Susan A. Walsh

Musclin is an activity-stimulated myokine that enhances physical endurance.

Significance Skeletal muscle is increasingly recognized as a secretory organ. Revealing the identity and function of myokines can improve our understanding of skeletal muscle function under sedentary or exercise conditions, as well as its coordination with other organs, tissues, and overall body metabolism. This study identifies musclin as an exercise-responsive myokine critical for skeletal muscle adaptation to physical activity. We develop a musclin-encoding gene (Ostn) knockout mouse, which allows us to determine a previously unrecognized physiologic function of musclin in regulation of skeletal muscle mitochondrial biogenesis and physical endurance. The demonstrated molecular mechanism for musclin-dependent skeletal muscle adaptation to exercise also transforms the perspective on natriuretic peptide signaling, particularly as it relates to physical activity and exercise-induced remodeling in different tissues. Exercise remains the most effective way to promote physical and metabolic wellbeing, but molecular mechanisms underlying exercise tolerance and its plasticity are only partially understood. In this study we identify musclin—a peptide with high homology to natriuretic peptides (NP)—as an exercise-responsive myokine that acts to enhance exercise capacity in mice. We use human primary myoblast culture and in vivo murine models to establish that the activity-related production of musclin is driven by Ca2+-dependent activation of Akt1 and the release of musclin-encoding gene (Ostn) transcription from forkhead box O1 transcription factor inhibition. Disruption of Ostn and elimination of musclin secretion in mice results in reduced exercise tolerance that can be rescued by treatment with recombinant musclin. Reduced exercise capacity in mice with disrupted musclin signaling is associated with a trend toward lower levels of plasma atrial NP (ANP) and significantly smaller levels of cyclic guanosine monophosphate (cGMP) and peroxisome proliferator-activated receptor gamma coactivator 1-α in skeletal muscles after exposure to exercise. Furthermore, in agreement with the established musclin ability to interact with NP clearance receptors, but not with NP guanyl cyclase-coupled signaling receptors, we demonstrate that musclin enhances cGMP production in cultured myoblasts only when applied together with ANP. Elimination of the activity-related musclin-dependent boost of ANP/cGMP signaling results in significantly lower maximum aerobic capacity, mitochondrial protein content, respiratory complex protein expression, and succinate dehydrogenase activity in skeletal muscles. Together, these data indicate that musclin enhances physical endurance by promoting mitochondrial biogenesis.

Regulation of Glucose Tolerance and Sympathetic Activity by MC4R Signaling in the Lateral Hypothalamus

Melanocortin 4 receptor (MC4R) signaling mediates diverse physiological functions, including energy balance, glucose homeostasis, and autonomic activity. Although the lateral hypothalamic area (LHA) is known to express MC4Rs and to receive input from leptin-responsive arcuate proopiomelanocortin neurons, the physiological functions of MC4Rs in the LHA are incompletely understood. We report that MC4RLHA signaling regulates glucose tolerance and sympathetic nerve activity. Restoring expression of MC4Rs specifically in the LHA improves glucose intolerance in obese MC4R-null mice without affecting body weight or circulating insulin levels. Fluorodeoxyglucose-mediated tracing of whole-body glucose uptake identifies the interscapular brown adipose tissue (iBAT) as a primary source where glucose uptake is increased in MC4RLHA mice. Direct multifiber sympathetic nerve recording further reveals that sympathetic traffic to iBAT is significantly increased in MC4RLHA mice, which accompanies a significant elevation of Glut4 expression in iBAT. Finally, bilateral iBAT denervation prevents the glucoregulatory effect of MC4RLHA signaling. These results identify a novel role for MC4RLHA signaling in the control of sympathetic nerve activity and glucose tolerance independent of energy balance.

FGF21 Regulates Metabolism Through Adipose-Dependent and -Independent Mechanisms

FGF21 is an endocrine hormone that regulates energy homeostasis and insulin sensitivity. The mechanism of FGF21 action and the tissues responsible for these effects have been controversial, with both adipose tissues and the central nervous system having been identified as the target site mediating FGF21-dependent increases in insulin sensitivity, energy expenditure, and weight loss. Here we show that, while FGF21 signaling to adipose tissue is required for the acute insulin-sensitizing effects of FGF21, FGF21 signaling to adipose tissue is not required for its chronic effects to increase energy expenditure and lower body weight. Also, in contrast to previous studies, we found that adiponectin is dispensable for the metabolic effects of FGF21 in increasing insulin sensitivity and energy expenditure. Instead, FGF21 acutely enhances insulin sensitivity through actions on brown adipose tissue. Our data reveal that the acute and chronic effects of FGF21 can be dissociated through adipose-dependent and -independent mechanisms.

Effect of Insulin and Dexamethasone on Fetal Assimilation of Maternal Glucose

The growing fetus depends upon transfer of glucose from maternal blood to fetal tissues. Insulin and glucocorticoid impact maternal glucose metabolism, but the effects of these hormones on fetal glucose assimilation in vivo are understudied. We thus used positron emission tomography imaging to determine the disposition of [(18)F]fluorodeoxyglucose (FDG) in rats on gestational d 20, quantifying the kinetic competition of maternal tissues and fetus for glucose. Three fasting maternal states were studied: after 2-d dexamethasone (DEX), during euglycemic hyperinsulinemic clamp insulin receiving (INS), and control (CON). In CON and DEX mothers, FDG accumulation in fetuses and placentae was substantial, rivaling that of maternal brain. By contrast, FDG accumulation was reduced in INS fetuses, placentae, and maternal brain by approximately 2-fold, despite no diminution in FDG extraction kinetics from maternal blood into these structures. The reduced FDG accumulation was due to more rapid clearance of FDG from the circulation in INS mothers, related to increased FDG avidity in INS select maternal tissues, including skeletal muscle, brown adipose tissue, and heart. DEX treatment of mothers reduced fetal weight by nearly 10%. Nonetheless, the accumulation of FDG into placentae and fetuses was similar in DEX and CON mothers. In our rat model, fetal growth restriction induced by DEX does not involve diminished glucose transport to the fetus. Maternal insulin action has little effect on the inherent avidity of the fetal-placental unit for glucose but increases glucose utilization by maternal tissues, thus indirectly reducing the glucose available to the fetus.

Hyperactive hypothalamus, motivated and non‐distractible chronic overeating in ADAR2 transgenic mice

ADAR2 transgenic mice misexpressing the RNA editing enzyme ADAR2 (Adenosine Deaminase that act on RNA) show characteristics of overeating and experience adult onset obesity. Behavioral patterns and brain changes related to a possible addictive overeating in these transgenic mice were explored as transgenic mice display chronic hyperphagia. ADAR2 transgenic mice were assessed in their food preference and motivation to overeat in a competing reward environment with ad lib access to a running wheel and food. Metabolic activity of brain and peripheral tissue were assessed with [(18) F] fluorodeoxyglucose positron emission tomography (FDG-PET) and RNA expression of feeding related genes, ADAR2, dopamine and opiate receptors from the hypothalamus and striatum were examined. The results indicate that ADAR2 transgenic mice exhibit, (1) a food preference for diets with higher fat content, (2) significantly increased food intake that is non-distractible in a competing reward environment, (3) significantly increased messenger RNA (mRNA) expressions of ADAR2, serotonin 2C receptor (5HT2C R), D1, D2 and mu opioid receptors and no change in corticotropin-releasing hormone mRNAs and significantly reduced ADAR2 protein expression in the hypothalamus, (4) significantly increased D1 receptor and altered bioamines with no change in ADAR2, mu opioid and D2 receptor mRNA expression in the striatum and (5) significantly greater glucose metabolism in the hypothalamus, brain stem, right hippocampus, left and right mid brain regions and suprascapular peripheral tissue than controls. These results suggest that highly motivated and goal-oriented overeating behaviors of ADAR2 transgenic mice are associated with altered feeding, reward-related mRNAs and hyperactive brain mesolimbic region.

Localized Fetomaternal Hyperglycemia: Spatial and Kinetic Definition by Positron Emission Tomography

Background Complex but common maternal diseases such as diabetes and obesity contribute to adverse fetal outcomes. Understanding of the mechanisms involved is hampered by difficulty in isolating individual elements of complex maternal states in vivo. We approached this problem in the context of maternal diabetes and sought an approach to expose the developing fetus in vivo to isolated hyperglycemia in the pregnant rat. Methodology and Principal Findings We hypothesized that glucose infused into the arterial supply of one uterine horn would more highly expose fetuses in the ipsilateral versus contralateral uterine horn. To test this, the glucose tracer [18F]fluorodeoxyglucose (FDG) was infused via the left uterine artery. Regional glucose uptake into maternal tissues and fetuses was quantified using positron emission tomography (PET). Upon infusion, FDG accumulation began in the left-sided placentae, subsequently spreading to the fetuses. Over two hours after completion of the infusion, FDG accumulation was significantly greater in left compared to right uterine horn fetuses, favoring the left by 1.9±0.1 and 2.8±0.3 fold under fasted and hyperinsulinemic conditions (p<10−11 nu200a=u200a32-35 and p<10−12 nu200a=u200a27–45) respectively. By contrast, centrally administered [3H]-2-deoxyglucose accumulated equally between the fetuses of the two uterine horns. Induction of significant hyperglycemia (103 mg/dL) localized to the left uterine artery was sustained for at least 48 hours while maternal euglycemia was maintained. Conclusions and Significance This approach exposes selected fetuses to localized hyperglycemia in vivo, minimizing exposure of the mother and thus secondary effects. Additionally, a set of less exposed internal control fetuses are maintained for comparison, allowing direct study of the in vivo fetal effects of isolated hyperglycemia. Broadly, this approach can be extended to study a variety of maternal-sided perturbations suspected to directly affect fetal health.

“Click”-Cyclized 68 Ga-Labeled Peptides for Molecular Imaging and Therapy: Synthesis and Preliminary In Vitro and In Vivo Evaluation in a Melanoma Model System

Cyclization techniques are used often to impart higher inxa0vivo stability and binding affinity to peptide targeting vectors for molecular imaging and therapy. The two most often used techniques to impart these qualities are lactam bridge construction and disulfide bond formation. While these techniques have been demonstrated to be effective, orthogonal protection/deprotection steps can limit achievable product yields. In the work described in this chapter, new α-melanocyte stimulating hormone (α-MSH) peptide analogs were synthesized and cyclized by copper-catalyzed terminal azide-alkyne cycloaddition click chemistry techniques. The α-MSH peptide and its cognate receptor (melanocortin receptor subtypexa01, MC1R) represent a well-characterized model system to examine the effect of the triazole linkage for peptide cyclization on receptor binding inxa0vitro and inxa0vivo. Four new DOTA-conjugated α-MSH analogs were cyclized and evaluated by inxa0vitro competitive binding assays, serum stability testing, and inxa0vivo imaging by positron emission tomography (PET) of tumor-bearing mice. These new DOTA-conjugated click-cyclized analogs exhibited selective high binding affinity (<2xa0nM) for MC1R on melanoma cells inxa0vitro, high stability in human serum, and produced high-contrast PET/CT images of tumor xenografts. (68)Ga-labeled DOTA bioconjugates displayed rapid pharmacokinetics with receptor-mediated tumor accumulation of up to 16xa0±xa05%xa0ID/g. The results indicate that the triazole ring is an effective bioisosteric replacement for the standard lactam bridge assemblage for peptide cyclization. Radiolabeling results confirm that Cu catalyst is sufficiently removed prior to DOTA chelator addition to enable insertion of radio metals or stable metals for molecular imaging and therapy. Thus, these click-chemistry-cyclized variants show promise as agents for melanocortin receptor-targeted imaging and radionuclide therapy.

Selective Deletion of Renin-b in the Brain Alters Drinking and MetabolismNovelty and Significance

The brain-specific isoform of renin (Ren-b) has been proposed as a negative regulator of the brain renin–angiotensin system (RAS). We analyzed mice with a selective deletion of Ren-b which preserved expression of the classical renin (Ren-a) isoform. We reported that Ren-bNull mice exhibited central RAS activation and hypertension through increased expression of Ren-a, but the dipsogenic and metabolic effects in Ren-bNull mice are unknown. Fluid intake was similar in control and Ren-bNull mice at baseline and both exhibited an equivalent dipsogenic response to deoxycorticosterone acetate–salt. Dehydration promoted increased water intake in Ren-bNull mice, particularly after deoxycorticosterone acetate–salt. Ren-bNull and control mice exhibited similar body weight when fed a chow diet. However, when fed a high-fat diet, male Ren-bNull mice gained significantly less weight than control mice, an effect blunted in females. This difference was not because of changes in food intake, energy absorption, or physical activity. Ren-bNull mice exhibited increased resting metabolic rate concomitant with increased uncoupled protein 1 expression and sympathetic nerve activity to the interscapular brown adipose tissue, suggesting increased thermogenesis. Ren-bNull mice were modestly intolerant to glucose and had normal insulin sensitivity. Another mouse model with markedly enhanced brain RAS activity (sRA mice) exhibited pronounced insulin sensitivity concomitant with increased brown adipose tissue glucose uptake. Altogether, these data support the hypothesis that the brain RAS regulates energy homeostasis by controlling resting metabolic rate, and that Ren-b deficiency increases brain RAS activity. Thus, the relative level of expression of Ren-b and Ren-a may control activity of the brain RAS.

PET/CT imaging reveals unrivaled placental avidity for glucose compared to other tissues.

INTRODUCTIONnThe goal of this study was to define the kinetics of glucose transport from maternal blood to placenta to fetus using real time imaging.nnnMETHODSnPositron emission tomography (PET) imaging of the glucose-tracer [(18)F]fluorodeoxyglucose (FDG) was used to temporally and spatially define, in vivo, the kinetics of glucose transport from maternal blood into placentae and fetuses, in the late gestational gravid rat. Computed tomography (CT), with intravenous contrast, co-registered to the PET images allowed anatomic differentiation of placentae from fetal and maternal tissues.nnnRESULTSnFDG was rapidly taken up by placentae and subsequently appeared in fetuses with minimal temporal lag. FDG standardized uptake values in placentae and fetuses approached that of maternal brain. In both anesthetized and awake dams, one quarter of the administered FDG ultimately was accrued in the collective fetuses and placentae. Accordingly, kinetic modeling demonstrated that the placentae had very high avidity for FDG, 2-fold greater than that of the fetus and maternal brain, when accounting for the fact that fetal FDG necessarily must first be taken up by placentae. Consistent with this, placental expression of glucose transporter 1 exceeded that of all other tissues.nnnDISCUSSIONnFetal and placental tissues place a substantial glucose metabolic burden on the mother, owing to very high avidity of placentae for glucose coupled with the large relative mass of fetal and placental tissues.nnnCONCLUSIONSnThe placenta has a tremendous capacity to uptake and transport glucose. PET/CT imaging is an ideal means to study metabolite transport kinetics in the fetoplacental unit.