Omega-3 Fatty Acids and Insulin Sensitivity

National Center for Advancing Translational Sciences (NCATS), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), Building Interdisciplinary Research Careers in Women's Health

This study is being done to understand the effects of dietary omega-3 fats on insulin sensitivity in adult men and women.

Dietary omega-3 polyunsaturated fatty acids (n-3 PUFA), which include eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) from fish oil, prevent insulin resistance in rodents, but data in humans is ambiguous. No existing studies have systematically evaluated the influence of n-3 PUFAs on insulin sensitivity and beta cell function in insulin resistant, non-diabetic humans. The Investigators hypothesize that 6 months of oral supplementation of purified EPA/DHA (3.9g/day) will significantly improve hepatic and peripheral insulin sensitivity and beta cell responsiveness in insulin-resistant, non-diabetic individuals. Based on recent work in mice, the investigators also hypothesize that EPA/DHA will increase the content and function of mitochondria in skeletal muscle, measured using a combination of in vivo and in vitro methods. Overall, the investigators hypothesize that EPA+DHA supplementation will improve hepatic and peripheral insulin sensitivity in insulin resistant humans, and this improvement will be associated with mitochondrial biogenesis and attenuated lipid accumulation in skeletal muscle and liver. A sub-study was added in which participants receiving dietary omega-3 fatty acids or placebo supplements underwent abdominal subcutaneous adipose tissue biopsies to measure the content of total, pro- (M1) and anti- (M2) inflammatory macrophages (immunohistochemistry), crown-like structures (immunohistochemistry), and senescent cells (β-galactosidase staining), as well as a two-step euglycemic, pancreatic clamp with a stable-isotope labeled precursor ((U-13C)palmitate) infusion to determine the insulin concentration needed to suppress palmitate flux by 50% (IC50(palmitate)f).

A 2-stage insulin clamp will be performed with titration of dextrose to maintain euglycemia. D2 glucose will be infused to evaluate hepatic glucose production at baseline and in response to insulin. Hyperinsulinemic-euglycemic clamp technique: The plasma insulin concentration is acutely raised and maintained by a continuous infusion of insulin. Meanwhile, the plasma glucose concentration is held constant at basal levels by a variable glucose infusion. When the steady-state is achieved, the glucose infusion rate (GIR) equals glucose uptake by all the tissues in the body and is therefore a measure of tissue insulin sensitivity.

Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up

Following consumption of a mixed meal, beta cell function will be evaluated from serial measurements of C-peptide. C-peptide was measured using a two-side immunometric assay using electrochemiluminescence detection.

Measurements of oxygen consumption in isolated mitochondria will be performed using a polarographic oxygen electrode.

Sensitivity of adipose tissue lipolysis to insulin suppression, was calculated as the insulin concentration needed to suppress palmitate appearance rates (ie, flux) by 50% (IC50(palmitate)f).

Tissue burden of senescent cells, which was measured by staining for senescence-associated B-galactosidase activity and expressed as the number per 100 nucleated positive cells.

One week after the pancreatic clamp study, participants were provided a standardized meal before an overnight fast. The next morning an abdominal adipose tissue biopsy was collected, and the samples were analyzed for adipocyte size. Immunohistochemistry was used to assess macrophage burden (total (CD68), M1 (CD14) and M2 (CD206) macrophages per 100 adipocytes).

Macrophages surrounding dying or dead adipocytes form crown-like structures (CLSs). One week after the pancreatic clamp study, participants were provided a standardized meal before an overnight fast. The next morning an abdominal adipose tissue biopsy was collected, and the samples were analyzed for adipocyte size. Immunohistochemistry was used to assess the number of crown-like structures per 10 images.

Inclusion criteria: Age 18-65 years Insulin resistant (Homeostasis Model Assessment (HOMA) Insulin Resistance (IR) ≥2.6) Exclusion criteria: Current use of omega-3 nutritional supplements Fasting plasma glucose ≥126 mg/dL Active coronary artery disease Participation in structured exercise (>2 times per week for 30 minutes or longer) Smoking Medications known to affect muscle metabolism (e.g., beta blockers, corticosteroids, tricyclic-antidepressants, benzodiazepines, opiates, barbiturates, anticoagulants) Renal failure (serum creatinine > 1.5mg/dl) Chronic active liver disease (AST>144 IU/L and alanine transaminase (ALT)>165 IU/L) Anti-coagulant therapy (warfarin/heparin) International normalized ratio (INR) >3 Use of systemic glucocorticoids Chronic use of NSAIDS or aspirin Pregnancy or breastfeeding Alcohol consumption greater than 2 glasses/day Hypothyroidism Fish or shellfish allergy

Lalia AZ, Johnson ML, Jensen MD, Hames KC, Port JD, Lanza IR. Effects of Dietary n-3 Fatty Acids on Hepatic and Peripheral Insulin Sensitivity in Insulin-Resistant Humans. Diabetes Care. 2015 Jul;38(7):1228-37. doi: 10.2337/dc14-3101. Epub 2015 Apr 7.

Hames KC, Morgan-Bathke M, Harteneck DA, Zhou L, Port JD, Lanza IR, Jensen MD. Very-long-chain omega-3 fatty acid supplements and adipose tissue functions: a randomized controlled trial. Am J Clin Nutr. 2017 Jun;105(6):1552-1558. doi: 10.3945/ajcn.116.148114. Epub 2017 Apr 19.