Evolution and functional characterisation of uncoupling proteins in vertebrates

The evolution of UCP1 in vertebrates – a summary This thesis unravels the evolution of UCP1, a crucial protein for heat generation recruited during adaptive NST. In eutherian mammals UCP1 uncouples the respiratory chain from ATP synthesis to dissipate proton motive force as heat but the question of when the protein emerged during evolution and what its original function was, has not been resolved. In the initial studies we show that standard molecular techniques are insufficient to detect UCP1 (Jastroch et al. 2004a, Chapter I) but the phylogeny of UCPs suggests an ancient origin of UCP1 (Jastroch et al. 2004b, Chapter II). The presence of UCP1 in fish demonstrates the emergence of UCP1 before the divergence of ray-finned and lobe-finned vertebrate lineages 420 MYA (Jastroch et al. 2005, Chapter III). UCP1 gene regulation in the common carp, Cyprinus carpio, indicates a physiological role other than heat production but biochemical studies suggests that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as eutherian UCP1 (Jastroch et al. 2007, Chapter IV). Studies in marsupials, which separated from eutherians 150 MYA, demonstrate that in these species transcriptional control targets UCP1 expression to adipose tissue (Jastroch et al. in preparation, Chapter V). In the gray short-tailed opossum, Monodelphis domestica, UCP1 is transiently expressed and restricted to the early development, similar to observations in larger eutherian neonates. In the fat-tailed dunnart, Sminthopsis crassicaudata, UCP1 is expressed during adulthood and is elevated in response to cold exposure. Although these experiments suggest the presence of UCP1-mediated NST, UCP1-dependent thermogenesis in the animal has yet to be shown in marsupials. However, the identification of a BAT-like tissue provides the molecular basis to reinvestigate adaptive NST in marsupials. In the phylogenetically ancient afrotherian rock elephant shrew, Elephantulus myurus, a species that diverged from modern eutherians about 100 MYA, we demonstrate the presence of functional BAT (Mzilikazi, Jastroch, Meyer, and Klingenspor, submitted, Chapter VI). Although NST, BAT and UCP1 are found in E. myurus, NST does not appear to be adaptive as demonstrated in modern eutherians. While adaptivity of NST seems not to be required in our experiments, the significance of NST during the seasons of the mild natural habitats of South Africa remains to be investigated in further studies. The focus of the thesis is the evolution of UCP1 but I also investigated if UCP3 might have a thermogenic role in the yellow-footed Antechinus, Antechinus flavipes, a marsupial lacking BAT (Jastroch et al., in preparation, Chapter VII). A thermogenic function of UCP3, as found for UCP1, can be excluded by measurements of uncoupling activity in myotubular mitochondria. However, I found evidence that mild uncoupling mediated by the ANT (adenine nucleotide translocase) occurs in myotubular mitochondria of cold-acclimated A. flavipes and may play a role in protection from oxidative stress during cold exposure. The search for UCPs in vertebrates has resulted in the identification of UCP2 and UCP3 in different vertebrate groups and might assist to resolve their physiological roles. By comparing phylogenetic branch lengths and gene regulation, I suggest that in contrast to UCP1, the function of UCP2 and UCP3 may be well conserved in all vertebrates. Finally, in order to compare different UCP orthologues and paralogues, we have established cell lines ectopically expressing mouse UCP1. In isolated mitochondria of this cell system, we demonstrate native function of mouse UCP1 (Jastroch et al. 2007, BBA, Chapter VIII and Results and Discussion). This cell system will serve in future studies to compare different UCPs in the presence of an identical mitochondrial and genetical background

Direct Substrate Delivery into Mitochondrial-Fission Deficient Pancreatic Islets Rescues Insulin Secretion

In pancreatic beta cells, mitochondrial bioenergetics control glucose-stimulated insulin secretion (GSIS). Mitochondrial dynamics are generally associated with quality control, maintaining the functionality of bioenergetics. By acute pharmacological inhibition of mitochondrial fission protein Drp1, we here demonstrate that mitochondrial fission is necessary for GSIS in mouse and human islets. We confirm that genetic silencing of Drp1 increases mitochondrial proton leak in MIN6 cells. However, our comprehensive analysis of pancreatic islet bioenergetics reveals that Drp1 does not control insulin secretion via its effect on proton leak but instead via modulation of glucose-fuelled respiration. Notably, pyruvate fully rescues the impaired insulin secretion of fission-deficient beta cells, demonstrating that defective mitochondrial dynamics solely impact substrate supply upstream of oxidative phosphorylation. The present findings provide novel insights in how mitochondrial dysfunction may cause pancreatic beta cell failure. In addition, the results will stimulate new thinking in the intersecting fields of mitochondrial dynamics and bioenergetics, as treatment of defective dynamics in mitochondrial diseases appears to be possible by improving metabolism upstream of mitochondria

12th International Hibernation Symposium

Nonshivering thermogenesis is dependent on the presence of UCP1 in brown adipose tissue. The function of UCP2 and UCP3 is most likely related to mitochondrial superoxide metabolism and/or fatty acid oxidation. These three members of the core UCP family are known in eutherians but had not been found in marsupials and monotremes so far. The objective of our search is to determine the origin of UCP1 and classical nonshivering thermogenesis. Furthermore, our approach to characterize UCP2/UCP3 in distantly related animal species will assist in the functional annotation of these proteins. We recently reported on the molecular identifi cation, tissue-distribution, and physiological regulation of UCP2 and UCP3 mRNA in the marsupial Antechinus fl avipes (yellow-footed Antechinus). Despite separate evolution of the marsupial lineage since 130 million years, our data suggest a conserved physiological role of these UCPs. Here, we present the immunological detection of marsupial UCP3 in skeletal muscle using antibodies raised against mouse/rat UCP3. A comprehensive phylogenetic analysis led us to hypothesize that all uncoupling proteins were already present at the evolutionary stage of modern teleost fi shes and questions the unique presence of UCP1 in placental mammals. However, the search for UCP1 in nonplacental mammals has been unsuccessful so far and is most likely hampered by the more rapid evolution of UCP1 as compared to other UCPs

Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis

<p>Abstract</p> <p>Background</p> <p>Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.</p> <p>Results</p> <p>Models of adaptive evolution through phylogenetic analysis of amino acid sequences by maximum likelihood were implemented to determine the mode of UCP1 protein evolution in Eutherians. An increase in the rate of amino acid substitutions on the branch leading to Eutherians is observed, but is best explained by relaxed constraints, not positive selection. Further, evidence for branch and site heterogeneity in selection pressures, as well as divergent selection pressures between UCP1 and its paralogs (UCP2-3) is observed.</p> <p>Conclusion</p> <p>We propose that the unique thermogenic function of UCP1 in Eutherians may be best explained by neutral processes. Along with other evidence, this suggests that the primary biochemical properties of UCP1 may not differ between Eutherians and non-Eutherians.</p

Molecular evolution of UCP1 and the evolutionary history of mammalian non-shivering thermogenesis

<p>Abstract</p> <p>Background</p> <p>Uncoupling protein 1 (UCP1) is a mitochondrial anion carrier, expressed in brown adipose tissue (BAT) of Eutherians. UCP1 is responsible for uncoupling mitochondrial proton transport from the production of ATP, thereby dissipating heat; it is essential for non-shivering thermogenesis (NST) in mammalian BAT. UCP1 orthologs have been identified in non-Eutherian mammals, fish and amphibians. Yet, UCP1 has a unique function in Eutherians in that it is necessary in the production of heat (NST). As such, this study aims to determine the evolutionary mode of UCP1 in Eutherians, where there is clear evidence of UCP1-dependent NST in BAT.</p> <p>Results</p> <p>Models of adaptive evolution through phylogenetic analysis of amino acid sequences by maximum likelihood were implemented to determine the mode of UCP1 protein evolution in Eutherians. An increase in the rate of amino acid substitutions on the branch leading to Eutherians is observed, but is best explained by relaxed constraints, not positive selection. Further, evidence for branch and site heterogeneity in selection pressures, as well as divergent selection pressures between UCP1 and its paralogs (UCP2-3) is observed.</p> <p>Conclusion</p> <p>We propose that the unique thermogenic function of UCP1 in Eutherians may be best explained by neutral processes. Along with other evidence, this suggests that the primary biochemical properties of UCP1 may not differ between Eutherians and non-Eutherians.</p

Adverse bioenergetic effects of N-acyl amino acids in human adipocytes overshadow beneficial mitochondrial uncoupling

Objective: Enhancing energy turnover via uncoupled mitochondrial respiration in adipose tissue has great potential to improve human obesity and other metabolic complications. However, the amount of human brown adipose tissue and its uncoupling protein 1 (UCP1) is low in obese patients. Recently, a class of endogenous molecules, N-acyl amino acids (NAAs), was identified as mitochondrial uncouplers in murine adipocytes, presumably acting via the adenine nucleotide translocator (ANT). Given the translational potential, we investigated the bioenergetic effects of NAAs in human adipocytes, characterizing beneficial and adverse effects, dose ranges, amino acid derivatives and underlying mechanisms. Method: NAAs with neutral (phenylalanine, leucine, isoleucine) and polar (lysine) residues were synthetized and assessed in intact and permeabilized human adipocytes using plate-based respirometry. The Seahorse technology was applied to measure bioenergetic parameters, dose-dependency, interference with UCP1 and adenine nucleotide translocase (ANT) activity, as well as differences to the established chemical uncouplers niclosamide ethanolamine (NEN) and 2,4-dinitrophenol (DNP). Result: NAAs with neutral amino acid residues potently induce uncoupled respiration in human adipocytes in a dose-dependent manner, even in the presence of the UCP1-inhibitor guanosine diphosphate (GDP) and the ANT-inhibitor carboxyatractylate (CAT). However, neutral NAAs significantly reduce maximal oxidation rates, mitochondrial ATP-production, coupling efficiency and reduce adipocyte viability at concentrations above 25 μM. The in vitro therapeutic index (using induced proton leak and viability as determinants) of NAAs is lower than that of NEN and DNP. Conclusion: NAAs are potent mitochondrial uncouplers in human adipocytes, independent of UCP1 and ANT. However, previously unnoticed adverse effects harm adipocyte functionality, reduce the therapeutic index of NAAs in vitro and therefore question their suitability as anti-obesity agents without further chemical modifications

Drp1 Overexpression Decreases Insulin Content in Pancreatic MIN6 Cells

Mitochondrial dynamics and bioenergetics are central to glucose-stimulated insulin secretion by pancreatic beta cells. Previously, we demonstrated that a disturbance in glucose-invoked fission impairs insulin secretion by compromising glucose catabolism. Here, we investigated whether the overexpression of mitochondrial fission regulator Drp1 in MIN6 cells can improve or rescue insulin secretion. Although Drp1 overexpression slightly improves the triggering mechanism of insulin secretion of the Drp1-knockdown cells and has no adverse effects on mitochondrial metabolism in wildtype MIN6 cells, the constitutive presence of Drp1 unexpectedly impairs insulin content, which leads to a reduction in the absolute values of secreted insulin. Coherent with previous studies in Drp1-overexpressing muscle cells, we found that the upregulation of ER stress-related genes (BiP, Chop, and Hsp60) possibly impacts insulin production in MIN6 cells. Collectively, we confirm the important role of Drp1 for the energy-coupling of insulin secretion but unravel off-targets effects by Drp1 overexpression on insulin content that warrant caution when manipulating Drp1 in disease therapy

Use of a fluorescence-based approach to assess short-term responses of the alga Pseudokirchneriella subcapitata to metal stress

This work explores the use of fluorescent probes to evaluate the responses of the green alga Pseudokirchneriella subcapitata to the action of three nominal concentrations of Cd(II), Cr(VI), Cu(II) and Zn(II) for a short time (6 h). The toxic effect of the metals on algal cells was monitored using the fluorochromes SYTOX Green (SG, membrane integrity), fluorescein diacetate (FDA, esterase activity) and rhodamine 123 (Rh123, mitochondrial membrane potential). The impact of metals on chlorophyll a (Chl a) autofluorescence was also evaluated. Esterase activity was the most sensitive parameter. At the concentrations studied, all metals induced the loss of esterase activity. SG could be used to effectively detect the loss of membrane integrity in algal cells exposed to 0.32 or 1.3 mol L1 Cu(II). Rh123 revealed a decrease in the mitochondrial membrane potential of algal cells exposed to 0.32 and 1.3 mol L1 Cu(II), indicating that mitochondrial activity was compromised. Chl a autofluorescence was also affected by the presence of Cr(VI) and Cu(II), suggesting perturbation of photosynthesis. In conclusion, the fluorescence-based approach was useful for detecting the disturbance of specific cellular characteristics. Fluorescent probes are a useful diagnostic tool for the assessment of the impact of toxicants on specific targets of P. subcapitata algal cells.The authors thank the FCT Strategic Project PEst-OE/EQB/LA0023/2013. Manuela D. Machado gratefully acknowledges the post-doctoral grant from FCT (SFRH/BPD/72816/2010)