The impact of operative approach and intraoperative imaging on leg length discrepancy and acetabular component angle in total hip arthroplasty: a retrospective cohort study.

PURPOSE: Operative approach in total hip arthroplasty (THA) has long been a topic of debate with each approach having unique benefits and disadvantages. One purported benefit of an anterior approach to THA is that it allows for intraoperative positioning using fluoroscopy rather than manual positioning. Proper positioning allows for improved outcomes including leg length discrepancy and acetabular component angle. This study aims to examine if operative approach and use of imaging in intraoperative positioning impact LLD and cup angle post-operatively. METHODS: A total of 300 hips were enrolled in the study with 100 hips per approach (anterior with fluoroscopy, lateral, and posterior). Retrospective chart review was conducted to assess patient demographics and radiographic analysis used to determine LLD and acetabular cup angle. RESULTS: Of the three groups, those receiving anterior approach THAs were on average older than those in the posterior group. Analysis comparing the LLD and acetabular angle across the three groups showed no statistically significant difference in LLD (p=0.091); this was also reflected when comparing hips that received fluoroscopy with those that did not (p=0.91). For acetabular angle, while no difference existed when comparing hips that received imaging versus those that did not, statistically significant differences were observed when comparing the three intraoperative approaches (p<0.0001). CONCLUSIONS: Neither intraoperative approach nor the use of intraoperative imaging in THA has a statistically significant effect on LLD post-operatively. However, approach did impact the acetabular cup angle across all three distinct approaches.

Hippocampal, Whole Midbrain, Red Nucleus, and Ventral Tegmental Area Volumes Are Increased by Selective Breeding for High Voluntary Wheel-Running Behavior.

Uncovering relationships between neuroanatomy, behavior, and evolution are important for understanding the factors that control brain function. Voluntary exercise is one key behavior that both affects, and may be affected by, neuroanatomical variation. Moreover, recent studies suggest an important role for physical activity in brain evolution. We used a unique and ongoing artificial selection model in which mice are bred for high voluntary wheel-running behavior, yielding four replicate lines of high runner (HR) mice that run ∼3-fold more revolutions per day than four replicate nonselected control (C) lines. Previous studies reported that, with body mass as a covariate, HR mice had heavier whole brains, non-cerebellar brains, and larger midbrains than C mice. We sampled mice from generation 66 and used high-resolution microscopy to test the hypothesis that HR mice have greater volumes and/or cell densities in nine key regions from either the midbrain or limbic system. In addition, half of the mice were given 10 weeks of wheel access from weaning, and we predicted that chronic exercise would increase the volumes of the examined brain regions via phenotypic plasticity. We replicated findings that both selective breeding and wheel access increased total brain mass, with no significant interaction between the two factors. In HR compared to C mice, adjusting for body mass, both the red nucleus (RN) of the midbrain and the hippocampus (HPC) were significantly larger, and the whole midbrain tended to be larger, with no effect of wheel access nor any interactions. Linetype and wheel access had an interactive effect on the volume of the periaqueductal gray (PAG), such that wheel access increased PAG volume in C mice but decreased volume in HR mice. Neither linetype nor wheel access affected volumes of the substantia nigra, ventral tegmental area, nucleus accumbens, ventral pallidum (VP), or basolateral amygdala. We found no main effect of either linetype or wheel access on neuronal densities (numbers of cells per unit area) for any of the regions examined. Taken together, our results suggest that the increased exercise phenotype of HR mice is related to increased RN and hippocampal volumes, but that chronic exercise alone does not produce such phenotypes.

Mast-Cell Tryptase Release Contributes to Disease Progression in Lymphangioleiomyomatosis.

Rationale: Lymphangioleiomyomatosis (LAM) is a multisystem disease that causes lung cysts and respiratory failure. Loss of TSC (tuberous sclerosis complex) gene function results in a clone of "LAM cells" with dysregulated mTOR (mechanistic target of rapamycin) activity. LAM cells and fibroblasts form lung nodules that also contain mast cells, although their significance is unknown. Objectives: To understand the mechanism of mast-cell accumulation and the role of mast cells in the pathogenesis of LAM. Methods: Gene expression was examined using transcriptional profiling and qRT-PCR. Mast cell/LAM nodule interactions were examined in vitro using spheroid TSC2-null cell/fibroblast cocultures and in vivo using an immunocompetent Tsc2-null murine homograft model. Measurements and Main Results: LAM-derived cell/fibroblast cocultures induced multiple CXC chemokines in fibroblasts. LAM lungs had increased tryptase-positive mast cells expressing CXCRs (CXC chemokine receptors) (P < 0.05). Mast cells located around the periphery of LAM nodules were positively associated with the rate of lung function loss (P = 0.016). LAM spheroids attracted mast cells, and this process was inhibited by pharmacologic and CRISPR/cas9 inhibition of CXCR1 and CXCR2. LAM spheroids caused mast-cell tryptase release, which induced fibroblast proliferation and increased LAM-spheroid size (1.36 ± 0.24-fold; P = 0.0019). The tryptase inhibitor APC366 and sodium cromoglycate (SCG) inhibited mast cell-induced spheroid growth. In vivo, SCG reduced mast-cell activation and Tsc2-null lung tumor burden (vehicle: 32.5.3% ± 23.6%; SCG: 5.5% ± 4.3%; P = 0.0035). Conclusions: LAM-cell/fibroblast interactions attract mast cells where tryptase release contributes to disease progression. Repurposing SCG for use in LAM should be studied as an alternative or adjunct to mTOR inhibitor therapy.

Associations between therapist factors and treatment efficacy in randomized controlled trials of trauma-focused cognitive behavioral therapy for children and youth: A systematic review and meta-analysis.

Previous research suggests that the effect of therapist factors on patient outcomes is significant. Yet, to date, no reviews have explored the potential effects of therapist characteristics on treatment outcomes for children and youth with posttraumatic stress disorder (PTSD). This systematic review and meta-analysis aimed to summarize the professional characteristics of trial therapists delivering trauma-focused cognitive behavioral interventions (TF-CBT) for child PTSD in clinical trials and understand the association between treatment efficacy and therapist factors. Systematic searches for randomized controlled trials (RCTs) published through November 3, 2020, were conducted; 40 RCTs were included in the full review. PTSD treatment outcome data were extracted from each publication along with any available data regarding trial therapists. Subgroup analyses were conducted to compare the outcomes of interventions conducted by different types of therapists. All therapist groups yielded significant effects for TF-CBT relative to active and passive control conditions, with the largest effect size, Hedges' g = -1.11, for RCTs that used clinical psychologists and psychiatrists. A significant moderating effect was found when comparing the treatment outcomes of clinical psychologists and psychiatrists versus other professionals, p = .044; however, this effect was no longer apparent when only studies with an active control arm were included. Further moderator analyses demonstrated no significant differences regarding therapists' educational and professional backgrounds and PTSD treatment outcomes. The current RCT evidence for TF-CBT for children and youth with PTSD does not suggest that therapist educational or professional background influences treatment efficacy. Limitations and implications for future research are discussed.

Development of low-cost cardiac and skeletal muscle laboratory activities to teach physiology concepts and the scientific method.

Anatomy and Physiology courses taught at community colleges tend to focus laboratory hours primarily on anatomy as opposed to physiology. However, research demonstrates that, when instructors utilize active learning approaches (such as in laboratory settings) where students participate in their own learning, students have improved outcomes, such as higher test scores and better retention of material. To provide community college students with opportunities for active learning in physiology, we developed two laboratory exercises to engage students in cardiac and skeletal muscle physiology. We utilized low-cost SpikerBox devices to measure electrical activity during cardiac (electrocardiogram) and skeletal muscle (electromyogram) contraction. Laboratory activities were employed in Anatomy and Physiology courses at two community colleges in southeast Michigan. A 2-h laboratory period was structured with a 20-min slide presentation covering background material on the subject and experiments to examine the effects of environmental variables on nervous system control of cardiac and skeletal muscle contraction. Students were asked to provide hypotheses and proposed mechanisms, complete a results section, and provide conclusions for the experiments based on their results. Our laboratory exercises improved student learning in physiology and knowledge of the scientific method and were well-received by community college students enrolled in Anatomy and Physiology. Our results demonstrate that the use of a SpikerBox for cardiac and skeletal muscle physiology concepts is a low-cost and effective approach to integrate physiology activities into an Anatomy and Physiology course.

The early- to medium-term results of a hemispherical, porous coated acetabular shell with multiple different bearing combinations are excellent with the exception of metal-on-metal.

PURPOSE: This study prospectively reports survivorship and radiographic and clinical outcomes following primary elective total hip arthroplasty (THA) using a novel single hemispherical, porous-coated acetabular cup with five different bearing combinations and a minimum of five year follow-up. METHODS: Continuing post-market release monitoring of this cup, we prospectively enrolled 108 patients (121 THA) between 2009 and 2015. We followed this cohort by examining survivorship, in addition to clinical and radiological outcomes for metal-on-metal (MoM) compared with non-MoM bearing combinations (ceramic-on-ceramic, oxinium-on-polyethylene, ceramic-on-metal, and metal-on-polyethylene). RESULTS: All 108 (121 hips) patients were followed up. Average age at time of surgery was 45.1 years (range 19 to 71 years) of which 42.1% were males. A total of seven (5.8%) cups were revised, all of which were MoM. No osteolysis was observed in any of the patients at the latest visit with a mean follow-up of 9.1 ± 1.7 years (range 4.4-10.7 years). With MoM excluded, survivorship of the cup at five  years is 97.8%. Survivorship for MoM implants was 90.0%. Validated hip scores showed significant improvements for all bearing types and no significant difference between groups at latest follow-up (p = 0.614). There was no cup migration with any bearing surface. CONCLUSION: This cup showed excellent survivorship at five year follow-up, except for patients receiving a MoM articulation. While there were concerns over the early survivorship of this cup, our cohort and joint registry data confirm excellent outcomes.

Effects of food restriction on voluntary wheel-running behavior and body mass in selectively bred High Runner lines of mice.

Food restriction can have profound effects on various aspects of behavior, physiology, and morphology. Such effects might be amplified in animals that are highly active, given that physical activity can represent a substantial fraction of the total daily energy budget. More specifically, some effects of food restriction could be associated with intrinsic, genetically based differences in the propensity or ability to perform physical activity. To address this possibility, we studied the effects of food restriction in four replicate lines of High Runner (HR) mice that have been selectively bred for high levels of voluntary wheel running. We hypothesized that HR mice would respond differently than mice from four non-selected Control (C) lines. Healthy adult females from generation 65 were housed individually with wheels and provided access to food and water ad libitum for experimental days 1-19 (Phase 1), which allowed mice to attain a plateau in daily running distances. Ad libitum food intake of each mouse was measured on days 20-22 (Phase 2). After this, each mouse experienced a 20 % food restriction for 7 days (days 24-30; Phase 3), and then a 40 % food restriction for 7 additional days (days 31-37; Phase 4). Mice were weighed on experimental days 1, 8, 9, 15, 20, and 23-37 and wheel-running activity was recorded continuously, in 1-minute bins, during the entire experiment. Repeated-measures ANOVA of daily wheel-running distance during Phases 2-4 indicated that HR mice always ran much more than C, with values being 3.29-fold higher during the ad libitum feeding trial, 3.58-fold higher with -20 % food, and 3.06-fold higher with -40 % food. Seven days of food restriction at -20 % did not significantly reduce wheel-running distance of either HR (-5.8 %, P = 0.0773) or C mice (-13.3 %, P = 0.2122). With 40 % restriction, HR mice showed a further decrease in daily wheel-running distance (P = 0.0797 vs. values at 20 % restriction), whereas C mice did not (P = 0.4068 vs. values at 20 % restriction) and recovered to levels similar to those on ad libitum food (P = 0.3634). For HR mice, daily running distances averaged 11.4 % lower at -40 % food versus baseline values (P = 0.0086), whereas for C mice no statistical difference existed (-4.8 %, P = 0.7004). Repeated-measures ANOVA of body mass during Phases 2-4 indicated a highly significant effect of food restriction (P = 0.0001), but no significant effect of linetype (P = 0.1764) and no interaction (P = 0.8524). Both HR and C mice had a significant reduction in body mass only when food rations were reduced by 40 % relative to ad libitum feeding, and even then the reductions averaged only -0.60 g for HR mice (-2.6 %) and -0.49 g (-2.0 %) for C mice. Overall, our results indicate a surprising insensitivity of body mass to food restriction in both high-activity (HR) and ordinary (C) mice, and also insensitivity of wheel running in the C lines of mice, thus calling for studies of compensatory mechanisms that allow this insensitivity.

A Geometric Analysis of Polyethylene Liners Exposed to Acrylic-based Bone Cement.

Background: Acrylic-based bone cement (polymethyl methacrylate [PMMA]) is a material commonly used in orthopaedic surgeries; however, during PMMA polymerization, a highly exothermic reaction occurs. The heat released in polymerization can damage nearby materials including poorly heat-resistant cross-linked polyethylene (XLPE). Both PMMA and XLPE are used in total hip arthroplasty and could interact during femoral stem fixation. We sought to determine if the exothermic polymerization of PMMA could alter the surface characteristics of XLPE acetabular liners. Methods: Six XLPE liners were assigned to one of 4 experimental categories with varying volumes of PMMA applied in a manner that mimicked how the 2 materials would come into contact intraoperatively. Measurements were taken both pre- and post-intervention using a coordinate measuring machine for geometric and gravimetric analysis. Light microscopy was conducted postintervention to examine the surface for damage. Results: Coordinate measuring machine measurements showed minimal gross deformation in all 6 liners, but there were isolated surface deposits in 4 of 6 liners. The average maximal surface deviations, when compared to the control, for liners exposed to 1 cc of cement, 2 cc of cement, or 1 cc of cement with a femoral head implant attached were 26.6 µm, 77.2 µm, and 26.4 µm, respectively. All but one liner showed an increase in volume following intervention when compared to the control. Subtle scratches were identified using light microscopy on all 6 liners. Conclusions: XLPE shows areas of isolated surface deformation in a dose-dependent manner but with minimal gross deformation after interacting with highly exothermic PMMA.

Effects of Selective Breeding, Voluntary Exercise, and Sex on Endocannabinoid Levels in the Mouse Small-Intestinal Epithelium.

The endocannabinoid (eCB) system in the gut communicates with the body and brain as part of the homeostatic mechanisms that affect energy balance. Although perhaps best known for its effects on energy intake, the eCB system also regulates voluntary locomotor behavior. Here, we examined gut eCB concentrations in relation to voluntary exercise, specifically in mice selectively bred for high voluntary wheel running behavior. We measured gut eCBs in four replicate non-selected Control (C) lines and four replicate lines of High Runner (HR) mice that had been selectively bred for 74 generations based on the average number of wheel revolutions on days 5 and 6 of a 6-day period of wheel access when young adults. On average, mice from HR lines run voluntarily on wheels ∼3-fold more than C mice on a daily basis. A recent study showed that circulating levels of primary endocannabinoids 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA) are altered by six days of wheel access, by acute wheel running, and differ between HR and C mice in sex-specific ways [1]. We hypothesized that eCBs in the upper small-intestinal epithelium (i.e., proximal jejunum), a region firmly implicated in eCB signaling, would differ between HR and C mice (linetype), between the sexes, between mice housed with vs. without wheels for six days, and would covary with amounts of acute running and/or home-cage activity (during the previous 30 minutes). We used the same 192 mice as in [1] , half males and half females, half HR and half C (all 8 lines), and half either given or not given access to wheels for six days. We assessed the eCBs, 2-AG and AEA, and their analogs docosahexaenoylglycerol (DHG), docosahexaenoylethanolamide (DHEA), and oleoylethanolamide (OEA). Both 2-AG and DHG showed a significant 3-way interaction of linetype, wheel access, and sex. In addition, HR mice had lower concentrations of 2-AG in the small-intestinal epithelium when compared to C mice, which may be functionally related to differences in locomotor activity or to differences in body composition and/or food consumption. Moreover, the amount of home-cage activity during the prior 30 min was a negative predictor of 2-AG and AEA concentrations in jejunum mucosa, particularly in the mice with no wheel access. Lastly, 2-AG, but not AEA, was significantly correlated with 2-AG in plasma in the same mice.

The large numbers of minicolumns in the primary visual cortex of humans, chimpanzees and gorillas are related to high visual acuity.

Minicolumns are thought to be a fundamental neural unit in the neocortex and their replication may have formed the basis of the rapid cortical expansion that occurred during primate evolution. We sought evidence of minicolumns in the primary visual cortex (V-1) of three great apes, three rodents and representatives from three other mammalian orders: Eulipotyphla (European hedgehog), Artiodactyla (domestic pig) and Carnivora (ferret). Minicolumns, identified by the presence of a long bundle of radial, myelinated fibers stretching from layer III to the white matter of silver-stained sections, were found in the human, chimpanzee, gorilla and guinea pig V-1. Shorter bundles confined to one or two layers were found in the other species but represent modules rather than minicolumns. The inter-bundle distance, and hence density of minicolumns, varied systematically both within a local area that might represent a hypercolumn but also across the whole visual field. The distance between all bundles had a similar range for human, chimpanzee, gorilla, ferret and guinea pig: most bundles were 20-45 µm apart. By contrast, the space between bundles was greater for the hedgehog and pig (20-140 µm). The mean density of minicolumns was greater in tangential sections of the gorilla and chimpanzee (1,243-1,287 bundles/mm2) than in human (314-422 bundles/mm2) or guinea pig (643 bundles/mm2). The minicolumnar bundles did not form a hexagonal lattice but were arranged in thin curving and branched bands separated by thicker bands of neuropil/somata. Estimates of the total number of modules/minicolumns within V-1 were strongly correlated with visual acuity.

Mitochondrial DNA quality control in the female germline requires a unique programmed mitophagy.

Mitochondria have their own DNA (mtDNA), which is susceptible to the accumulation of disease-causing mutations. To prevent deleterious mutations from being inherited, the female germline has evolved a conserved quality control mechanism that remains poorly understood. Here, through a large-scale screen, we uncover a unique programmed germline mitophagy (PGM) that is essential for mtDNA quality control. We find that PGM is developmentally triggered as germ cells enter meiosis by inhibition of the target of rapamycin complex 1 (TORC1). We identify a role for the RNA-binding protein Ataxin-2 (Atx2) in coordinating the timing of PGM with meiosis. We show that PGM requires the mitophagy receptor BNIP3, mitochondrial fission and translation factors, and members of the Atg1 complex, but not the mitophagy factors PINK1 and Parkin. Additionally, we report several factors that are critical for germline mtDNA quality control and show that pharmacological manipulation of one of these factors promotes mtDNA quality control.

Juxtacellular Labeling of Stellate, Disk and Basket Neurons in the Central Nucleus of the Guinea Pig Inferior Colliculus.

We reconstructed the intrinsic axons of 32 neurons in the guinea pig inferior colliculus (IC) following juxtacellular labeling. Biocytin was injected into cells in vivo, after first analyzing physiological response properties. Based on axonal morphology there were two classes of neuron: (1) laminar cells (14/32, 44%) with an intrinsic axon and flattened dendrites confined to a single fibrodendritic lamina and (2) translaminar cells (18/32, 56%) with axons that terminated in two or more laminae in the central nucleus (ICc) or the surrounding cortex. There was also one small, low-frequency cell with bushy-like dendrites that was very sensitive to interaural timing differences. The translaminar cells were subdivided into three groups of cells with: (a) stellate dendrites that crossed at least two laminae (8/32, 25%); (b) flattened dendrites confined to one lamina and that had mainly en passant axonal swellings (7/32, 22%) and (c) short, flattened dendrites and axons with distinctive clusters of large terminal boutons in the ICc (3/32, 9%). These terminal clusters were similar to those of cortical basket cells. The 14 laminar cells all had sustained responses apart from one offset response. Almost half the non-basket type translaminar cells (7/15) had onset responses while the others had sustained responses. The basket cells were the only ones to have short-latency (7-9 ms), chopper responses and this distinctive temporal response should allow them to be studied in more detail in future. This is the first description of basket cells in the auditory brainstem, but more work is required to confirm their neurotransmitter and precise post-synaptic targets.

Conditioned place preference for cocaine and methylphenidate in female mice from lines selectively bred for high voluntary wheel-running behavior.

Behavioral addictions can come in many forms, including overeating, gambling and overexercising. All addictions share a common mechanism involving activation of the natural reward circuit and reinforcement learning, but the extent to which motivation for natural and drug rewards share similar neurogenetic mechanisms remains unknown. A unique mouse genetic model in which four replicate lines of female mice were selectively bred (>76 generations) for high voluntary wheel running (High Runner or HR lines) alongside four non-selected control (C) lines were used to test the hypothesis that high motivation for exercise is associated with greater reward for cocaine (20 mg/kg) and methylphenidate (10 mg/kg) using the conditioned place preference (CPP) test. HR mice run ~three times as many revolutions/day as C mice, but the extent to which they have increased motivation for other rewards is unknown. Both HR and C mice displayed significant CPP for cocaine and methylphenidate, but with no statistical difference between linetypes for either drug. Taken together, results suggest that selective breeding for increased voluntary running has modified the reward circuit in the brain in a way that increases motivation for running without affecting cocaine or methylphenidate reward.

Effects of early-life exposure to Western diet and voluntary exercise on adult activity levels, exercise physiology, and associated traits in selectively bred High Runner mice.

Exercise behavior is under partial genetic control, but it is also affected by numerous environmental factors, potentially including early-life experiences whose effects persist into adulthood. We studied genetic and early-life environmental effects on wheel-running behavior in a mouse model that includes four replicate high runner (HR) lines selectively bred for increased voluntary wheel running as young adults and four non-selected control (C) lines. In a full factorial design, mice from each line were granted wheel access or not and administered either standard or Western diet (WD) from weaning (3 weeks old) to 6 weeks of age (sexual maturity). In addition to acute effects, after a washout period of 8 weeks (∼6 human years) in which all mice had standard diet and no wheel access, we found both beneficial and detrimental effects of these early-life exposures. During the first week of treatments, WD increased distance run by 29% in C mice and 48% in HR mice (significant Diet × Linetype interaction), but diet effects disappeared by the third week. Across the three weeks of juvenile treatment, WD significantly increased fat mass (with lean mass as a covariate). Tested as adults, early-life exercise increased wheel running of C mice but not HR mice in the first week. Early-life exercise also reduced adult anxiety-like behavior and increased adult fasted blood glucose levels, triceps surae mass, subdermal fat pad mass, and brain mass, but decreased heart ventricle mass. Using fat mass as a covariate, early-life exercise treatment increased adult leptin concentration. In contrast, early-life WD increased adult wheel running of HR mice but not C mice. Early-life WD also increased adult lean mass and adult preference for Western diet in all groups. Surprisingly, early-life treatment had no significant effect on adult body fat or maximal aerobic capacity (VO2max). No previous study has tested for combined or interactive effects of early-life WD and exercise. Our results demonstrate that both factors can have long-lasting effects on adult voluntary exercise and related phenotypes, and that these effects are modulated by genetic background. Overall, the long-lasting effects of early-life exercise were more pervasive than those of WD, suggesting critical opportunities for health intervention in childhood habits, as well as possible threats from modern challenges. These results may be relevant for understanding potential effects of activity reductions and dietary changes associated with the obesity epidemic and COVID-19 pandemic.

Hypothalamic POMC deficiency increases circulating adiponectin despite obesity.

OBJECTIVE: The steep rise in the prevalence of obesity and its related metabolic syndrome have become a major worldwide health concerns. Melanocortin peptides from hypothalamic arcuate nucleus (Arc) POMC neurons induce satiety to limit food intake. Consequently, Arc Pomc-deficient mice (ArcPomc-/-) exhibit hyperphagia and obesity. Previous studies demonstrated that the circulating levels of adiponectin, a protein abundantly produced and secreted by fat cells, negatively correlate with obesity in both rodents and humans. However, we found that ArcPomc-/- mice have increased circulating adiponectin levels despite obesity. Therefore, we investigated the physiological function and underlying mechanisms of hypothalamic POMC in regulating systemic adiponectin levels. METHODS: Circulating adiponectin was measured in obese ArcPomc-/- mice at ages 4-52 weeks. To determine whether increased adiponectin was a direct result of ArcPomc deficiency or a secondary effect of obesity, we examined plasma adiponectin levels in calorie-restricted mice with or without a history of obesity and in ArcPomc-/- mice before and after genetic restoration of Pomc expression in the hypothalamus. To delineate the mechanisms causing increased adiponectin in ArcPomc-/- mice, we determined sympathetic outflow to adipose tissue by assessing epinephrine, norepinephrine, and tyrosine hydroxylase protein levels and measured the circulating adiponectin in the mice after acute norepinephrine or propranolol treatments. In addition, adiponectin mRNA and protein levels were measured in discrete adipose tissue depots to ascertain which fat depots contributed the most to the high level of adiponectin in the ArcPomc-/- mice. Finally, we generated compound Adiopoq-/-:ArcPomc-/- mice and compared their growth, body composition, and glucose homeostasis to the individual knockout mouse strains and their wild-type controls. RESULTS: Obese ArcPomc-/- female mice had unexpectedly increased plasma adiponectin compared to wild-type siblings at all ages greater than 8 weeks. Despite chronic calorie restriction to achieve normal body weights, higher adiponectin levels persisted in the ArcPomc-/- female mice. Genetic restoration of Pomc expression in the Arc or acute treatment of the ArcPomc-/- female mice with melanotan II reduced adiponectin levels to control littermate values. The ArcPomc-/- mice had defective thermogenesis and decreased epinephrine, norepinephrine, and tyrosine hydroxylase protein levels in their fat pads, indicating reduced sympathetic outflow to adipose tissue. Injections of norepinephrine into the ArcPomc-/- female mice reduced circulating adiponectin levels, whereas injections of propranolol significantly increased adiponectin levels. Despite the beneficial effects of adiponectin on metabolism, the deletion of adiponectin alleles in the ArcPomc-/- mice did not exacerbate their metabolic abnormalities. CONCLUSION: In summary, to the best of our knowledge, this study provides the first evidence that despite obesity, the ArcPomc-/- mouse model has high circulating adiponectin levels, which demonstrated that increased fat mass is not necessarily correlated with hypoadiponectinemia. Our investigation also found a previously unknown physiological pathway connecting POMC neurons via the sympathetic nervous system to circulating adiponectin, thereby shedding light on the biological regulation of adiponectin.

Expression of a hypomorphic Pomc allele alters leptin dynamics during late pregnancy.

Proopiomelanocortin (POMC) neurons in the hypothalamic arcuate nucleus (ARC) are essential for normal energy homeostasis. Maximal ARC Pomc transcription is dependent on neuronal Pomc enhancer 1 (nPE1), located 12 kb upstream from the promoter. Selective deletion of nPE1 in mice decreases ARC Pomc expression by 70%, sufficient to induce mild obesity. Because nPE1 is located exclusively in the genomes of placental mammals, we questioned whether its hypomorphic mutation would also alter placental Pomc expression and the metabolic adaptations associated with pregnancy and lactation. We assessed placental development, pup growth, circulating leptin and expression of Pomc, Agrp and alternatively spliced leptin receptor (LepR) isoforms in the ARC and placenta of Pomc∆1/∆1 and Pomc+/+ dams. Despite indistinguishable body weights, lean mass, food intake, placental histology and Pomc expression and overall pregnancy outcomes between the genotypes, Pomc ∆1/∆1 females had increased pre-pregnancy fat mass that paradoxically decreased to control levels by parturition. However, Pomc∆1/∆1 dams had exaggerated increases in circulating leptin, up to twice of that of the typically elevated levels in Pomc+/+ mice at the end of pregnancy, despite their equivalent fat mass. Pomc∆1/∆1dams also had increased placental expression of soluble leptin receptor (LepRe), although the protein levels of LEPRE in circulation were the same as Pomc+/+ controls. Together, these data suggest that the hypomorphic Pomc∆1/∆1 allele is responsible for the perinatal super hyperleptinemia of Pomc∆1/∆1 dams, possibly due to upregulated leptin secretion from individual adipocytes.

Electrocardiograms of mice selectively bred for high levels of voluntary exercise: Effects of short-term exercise training and the mini-muscle phenotype.

Changes in cardiac function that occur with exercise training have been studied in detail, but those accompanying evolved increases in the duration or intensity of physical activity are poorly understood. To address this gap, we studied electrocardiograms (ECGs) of mice from an artificial selection experiment in which four replicate lines are bred for high voluntary wheel running (HR) while four non-selected lines are maintained as controls (C). ECGs were recorded using an ECGenie (Mouse Specifics, Inc.) both before and after six days of wheel access (as used in the standard protocol to select breeders). We hypothesized that HR mice would show innate differences in ECG characteristics and that the response to training would be greater in HR mice relative to C mice because the former run more. After wheel access, in statistical analyses controlling for variation in body mass, all mice had lower heart rates, and mice from HR lines had longer PR intervals than C lines. Also after wheel access, male mice had increased heart rate variability, whereas females had decreased heart rate variability. With body mass as a covariate, six days of wheel access significantly increased ventricle mass in both HR and C males. Within the HR lines, a subset of mice known as mini-muscle individuals have a 50% reduction in hindlimb muscle mass and generally larger internal organs, including the heart ventricles. As compared with normal-muscled individuals, mini-muscle individuals had a longer QRS complex, both before and after wheel access. Some studies in other species of mammals have shown correlations between athletic performance and QRS duration. Correlations between wheel running and either heart rate or QRS duration (before wheel running) among the eight individual lines of the HR selection experiment or among 17 inbred mouse strains taken from the literature were not statistically significant. However, total revolutions and average speed were negatively correlated with PR duration among lines of the HR selection experiment for males, and duration of running was negatively correlated with PR duration among 17 inbred strains for females. We conclude that HR mice have enhanced trainability of cardiac function as compared with C mice (as indicated by their longer PR duration after wheel access), and that the mini-muscle phenotype causes cardiac changes that have been associated with increased athletic performance in previous studies of mammals.

High-runner mice have reduced incentive salience for a sweet-taste reward when housed with wheel access.

To explore reward substitution in the context of voluntary exercise, female mice from four replicate high-runner (HR) lines (bred for wheel running) and four non-selected control (C) lines were given simultaneous access to wheels and palatable solutions as competing rewards (two doses of saccharin [0.1, 0.2% w/v]; two doses of common artificial sweetener blends containing saccharin [Sweet 'N Low®: 0.1, 0.2% w/v], aspartame [Equal®: 0.04, 0.08% w/v], or sucralose [Splenda®: 0.08, 0.16% w/v]; or two doses of sucrose [3.5, 10.5% w/v]). Wheel running and fluid consumption were measured daily, with each dose (including plain water) lasting two days and two "washout" days between solutions. In a separate set of mice, the experiment was repeated without wheel access. The artificial sweeteners had no statistical effect on wheel running. However, based on proportional responses, both doses of sucrose significantly elevated wheel running in C but not HR mice. In contrast, the high dose of sucrose suppressed home-cage activity for both linetypes. Both sucrose and the artificial blends generally increased fluid consumption in a dose-dependent manner. When they had access to wheels, HR had a significantly smaller increase in consumption of artificial sweetener blends when compared with C mice, but not when housed without wheels. Overall, these results provide further evidence that the reward system of HR mice has evolved, and specifically suggest that HR mice have a reduced incentive salience for some artificial sweetener blends, likely attributable to the stronger competing reward of wheel running that has evolved in these lines.

Circulating levels of endocannabinoids respond acutely to voluntary exercise, are altered in mice selectively bred for high voluntary wheel running, and differ between the sexes.

The endocannabinoid system serves many physiological roles, including in the regulation of energy balance, food reward, and voluntary locomotion. Signaling at the cannabinoid type 1 receptor has been specifically implicated in motivation for rodent voluntary exercise on wheels. We studied four replicate lines of high runner (HR) mice that have been selectively bred for 81 generations based on average number of wheel revolutions on days five and six of a six-day period of wheel access. Four additional replicate lines are bred without regard to wheel running, and serve as controls (C) for random genetic effects that may cause divergence among lines. On average, mice from HR lines voluntarily run on wheels three times more than C mice on a daily basis. We tested the general hypothesis that circulating levels of endocannabinoids (i.e., 2-arachidonoylglycerol [2-AG] and anandamide [AEA]) differ between HR and C mice in a sex-specific manner. Fifty male and 50 female mice were allowed access to wheels for six days, while another 50 males and 50 females were kept without access to wheels (half HR, half C for all groups). Blood was collected by cardiac puncture during the time of peak running on the sixth night of wheel access or no wheel access, and later analyzed for 2-AG and AEA content by ultra-performance liquid chromatography coupled to tandem mass spectrometry. We observed a significant three-way interaction among sex, linetype, and wheel access for 2-AG concentrations, with females generally having lower levels than males and wheel access lowering 2-AG levels in some but not all subgroups. The number of wheel revolutions in the minutes or hours immediately prior to sampling did not quantitatively predict plasma 2-AG levels within groups. We also observed a trend for a linetype-by-wheel access interaction for AEA levels, with wheel access lowering plasma concentrations of AEA in HR mice, while raising them in C mice. In addition, females tended to have higher AEA concentrations than males. For mice housed with wheels, the amount of running during the 30min before sampling was a significant positive predictor of plasma AEA within groups, and HR mice had significantly lower levels of AEA than C mice. Our results suggest that voluntary exercise alters circulating levels of endocannabinoids, and further demonstrate that selective breeding for voluntary exercise is associated with evolutionary changes in the endocannabinoid system.