Myoglobin‐mediated lipid shuttling increases adrenergic activation of brown and white adipocyte metabolism and is as a marker of thermogenic adipocytes in humans

Background: Recruitment and activation of brown adipose tissue (BAT) results in increased energy expenditure (EE) via thermogenesis and represents an intriguing therapeutic approach to combat obesity and treat associated diseases. Thermogenesis requires an increased and efficient supply of energy substrates and oxygen to the BAT. The hemoprotein myoglobin (MB) is primarily expressed in heart and skeletal muscle fibres, where it facilitates oxygen storage and flux to the mitochondria during exercise. In the last years, further contributions of MB have been assigned to the scavenging of reactive oxygen species (ROS), the regulation of cellular nitric oxide (NO) levels and also lipid binding. There is a substantial expression of MB in BAT, which is induced during brown adipocyte differentiation and BAT activation. This suggests MB as a previously unrecognized player in BAT contributing to thermogenesis. Methods and results: This study analyzed the consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo. Using MB overexpressing, knockdown or knockout adipocytes, we show that expression levels of MB control brown adipocyte mitochondrial respiratory capacity and acute response to adrenergic stimulation, signalling and lipolysis. Overexpression in white adipocytes also increases their metabolic activity. Mutation of lipid interacting residues in MB abolished these beneficial effects of MB. In vivo, whole-body MB knockout resulted in impaired thermoregulation and cold- as well as drug-induced BAT activation in mice. In humans, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) adipose tissue (AT) depots, differentially regulated by the state of obesity and higher expressed in AT samples that exhibit higher thermogenic potential. Conclusions: These data demonstrate for the first time a functional relevance of MBs lipid binding properties and establish MB as an important regulatory element of thermogenic capacity in brown and likely beige adipocytes. Keywords: energy expenditure; hemoprotein; metabolism; obesity; oxphos; uncoupling protein

Crosstalk between nitrite, myoglobin and reactive oxygen species to regulate vasodilation under hypoxia.

The systemic response to decreasing oxygen levels is hypoxic vasodilation. While this mechanism has been known for more than a century, the underlying cellular events have remained incompletely understood. Nitrite signaling is critically involved in vessel relaxation under hypoxia. This can be attributed to the presence of myoglobin in the vessel wall together with other potential nitrite reductases, which generate nitric oxide, one of the most potent vasodilatory signaling molecules. Questions remain relating to the precise concentration of nitrite and the exact dose-response relations between nitrite and myoglobin under hypoxia. It is furthermore unclear whether regulatory mechanisms exist which balance this interaction. Nitrite tissue levels were similar across all species investigated. We then investigated the exact fractional myoglobin desaturation in an ex vivo approach when gassing with 1% oxygen. Within a short time frame myoglobin desaturated to 58±12%. Given that myoglobin significantly contributes to nitrite reduction under hypoxia, dose-response experiments using physiological to pharmacological nitrite concentrations were conducted. Along all concentrations, abrogation of myoglobin in mice impaired vasodilation. As reactive oxygen species may counteract the vasodilatory response, we used superoxide dismutase and its mimic tempol as well as catalase and ebselen to reduce the levels of reactive oxygen species during hypoxic vasodilation. Incubation of tempol in conjunction with catalase alone and catalase/ebselen increased the vasodilatory response to nitrite. Our study shows that modest hypoxia leads to a significant nitrite-dependent vessel relaxation. This requires the presence of vascular myoglobin for both physiological and pharmacological nitrite levels. Reactive oxygen species, in turn, modulate this vasodilation response

Nitric Oxide Generating Formulation as an Innovative Approach to Topical Skin Care: An Open-Label Pilot Study

Nitric oxide (NO) plays multiple roles in both normal and abnormal skin processes. Its deranging disbalance is involved in the pathogenesis of multiple dermatologic diseases such as acne vulgaris, pointing towards beneficial therapeutic directions. A novel NO-producing gel-formulation was tested beneficial in the treatment of acne vulgaris in an open-label pilot study using clinical evaluation scores. It showed a decrease of comedones and inflammatory pustulae and reduced the Global Acne Grading System score by 50% within eight weeks. In addition, we demonstrate a potential use as cosmetic agent where NO therapy leads to an increase of skin integrity and a reduction of skin ageing processes

A practical approach to remote ischemic preconditioning and ischemic preconditioning against myocardial ischemia/reperfusion injury

Although urgently needed in clinical practice, a cardioprotective therapeutic approach against myocardial ischemia/reperfusion injury remains to be established. Remote ischemic preconditioning (rIPC) and ischemic preconditioning (IPC) represent promising tools comprising three entities: the generation of a protective signal, the transfer of the signal to the target organ, and the response to the transferred signal resulting in cardioprotection. However, in light of recent scientific advances, many controversies arise regarding the efficacy of the underlying signaling. We here show methods for the generation of the signaling cascade by rIPC as well as IPC in a mouse model for in vivo myocardial ischemia/reperfusion injury using highly reproducible approaches. This is accomplished by taking advantage of easily applicable preconditioning strategies compatible with the clinical setting. We describe methods for using laser Doppler perfusion imaging to monitor the cessation and recovery of perfusion in real time. The effects of preconditioning on cardiac function can also be assessed using ultrasound or magnetic resonance imaging approaches. On a cellular level, we confirm how tissue injury can be monitored using histological assessment of infarct size in conjunction with immunohistochemistry to assess both aspects in a single specimen. Finally, we outline, how the rIPC-associated signaling can be transferred to the target cell via conservation of the signal in the humoral (blood) compartment. This compilation of experimental protocols including a conditioning regimen comparable to the clinical setting should proof useful to both beginners and experts in the field of myocardial infarction, supplying information for the detailed procedures as well as troubleshooting guides

Dose-dependency for nitrite-induced hypoxic vasodilation in the presence and absence of myoglobin (Mb).

<p>(A) Experimental schema. After equilibration, normoxic gassing was either continued or changed to hypoxia (1% O₂). Isolated aortic rings of <i>Mb^+/+</i> and <i>Mb^−/−</i> mice were then pre-constricted using phenylephrine (Phe) and subsequently challenged with cumulating doses of nitrite from physiological to pharmacological levels. Under normoxia, nitrite-vasodilation response were identical in both mouse types (B) leading to similar EC₅₀ levels (C). On the contrary, under hypoxia, nitrite-induced vasodilation was significantly impaired in <i>Mb^−/−</i> (D) with significantly higher resulting EC₅₀ levels (E). All values are means±s.e.m.</p

Role of nitrite and reactive oxygen species (ROS) in hypoxic vasodilation – proposed mechanism.

<p>Nitrite derives from NO^• synthesis dietary sources. NO^• to nitrite reactions occur by autoxidation or by reaction with ceruloplasmin (CP). Under hypoxia, nitrite levels in the vessel wall are increased. Nitrite can then be reduced to vasodilatory NO^• particularly by reaction with myoglobin (Mb). ROS modulate this response.</p

Endogenous ROS modulate the nitrite-induced hypoxic vasodilation response.

<p>(A) Experimental schema. After an equilibration period, SOD, SOD mimic tempol, catalase and gluthation peroxidase mimic ebselen were added to the organ bath in order to decompose endogenously formed ROS. The nitrite concentration in the organ bath was 300 nM. Vessels were then preconstricted with phenylephrine (Phe). After stabilization of constriction, hypoxic was induced and vasodilation observed for the following 15 min. (B) Graph shows the decrease in intention for the 15 min of hypoxia for controls and treated rings. Incubation of SOD mimic tempol with catalase and with catalase/ebselen significantly increased the vasodilation response at 10 and 15 min (*<i>P</i><0.05, n = 3–5). Values are means±s.e.m.</p