Hyperglycemia and Mitochondrial Function in The Endothelium of Humans
Conditions: Diabetes, Hyperglycemia
Sponsor: Medical College of Wisconsin

Hyperglycemia and Mitochondrial Function 
 
9/4/2018 
NCT02682342 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

 
 
 
Goal:To determine if acute exposure to abnormal glucose levels induces mitochondrial fission and 
endothelial dysfunction through increased Drp1-Fis1 activity in humans. 
 
 
Rationale: While DM often includes multiple metabolic abnormalities, the central phenotypical attribute of DM is 
abnormal systemic glycemic exposure.  Hyperglycemia and hypoglycemia (occurring secondary to therapies), 
both acutely and chronically, are critical to both chronic and acute-on-chronic endothelial dysfunction in DM.1-9  
Both exposures rapidly impair human vascular endothelial dysfunction through increased oxidative stress, 
inflammation, and a loss of NO bioavailability in both healthy non-DM subjects and patients with DM. 5-10  Hyper- 
and hypoglycemia driven mitochondrial ROS production critically contribute to the development of endothelial 
dysfunction.4, 11   However, the mechanisms behind the acute phenotypical change in the endothelium with acute 
hyperglycemic exposure remain poorly understood.  Better understanding the mechanisms underlying the 
adverse impact of acute dysglycemia on the endothelium could lead to novel treatments to mitigate the burden 
and complications of CV events in patients both with and without DM.12-15 
Hyperglycemia also drives over-expression of Drp1 and Fis1 in the endothelium.16 Drp1 and Fis1 interact 
to drive mitochondrial fission leading to subsequent excessive mitochondrial superoxide production.16, 17  
Molecular suppression of Drp1 or Fis1 blocks hyperglycemia’s adverse impact cultured endothelial cells.16 Our 
preliminary data show that Drp1 and Fis1 also control low glucose driven mitochondrial fission and excessive 
mitochondrial ROS production (Fig X or not shown).  While there are multiple docking proteins for Drp1 on the 
outer mitochondrial membrane besides Fis1 (Mff, MiD49, MiD51), Fis1 is the preferred Drp1 docking protein in 
the setting of cellular stressors, including high glucose, in cell culture.18-21 These foundational data and our 
preliminary data strongly support our initial strategy of concentrating on Drp1 and Fis1. 
 In this study, we will test the hypothesis that acute in vivo exposure to hyperglycemia increases 
mitochondrial network fragmentation and 
mitochondrial ROS production in human 
arterial endothelial cells.  Affirmative 
findings will critically establish that acute in 
vivo 
exposure 
to 
clinically 
relevant 
hyperglycemia levels in humans triggers 
mitochondrial 
fission 
and 
excess 
mitochondrial ROS production. Further, 
affirmative findings will establish that 
inhibition of mitochondrial fission proteins 
Fis1 and Drp1 reverses acute dysglycemia-
related endothelial dysfunction.  
 
Protocol 
Subjects will be recruited according to our 
inclusion/exclusion criteria listed in Table 1.  
We employ a multifaceted approach to 
recruitment, including flyers, community 
newspaper ads, internet ad postings, and 
direct 
recruitment 
from 
the 
Internal 
Medicine Clinics of Froedtert Memorial 
Lutheran 
Hospital/Medical 
College 
of 
Wisconsin which saw approximately 50,000 
unique outpatient visits over last year.   The electronic health record is leveraged to identify potential subjects by 
HIPAA guidelines and MCW policies. .Potential subjects will undergo phenotyping that includes a detailed 
medical history, anthropomorphic measurements, blood pressure and heart rate measurements, and a blood 
draw for measurements that include fasting lipids, glucose, and glycosylated hemoglobin, creatinine, and liver 
function tests.  Healthy, non-DM subjects who pass the screening as per the inclusion/exclusion criteria in Table 
2 will be enrolled in the study protocol.   
Table 1: Inclusion/Exclusion Criteria for Aims 1 and 2 
T2DM Inclusion Criteria  
1. 
Adult age 21-70  
2. 
If on HMG-CoA reductase therapy, must be on a stable dose for at least 6 
weeks prior to enrollment 
3. 
Diagnosis of type 2 diabetes by a physician as defined by the American 
Diabetes Association standard criteria 
Inclusion Criteria for Healthy subjects 
1. 
Adult age 21 to 70 
2. 
No evidence of metabolic syndrome or diabetes, hypertension (BP≥140/90), 
or high cholesterol (LDL≥160) at the time of screening. 
T2DM Subject Exclusion Criteria: 
        1.      History of stroke, peripheral arterial disease, or coronary artery disease (as   
                 defined by the presence of at least one coronary stenosis ≥ 40% on  
                 angiography or by confirmed history of myocardial infarction by standard  
                criteria). History of bleeding disorders. 
        2.      Evidence of other evident major illness including chronic renal  
                 insufficiency(plasma creatinine > 1.4 for women or 1.5 for men), liver  
                 disease (AST or ALT greater than 2.5 x normal), and cancer currently  
                 undergoing therapy or had therapy for cancer within 1 year of enrollment.   
3. 
Pregnancy as determined by urinary beta-HCG test    
4. 
Thienopyridine, anti-thrombin/Xa, or warfarin therapy at time of screening 
Exclusion Criteria for Healthy Subjects: 
1. 
Meet any of the exclusion criteria for diabetic or metabolic syndrome group  
2. 
Meet criteria for type 2 diabetes or metabolic syndrome 
3. 
On medication for cholesterol or blood pressure 
4. 
History of superficial thrombophlebitis, deep vein thrombosis and pulmonary 
embolism 
5. 
Women of childbearing potential using systemic birth control 

 
Following an overnight fast (12 hours), subjects will come in the morning to our Adult Translational 
Research Unit (A-TRU) which is part of the Clinical Translational Research Initiative of Southeast Wisconsin 
(8UL1TR000055).  An antecubital intravenous line will be placed to facilitate blood glucose sampling during the 
hyperglycemic challenge.  An antecubital intravenous catheter will be placed to facilitate obtaining endothelial 
cells from the vein by J-wire biopsy for of mitochondrial testing.  The PI has published experience with the J-wire 
endothelial cell biopsy technique, and the technique has been extensively validated technique.22-27    An initial 
venous glucose sample will be taken from the antecubital vein and an initial J-wire biopsy of the radial artery 
endothelium will be performed through the radial arterial line.  Subjects will then be asked to drink a standardized 
75 g glucose drink created by the A-TRU nutritionist- a standard oral glucose challenge as used clinically.  Blood 
glucose samples will be taken hourly after that until 2 hours post drink ingestion. At one and 2 hours post-
ingestion, J-wire endothelial biopsies of the radial artery will be repeated. Three separate J wires will be passed 
into the radial artery for each measurement time point (0,1, and 2 hours). 
 
Because of the prolonged fast, subjects may have a decrease in blood sugar to <70 during this study.  
To mitigate this phenomenon, subjects will be given 6 ounces of juice to drink at the 2-hour mark.  If subjects 
report symptoms of hypoglycemia, which include but are not limited to feeling tremulous, headache, 
lightheadedness, feeling clammy or sweaty and nausea, the blood glucose level will be checked via a fingerstick.  
If the blood glucose level is < 70, they will be treated with juice and/or food.  The fingerstick will also be repeated 
periodically to ensure adequate response to the intervention. 
 
In non-DM subjects, the 75 g oral challenge induces a peak increase in systemic glucose at 1-hour post-
administration at which time endothelial dysfunction is concomitantly detectable.28  While we could achieve a 
greater degree of hyperglycemia for a longer period using a hyperinsulinemic-hyperglycemic clamp procedure, 
the hyperinsulinemic-hyperglycemic clamp procedure fails to induce endothelial dysfunction, likely secondary to 
the pharmacological levels of insulin required for the protocol.29  Therefore, the oral glucose tolerance test is 
better suited for the purposes of this Sub-Aim.   
 
A portion of the endothelial cells isolated from the J-wires will be used to measure mitochondrial network 
complexity prior hyperglycemic challenge, 1-hour post challenge, and 2 hours post challenge.  We will visualize 
the mitochondrial networks in these cells  using our previously reported and validated immunofluorescence 
method using  cytochrome c antibodies to tag the mitochondria.27  Network fragmentation (high number=greater 
fragmentation and fission) will be quantified by network fragmentation count calculated with ImageJ (NIH, 
Bethesda) using a validated protocol adapted in collaboration with Dr. Jalees Rehman from the University of 
Illinois-Chicago.30, 31   
 
SUB-STUDY: The J-wire procedure proposed in this protocol is used to isolate endothelial cells and used to 
measure mitochondrial network complexity. In order to train (new) study staff on this procedure, as well as refine 
the best way to isolate these cells off the wires after they have been obtained from a subject approximately 20 
subjects will be enrolled into a “sub-study” under this protocol. In this sub-study, subjects will sign informed 
consent one or two antecubital IV lines will be paced. The Research Fellow or PI will obtain endothelial cells by 
passing 1 to 3 J-Wires through the IV. No fasting is required of the subject and we will not need to collect any 
medical history on the subject. Subjects in this sub-study could enroll in the main study if desired, they would be 
consented again with the main study consent.  
 
 
PRELIMINARY DATA: Our published work supports the feasibility of measuring mitochondrial network 
fragmentation in endothelial cells captured by J-wire biopsy.16    
 
Mitochondrial superoxide production using MitoSox™(Life Technologies). Cells are re-suspended in PBS 
buffer and incubated for 30 minutes at 37ºC with 5 µM of MitoSox™ (Invitrogen), a fluorescent probe specific for 
mitochondrial superoxide production. Cells are then immediately fixed with 2% paraformaldehyde and plated on 
poly-lysine coated slides (Sigma, St. Louis) and allowed to settle.  Following the 
addition of DAPI (4’, 6’-diamidino-2-phenylindole hydrochloride) for nuclear 
identification to help discern endothelial cells from polymorphonuclear cells, 
fluorescence intensity of endothelial cells are measured by fluorescence 
microscopy (Nikon E55i).  Images are analyzed using MetaMorph 6.1 (Universal 
Imaging).  Fifteen to twenty cells are measured, and the average fluorescence 
intensity reported for each sample. All images are captured with the same 
exposure time. Values are reported ratios of subject fluorescence to that of 
human umbilical vein endothelial cells (HUVECs) stained simultaneously for 
Figure 2: Sample endothelial cells 
taken from a human volunteer after 60 
minutes of  euglycemia (a) and 
hypoglycemia (b).  Red- MitoSox™, 
Blue- DAPI. 

interexperimental normalization.23, 24, 32  PRELIMINARY DATA: We have successfully obtained vascular 
endothelial cells using the J-wire technique and measured mitochondrial superoxide production prior to and 
following lowering blood glucose levels for 1 hour, proving the feasibility of these measurements (Figure. 2).  
 
Data Analysis and Sample Sizes: The primary outcome for this sub-aim is the measurement of mitochondrial 
network complexity from before oral glucose administration to 1 hour afterward measurements.  These will be 
compared by paired t-tests with log transformation for normality or non-parametric methods if needed. A 
secondary analysis using repeated measures ANOVA will also be performed using data from all 3 time points. 
Marginal/conditional residual diagnostics and influence diagnostics will be performed. If some of them are 
violated, log-transformation or non-parametric methods will be considered.  Sample size calculations for 
mitochondrial network complexity measurements were derived from our observations of HUVECs described in 
our preliminary data section.  The mean difference in network fragmentation in the preliminary data was 1.5±0.8 
between normal glucose and high glucose exposure. Power analysis was performed for paired test and repeated 
measures ANOVA assuming 30% inflation in the number need to account for dropouts and failed J-wire captures. 
A mean difference between before oral glucose administration and 1 hour afterward measurements in network 
complexity was assumed to be 40% lower than our preliminary data in light of our less aggressive in vivo 
hyperglycemic exposure and the same standard deviation. Also, 4 hour afterward measurements were assumed 
to be back to the level of prior oral glucose administration. The correlation between time points was assumed to 
be 0.5. Then, we will need to recruit 15 subjects to have at least 90% power at α=0.05 for paired t-test and 
repeated measures ANOVA. . Mitochondrial superoxide measurements are a secondary outcome whose results 
will be analyzed in the same manner as network fragmentation.  
 
 

 
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