Mitochondrial Function of Cerebral Vasculature in Insulin Resistant Zucker Obese Rats
Authors: Merdzo Ivan , Rutkai Ibolya , Tokes Tunde , Sure Venkata N.L.R. , Katakam Prasad V.G. , Busija David W. 
Published: November 2023

We previously have shown that dilation of large cerebral arteries to mitochondrial activators is reduced in insulin resistant rats (Am, J, Physiol, 296:R289–298, 2009 & 307:H493–503, 2014). However, little is known about other effects of insulin resistance on mitochondria in the cerebral vasculature. We determined the effects of insulin resistance on mitochondrial respiration and expression of mitochondrial and related non‐mitochondrial proteins in major cerebral arteries of insulin resistant Zucker obese (ZO) and phenotypically normal Zucker lean (ZL) rats. We also examined the cerebral microvessels of these rats to determine mitochondrial and related non‐mitochondrial protein expression and reactive oxygen species (ROS) production. We studied 10–12 week old ZO and ZL rats. Oxygen consumption rate (OCR) measured mitochondrial respiration using the Seahorse XFe24 analyzer under basal conditions and during treatments with diazoxide (DZ), a mitochondrial ATP sensitive potassium channel opener, and L‐NAME, a nitric oxide synthase inhibitor. Electron spin resonance spectroscopy measured ROS levels. Western blotting was used for protein expression studies. Under basal conditions, maximal mitochondrial respiration of large cerebral arteries was significantly higher (p<0.05) in the ZO (375±6.5 pM/min/μg protein) compared with the ZL (334.6±6.66 pM/min/μg protein). Other components of mitochondrial respiration (basal respiration, ATP production, spare capacity, and proton leak) and non‐mitochondrial respiration were not significantly different between the groups. However, L‐NAME treatment significantly increased proton leak in the ZL group (94.94±9.4 pM/min/μg protein vs. 65.24±8.5 pM/min/μg protein; p‐<0.05) but did not significantly affect mitochondrial and non‐mitochondrial components in either group. Diazoxide treatment significantly decreased non‐mitochondrial respiration in the ZL (53.24±5.81 pM/min/μg protein vs. 73.15±6.89 pM/min/μg protein; p<0.05) but not in ZO and did not significantly affect other components of mitochondrial respiration in either group. Mitochondrial proteins levels were not significantly different in large cerebral arteries and microvessels of ZO compared with ZL rats. Levels of endothelial nitric oxide synthase (eNOS) were significantly higher in microvessels of ZO compared with ZL (132.7±20.84% vs. 76.67±10.54%, p<0.05). Finally, ROS levels, specifically superoxide (O2−), were significantly higher in microvessels of ZO compared with ZL rats (1397.33±317.86 vs. 560.6±63.43 arbitrary units; p<0.05). Our results suggest that mitochondrial protein mass and respiration in large cerebral arteries and mitochondrial protein mass in microvessels are not significantly altered in young, insulin resistant rats. However, increased ROS production in microvessels of ZO rats is consistent with our previous findings in larger arteries and thus confirms the potential for ROS‐dependent vascular dysfunction throughout the cerebral vasculature in the pre‐diabetic period. Furthermore, it is possible that uncoupled eNOS may be the source of increased ROS production in cerebral microcirculation during insulin resistance.Support or Funding InformationThis work was supported by National Institutes of Health Grants (D. W. Busija: HL‐077731 and HL‐093554), Louisiana Board of Regents Support Fund‐Research Competitiveness Subprogram [P. V. G. Katakam: LEQSF‐(2014–17)‐RD‐A‐11], American Heart Association National Center NRCP Scientist Development Grant (P. V. G. Katakam: 14SDG20490359), and American Heart Association Postdoctoral Fellowship Grant (I. Rutkai: 15POST23040005). This research was supported in whole or in part by the Louisiana Board of Regents Endowed Chairs for Eminent Scholars program.