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Show * SCHOOL OF MEDICINE AND HEALTH SCIENCES UNDERGRADUATE RESEARCH ABSTRACTS TYPETWO DIABETES MELLITUS AND MITOCHONDRIAL DEGRADATION VIA AUTOPHOGY Spencer Thompson (Timothy E. Graham) Department of Molecular Medicine University of Utah Mechanisms and metabolic consequences of the mitochondrial dysfunction that characterizes type 2 diabetes mellitus (T2DM) are not fully understood. Autophagy is the sole mechanism through which old or damaged mitochondria are removed from cells. The aim of this work is to determine whether an impairment of mitochondrial degradation via autophagy plays a role in T2DM-associated mitochondrial dysfunction. We hypothesize that impaired mitochondrial degradation could cause accumulation of dysfunctional mitochondria in T2DM. To test this, w e engineered mice expressing a novel mitochondria-targeted autophagy "reporter" protein, referred to as"mitoRosella"in all of their tissues. MitoRosella is a fusion protein containing a mitochondrial targeting sequence fused to a pH-stable red fluorescent protein (dsRed) and a pH-sensitive green fluorescent protein (pHluorin). During mitophagy, mitoRosella-labeled mitochondria are engulfed by autophagosomes and delivered to acidic lysosomes, where they are degraded by acid hydrolyses. Fluorescence of the pH-sensitive pHluorin in the mitoRosella is completely quenched in the acidic lysosome, whereas dsRed fluorescence remains intact. Hence, the flux of mitochondria delivered to lysosomes via autophagy can be quantified by the change in ratio of green to red fluorescence over time. To generate the mice, we engineered a DNA construct containing the mitoRosella sequence under control of the ubiquitous CMV/CBA promoter sequence, and delivered it to mouse oocytes by direct pronuclear injection. Successful progeny of in vitro fertilization were screened for mitoRosella expression using qRTPCR, and positive mice were further bred to confirm germ line transmission of mitoRosella. W e are presently screening a first cohort of germ line "founder" mice for stable Rosella expression in tissues using Western blotting and confocal microscopy. In future studies, we will subject mitoRosella transgenic mice to high fat feeding to induce obesity and insulin resistance, an early feature of T2DM. W e will use confocal fluorescence microscopy to image mitochondria in metabolically relevant tissues (e.g., liver, skeletal muscle, and adipose tissue), and calculate rates of mitochondrial degradation based on the ratio of green to red mitoRosella fluorescence. We will compare observed rates of mitochondrial degradation with severity of mitochondrial dysfunction such as decreased respiratory rate or increased reactive oxygen species production. Spencer Thompson 133 |