In this study, the investigators will test the hypothesis that acute in vivo exposure to hyperglycemia increases mitochondrial network fragmentation and mitochondrial reactive oxygen species production (ROS) production in human arterial endothelial cells.

Subjects will be recruited according to our inclusion/exclusion criteria. The investigators 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 Medical College of Wisconsin 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.

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 principal investigator has published experience with the J-wire endothelial cell biopsy technique, and the technique has been extensively validated technique. 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 4 hours post drink ingestion. At one and 4 hours post-ingestion, J-wire endothelial biopsies of the radial artery will be repeated. Four separate J wires will be passed into the radial artery for each measurement time point (0,1, and 4 hours).

In non-diabetic 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. Endothelial cells will be obtained via J-wire capture technique. 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 4 hours post challenge. The investigators will visualize the mitochondrial networks in these cells using our previously reported and validated immunofluorescence method using cytochrome c antibodies to tag the mitochondria. Network fragmentation (high number=greater fragmentation and fission) will be quantified by network fragmentation count calculated with ImageJ (NIH, Bethesda) using a validated protocol.

Inclusion Criteria:

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.

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).
2. History of bleeding disorders.
3. Evidence of other evident major illness including chronic renal insufficiency (plasma creatinine > 1.4 for women or 1.5 for men), liver disease (liver enzymes greater than 2.5 x normal), and cancer
4. currently undergoing therapy or had therapy for cancer within 1 year of enrollment.
5. Pregnancy as determined by urinary human chorionic gonadotropin beta test
6. Thienopyridine, anti-thrombin/Xa, or warfarin therapy at time of screening
7. On medication for cholesterol or blood pressure

Healthy subjects without evidence of diabetes, insulin resistance, hypertension, or elevated cholesterol. Subjects will also be without prevalent cardiovascular disease, chronic renal insufficiency, or chronic liver disease.

The data may be available to qualified investigators following study completion and initial publication of the data with collaboration with the study PI.

Adverse event data were collected over the patient enrollment period of the study (from 2016 through 2023). Each subject was only followed for adverse events for 2 hours following the completion of study protocol

Asymptomatic hypoglycemia