Methods for Assessing Mitochondrial Function in Diabetes

Abstract

A growing body of research is investigating the potential contribution of mitochondrial function to the etiology of type 2 diabetes. Numerous in vitro, in situ, and in vivo methodologies are available to examine various aspects of mitochondrial function, each requiring an understanding of their principles, advantages, and limitations. This review provides investigators with a critical overview of the strengths, limitations and critical experimental parameters to consider when selecting and conducting studies on mitochondrial function. In vitro (isolated mitochondria) and in situ (permeabilized cells/tissue) approaches provide direct access to the mitochondria, allowing for study of mitochondrial bioenergetics and redox function under defined substrate conditions. Several experimental parameters must be tightly controlled, including assay media, temperature, oxygen concentration, and in the case of permeabilized skeletal muscle, the contractile state of the fibers. Recently developed technology now offers the opportunity to measure oxygen consumption in intact cultured cells. Magnetic resonance spectroscopy provides the most direct way of assessing mitochondrial function in vivo with interpretations based on specific modeling approaches. The continuing rapid evolution of these technologies offers new and exciting opportunities for deciphering the potential role of mitochondrial function in the etiology and treatment of diabetes.

Perspectives on the membrane fatty acid unsaturation/pacemaker hypotheses of metabolism and aging

“…Some studies have done this (Brand et al, 1991;Hulbert et al, 2002a;Pamplona et al, 1999aPamplona et al, , 1999cPamplona et al, , 1996, but the methods used in these studies have their own problems, including a lack of reliability in measures of lipid peroxidation used and disruption of mitochondrial structure in isolated mitochondria (Halliwell and Whiteman, 2004;Perry et al, 2013;Zhang et al, 2012).…”

Lactate is oxidized outside of the mitochondrial matrix in rodent brain

“…1). Further, where it has been previously hypothesized that 60 mmol·L −1 lactobionate used in the respiration buffer may inhibit LDH (Passarella et al 2014), we replaced our buffer with the frequently employed buffer Z (Perry et al 2013) and observed no increase in respiration ( Supplementary Fig. S1 1 ), demonstrating that lactobionate does not have an inhibitory effect on lactate metabolism.…”

“…The method was developed to ensure H 2 O 2 could be determined, and therefore taurine was removed from the buffer, as originally reported (Herbst and Holloway 2015); taurine was excluded in the current study to be consistent with those experiments. MiR05 media was chosen following preliminary experiments comparing it to another popular, but notably sucrose-and lactobionate-free potassium salt buffer (buffer Z (Perry et al 2013)), owing to differences in maximal respiration ( Supplementary Fig. S1 1 ).…”

The Pancreatic β-Cell: A Bioenergetic Perspective

“…Permeabilization of insulinoma cell lines has been performed with saponin (89,91,96,98,139,269), staphylococcal ␣-toxin (246,323,324,326), streptolysin-O (117), or perfringolysin-O (260) or by electropermeabilization (459). The advantages and limitations inherent in these permeabilization strategies have been recently reviewed, with a focus on mitochondrial function in skeletal muscle (408).…”