SUMMARY Hepatic lipid accumulation in obesity correlates with the severity of hyperinsulinemia and systemic insulin resistance. Obesity-induced hepatocellular lipid accumulation results in hepatocyte depolarization. We have established that hepatocyte depolarization depresses hepatic afferent vagal nerve firing, increases GABA release from liver slices, and causes hyperinsulinemia. Preventing hepatic GABA release or eliminating the ability of the liver to communicate to the hepatic vagal nerve ameliorates the hyperinsulinemia and insulin resistance associated with diet-induced obesity. In people with obesity, hepatic expression of GABA transporters is associated with glucose infusion and disposal rates during a hyperinsulinemic euglycemic clamp. Single-nucleotide polymorphisms in hepatic GABA re-uptake transporters are associated with an increased incidence of type 2 diabetes mellitus. Herein, we identify GABA as a neuro-hepatokine that is dysregulated in obesity and whose release can be manipulated to mute or exacerbate the glucoregulatory dysfunction common to obesity.

Mice are a valuable model for elegant studies of complex, systems-dependent diseases, including pulmonary diseases. Current tools to assess lung function in mice are either terminal or lack accuracy. We set out to develop a low-cost, accurate, head-out variable-pressure plethysmography system to allow for repeated, non-terminal measurements of lung function in mice. Current head-out plethysmography systems are limited by air leaks that prevent accurate measures of volume and flow. We designed an inflatable cuff that encompasses the mouse's neck preventing air leak. We wrote corresponding software to collect and analyze the data, remove movement artifacts, and automatically calibrate each dataset. This software calculates inspiratory/expiratory volume, inspiratory/expiratory time, breaths per minute, mid-expiratory flow, and end-inspiratory pause. To validate the use, we established that our plethysmography system accurately measured tidal breathing, the bronchoconstrictive response to methacholine, sex and age associated changes in breathing, and breathing changes associated with house dust mite sensitization. Our estimates of volume, flow, and timing of breaths are in line with published estimates, we observed dose-dependent decreases in volume and flow in response to methacholine (P < 0.05), increased lung volume and decreased breathing rate with aging (P < 0.05), and that house dust mite sensitization decreased volume and flow (P <0.05) while exacerbating the methacholine induced increases in inspiratory and expiratory time (P < 0.05). We describe an accurate, sensitive, low-cost, head-out plethysmography system that allows for longitudinal studies of pulmonary disease in mice.

Animals store metabolic energy as electrochemical gradients. At least 50% of mammalian energy is expended to maintain electrochemical gradients across the inner mitochondrial membrane (H+), the sarcoplasmic reticulum (Ca++), and the plasma membrane (Na+/K+). The potential energy of these gradients can be used to perform work (e.g., transport molecules, stimulate contraction, and release hormones) or can be released as heat. Because ectothermic species adapt their body temperature to the environment, they are not constrained by energetic demands that are required to maintain a constant body temperature. In fact, ectothermic species expend seven to eight times less energy than similarly sized homeotherms. Accordingly, ectotherms adopt low metabolic rates to survive cold, hypoxia, and extreme bouts of fasting that would result in energy wasting, lactic acidosis and apoptosis, or starvation in homeotherms, respectively. Ectotherms have also evolved unique applications of ion gradients to allow for localized endothermy. Endothermic avian species, which lack brown adipose tissue, have been integral in assessing the role of H+ and Ca++ cycling in skeletal muscle thermogenesis. Accordingly, the diversity of non-mammalian vertebrate species allows them to serve as unique models to better understand the role of ion gradients in heat production, metabolic flux, and adaptation to stressors, including obesity, starvation, cold, and hypoxia.

In the U.S., one in thirteen individuals have been diagnosed with asthma. Obesity increases the incidence of asthma by 56%. The degree of obesity is associated with the severity of reduction in lung volume and lung volume can be restored with weight loss. Obesity also increases the risk of hospitalization by 460%. Although asthma and altered lung function is commonly assessed with spirometry in humans, plethysmography is recommended by the American Thoracic Society for assessing lung volume. The most common and effective method for assessing lung function in mice, forced ventilation, is terminal and limited to assessing lower airway function, preventing repeated measures. We aimed to develop a head‐out, variable pressure plethysmography system that would allow for repeated sensitive assessment of lung function and volume in lean and diet‐induced obese mice while mimicking the measures in human clinical practice. Preventing air leak from the chamber is vital for recording accurate measurements in a variable pressure plethysmography system. To eliminate air leak, we created an inflatable cuff that encompassed the mouse’s neck. This allowed us to repeatedly and accurately assess airway function in mice. To validate the head‐out, variable pressure plethysmography system, we used a series of models with altered muscarinic signaling. First, we assessed the response to a nebulized bronchoconstrictor, methacholine, establishing that methacholine resulted in the expected decreased breaths/minute, increased expiratory and inspiratory time, and decreased the rate of air flow at 50% of exhalation (EF50). To assess chronic muscarinic signaling, we used an adeno‐associated viral delivery to induce expression of a mutated constitutively active muscarinic 3 receptor (Q409L M3R) in airway smooth muscle, creating a model of chronic bronchoconstriction. In mice that expressed the Q490L M3R, we similarly found a decrease in breaths/minute and an increase in expiratory and inspiratory time at tidal breathing. Finally, we observed a decrease in breaths per minute at tidal breathing in muscarinic 3 receptor knock‐out mice. In our model of diet‐induced obesity we showed that obesity increased breaths per minute and decreased expiratory time at tidal breathing relative to that observed in lean mice. We have developed a reliable tool to repeatedly assess lung function in mice using the same clinically relevant measure applied in human asthmatics. Future research will apply this tool to better understand the mechanism by which obesity alters lung function and tidal breathing volume in mice. Support or Funding Information ABRC ADHS14‐082986ABRC ADHS17‐00002043T32 HL 007249

Mice are a valuable model for elegant studies of complex, systems-dependent diseases, including pulmonary diseases. Current tools to assess lung function in mice are either terminal or lack accuracy. We set out to develop a low-cost, accurate, head-out variable-pressure plethysmography system to allow for repeated, non-terminal measurements of lung function in mice. Current head-out plethysmography systems are limited by air leaks that prevent accurate measures of volume and flow. We designed an inflatable cuff that encompasses the mouse’s neck preventing air leak. We wrote corresponding software to collect and analyze the data, remove movement artifacts, and automatically calibrate each dataset. This software calculates inspiratory/expiratory volume, inspiratory/expiratory time, breaths per minute, enhanced pause, mid-expiratory flow, and end-inspiratory pause. To validate the use, we established our plethysmography system accurately measured tidal breathing, the bronchoconstrictive response to methacholine, sex and age associated changes in breathing, and breathing changes associated with house dust mite sensitization. Our estimates of volume, flow, and timing of breaths are in line with published estimates, we observed dose-dependent decreases in volume and flow in response to methacholine (P < 0.05), increased lung volume and decreased breathing rate with aging (P < 0.05), and that house dust mite sensitization decreased tidal volume and flow (P <0.05) while exacerbating the methacholine induced increases in inspiratory and expiratory time (P < 0.05). We describe an accurate, sensitive, low-cost, head-out plethysmography system that allows for longitudinal studies of pulmonary disease in mice.New & NoteworthyWe describe a variable-pressure head-out plethysmography system that can be used to assess lung function in mice. A balloon cuff that inflates around the mouse’s neck prevents air leak, allowing for accurate measurements of lung volume and air flow. Custom software facilitates system calibration, removes movement artifacts, and eases data analysis. The system was validated by measuring tidal breathing, responses to methacholine, and changes associated with house dust mite sensitization, sex, and aging.Contributions to StudyStephanie Bruggink: development of head-out plethysmography chamber, measurement of breathing, data analysis, prepared manuscriptKyle Kentch: development of head-out plethysmography chamber, programmed software to collect and analyze data, prepared manuscriptJason Kronenfeld: development of tools to analyze data, analysis of dataBenjamin Renquist: development of head-out plethysmography chamber, statistical analysis, prepared manuscript