Examining the Effect of Exogenous Ketone Supplementation on Glucose Control in Type 2 Diabetes

Effect of 14 Days of Exogenous Ketone Supplementation on Glycemic Control in Type 2 Diabetes: a Randomized Placebo-controlled Crossover Trial

Ketone bodies are a fuel source and signaling molecule that are produced by the body during prolonged fasting or if an individuals consistently eats a low-carbohydrate "keto" diet. Blood ketones can be used as a source of energy by the body, but they may also act as signals that impact the functioning of different cells in the body. Recently, the availability of ketone supplements that can be taken orally allows for raising blood ketones without having to fast or eat a "keto" diet. The investigators' studies and those of other researchers have shown that ketone supplementation can lower blood sugar without having to make any other dietary changes. Oral ingestion of ketones may therefore be an effective strategy to improve blood sugar control and influence how cells function. The main objective of this study is to determine if consuming a ketone supplement 3 times per day (before meals) for 14 days lowers blood sugar and impacts how the body's cells function. The results of this study will be used to guide future recommendations on the utility of ketone supplements for improving health in individuals with, or at elevated risk of, type 2 diabetes.

3-Hydroxybutyric Acid, Glycemic Control, Hyperglycemia, Ketones, Glucose Control, Inflammation, Immune cell function, Cognition

Participants will consume 15 g of an active oral exogenous ketone monoester supplement 15 minutes prior to each meal of the day for a 14-day period. Pre-intervention (baseline) and post-intervention measurements will be obtained before and immediately after the 14-day period. All meals will be provided throughout the supplementation period Participants will wear a continuous glucose monitor for the first 10 consecutive days during the supplementation period.

Participants will consume a flavor-matched placebo drink and undergo the same procedures described in the Experimental Arm

Participants will consume 15g of the oral ketone monoester supplement 15 minutes prior to each meal of the day for 14 days. All meals will be provided throughout the 14-day supplementation period.

Participants will consume an equivalent volume (30ml) of the active intervention supplement 15 minutes prior to each meal for 14 days. All meals will be provided throughout the 14-day placebo supplementation period.

Change in glucose control (from pre-intervention Day 0) will be quantified by serum fructosamine obtained by fasting blood sample in both conditions.

Vascular function will be assessed by flow mediated dilation of the brachial artery using vascular ultrasound. A cuff will affixed on the forearm, distal to the brachial artery and will be inflated for 5 minutes. Flow mediation dilation will be measured over a 3-minute period following cuff release.

Cognition will be assessed using a customized battery of psychometrically validated tests within the domain of executive functions using the computer-based app Inqisit6 Lab (Millisecond). The test will be the n-back test.

Cognition will be assessed using a customized battery of psychometrically validated tests within the domain of executive functions using the computer-based app Inquisit6 Lab (Millisecond). The test will be the digit-symbol substitution test.

Plasma insulin from venous blood samples will be measured using a high-sensitivity human insulin enzyme-like immunosorbent assay (ELISA) run in duplicate.

Key inflammatory cytokines including CRP will be quantified by Mesoscale Discovery U-PLEX run in duplicate.

Phagocytosis of fluorescent-labelled E. coli by immune cells from whole blood will be quantified by flow cytometry

Immune cell degranulation will be quantified by enzyme-linked immunosorbent assay run in duplicate (quantifying myeloperoxidase and elastase in whole blood cell culture supernatants).

Phenotyping of macrophages and T cells will be quantified by surface and intracellular staining by flow cytometry.

A 5-part white blood cell differential and complete blood count will be quantified by hematology analyzer.

Glycemic control will be measured by continuous glucose monitoring using the G6 CGM (Dexcom) in both the active and placebo supplement conditions. Glycemic control will be quantified by assessing 2hr postprandial hyperglycemia.

Glycemic control will be measured by continuous glucose monitoring using the G6 CGM (Dexcom) in both the active and placebo supplement conditions. Glycemic control will be quantified by assessing 24hr average glucose AUC.

Glycemic control will be measured by continuous glucose monitoring using the G6 CGM (Dexcom) in both the active and placebo supplement conditions. Glycemic control will be quantified by assessing fasting plasma glucose.

Change in fasting plasma glucose (from pre-intervention Day 0) will be measured by fasting blood sample in both the active and placebo supplement conditions.

Glycemic control will be measured by continuous glucose monitoring using the G6 CGM (Dexcom) in both the active and placebo supplement conditions. Glycemic control will be quantified by assessing glycemic variability.

Glycemic control will be measured by continuous glucose monitoring using the G6 CGM (Dexcom) in both the active and placebo supplement conditions. Glycemic control will be quantified by assessing time in target range.

Lipid panel (total cholesterol, high-density cholesterol, low-density cholesterol, triglycerides, non-HDL cholesterol, cholesterol/HDL ratio) will be measured using a point-of-care analyzer.

Change in blood pressure will be measured using an automated blood pressure device. Both systolic and diastolic blood pressure will be measured.

Physical activity will be assessed using an accelerometer (activePal) worn throughout the entire intervention period.

Sedentary time will be assessed using an accelerometer (activePal) worn throughout the entire intervention period.

Sleeping time will be assessed using an accelerometer (activePal) worn throughout the entire intervention period.

Acceptability of the supplement (easy of compliance, taste etc.) will be assessed via questionnaire. A 7-point Likert scale will be used.

Perceived hunger will be measured on a visual analogue scale questionnaire assessing hunger and fullness. The questions assessed are: How hungry do you feel? (0 = I am not hungry at all; 10 = I have never been more hungry) How satisfied do you feel? (0 = I am completely empty; 10 = I cannot eat another bite) How full do you feel? (0 = Not at all full; 10 = Totally full) How much more do you think you can eat? (0 = Nothing at all; 10 = A lot)

Participants will be asked to report their desire to eat a particular type of food. A visual analogue scale will be used. The questions assessing cravings are: How often do you experience strong urges to eat particular types of food? (0 = Never; 10 = All the time) On average how often do you experience a strong urge to eat a particular type of food? (0 = Several times per day; 10 = Once per month) How strong are these urges you experience to eat particular types of food? (0 = Extremely weak; 10 = Extremely strong) Are the experiences of strong urges to eat a particular food always of the same strength? (0 = Never; 10 = Always) How easy is it to ignore this strong urge to eat a particular food? (0 = Very easy; 10 = Impossible) Is a strong urge to eat a particular food the same as a craving for food? (0 = No; 10 = Yes)

Inclusion Criteria: Have a type 2 diabetes diagnosis from a physician Have stable use of glucose-lowering medications for at least 3 months Exclusion Criteria: Are a competitively trained endurance athlete Are actively attempting to gain or lose weight Have a history of mental illness or existing neurological disease(s), cardiovascular events (i.e., heart attack, stroke) in the last 2 years Have hypoglycemia, irritable bowel syndrome or inflammatory bowel disease Are currently using insulin or SGLT2 inhibitors Are using more than 2 classes of glucose-lowering medication Are currently following a ketogenic diet or taking ketone supplements Are unable to commit for a 29-day trial Are unable to follow a controlled diet

The investigators will share individual patient data (de-identified) with researchers upon reasonable request.

The de-identified data and associated documents will be made available to researchers upon reasonable request for the duration that is required by the researchers.

Researchers from accredited institutions will be granted access to the de-identified data and associated documents provided they can show that it will be used for a research-related purpose (e.g., meta-analysis).