To characterize tissue-specific differences in insulin signaling, we compared the mechanisms of mitogen-activated protein (MAP) kinase activation by insulin in the mitogenically active 3T3-L1 fibroblasts with the metabolically active 3T3-L1 adipocytes. In both cell lines, insulin significantly increased p21 ras ⅐GTP loading (1.5-2-fold) and MAP kinase activity (5-8-fold). Inhibition of Ras farnesylation with lovastatin blocked activation of p21 ras and Raf-1 kinase in both 3T3-L1 fibroblasts and 3T3-L1 adipocytes. In 3T3-L1 fibroblasts, this was accompanied by an inhibition of the stimulatory effect of insulin on MAP kinase. In contrast, in 3T3-L1 adipocytes, despite an inhibition of activation of p21 ras and Raf-1 by lovastatin, insulin continued to stimulate MAP kinase activity. Fractionation of the cell lysates on the FPLC Mono-Q column revealed that lovastatin inhibited insulin stimulation of ERK2 (and, to a lesser extent, ERK1) in 3T3-L1 fibroblasts and had no effect on the insulin-stimulated ERK2 in 3T3-L1 adipocytes. These results demonstrate an important distinction between the mechanism of insulin signaling in the metabolically and mitogenically active cells. Insulin activates MAP kinase by the Ras-dependent pathway in the 3T3-L1 fibroblasts and by the Ras-independent pathway in the 3T3-L1 adipocytes.Insulin's interaction with its cell surface receptor triggers both metabolic and mitogenic cellular responses. Insulin binding activates the tyrosine kinase of the ␤-subunit of the insulin receptor, which immediately phosphorylates insulin receptor substrate-1 and Shc. Phosphorylated sites of insulin receptor substrate-1 and Shc bind Src homology-2 domain-containing intermediates such as phosphatidylinositol 3-kinase (PI 3-kinase) 1 and Grb-2, which, in turn, propagate insulin signaling downstream. PI 3-kinase appears to be involved in regulation of glucose transport and protein synthesis, while Grb-2 association with Sos appears to be an important step in activating the Ras-Raf-MEK-MAP kinase pathway (reviewed in Ref. 1).Although many intermediates of the insulin signaling have been identified, numerous questions remain unanswered. In particular, most of the signaling molecules activated by insulin are also significantly stimulated by other growth factors, such as platelet-derived growth factor and EGF (2). However, in contrast to these growth factors, only insulin elicits defined metabolic responses, suggesting that additional steps must be involved in the mechanism of insulin action.Recent studies from our laboratory have identified significant differences between the regulation of certain aspects of insulin signaling in metabolically responsive 3T3-L1 adipocytes and mitogenically responsive 3T3-L1 fibroblasts (3, 4). For example, in 3T3-L1 adipocytes, PI 3-kinase and PKC exert a constitutively inhibitory influence on GTPase-activating protein (GAP), allowing insulin signaling to proceed through Sos and p21ras . Removal of this inhibitory influence results in activation of GAP and inhibition of the p21 r...

Background Few studies have examined how developing obesity in early adulthood affects the course of asthma. Objective We analyzed lung function and asthma impairment and risk among non-obese children with asthma, comparing those who were obese in young adulthood to those who remained non-obese. Methods Post-hoc analysis of 771 subjects with mild-moderate asthma who were not obese (pediatric definition, body mass index (BMI) <95th percentile) when enrolled in the Childhood Asthma Management Program at ages 5–12 years. Subjects were then followed to age ≥ 20 years. For visits at ages ≥ 20 years, spirometry values as percent predicted and recent asthma symptom scores and prednisone exposure were compared between 579 subjects who were non-obese at all visits and 151 who obese (adult definition of BMI ≥ 30 kg/m2) on at least one visit (median number of visits when obese = 4, IQR 2–7). Results Compared to participants who were non-obese (BMI 23.4 ± 2.6 kg/m2), those who became obese (BMI 31.5 ± 3.8 kg/m2) had significant decreases in FEV1/FVC (p<0.0003) and FEV1 (p = 0.001), without differences in FVC (p=0.15) during visits at ages ≥ 20 years. For each unit increase of BMI, FEV1 percent predicted decreased by 0.29 (p=0.0009). The relationship between BMI and lung function was not confounded by sex or BMI at baseline. Asthma impairment (symptom scores) and risk (prednisone use) did not differ between the two groups. Conclusion Becoming obese in early adulthood was associated with increased airway obstruction, without impact on asthma impairment or risk.

The role of phosphatidylinositol (PI) 3-kinase in specific aspects of insulin signaling was explored in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity by LY294002 or wortmannin significantly enhanced basal and insulin-stimulated GTPase-activating protein (GAP) activity in 3T3-L1 adipocytes. Furthermore, removal of the inhibitory influence of PI 3-kinase on GAP resulted in dose-dependent decreases in the ability of insulin to stimulate p21ras. This effect was specific to adipocytes, as inhibition of PI 3-kinase did not influence GAP in either 3T3-L1 fibroblasts, Rat-1 fibroblasts, or CHO cells. Immunodepletion of either of the two subunits of the PI 3-kinase (p85 or p110) yielded similar activation of GAP, suggesting that catalytic activity of p110 plays an important role in controlling GAP activity in 3T3-L1 adipocytes. Inhibition of PI 3-kinase activity in 3T3-L1 adipocytes resulted in abrogation of insulin-stimulated glucose uptake and thymidine incorporation. In contrast, effects of insulin on glycogen synthase and mitogen-activated protein kinase activity were inhibited only at higher concentrations of LY294002. It appears that in adipocytes, P1 3-kinase prevents activation of GAP. Inhibition of PI 3-kinase activity or immunodepletion of either one of its subunits results in activation of GAP and decreases in GTP loading of p21ras.

BACKGROUND: Penicillin allergy is reported in up to 10% of the general population; however, .90% of patients reporting an allergy are tolerant. Patients labeled as penicillin allergic have longer hospital stays, increased exposure to suboptimal antibiotics, and an increased risk of methicillin-resistant Staphylococcus aureus and Clostridioides difficile. The primary aim with our quality improvement initiative was to increase penicillin allergy delabeling to at least 10% among all hospitalized pediatric patients reporting a penicillin allergy with efforts directed toward patients determined to be low risk for true allergic reaction.METHODS: Our quality improvement project included several interventions: the development of a multidisciplinary clinical care pathway to identify eligible patients, workflow optimization to support delabeling, an educational intervention, and participation in our institution's quality improvement incentive program. Our interventions were targeted to facilitate appropriate delabeling by the primary hospital medicine team. Statistical process control charts were used to assess the impact of this intervention pre-and postpathway implementation.RESULTS: After implementation of the clinical pathway, the percentage of patients admitted to hospital medicine delabeled of their penicillin allergy by discharge increased to 11.7%. More than one-half of those delabeled (51.2%) received a penicillin-based antimicrobial at time of discharge. There have been no adverse events or allergic reactions requiring emergency medication administration since pathway implementation.CONCLUSIONS: Our quality improvement initiative successfully increased the rate of penicillin allergy delabeling among low-risk hospitalized pediatric patients, allowing for increased use of optimal antibiotics.

To evaluate the role of the "Ras pathway" in mediating metabolic signaling by insulin, we employed lovastatin to exhibit isoprenilation of Ras proteins in Rat-1 fibroblasts transfected with human insulin receptors (HIRc cells) and in differentiated 3T3-L1 adipocytes. Lovastatin blocked an ability of insulin to activate p21ras and mitogen-activated protein kinase. Lovastatin also significantly (p < 0.01) reduced insulin effects on thymidine incorporation and glucose incorporation into glycogen. Nevertheless, an effect of insulin on glucose uptake remained unaffected. It appears that in contrast to its mitogenic action and to its effect on glycogenesis, an effect of insulin on glucose uptake does not require p21ras activation.