MtDNA is usually present in multiple identical copies in the

MtDNA is usually present in multiple identical copies in the cells, but the location of MtDNA close to the electron transport chain renders it more susceptible to ROS induced damage and mutations. In diabetes patients, MtDNA may be exposed to hyperglycaemia induced oxidative stress (Singh et al., 2011) resulting in random mutations in the mitochondrial genome leading to heteroplasmy, the presence of normal and mutated MtDNA in the same cell. The damaged MtDNA in such cases would only have an impact once it reached a threshold where the normal non-mutated MtDNA is no longer able to compensate for mitochondrial dysfunction (Chinnery, 2002). It has recently been demonstrated that MtDNA heteroplasmic mutations are associated with widely spread chronic diseases, including atherosclerosis and cancer (Sobenin et al., 2014; Ye et al., 2014). We used two methods to detect MtDNA damage, both of which suggest that MtDNA from diabetes samples shows increased damage, with heteroplasmy being detected only in DN samples. Furthermore, HMCs showed a significant increase in MtDNA damage after 4days of growth in hyperglycaemic conditions, suggesting MtDNA damage in HMCs could play a role in the subsequent bioenergetic deficit observed in these btk inhibitor after 8 and 12days of hyperglycaemia. The combination of the data presented in this paper paint a picture of severely compromised mitochondria in patients with DN. These patients have reduced MtDNA in circulation, with increased MtDNA damage, and decreased mitophagy, the functional impact of these changes is the reduced metabolic flexibility of these cells. Furthermore if we assume that the PBMCs are representative of systemic changes in the body, then it could be predicted that kidney cells in the DN patients will show a similar compromised response, which could play a major role in progression of pathology. Our results support a recent metabolomics study showing that patients with DN have reduced mitochondrial metabolites in urine (Sharma et al., 2013) and we propose that DN could be viewed as a disease of acquired mitochondrial dysfunction. In conclusion, we have shown that metabolic dysfunction can be detected in peripheral blood samples of patients with DN. We have also shown using renal cells in-vitro that hyperglycaemia affects mitochondria, with MtDNA levels changing before other indicators of mitochondrial dysfunction. Our data suggests that the BHI formula can indicate mitochondrial dysfunction in live PBMCs from patients, and therefore could be developed into a non-invasive translational measure of mitochondrial function. It is of importance to determine if the changes we observe in clinical samples precede the onset of DN, and therefore longitudinal studies should be carried out to determine this. The potential of regular and routine monitoring of MtDNA content, integrity and BHI as indicators of DN should be further evaluated.
Acknowledgements Special thanks to the patients, and the volunteers without whose samples this work could not have been done, we are indebted to the nursing/clinical staff at the Diabetes clinic at Guy\'s Hospital, especially Nurse Siew Cohen for her tireless help and constant encouragement. Thanks to Dr Sylvie Bannwarth (Laboratoire de GénétiqueMoléculaire, Nice France) for the help with the surveyor nuclease method, Dr John Harris and the Nikon Imaging Centre at KCL, Alex Liversage of Seahorse Biosciences for loaning us a Seahorse XFe96, Professor Kinya Otsu, KCL, Professor Victor Darley-Usmar (University of Alabama, USA) and Dr David Ferrick, Seahorse Biosciences, for the critical evaluation of this manuscript.
Introduction Therapeutic cooling offers robust protection against ischaemic brain damage, but its practical challenges and risks have limited its application to specific patient groups (Choi et al., 2012; Yenari and Han, 2012). Advancing our insight into cooling-induced neuroprotection at the cellular level could provide new molecular targets to bypass the need for cooling — whilst expanding its therapeutic potential. Preconditioning describes the tolerance achieved against an intensively toxic insult by subjecting cells or tissue to a sublethal stress (Stetler et al., 2014). Neuronal preconditioning can be effected by many and varied stimuli, including hypothermia (Dirnagl et al., 2003; Yuan et al., 2004; Stetler et al., 2014). In rodents, this cooling-induced tolerance requires de novo protein synthesis (Nishio et al., 2000) — a fundamental arm of the cold-shock response (Fujita, 1999), for which data in human neurons is lacking. Depending on the depth of cooling, this response leads to cell-cycle arrest with shut-down of transcription and translation (Yenari and Han, 2012). Simultaneously, a subset of highly conserved ‘cold-inducible’ RNA chaperones including RNA binding motif 3 (RBM3) and cold-inducible RNA binding protein (CIRBP) is rapidly upregulated (Lleonart, 2010). These ‘cold-shock’ proteins mediate important survival functions including facilitated translation of essential mRNAs and suppression of apoptosis (Lleonart, 2010; Saito et al., 2010).