1. amanda white proposal

Effects of doxycycline treatment on skeletal muscle mitochondrial content and function from
obese, diabetic subjects
Amanda White
Med-Into-Grad Area: Diabetes
Winter 2010
University of California San Diego

The increasing prevalence of the metabolic syndrome, which includes obesity and type 2 diabetes, is a major
health crisis in the Western world. This disease affects approximately 24 mil ion Americans and this number is
projected to grow to almost 65 mil ion by 20506. In 2007 alone, costs associated with diabetes and its
complications in the US totaled 174 bil ion dol ars6. Better strategies for therapeutic intervention are necessary
to deal with this increasing threat. Insulin resistance, an impaired ability of muscle and other tissues to take up
glucose in response to insulin, is an early abnormality of type 2 diabetes. It is known that mitochondrial defects
lead to impairment of glucose-stimulated insulin secretion4 and there is a growing body of evidence suggesting
that mitochondrial dysfunction contributes to insulin resistance in type 2 diabetes3. Decreased expression of
mitochondrial genes, such as those for oxidative phosphorylation, has been implicated in the metabolic
perturbations that lead to insulin resistance and type 2 diabetes. Additional y, tissues from diabetic and insulin
resistant patients display decreased oxidative capacity of the mitochondrial electron transport chain2. Thus, it
is important to better understand the quality of mitochondria in obese, diabetic patients and it is possible that
mitochondria may be a target for novel therapeutics.
My Med-Into-Grad training included interacting with Dr. Karen Herbst, an endocrinologist at the VA who
specializes in metabolic syndrome. In my conversations with her, we discussed the various clinical studies in
which she is currently involved. One of these studies involves the treatment of obese, diabetic patients with
doxycycline, a member of the tetracycline antibiotic group and a matrix metal oproteinase (MMP) inhibitor.
MMPs are enzymes that are involved in adipose tissue remodeling (i.e. adipogenesis, angiogenesis and
extracel ular matrix proteolysis) that occurs as a result of obesity and may be involved in the cleavage of the
extracel ular domain of the insulin receptor resulting in hyperglycemia, insulin resistance and type 2 diabetes.
The hypotheses of Dr. Herbst and her col aborators include that patients with obesity and type 2 diabetes wil
have higher levels of MMPs than control subjects that wil then be suppressed after 12 weeks of doxycycline
treatment (100mg twice daily) and that these subjects wil have improved measures of diabetes.
Endpoints of the clinical study include fingerstick glucose monitoring throughout the 12 weeks of the study,
dual x-ray absorptiometry scans, oral glucose tolerance tests, hemoglobin A1C, skin and fat punch biopsies (1-
10 grams from peri-umbilical region), and muscle biopsies of the vastus lateralis muscle before and after
doxycycline treatment.
My motivation for getting involved in this study is due to our lab’s interest in the effects of doxycycline on
mitochondria. Whether tetracyclines are toxic or beneficial to mitochondria is controversial. Doxycycline is
known to inhibit mitochondrial protein synthesis which would presumably contribute to toxicity. However, it is
possible that mild inhibition of protein synthesis may result in a compensatory response by the tissue to
produce additional mitochondria (mitochondrial biogenesis). In fact, there have been reports of both
mitochondrial biogenesis and inhibition of the mitochondrial permeability transition (MPT) by tetracyclines,
which could benefit the patient. The MPT is associated with oxidative and Ca2+-mediated injury to
mitochondria, and results in uncoupling of oxidative phosphorylation and osmotic swel ing7. The resulting
adenosine triphosphate (ATP) depletion and cytochrome c release can lead to tissue dysfunction, or necrotic
or apoptotic cel death. As oxidative stress and inflammation affects multiple tissues in type 2 diabetes, it is
possible that the MPT participates in disease pathogenesis. Minocycline, another drug in the tetracycline
family, has been observed to block the mitochondrial permeability transition by decreasing mitochondrial Ca2+
uptake, and has been shown to have effects on mitochondrial respiration7.
I propose to probe for evidence of mitochondrial biogenesis, changes in respiration, alteration of mitochondrial
membrane potential and/or prevention of MPT, and changes in oxidative stress in the skeletal muscle biopsy
samples that wil be taken from the obese, diabetic subjects before and after doxycycline treatment. My
hypothesis is that skeletal muscle mitochondria from obese, diabetic patients wil be impaired, either
functional y or via decreased mitochondrial mass and that doxycycline treatment wil improve either
mitochondrial function (i.e. blocking the mitochondrial permeability transition or decreasing oxidative stress) or
wil increase mitochondrial number.
To assess the functionality of skeletal muscle mitochondria from diabetic subjects treated with doxycycline, I
would prepare the muscle biopsies in three ways: 1) use fresh material from the biopsy to isolate functional
mitochondria on which I wil perform respiration and membrane potential analyses, 2) flash freeze the sample
to be used for ATP/ADP, lactate/pyruvate assays and Western blotting, and 3) fix the samples for electron
microscopy for the quantitation of mitochondrial number and the evaluation of mitochondrial morphology.
Respiratory measurements wil be made on isolated mitochondria using the XF24 Analyzer from Seahorse
Biosciences. Mitochondrial membrane potential wil be measured in isolated mitochondria using a TPP+
electrode inserted into a Hansatech Oxytherm oxygen electrode chamber through a modified stopper, a
method that has been used successful y by our lab members. Protection from MPT wil be measured
fluorometrical y as described by Murphy et. al.5 To investigate whether doxycycline protects mitochondria from
oxidative stress, we can examine the ratio of oxidized (GSSG) to reduced glutathione (GSH) as a marker of
oxidative stress as described by Tietze8 and analyze the activity of mitochondrial aconitase, an enzyme
sensitive to inactivation by reactive oxygen. These experiments wil be carried out in muscle biopsies from
control and doxycycline-treated patients to determine the effect of the drug on respiration, mitochondrial
membrane potential, induction of MPT and oxidative stress.

Mitochondria are the major source in the cel for ATP synthesis. Measurement of ATP/ADP ratios and
lactate/pyruvate ratios are essential tools in the understanding of cel ular energy metabolism and the balance
between glycolysis and oxidative phosphorylation. To measure cel ular ATP/ADP ratios we wil use a
luciferin/luciferase based approach as previously described1. Lactate/pyruvate ratios and mitochondrial
aconitase activity wil be measured by spectrophotometric enzyme assays (BioVision).
There have been some observations of the effects of tetracyclines on mitochondrial respiration and
biogenesis7. We wil use western analysis with antibodies for known markers of mitochondrial biogenesis such
as PGC-1α and subunits of the respiratory complexes to determine if any observed effects on respiration are
due to a biogenesis response. Additionally, cel s from the muscle biopsies wil be fixed and mitochondrial
number and morphology wil be quantitated by electron microscopy by Dr. Guy Perkins at the National Center
for Microscopy and Imaging Research, at UCSD. By comparing the results of these assays with the clinical
outcomes of the study we wil be able to determine if any of the observed therapeutic effects of doxycycline
correlate with alterations in skeletal muscle mitochondria.
References:

1. Budd SL and Nicholls DG. Mitochondria, calcium regulation, and acute glutamate excitotoxicity in
cultured cerebel ar granule cel s. J Neurochem 67: 2282-2291, 1996. 2. Kelley DE, He J, Menshikova EV, and Ritov VB. Dysfunction of mitochondria in human skeletal
muscle in type 2 diabetes. Diabetes 51: 2944-2950, 2002. 3. Lowell BB and Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science 307: 384-387,
4. Maechler P and de Andrade PB. Mitochondrial damages and the regulation of insulin secretion.
Biochem Soc Trans 34: 824-827, 2006. 5. Murphy AN, Bredesen DE, Cortopassi G, Wang E, Fiskum, G. Bcl-2 potentiates the maximal
calcium uptake capacity of neural cel mitochondria. Proc Natl Acad Sci U S A 93: 9893-9898, 1996. 6. Narayan KM, Boyle JP, Geiss LS, Saaddine JB, and Thompson TJ. Impact of recent increase in
incidence on future diabetes burden: U.S., 2005-2050. Diabetes Care 29: 2114-2116, 2006. 7. Theruvath TP, Zhong Z, Pediaditakis P, Ramshesh VK, Currin RT, Tikunov A, Holmuhamedov E,
Lemasters JJ. Minocycline and N-methyl-4-isoleucine cyclosporin (NIM811) mitigate
storage/reperfusion injury after rat liver transplantation through suppression of the mitochondrial
permeability transition. Hepatology 47: 236-246, 2008.
8. Tietze F. Disulfide reduction in rat liver. I. Evidence for the presence of nonspecific nucleotide-
dependent disulfide reductase and GSH-disulfide transhydrogenase activities in the high-speed supernatant fraction. Arch Biochem Biophys 138: 177-188, 1970.

Source: http://molpath.ucsd.edu/HHMI/Research%20Props%2009-10/1.%20Amanda%20White%20Proposal.pdf