE-46, which exemplifies neuronal function and expresses the components of the insulin signaling pathway, to determine how hyperinsulinemia modifies neuronal function. Western blot analysis indicated that prolonged insulin treatment of mHypoE-46 cells attenuated insulin signaling through phospho-Akt. To understand the mechanisms involved, time-course analysis was performed. Insulin exposure for 4 and 8 h phosphorylated Akt and p70-S6 kinase (S6K1), whereas 8 and 24 h treatment decreased insulin receptor (IR) and IR substrate 1 (IRS-1) protein levels. Insulin phosphorylation of S6K1 correlated with IRS-1 ser1101 phosphorylation and the mTOR-S6K1 pathway inhibitor rapamycin prevented IRS-1 serine phosphorylation. The proteasomal inhibitor epoxomicin and the lysosomal pathway inhibitor 3-methyladenine prevented the degradation of IRS-1 and IR by insulin, respectively, and pretreatment with rapamycin, epoxomicin, or 3-methyladenine prevented attenuation of insulin signaling by long-term insulin exposure. Thus, a sustained elevation of insulin levels diminishes neuronal insulin signaling through mTOR-S6K1-mediated IRS-1 serine phosphorylation, proteasomal degradation of IRS-1 and lysosomal degradation of the IR.

It is established that increases in neuropeptide Y (NPY) expression are associated with hyperphagia and obesity. These effects can be reversed by estrogen, a recognized anorexigen. We found that 17␤-estradiol (E 2 ) regulates biphasic NPY gene expression in a clonal, immortalized hypothalamic cell line, N-38, through estrogen receptor (ER) action at the level of the NPY promoter. However, rapid, nongenomic actions of estrogen, linked to the phosphatidylinositol 3-kinase (PI3-K)/Akt and ERK1/2 mitogen-activated protein kinase (MAPK) pathways, may also play a role. We therefore examined the changes in the phosphorylation status of Akt, ERK1/2, and cAMP response element-binding protein (CREB) after treatment with 10 nM E 2 in the N-38 neurons and found activation of these signaling proteins within 5-30 min. We also demonstrated possible cross talk between the estrogen-activated PI3-K/Akt and MAPK/extracellular signal-regulated kinase pathways using pharmacological inhibitors. We find that only ER␣ is involved in the early signaling events using the ER␣ agonist 4,4Ј,4Љ-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol and the ER␤ agonist 2,3-bis(4-hydroxyphenyl)-propionitrile. Furthermore, we can detect colocalization of ER␣ and caveolin-1, a membrane-associated signaling protein. Remarkably, we find that the membrane-mediated events are critical for the long-term estrogen-mediated repression of NPY gene expression that can be mapped to within Ϫ97 bp of the NPY promoter. To link the early signaling events to downstream effectors, we detected induction of c-fos and inactivation of MSK-1 by estrogen and binding of CREB to this minimal promoter region. These observations suggest that rapid estrogenmediated signaling is mediated by ER␣, and the signal transduction events potentiate the genomic actions of estrogen on NPY gene expression in the N-38 NPY neurons.

Introduction Geographic and transportation barriers are associated with poorer HIV ‐related health outcomes in sub‐Saharan Africa, but data on the impact of these barriers on prevention interventions are limited. We estimated the association between distance to clinic and other transportation‐related barriers on pre‐exposure prophylaxis (Pr EP ) uptake and initial clinic visit attendance in a rural community in southwestern Uganda enrolled in the ongoing SEARCH study ( NCT 01864603). Methods Community‐wide HIV testing was conducted and offered to adult (≥15 years) participants in Ruhoko. Participants were eligible for Pr EP based on an empiric risk score, having an HIV ‐discordant partner, or self‐referral at either the community health campaign or during home‐based testing from March to April 2017. We collected data from Pr EP ‐eligible households on GPS ‐measured distance to clinic, walking time to clinic and road difficulty. A sample of participants was also asked to identify their primary barriers to Pr EP use with a semi‐quantitative questionnaire. We used multivariable logistic regression to evaluate the association between transportation barriers and (1) Pr EP uptake among Pr EP ‐eligible individuals and (2) four‐week clinic visit attendance among Pr EP initiators. Results Of the 701 Pr EP ‐eligible participants, 272 (39%) started Pr EP within four weeks; of these, 45 (17%) were retained at four weeks. Participants with a distance to clinic of ≥2 km were less likely to start Pr EP ( aOR 0.34; 95% CI 0.15 to 0.79, p = 0.012) and less likely to be retained on Pr EP once initiated ( aOR 0.29; 95% CI 0.10 to 0.84; p = 0.024). Participants who were deemed eligible during home‐based testing and did not have the option of same‐day Pr EP start were also substantially less likely to initiate Pr EP ( aOR 0.16, 95% CI 0.07 to 0.37, p < 0.001). Of participants asked to name barriers to Pr EP use (N = 98), the most frequently cited were “needing to take Pr EP every day” (N = 18) and “low/no risk of getting HIV ” (N = 18). Transportation‐related barriers, including “clinic is too far away” (N = 6) and “t...