Pancreatic Clamp in NAFLD

Study Overview
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Brief Summary
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This is a single-center, prospective, randomized, controlled (crossover) clinical study designed to investigate the specific dose-response impact of insulin infusion rate (IIR) on blood glucose levels during a pancreatic clamp study. The investigators will recruit participants with a history of overweight/obesity and evidence of insulin resistance (i.e., fasting hyperinsulinemia plus prediabetes and/or impaired fasting glucose and/or Homeostasis Model Assessment of Insulin Resistance [HOMA-IR] score >=2.73), and with evidence of, or clinically judged to be at high risk for, uncomplicated non-alcoholic fatty liver disease (NAFLD). Participants will undergo two pancreatic clamp procedures in which individualized basal IIR are identified, followed in one by maintenance of basal IIR (maintenance hyperinsulinemia, MH) and in the other by a stepped decline in IIR (reduction toward euinsulinemia, RE). In both clamps the investigators will closely monitor plasma glucose and various metabolic parameters. The primary outcome will be the absolute and relative changes in steady-state plasma glucose levels at each stepped decline in IIR.

Detailed Description
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Although high blood sugar and risk of heart disease are the most well-known health effects of type 2 diabetes (T2DM), nonalcoholic fatty liver disease (NAFLD), in which too much fat accumulates in the liver, has come to be recognized as another important complication. Unchecked, NAFLD can progress to severe liver inflammation, liver failure, and even liver cancer. The investigators suspect that high levels of the blood sugar-lowering hormone insulin leads to excessive fat production by the liver, and so lowering insulin levels might help to improve NAFLD. In order to answer this question, the investigators will recruit people at risk for T2DM and NAFLD to perform a pancreatic clamp - a procedure in which the body's production of insulin is temporarily shut off and then replaced at the same or lower levels. Again, the investigators expect that lowering insulin levels will lower fat production. Because this is a new research approach, the investigators first need to understand how lowering insulin levels affects blood sugar. Research participants in this pilot study will therefore undergo two pancreatic clamps in random order: one roughly maintaining their own internal (basal) insulin level and one in which the investigators lower that basal insulin level by 10%, 25%, and 40%. In each case, the investigators will observe the absolute and relative changes in blood sugar and the levels of certain fats as the investigators change the insulin level. Once the investigators have found a lower insulin level that they can safely maintain, the investigators will go on to study its effect on fat production in a later study.

Official Title
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Role of Hyperinsulinemia in Non-Alcoholic Fatty Liver Disease (NAFLD) Pathogenesis: Pancreatic Clamp Pilot & Feasibility Study

Conditions
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Insulin Resistance, Prediabetic State, Hyperinsulinemia, Non-Alcoholic Fatty Liver Disease, Obesity

Intervention / Treatment
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* Drug: Insulin human
* Drug: Octreotide Acetate
* Drug: Glucagon
* Drug: Growth Hormone, Human
* Other: [6,6-2H2] D-glucose
* Drug: 20% D-glucose (aq)
* Dietary Supplement: BOOST Plus
* Dietary Supplement: CORE Bar
* Device: Harvard Apparatus PHD ULTRA CP syringe pump
* Device: Yellow Springs Instruments (YSI) 2500 Biochemistry Glucose/Lactate Analyzer


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Men and women (using highly effective contraception if of childbearing potential, aged 18-65 years Body mass index of 25.0-39.9 kg/m2 Able to understand written and spoken English and/or Spanish Evidence of insulin resistance, represented by any or all of the following criteria: a. Meeting either of the American Diabetes Association's definitions for prediabetes or IFG within the previous year* and on screening labs: i. Prediabetes: Hemoglobin A1c 5.7-6.4% ii. IFG: plasma glucose of 100-125 mg dL-1 after 8-h fast b. Homeostasis Model of Insulin Resistance (HOMA-IR) score ≥ 2.73 Fasting hyperinsulinemia (fasting insulin level ≥ 15 µIU/mL) on screening labs Diagnosed with, or clinically judged to be at high risk for, non-alcoholic fatty liver disease (NAFLD), also known as metabolic-associated fatty liver disease (MAFLD), by hepatologist or other qualified specialist physician and the condition is listed as an active problem in the electronic medical record Written informed consent (in English or Spanish) and any locally required authorization (e.g., Health Insurance Portability and Accountability Act) obtained from the participant prior to performing any protocol-related procedures, including screening evaluations. Exclusion Criteria: Unable to provide informed consent in English or Spanish Concerns arising at screening visit (any of the following): i. Unwillingness to use only bedpan or urinal to void during the clamp ii. Unwillingness to fast (except water) for up to 22 hours iii. Documented weight loss of ≥ 5% of baseline within the previous 6 months iv. Abnormal blood pressure (including on treatment, if prescribed) Systolic blood pressure < 90 mm Hg or > 160 mm Hg, and/or Diastolic blood pressure < 60 mm Hg or > 100 mm Hg v. Abnormal resting heart rate: <60 or ≥100 bpm Sinus brady- or tachycardia that has been extensively worked up and considered benign by the recruit's personal physician may be permitted at the Principal Investigator's discretion vi. Abnormal screening electrocardiogram (or if on file, performed within previous 90 d) Non-sinus rhythm Significant corrected QT segment (QTc) prolongation (≥ 480 ms) New or previously unknown ischemic changes that persist on repeat EKG: ST segment elevations T-wave inversions vii. Laboratory evidence of diabetes mellitus: Hemoglobin A1c ≥ 6.5%, and/or Fasting plasma glucose ≥ 126 mg/dL viii. Positive qualitative β-hCG (i.e., pregnancy test) in women of childbearing potential ix. Liver function abnormalities (either of the following) Transaminases (AST or ALT) > 2.0 x the upper limit of normal Total bilirubin > 1.25 x the upper limit of normal x. Abnormal fasting lipids at screening (either of the following) Triglycerides ≥ 400 mg/dL LDL-cholesterol ≥ 190 mg/dL xi. Abnormal screening serum electrolytes (any of the following) Sodium, potassium, or bicarbonate outside of the reference range Creatinine equating to estimated glomerular filtration rate < 60 mL min-1 1.73 m-2 xii. Abnormal complete blood count (CBC) (any of the following) Hemoglobin < 10 g dL-1 or hematocrit < 30% Platelet count < 100,000 µL-1 Exempt from CBC requirement if previously obtained value within 2 months of screening is available COVID-19 precautions i. Not fully vaccinated against COVID-19 (4 doses if ages 50-65, 3 doses if ages 18-49) ii. Unwillingness to comply with masking requirements per hospital policy iii. Active, documented COVID-19 at any time after screening Reproductive concerns i. Women of childbearing potential not using highly effective contraception, defined as: Surgical sterilization (e.g., bilateral tubal occlusion, bilateral oophorectomy and/or salpingectomy, hysterectomy) Combined oral contraceptive pills taken daily, including during the study Intrauterine device (levonorgestrel-eluting or copper) active at the time of study Medroxyprogesterone acetate (Depo-Provera®) injection active at the time of study Etonogestrel implants (e.g., Implanon®, etc.) active at the time of the study Norelgestromin/ethinyl estradiol transdermal system (e.g., Ortho-Evra®) active at the time of the study ii. Women currently pregnant, measured by serum and/or urine human chorionic gonadotropin, beta subunit (β-hCG) iii. Women currently breastfeeding Concerns related to glucose metabolism i. History of having met any of the American Diabetes Association's definitions of diabetes mellitus (i.e., overt diabetes): Hemoglobin A1c ≥ 6.5%, or rapid rise in documented HbA1c values causing clinical concern for evolving insulin deficiency Plasma glucose ≥ 126 mg/dL after 8-h fast Plasma glucose of ≥ 200 mg/dL at 2 h after ingestion of a 75-g glucose load Random plasma glucose ≥ 200 mg/dL associated with typical hyperglycemic symptoms, diabetic ketoacidosis, or hyperglycemic-hyperosmolar state ii. History of gestational diabetes mellitus within the previous 5 years iii. Use of most antidiabetic medications within the 90 days prior to screening, including those prescribed for other indications (e.g., weight control, restoration of ovulation in of polycystic ovarian syndrome): Excluded: thiazolidinediones, sulfonylureas, meglitinides, DPP4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, amylin mimetics, acarbose, insulin Metformin is acceptable provided that recruits meet all of the inclusion criteria at screening iv. Clinical concern for absolute insulin deficiency (e.g., type 1 diabetes, pancreatic disease) v. Fasting plasma glucose < 89 mg/dL at screening Concerns related to lipid metabolism i. Known diagnoses of familial hypercholesterolemia, familial combined hyperlipidemia, or familial hyperchylomicronemia in the participant or a first-degree relative ii. Use of certain lipid-lowering drugs other than statins for primary prevention within 90 d prior to screening visit, including: Statins or PCSK9 inhibitors for secondary prevention or treatment of familial hypercholesterolemia. Statins or PCSK9 inhibitors for primary prevention of ASCVD are acceptable. Fibrates (e.g., fenofibrate, clofibrate, gemfibrozil) High-dose niacin (>100 mg daily) Known, documented history, at the time of screening, of any of the following medical conditions: i. Pancreatic pathology, including but not limited to: Pancreatic neoplasia, unless appropriately evaluated and considered benign and not producing hormones Chronic pancreatitis Acute pancreatitis (history of) Autoimmune pancreatitis Surgical removal of any portion of the pancreas ii. Cardiovascular diseases (N.B. uncomplicated hypertension is not exclusionary) Atherosclerotic cardiovascular disease Stable or unstable angina Myocardial infarction Ischaemic or hemorrhagic stroke, or transient ischaemic attack Peripheral arterial disease (claudication) Use of dual antiplatelet therapy (aspirin + P2Y12 inhibitor) History of percutaneous coronary intervention Heart rhythm abnormalities Congestive heart failure of any New York Heart Association class Severe valvular heart disease (e.g., aortic stenosis) Pulmonary hypertension iii. Chronic kidney disease, Stage 3 or higher (estimated glomerular filtration rate < 60 mL / min / 1.73 m2), of any cause iv. Advanced or severe liver disease, including but not limited to: Advanced liver fibrosis, as determined by non-invasive testing Cirrhosis of any etiology Autoimmune hepatitis or other rheumatologic disorder affecting the liver Biliopathy (e.g., progressive sclerosing cholangitis, primary biliary cholangitis) Chronic liver infection (e.g., viral hepatitis, parasitic infestation) Hepatocellular carcinoma Infiltrative disorders (e.g., sarcoidosis, hemochromatosis, Wilson disease) v. Gallstone disease, including: Biliary colic (active) History of acute cholecystitis not treated with cholecystectomy History of other gallstone complications (e.g., pancreatitis, cholangitis) vi. Chronic viral illness (N.B. diagnosis based only on medical history; we will not test for any of these viruses at any point in this study) Hepatitis B virus (HBV), unless previously successfully eradicated with antiviral drugs that have been discontinued for at least 90 d prior to screening Hepatitis C virus (HCV) infection, unless previously successfully eradicated with antiviral drugs that have been discontinued for at least 90 d prior to screening Human immunodeficiency virus (HIV) infection vii. Malabsorptive conditions (active) Active inflammatory bowel disease (quiescent and off medication is acceptable) Celiac disease (in remission with gluten-free diet is acceptable) Surgical removal of a significant length of intestine viii. Active seizure disorder (including controlled with antiepileptic drugs) ix. Psychiatric diseases causing functional impairment that… Are or have been decompensated within 1 year of screening, and/or Require use of anti-dopaminergic antipsychotic drugs associated with significant weight gain/metabolic dysfunction (e.g., clozapine, olanzapine), monoamine oxidase inhibitors, tricyclic antidepressants, or lithium x. Other endocrinopathies: Cushing syndrome (okay if considered in remission after treatment, provided that no exogenous corticosteroids or other ongoing treatment are required) Adrenal insufficiency Primary aldosteronism xi. Venous thromboembolic disease (deep vein thrombosis or pulmonary embolism) or any required use of therapeutic anticoagulation xii. Bleeding disorders, including due to anticoagulation, or significant anemia (see above) xiii. Dysautonomia, including post-vagotomy xiv. Active malignancy, or hormonally active benign neoplasm, except allowances for: Non-melanoma skin cancer Differentiated thyroid cancer (AJCC Stage I only) Clinical concern for increased risk of volume overload, including due to medications and/or heart/liver/kidney problems, as listed above Clinical concern for increased risk of hypokalemia, including low potassium on screening labs (i.e., below lower limit of normal), use of certain medications, or any medical conditions listed above Use of certain medications currently or within 90 d prior to screening: i. Prescribed medications used for any of the indications in the preceding list (§5.3.7) of excluded conditions, or their use within 90 d prior to screening, except allowances for: Use of drugs prescribed for indications other than the exclusionary diagnoses/purposes listed above (e.g., antiepileptic drugs used for non-seizure indications, angiotensin converting enzyme inhibitor (ACEi)/angiotensin receptor blocker (ARB) used for uncomplicated hypertension rather than for congestive heart failure, etc.) •• Note, as above, that antidiabetic drugs except metformin for any indication within 90 d of screening are excluded ii. Thiazide or loop diuretics for any indication Note, as above, that other antihypertensive drugs (e.g., ACEi/ARB, calcium channel blockers, alpha/beta blockers) iii. Oral or parenteral corticosteroids (at greater than prednisone 5 mg daily, or equivalent) for more than 3 days within the previous 90 days; topical and inhaled formulations are permitted iv. Fludrocortisone v. Opioids other than dextromethorphan for cough History of certain weight-loss (bariatric) surgery, including: i. Roux-en-Y gastric bypass ii. Biliopancreatic diversion iii. Restrictive procedures (lap band, sleeve gastrectomy) performed within the past 6 months Clinical concern for alcohol overuse, including recent documented history or phosphatidylethanol ≥ 0.05 µmol/L at screening and/or participant report of regularly consuming more than 2 drinks per day for males or 1 drink per day for females. Positive urine drug screen, with exceptions for: i. Lawfully prescribed medications ii. Marijuana/THC positivity, provided that the participant agrees not to use it during the same period that they will abstain from alcohol History of severe infection or ongoing febrile illness within 30 days of screening Any other disease, condition, or laboratory value that, in the opinion of the investigator, would place the participant at an unacceptable risk and/or interfere with the analysis of study data. Known allergy/hypersensitivity to any component of the medicinal product formulations (including soy or dairy), IV infusion equipment, plastics, adhesive or silicone, history of infusion site reactions with IV administration of other medicines, or ongoing clinically important allergy/hypersensitivity as judged by the investigator. Concurrent enrollment in another clinical study of any investigational drug therapy within 6 months prior to screening or within 5 half-lives of an investigational agent, whichever is longer.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 65 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Basic Science
Allocation: Randomized
Intervention Model: Crossover Assignment
Interventional Model Description: All participants will undergo (i.e., cross over between) both pancreatic clamp protocols (MH, RE) separated by 2-4 weeks. The order of the clamp protocols (i.e., MH > RE, RE > MH) will be randomized.
Masking: Single


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Active Comparator: Maintenance hyperinsulinemia (MH) protocol<br>The basal insulin infusion rate (IIR) necessary to maintain participants' mean basal fasting plasma glucose (mbFPG) will be determined during the basal titration period. Then, during the intervention period, the IIR will remain at 100% of basal for the full duration (225 min). The IIR and resulting insulin levels are expected to be relatively high (cf. hyperinsulinemia) because of underlying insulin resistance. | Drug: Insulin human<br>* Insulin infusion rate (IIR) will be determined empirically first to maintain mean basal fasting plasma glucose, and then either maintained at the basal rate (MH protocol) or be reduced stepwise toward euinsulinemia (RE protocol).<br>Drug: Octreotide Acetate<br>* Octreotide will be infused at 30 ng/kg/min to suppress endogenous insulin, glucagon, and growth hormone secretion. Co-administered with glucagon and rhGH.<br>Drug: Glucagon<br>* Glucagon will be replaced at a constant rate of up to 0.65 ng/kg/min to maintain baseline counterregulatory response. Co-administered with octreotide and rhGH.<br>Drug: Growth Hormone, Human<br>* Recombinant human growth hormone (rhGH) will be replaced at a constant rate of up to 3 ng/kg/min to maintain baseline counterregulatory response. Co-administered with octreotide and glucagon.<br>Other: [6,6-2H2] D-glucose<br>* Stable isotope tracer administered to calculate glucose kinetics during pancreatic clamp.<br>* Other names: D2-glucose, D2G;Drug: 20% D-glucose (aq)<br>* 20% D-glucose (aq) (D20W) will be administered to counteract hypoglycemia or strongly downward blood glucose trends, as needed.<br>* Other names: D20W;Dietary Supplement: BOOST Plus<br>* Nutritional supplement will be administered to provide three standardized mixed meals on the day before the pancreatic clamp.<br>Dietary Supplement: CORE Bar<br>* Energy bar used as a standardized snack on the day before the pancreatic clamp.<br>Device: Harvard Apparatus PHD ULTRA CP syringe pump<br>* Syringe pump used for highly precise administration of insulin, octreotide/glucagon/rhGH, and D20W (as needed) even at low infusion rates.<br>Device: Yellow Springs Instruments (YSI) 2500 Biochemistry Glucose/Lactate Analyzer<br>* Glucose oxidase analyzer used to detect plasma glucose levels at the point of care. YSI have been the gold standard in clamp studies for many years. Two machines will run in parallel to ensure accuracy of results.<br>|
| Experimental: Reduction toward euinsulinemia (RE) protocol<br>The basal insulin infusion rate (IIR) necessary to maintain participants' mean basal fasting plasma glucose (mbFPG) will be determined during the basal titration period. Then, during the intervention period, the IIR will be reduced progressively, at 75-min intervals, to 90%, 75%, and 60% of basal IIR. Thus, the basal hyperinsulinemia expected due to underlying insulin resistance will be reduced toward euinsulinemia. | Drug: Insulin human<br>* Insulin infusion rate (IIR) will be determined empirically first to maintain mean basal fasting plasma glucose, and then either maintained at the basal rate (MH protocol) or be reduced stepwise toward euinsulinemia (RE protocol).<br>Drug: Octreotide Acetate<br>* Octreotide will be infused at 30 ng/kg/min to suppress endogenous insulin, glucagon, and growth hormone secretion. Co-administered with glucagon and rhGH.<br>Drug: Glucagon<br>* Glucagon will be replaced at a constant rate of up to 0.65 ng/kg/min to maintain baseline counterregulatory response. Co-administered with octreotide and rhGH.<br>Drug: Growth Hormone, Human<br>* Recombinant human growth hormone (rhGH) will be replaced at a constant rate of up to 3 ng/kg/min to maintain baseline counterregulatory response. Co-administered with octreotide and glucagon.<br>Other: [6,6-2H2] D-glucose<br>* Stable isotope tracer administered to calculate glucose kinetics during pancreatic clamp.<br>* Other names: D2-glucose, D2G;Drug: 20% D-glucose (aq)<br>* 20% D-glucose (aq) (D20W) will be administered to counteract hypoglycemia or strongly downward blood glucose trends, as needed.<br>* Other names: D20W;Dietary Supplement: BOOST Plus<br>* Nutritional supplement will be administered to provide three standardized mixed meals on the day before the pancreatic clamp.<br>Dietary Supplement: CORE Bar<br>* Energy bar used as a standardized snack on the day before the pancreatic clamp.<br>Device: Harvard Apparatus PHD ULTRA CP syringe pump<br>* Syringe pump used for highly precise administration of insulin, octreotide/glucagon/rhGH, and D20W (as needed) even at low infusion rates.<br>Device: Yellow Springs Instruments (YSI) 2500 Biochemistry Glucose/Lactate Analyzer<br>* Glucose oxidase analyzer used to detect plasma glucose levels at the point of care. YSI have been the gold standard in clamp studies for many years. Two machines will run in parallel to ensure accuracy of results.<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Plasma glucose (absolute values) (units: mg/dL) | Goal is first to clamp insulin infusion rate to maintain mean basal fasting plasma glucose during the basal titration phase, and then during the intervention phase to observe the glucose levels that result from altering the basal IIR. | Up to 425 minutes from the start of the procedure. | 
| Plasma glucose (relative/change) (units: fold difference and/or ∆mg/dL relative to previous time points) | Goal is first to clamp insulin infusion rate to maintain mean basal fasting plasma glucose during the basal titration phase, and then during the intervention phase to observe the impact of altering the basal IIR on glycemia. | Up to 425 minutes from the start of the procedure. | 
| Serum insulin (absolute values) (units: micro-international units per milliliter (µIU/mL)) | Investigators will assess the insulin levels attained at the basal IIR, and at each stepwise reduction in IIR during the intervention phase. | Up to 425 minutes from the start of the procedure. | 
| Serum insulin (relative/change) (units: fold difference and/or ∆ µIU/mL relative to previous time points) | Investigators will compare the baseline insulin level to that attained at the basal IIR, as well as comparing to the change in insulin level that occurs with alterations in the IIR during the intervention phase. | Up to 425 minutes from the start of the procedure. | 
| Serum C-peptide (absolute values) (units: ng/mL) | Suppression of endogenous insulin by octreotide during pancreatic clamp is expected to result in a fall in C-peptide levels to near zero. | Up to 425 minutes from the start of the procedure | 
| Serum C-peptide (relative/change) (units: fold difference and/or ∆ µIU/mL relative to previous time points) | Suppression of endogenous insulin by octreotide during pancreatic clamp is expected to result in a fall in C-peptide levels to near zero. | Up to 425 minutes from the start of the procedure | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Serum or plasma triglyceride (TG) (absolute values) (units: mg/dL) | TG levels in serum reflect hepatic synthesis/storage and very low-density lipoprotein (VLDL) secretion. | Up to 425 minutes from the start of the procedure | 
| Serum or plasma triglyceride (TG) (relative/change) (units: fold difference and/or ∆mg/dL relative to previous time points) | TG levels in serum reflect hepatic synthesis/storage and VLDL secretion. | Up to 425 minutes from the start of the procedure | 
| Serum or plasma free fatty acid (FFA) (absolute values) (units: mg/dL) | FFA levels reflect adipose tissue lipolysis and its response to insulin and counterregulatory hormones. | Up to 425 minutes from the start of the procedure | 
| Serum or plasma free fatty acid (FFA) (relative/change) (units: fold difference and/or ∆mg/dL relative to previous time points) | FFA levels reflect adipose tissue lipolysis and its response to insulin and counterregulatory hormones. | Up to 425 minutes from the start of the procedure | 
| Serum or plasma apolipoprotein B (ApoB) (absolute values) (units: mg/dL) | ApoB level is a surrogate for triglyceride-rich lipoproteins, especially hepatic VLDL. | Up to 425 minutes from the start of the procedure | 
| Serum or plasma apolipoprotein B (ApoB) (relative/change) (units: fold difference and/or ∆mg/dL relative to previous time points) | ApoB level is a surrogate for triglyceride-rich lipoproteins, especially hepatic VLDL. | Up to 425 minutes from the start of the procedure | 
| Plasma glucose kinetics: rate of appearance (units: mg/kg/min) | Calculated from D2G tracer enrichment by the Steele equations. | Measured every 5 minutes x 4 at the end of each steady-state IIR period, up to 425 minutes from the start of the procedure | 
| Plasma glucose kinetics: rate of disappearance (units: mg/kg/min) | Calculated from D2G tracer enrichment by the Steele equations | Measured every 5 minutes x 4 at the end of each steady-state IIR period, up to 425 minutes from the start of the procedure | 
| Plasma glucose kinetics: endogenous glucose production (units: mg/kg/min) | Calculated from D2G tracer enrichment by the Steele equations | Measured every 5 minutes x 4 at the end of each steady-state IIR period, up to 425 minutes from the start of the procedure | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Insulin resistance, Hyperinsulinemia, Diabetes, Non-alcoholic fatty liver disease