Obesity is a chronic disease caused by an imbalance of energy intake and expenditure resulting in excessive accumulation of adipose tissue (AT) either in major adipose depots like subcutaneous (SAT) or visceral (VAT), or ectopic lipid deposition in other organs and tissues such as liver or muscle. In 2019 obesity was ranked globally under the top five death causes and increases the risk for suffering from non-communicable diseases such as stroke, diabetes and various types of cancer. Current therapeutic strategies implement dietary interventions and increased physical activity, application of incretin-based drugs such as dual or triple agonists, or bariatric surgery.
The recruitment and activation of brown adipose tissue (BAT) represents an intriguing therapeutic approach to combat obesity by increasing energy expenditure via thermogenesis. BAT is a highly metabolically active organ and its activity is induced by cold. To maintain body temperature, BAT is specialized in the dissipation of energy to produce heat by a high demand of oxygen and substrates such as lipids and glucose.
Myoglobin (MB) expression was detected in BAT of cold-exposed rodents and is increased during brown adipocyte differentiation suggesting an unrecognized physiological role in BAT contributing to thermogenesis. Since BAT and muscle are both highly metabolic active organs and are derived from the same myogenic factor 5 positive progenitor, it is likely, that BAT MB might exert similar functions as in muscle tissue. In addition to facilitating oxygen supply, further contributions of MB have been assigned to scavenging ROS and regulating cellular NO levels. Furthermore, a role of MB as a lipid shuttle was proposed, as MB seems to enable energy production via beta-oxidation and prevent myocardial lipid accumulation.
This project addressed the hypothesis that MB expression is upregulated in active (brown) AT to support thermogenesis by serving as lipid shuttle from the cytosol into the mitochondria, by contributing and sustaining oxygen supply and/ or by acting as ROS scavenger during thermogenesis. To investigate consequences of MB expression in BAT on mitochondrial function and thermogenesis in vitro and in vivo, MB overexpressing, knockdown or knockout adipocytes and global myoglobin-knockout (Mb-KO) mice were used. Initially, temperature- and differentiation-dependent changes in MB gene and protein expression were investigated in vivo and in vitro. MB expression was upregulated in BAT of cold-exposed C57BL/6N mice and during adipogenesis of brown adipocytes as confirmed in previous findings. Furthermore, BAT Mb gene expression correlated positively with Ucp1 suggesting MB in vivo being regulated by -adrenergic signaling. Surprisingly, in vitro Mb expression was inversely correlated to Ucp1 expression in immortalized and primary brown adipocytes after -adrenergic stimulation with norepinephrine or CL316,243. Since MB expression is increased during adipogenesis, the regulation by PPARG was investigated in immortalized brown adipocytes. Neither the stimulation by PPARG agonists such rosiglitazone or fatty acids nor cell-autonomous effects induced by hypothermia changed Mb gene expression concluding that other pathways regulate MB expression.
Evaluating various MB expression levels (high, low, none) on mitochondrial respiration and responsiveness to adrenergic stimulation, Mb knockdown, knockout and overexpression experiments in immortalized and primary brown adipocytes were performed. Herein, a MB expression level dependent increase in maximal mitochondrial respiratory capacity and acute response to adrenergic stimulation, signaling and lipolysis was observed. Also in white adipocytes, metabolic activity was improved by MB overexpression.
Since -adrenergic stimulation is accompanied with enhanced ROS production and MB acts as ROS scavenger in cardio myocytes, effects of MB expression on ROS and superoxide levels were determined. However, no impacts were detected, although cold-induced genes were found related to ROS in BAT of Mb-KO mice in vivo, thus a function as ROS scavenger in BAT cannot be excluded.
MB binds fatty acids and acylcarnitines, therefore proposed lipid binding residues of MB were mutated. At first, the ability of MB’s lipid binding property was evaluated by dot blot lipid overlay assays. As a result, palmitic and oleic acid were bound by oxygen-carrying MB. The mutant instead showed a reduced binding capacity. In functional assays, the non-lipid binding property abolished the beneficial effects in substrate flux, mitochondrial respiration and thermogenesis of MB in immortalized brown adipocytes. At the end, this data clearly demonstrated that MB’s lipid binding is essential to augment substrate flux and permit increased mitochondrial respiration and thermogenesis.
To investigate consequences of MB-deficiency on thermogenesis in vivo, whole-body Mb-KO mice models were exposed to thermoneutrality (30 °C), room temperature (23 °C, mild cold stress) and cold (8 °C) for seven days. Lack of MB resulted in impaired thermoregulation at temperatures below thermoneutrality and diminished the response to pharmacological BAT activation after intraperitoneal CL316,243 application.
To address the translational potential, MB is differentially expressed in subcutaneous (SC) and visceral (VIS) depots of human adipose tissue (AT). In lean patients MB was significantly lower expressed in SC AT compared to VIS AT, whereas in obese patients the opposite was overserved. Further analyses revealed that MB expression was more pronounced in AT samples with higher thermogenic potential.
As a conclusion, the present study demonstrates for the first time a functional relevance of MB’s lipid binding property and suggests MB as a previously unrecognized player in BAT biology that increases mitochondrial respiratory capacity, a crucial aspect for rapid adaptation to metabolic changes. The exact mechanisms how MB contributes to lipid transport, remain to be elucidated.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:90365 |
| Date | 07 March 2024 |
| Creators | Christen, Lisa |
| Contributors | Universität Leipzig |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | 10.1002/ctm2.1108 |
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