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Background A multitude of studies have suggested that there is a link between obesity and inflammation. Various in vitro and animal studies have shown that saturated fatty acids are largely responsible for the induction of various pro‐inflammatory pathways. As vegetarians consume about half the amount of saturated fat as omnivores, we hypothesized that they would have less inflammation for any given amount of obesity. Objective To quantitate the inflammatory status of adipose tissue from obese vegetarians and omnivores. Methods The study included 8 obese vegetarians (1 male) and 8 obese omnivores matched for age, sex, and BMI. Body composition was measured by DXA. A blood sample to measure plasma free fatty acids (FFA) and abdominal and femoral subcutaneous adipose tissue biopsies to measure macrophage content, and the fatty acid composition were collected. Each participant also completed a Food Frequency Questionnaire (FFQ) to determine their average dietary intake. Results By design there were no differences in age, weight, BMI, percentage body fat, fat free mass or adipocyte size between the omnivore and vegetarian group. The plasma FFA of vegetarians revealed that they had greater proportions of the polyunsaturated fatty acids linoleic, linolenic and DHA ( p = 0.04, 0.05, 0.05 respectively ). Surprisingly, there was no difference in the fatty acid composition of the adipose tissue samples (abdominal or femoral) between the two groups. The FFQ data showed no significant difference in total fat, protein, or carbohydrate intake between the groups. However, the omnivore group had significantly greater intake of saturated fat and cholesterol than the vegetarian group ( p = 0.05, 0.04, respectively). The inflammatory status of adipose tissue was quantified by measuring macrophage content (total, pro‐inflammatory M1, and anti‐inflammatory M2 macrophages). There was no difference in total, M1 or M2 macrophage content between the vegetarian and omnivore groups. We also quantified the senescent cell population within the adipose tissue and again found no difference between the groups. Conclusions While vegetarians have significantly greater proportions of polyunsaturated fatty acids in plasma FFA than omnivores, this was not true for adipose tissue fatty acid composition. As expected, omnivores consume significantly greater amounts of saturated fatty acids, but there was no difference in adipose tissue inflammatory status between the two groups when measured using flow cytometry and senescent cell population. It is possible that the burden of obesity is more important than the consumption of saturated fatty acids in terms of human adipose tissue inflammation.

Aging‐related changes in lung mechanics of human subjects are characterized by a decrease in resistance and an increase in static compliance (decrease in elastic recoil). Together, these changes result in an increase in residual volume in the absence of an increase in total lung capacity. In C57BL/6x129 mice, it was reported that lung compliance increases with aging while elastic recoil pressure does not change. However, in these studies, “old” mice at 24 months of age were compared to “young” mice at 2 months of age, but at this younger age, alveologenesis is incomplete (completion does not occur until ~6 months of age). Accordingly, it remains to be established if the same aging‐related changes in lung mechanics are observed using fully developed adult C57BL/6x129 mice for comparison. To this end, we compared lung mechanics in C57BL/6x129 mice at 6 months (100% survival, n = 6), 18 months (90% survival, n = 6), and 24 months (75% survival, n = 6) of age. Respiratory system static mechanics (Resistance, R rs ; Compliance, C rs ; Elastance, E rs ) were assessed following tracheal intubation using the FlexiVent. There was a progressive aging‐related decrease in R rs (6 month, 0.47 ± 0.02; 18 month, 0.41 ± 0.02; 24 month, 0.35 ± 0.02 cmH 2 O.s/ml), an increase in C rs (6 month, 0.046 ± 0.002; 18 month, 0.052 ± 0.002; 24 month, 0.062 ± 0.002 ml/cmH 2 O), and a decrease in E rs (6 month, 21.73 ± 1.19; 18 month, 19.30 ± 0.87; 24 month, 16.62 ± 1.25 cmH 2 O/ml). Additionally, lungs were fixed following tracheal instillation of 4% paraformaldehyde at 25 cmH 2 O. Thereafter, lungs were dehydrated, paraffin embedded, sectioned (6 μm thick), and stained (hematoxylin and eosin). Using standard stereological approaches, lung sections were analyzed in a uniform systematic but random fashion to assess alveolar morphology (measurement of average distance between alveolar walls based on mean linear intercept, L m ). There was a progressive aging‐related increase in L m (6 month, 34 ± 2; 18 month, 40 ± 2; 24 month, 45 ± 2 μm) consistent with an aging‐related emphysematous‐like change in lung morphometry. Taken together, these results in appropriately aged C57BL/6x129 mice demonstrate an aging‐related change in lung mechanics and morphometry consistent with what is observed in aging humans. Therefore, the results of the present study confirm the use of rodent models for studying aging‐related changes in lung mechanics and morphometry. Support or Funding Information National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

Neurotrophic signaling is essential to the maintenance of diaphragm muscle function across the lifespan. With aging, there is an age‐related loss of neurotrophic signaling and a resultant increase in diaphragm muscle denervation and impairment in neuromuscular transmission. We hypothesized that enhancing neurotrophic signaling via the tropomyosin‐related kinase receptor subtype B (TrkB) using the small molecule TrkB agonist 7,8‐dihydroxyflavone (7,8‐DHF) would mitigate age‐related changes in diaphragm neuromuscular transmission and possibly sarcopenia (muscle atrophy and force loss). Adult male TrkB F616A mice (n=32) were randomized to receive 6‐month oral treatment with vehicle (control), 7,8‐DHF, or co‐treatment with 7,8‐DHF and 1NMPP1(a specific inhibitor of TrkB kinase activity in TrkB F616A mice) beginning at 18 and ending at 24 months of age. Mice were analyzed for neuromuscular transmission failure using diaphragm muscle‐phrenic nerve preparations, diaphragm muscle force, and morphologic characteristics of sarcopenia. Chronic 7,8‐DHF treatment did not improve diaphragm neuromuscular function or force (P≥0.25). Co‐treatment with 7,8‐DHF and 1NMPP1 decreased the ratio of the initial nerve stimulation‐derived to initial muscle stimulation‐derived force (~22%) compared to vehicle (p=0.01), consistent with a deleterious effect of inhibiting TrkB kinase activity on aging diaphragm muscle. With age, there was fiber type specific atrophy of type IIa and type IIx and/or IIb diaphragm muscle fibers. There was no evidence of a protective effect of 7,8‐DHF on muscle fiber cross‐sectional area. Taken together, these results support a role for BDNF/TrkB signaling in old age but do not support 7,8‐DHF as a therapeutic agent to mitigate age‐related effects on diaphragm muscle function. Support or Funding Information Supported by R01‐AG044615, T32‐HL105355, and the Mayo Clinic.

At present, there are limited delivery systems to target neurons in the central nervous system. Neurological diseases are commonly hindered by treatments lacking drug selectivity to the neurons of interest. In particular, motoneuron diseases will likely require selective targeting of therapeutic interventions, which can be delivered via the peripheral nervous system. Nanoparticles are novel drug delivery systems with exceptional therapeutic potential. Specifically, mesoporous silica nanoparticles coated with a lipid bilayer (protocells) are a suitable treatment vehicle to target the peripheral and central nervous system because they offer advantageous fusion characteristics including 1) increased surface area with larger loading capacity, 2) stability and 3) improved biocompatibility. A number of natural toxins exist that already target the nervous system and could be used as a targeting strategy. Cholera toxin produced by the bacterium Vibrio cholerae has an atoxic subunit (CTB) that binds to gangliosides present on neuronal membranes and is a reliable motoneuron tracer. We hypothesized that CTB‐coated protocells specifically target motoneurons. In culture, CTB uptake is evident in motoneuron‐like NSC‐34 cells but not in L6 muscle cells, consistent with the suitability of CTB as a motoneuron targeting strategy. Fluorescently labeled protocells (130 nm in diameter) were bound to CTB using biotin‐neutravidin. CTB‐protocells showed significantly greater motoneuron uptake compared to non‐CTB coated protocells when quantified by measurements of intracellular protocell fluorescence intensity. Confocal images were used for co‐localization of fluorescently labeled protocells and lysosomes. Protocells effectively escape endo‐lysosomal degradation delivering their cargo intracellularly. Biocompatibility was evidenced by lack of cytotoxicity in cultured motoneurons and unimpaired cellular proliferation. To evaluate the targeting efficacy of CTB‐protocells in tissue, we utilized isolated diaphragm muscle‐phrenic nerve preparations. Evidence of protocell uptake at diaphragm neuromuscular junctions measured by protocell fluorescence intensity supports uptake into phrenic motoneuron axon terminals. In conclusion, CTB‐protocells are a promising, customizable approach for targeting and delivering drugs to motoneurons in vivo.

The risk for respiratory diseases is significantly increased in the elderly population (>65 years of age), which may be partially due to an inability to adequately clear the airways resulting from a progressive aging‐related atrophy and weakening (i.e., sarcopenia) of the diaphragm muscle (DIAm). In humans, reduced maximum inspiratory and transdiaphragmatic pressures suggest DIAm weakening, but muscle fiber atrophy has not been examined. In a National Institute on Aging (NIA) supported mouse model of aging (C57BL/6J strain), we recently showed that DIAm sarcopenia exists and affects non‐ventilatory functions (Greising et al., Exp Gerontol 48:881–887, 2013; Greising et al., Am J Physiol Lung Cell Mol Physiol 309:46–52, 2015). Importantly, rodent models such as this provide a valuable tool to explore the mechanisms underlying DIAm sarcopenia. Currently, the NIA also provides two inbred rat models for aging‐related research: the Fischer 344 (F344) and Brown Norway (BN) rat strains. The purpose of this study was to comprehensively examine whether sarcopenia also exists in the DIAm of F344 and BN rat strains provided by the NIA. Young (6 month, 100% survival) and old (24 month, 50% survival) F344 and young (6 month, 100% survival) and old (32 month, 50% survival) BN male, pathogen free, rats were obtained from the NIA rodent colony. In DIAm strips from the right midcostal region, measurements of contractility, contractile protein concentration, relative myosin heavy chain (MyHC) isoform expression, fiber type distribution and fiber cross‐sectional area (CSA) were obtained. Peak twitch specific force (P t ) was reduced by ~17% and ~14% in the aged F344 and BN rats, respectively. Additionally, maximal specific force (P o ) was reduced by ~24% and ~12% in the aged F344 and BN rats, respectively. Thus, although there was significant aging‐related DIAm weakness in both rat strains, reductions in both P t and P o were more pronounced in old F344 compared to BN rats. In F344 rats, aging also affected DIAm fiber type proportions with a significant increase in the proportion of type I fibers in old (45 ± 3%), compared to young (35 ± 3%) rats, with a concomitant decrease in the relative proportions of both type IIa fibres and type IIx and/or IIb fibers. In BN rats, DIAm fiber type proportions were not affected by aging. In both F344 and BN rats, the CSA of type I and IIa fibers were unchanged, whereas the CSA of type IIx and/or IIb DIAm fibers were reduced by ~20% in F344 rats and by ~15% is BN rats. Similar to DIAm weakness, the selective aging‐related atrophy of type IIx and/or IIb DIAm fibers was more pronounced in F344 rats. In conclusion, DIAm sarcopenia is clearly present in both F344 and BN rat strains, but more pronounced in the F344 strain. These results indicate that in addition to the C57BL/6J mouse model, the F344 rat strain is an appropriate model for research pertaining to DIAm sarcopenia. Support or Funding Information Support: National Institute of Health grants R01‐AG044615 (CBM & GCS) and T32‐HL105355 (JEE).

Background We have previously shown that increased caveolin‐1 expression impairs leptin‐dependent activation of cellular signaling pathways and may be responsible for attenuating leptin‐mediated increases in adiponectin expression in obesity. Further, insulin and leptin cellular signaling mechanisms are closely related such that molecules known to impair insulin signaling also alter leptin signaling. Conversely, known insulin‐sensitizing drugs may improve leptin‐signaling via reducing caveolin‐1 expression. We therefore tested the hypothesis that insulin‐sensitizing drugs decrease caveolin‐1 expression. Methods We used an in‐vitro approach to examine the effects of different insulin‐sensitizing drugs on caveolin‐1 expression. Human white preadipocytes were cultured to confluence and differentiated for 15 days. After differentiation, cells were then treated with increasing concentrations of pioglitazone (5–30 μM), troglitazone (10–30 μM), metformin (1–5 mM), or glimepiride (20–100 μM) for 24 hours. Caveolin‐1 protein expression was determined by Western blot analysis. Results Among the insulin‐sensitizers used in our experiment, only treatment with increasing concentrations of metformin resulted in a dose‐dependent decease in caveolin‐1 expression (P =0.007). Treatment with increasing concentration of troglitazone upregulated caveolin‐1 expression (p=0.005), while pioglitazone (p=0.5020) and glimepiride (p=0.0965) had no effect of caveolin‐1 protein expression. Conclusions Metformin reduces caveolin‐1 expression in differentiated human white preadipocytes. In obesity, caveolin‐1 expression is increased which may contribute to impaired leptin signaling. Therefore, metformin may be used to decrease caveolin‐1 expression and consequently improve leptin signaling and leptin‐dependent increases in adiponectin expression in obese individuals. Support or Funding Information Michaela Pfeifer was supported by the American Physiological Society Stride Summer Undergraduate Research Fellowship NHLBI 1 R25 HL115473‐01; Prachi Singh was supported by the American Heart Association Scientist Development Grant 11SDG7260046.

Hypothermia/Rewarming (H/R) is poorly tolerated by the myocardium; however, the underlying intracellular basis of H/R‐induced cardiac injury remains elusive. We hypothesized that in cardiomyocytes, H/R leads to contractile dysfunction primarily through reduced Ca 2+ sensitivity of the myofilaments. To test this hypothesis, isolated rat cardiomyocytes (13–15 cells from 6 rats per group) were electrically stimulated to evoke both cytosolic Ca 2+ ([Ca 2+ ] cyto ) and contractile (sarcomere shortening) responses that were simultaneously measured using an IonOptix system. These measurements were subjected to the following temperature protocols: 1) hypothermia (15°C) followed by rewarming (35°C) or 2) time‐matched control (35°C). Contractile dysfunction after H/R was indicated by reduced shortening velocity and reduced extent of shortening of sarcomere length (SL) compared to time‐matched controls. Throughout hypothermia, basal [Ca 2+ ] cyto increased and the duration of the evoked [Ca 2+ ] cyto transients was prolonged. Phase‐loop plots of [Ca 2+ ] cyto versus contraction in cells exposed to H/R indicate that Ca 2+ sensitivity of contraction is reduced. Consistent with decreased Ca 2+ sensitivity, we found that cardiac troponin I (cTnI) phosphorylation (measured by Western blot) increased after H/R. Our results support our overall hypothesis and suggest that reduced Ca 2+ sensitivity and increased cTnI phosphorylation contributes to the altered excitation‐contraction coupling of cardiomyocytes following H/R. The clinical importance of our results brings focus to Ca 2+ sensitivity as a plausible therapeutic target in treating patients exposed to accidental hypothermia. Support or Funding Information NIH Grant 5T32HL10535 (NSS)

Background The baroreflex maintains blood pressure homeostasis by modulating sympathetic activity in response to changes in arterial pressure. Baroreflex sensitivity (BRS) is blunted by activation of either the chemo‐or cardiopulmonary reflex; however, little is known about the interaction of these two reflexes on BRS. We hypothesized that activation of the chemoreflex during stimulation of the cardiopulmonary reflex would attenuate the reduction in BRS. Methods 10 healthy male volunteers (35±3 years, BMI: 25±1 kg/m 2 ) underwent lower body negative pressure (LBNP) at −15mmHg for 5 minutes to stimulate the cardiopulmonary reflex, with chemoreflex activation (hypoxia; arterial oxygen saturation: 85±1%) and without (normoxia; arterial oxygen saturation: 98±0%). Heart rate (HR; ECG), blood pressure (BP; brachial arterial catheter), oxygen saturation (finger pulse oximeter), and ventilation (expired gases) were recorded continuously. BRS was calculated by arterial pressure waveform analysis (spectrum; WinCPRS) during baseline (BL) and LBNP. Results Baseline HR was higher during hypoxia than normoxia (62±2 vs 77±3 bpm, p<0.001) while baseline systolic BP (SBP) was not statistically differentiated between conditions (151±3 vs 149±5 mmHg, normoxia vs hypoxia, p=0.669). During normoxic LBNP, HR increased (62±2 vs 66±3 bpm, p=0.001) and SBP was unchanged (151±3 vs 151±4 mmHg, BL vs LBNP, p=0.464). During hypoxic LBNP, HR remained unchanged (77±3 vs 76±4 bpm, BL vs LBNP, p=0.693) and SBP tended to decrease (149±5 vs 139±6, BL vs LBNP, p=0.067). Baseline BRS was lower during hypoxia than normoxia (15±2 vs 9±1 ms/mmHg, p=0.012). LBNP caused a similar % reduction in BRS during normoxia and hypoxia (−14±9% vs −10±7%, p=0.772). Conclusions Contrary to our hypothesis, chemoreflex activation during LBNP does not attenuate the reduction in BRS. This suggests that the cardiopulmonary reflex and the chemoreflex blunt BRS through independent mechanisms. Support or Funding Information Funding: W81XWH‐13‐2‐0038

Background Evidence indicates that the carotid chemoreceptors play a minimal role in regulating cerebral blood flow under normoxic resting conditions. However, studies in heart failure suggest over‐active carotid chemoreceptors may decrease cerebral blood flow. With this information in mind, we hypothesized that individuals with the highest resting chemosensitivity will have the greatest increase in cerebral blood flow when the carotid chemoreceptors are desensitized with low‐dose IV dopamine. Methods Seven young, healthy men (33±3 y) participated in two randomized, double‐blinded study days separated by at least 4 weeks. Middle cerebral artery blood velocity (MCAv, transcranial Doppler) was measured continuously as a surrogate for cerebral blood flow. Cerebral vascular conductance index (CVCi) was calculated by normalizing MCAv for blood pressure as measured by brachial arterial catheter (MCAv/mean arterial blood pressure) and blood gasses were drawn. Subjects rested quietly in the supine position for 5‐minutes during intravenous saline infusion (saline and dopamine baseline). This was followed by a 25‐minute continuous IV infusion of either saline or dopamine. The dose of dopamine was individualized (1–2 μg/kg/min) such that it resulted in a nadir hypoxic ventilatory response. Data were analyzed from the last 5‐minutes of the infusion period (saline infusion or dopamine infusion) and compared to baseline data. Results Saline and dopamine infusion did not affect mean arterial blood pressure (% change saline vs dopamine day: 1.3±1.2 vs 0.9±1.3%; p>0.05) or arterial blood CO 2 (% change saline vs dopamine day: −2.6±1.7 vs 1.6±1.9%; p>0.05). Dopamine infusion resulted in a significant reduction in carotid body chemosensitivity as assessed by the hypoxic ventilatory response (dopamine baseline vs infusion HVR: 0.33±0.06 vs 0.17±0.03 L/min/%SpO 2 ; p=0.027). No changes in MCAv or CVCi were observed with saline infusion (saline baseline vs infusion: p>0.05). Dopamine did not alter MCAv (dopamine baseline vs infusion: 62±5 vs 64±5 cm/s; p>0.05). Similarly, no changes in CVCi were observed with dopamine infusion (dopamine baseline vs infusion: 0.68±0.07 vs 0.68±0.06 cm/s/mmHg; p>0.05). Although differences in group means for MCAv and CVCi were not observed with dopamine infusion, a significant negative relationship was found between baseline carotid chemosensitivity and the change in MCAv (r 2 =0.594, p=0.043), and CVCi (r 2 =0.741, p=0.013). With saline infusion, no significant relationship was found between baseline carotid chemosensitivity and the change in MCAv (r 2 =0.091, p>0.05), and CVCi (r 2 =0.089, p>0.05). Conclusions Blunting of the carotid chemoreceptors using IV dopamine has no group effect on MCAv or CVCi. However, contrary to our hypothesis, individuals with highest resting chemosensitivity had either a decrease or no change in cerebral blood flow after carotid body desensitization. Support or Funding Information W81XWH‐13‐2‐003