Insulin Resistance
Conditions
Brief summary
This study is being done to understand the effects of dietary omega-3 fats on insulin sensitivity in adult men and women.
Detailed description
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).
Interventions
DRUGOmega-3
Patients in this group will receive oral supplementation with EPA+DHA (3.9grams/day) for 6 months.
DRUGplacebo
Sponsors
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
Mayo Clinic
Study design
Allocation
RANDOMIZED
Intervention model
PARALLEL
Primary purpose
TREATMENT
Masking
QUADRUPLE (Subject, Caregiver, Investigator, Outcomes Assessor)
Eligibility
Sex/Gender
ALL
Age
18 Years to 65 Years
Healthy volunteers
Yes
Inclusion criteria
1. Age 18-65 years 2. Insulin resistant (Homeostasis Model Assessment (HOMA) Insulin Resistance (IR) ≥2.6)
Exclusion criteria
1. Current use of omega-3 nutritional supplements 2. Fasting plasma glucose ≥126 mg/dL 3. Active coronary artery disease 4. Participation in structured exercise (\>2 times per week for 30 minutes or longer) 5. Smoking 6. Medications known to affect muscle metabolism (e.g., beta blockers, corticosteroids, tricyclic-antidepressants, benzodiazepines, opiates, barbiturates, anticoagulants) 7. Renal failure (serum creatinine \> 1.5mg/dl) 8. Chronic active liver disease (AST\>144 IU/L and alanine transaminase (ALT)\>165 IU/L) 9. Anti-coagulant therapy (warfarin/heparin) 10. International normalized ratio (INR) \>3 11. Use of systemic glucocorticoids 12. Chronic use of NSAIDS or aspirin 13. Pregnancy or breastfeeding 14. Alcohol consumption greater than 2 glasses/day 15. Hypothyroidism 16. Fish or shellfish allergy
Design outcomes
Primary
| Measure | Time frame | Description |
|---|---|---|
| Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up | Baseline, after 6 months of treatment | 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. |
Secondary
| Measure | Time frame | Description |
|---|---|---|
| Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up | Baseline, after 6 months of treatment | Measurements of oxygen consumption in isolated mitochondria will be performed using a polarographic oxygen electrode. |
| Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f) | approximately after 6 months of treatment | 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). |
| Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up | baseline, after 6 months of treatment | 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. |
| Immunohistochemistry Assessments of Macrophage Burden | approximately after 6 months of treatment | 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). |
| Macrophage Crown-like Structures | approximately after 6 months of treatment | 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. |
| Senescent Cells | approximately after 6 months of treatment | 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. |
Countries
United States
Participant flow
Recruitment details
Participants were recruited from the Mayo Clinic in Rochester, Minnesota.
Participants by arm
| Arm | Count |
|---|---|
| Omega-3 Patients in this group will receive oral supplementation with EPA+DHA (3.9grams/day) for 6 months. | 14 |
| Placebo Patients in this group will be supplemented with placebo capsules containing ethyl oleate. | 11 |
| Total | 25 |
Withdrawals & dropouts
| Period | Reason | FG000 | FG001 |
|---|---|---|---|
| Main Study | Liver enzymes above range of exclusion | 1 | 0 |
| Main Study | Noncompliance with protocol | 0 | 1 |
| Main Study | Withdrawal by Subject | 1 | 3 |
| Sub-Study | Not consented for sub-study | 2 | 2 |
Baseline characteristics
| Characteristic | Omega-3 | Placebo | Total |
|---|---|---|---|
| Age, Continuous | 35.3 years STANDARD_DEVIATION 2.9 | 32.6 years STANDARD_DEVIATION 2.5 | 34.1 years STANDARD_DEVIATION 9.7 |
| Region of Enrollment United States | 14 participants | 11 participants | 25 participants |
| Sex: Female, Male Female | 9 Participants | 9 Participants | 18 Participants |
| Sex: Female, Male Male | 5 Participants | 2 Participants | 7 Participants |
Adverse events
| Event type | EG000 affected / at risk | EG001 affected / at risk |
|---|---|---|
| deaths Total, all-cause mortality | — / — | — / — |
| other Total, other adverse events | 0 / 14 | 0 / 11 |
| serious Total, serious adverse events | 0 / 14 | 0 / 11 |
Outcome results
Primary
Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up
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.
Time frame: Baseline, after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up | Baseline | 10.92 mg/kg FFM/min | Standard Error 1.04 |
| Omega-3 | Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up | 6 Month Follow Up | 10.16 mg/kg FFM/min | Standard Error 1.02 |
| Placebo | Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up | Baseline | 10.39 mg/kg FFM/min | Standard Error 0.76 |
| Placebo | Insulin Sensitivity by Hyperinsulinemic-euglycemic Clamp at Baseline and 6 Month Follow up | 6 Month Follow Up | 10.80 mg/kg FFM/min | Standard Error 0.73 |
Secondary
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.
Time frame: baseline, after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up | Baseline | 537.17 nmol/L | Standard Error 45.33 |
| Omega-3 | Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up | 6 Month Follow Up | 561.33 nmol/L | Standard Error 48.21 |
| Placebo | Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up | Baseline | 488.90 nmol/L | Standard Error 45.47 |
| Placebo | Beta Cell Function From Insulin Secretion Following Ingestion of a Mixed Meal at Baseline and 6 Month Follow up | 6 Month Follow Up | 504.39 nmol/L | Standard Error 35.93 |
Secondary
Immunohistochemistry Assessments of Macrophage Burden
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).
Time frame: approximately after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | Total (CD68) baseline | 31 macrophages per 100 adipocytes | Standard Deviation 8 |
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | Total (CD68) post intervention | 33 macrophages per 100 adipocytes | Standard Deviation 8 |
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | M1 (CD14) baseline | 11 macrophages per 100 adipocytes | Standard Deviation 6 |
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | M1 (CD14) post intervention | 14 macrophages per 100 adipocytes | Standard Deviation 6 |
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | M2 (CD206) baseline | 28 macrophages per 100 adipocytes | Standard Deviation 5 |
| Omega-3 | Immunohistochemistry Assessments of Macrophage Burden | M2 (CD206) post intervention | 29 macrophages per 100 adipocytes | Standard Deviation 9 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | M2 (CD206) baseline | 29 macrophages per 100 adipocytes | Standard Deviation 7 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | Total (CD68) baseline | 33 macrophages per 100 adipocytes | Standard Deviation 5 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | M1 (CD14) post intervention | 12 macrophages per 100 adipocytes | Standard Deviation 5 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | Total (CD68) post intervention | 31 macrophages per 100 adipocytes | Standard Deviation 5 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | M2 (CD206) post intervention | 29 macrophages per 100 adipocytes | Standard Deviation 5 |
| Placebo | Immunohistochemistry Assessments of Macrophage Burden | M1 (CD14) baseline | 13 macrophages per 100 adipocytes | Standard Deviation 4 |
Secondary
Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f)
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).
Time frame: approximately after 6 months of treatment
Population: The number of subjects analyzed for this outcome measure for the placebo arm was 8 instead of 9. One subject did not have blood drawn for this outcome measure.
| Arm | Measure | Group | Value (MEDIAN) |
|---|---|---|---|
| Omega-3 | Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f) | Baseline | 22 µU/mL |
| Omega-3 | Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f) | Post-intervention | 18 µU/mL |
| Placebo | Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f) | Baseline | 25 µU/mL |
| Placebo | Insulin Concentration Needed to Suppress Palmitate Appearance Rates (IC50(Palmitate)f) | Post-intervention | 19 µU/mL |
Secondary
Macrophage Crown-like Structures
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.
Time frame: approximately after 6 months of treatment
| Arm | Measure | Group | Value (MEDIAN) |
|---|---|---|---|
| Omega-3 | Macrophage Crown-like Structures | Baseline | 0 crown-like structures per 10 images |
| Omega-3 | Macrophage Crown-like Structures | Post-intervention | 0 crown-like structures per 10 images |
| Placebo | Macrophage Crown-like Structures | Baseline | 1 crown-like structures per 10 images |
| Placebo | Macrophage Crown-like Structures | Post-intervention | 1 crown-like structures per 10 images |
Secondary
Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up
Measurements of oxygen consumption in isolated mitochondria will be performed using a polarographic oxygen electrode.
Time frame: Baseline, after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up | Baseline | 496.81 pmol/s/mg tissue | Standard Error 26.54 |
| Omega-3 | Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up | 6 Month Follow Up | 406.38 pmol/s/mg tissue | Standard Error 40.39 |
| Placebo | Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up | Baseline | 564.86 pmol/s/mg tissue | Standard Error 42.1 |
| Placebo | Mitochondrial Function Determined by Muscle Biopsy at Baseline and 6 Month Follow up | 6 Month Follow Up | 495.12 pmol/s/mg tissue | Standard Error 39.79 |
Secondary
Senescent Cells
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.
Time frame: approximately after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | Senescent Cells | Baseline | 4 number positive cells/100 total cells | Standard Deviation 3 |
| Omega-3 | Senescent Cells | Post-intervention | 4 number positive cells/100 total cells | Standard Deviation 3 |
| Placebo | Senescent Cells | Baseline | 4 number positive cells/100 total cells | Standard Deviation 3 |
| Placebo | Senescent Cells | Post-intervention | 4 number positive cells/100 total cells | Standard Deviation 2 |
Post Hoc
EPA and DHA Concentrations in Adipose Tissue
Post hoc analyses were conducted to test whether EPA and DHA concentrations in subcutaneous abdominal adipose tissue in response to intervention explained variation in outcome measures of adipose tissue lipolysis insulin sensitivity and inflammatory markers post-intervention.
Time frame: approximately after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | EPA and DHA Concentrations in Adipose Tissue | EPA baseline | 0.06 percentage of total free fatty acid | Standard Error 0 |
| Omega-3 | EPA and DHA Concentrations in Adipose Tissue | EPA Post-Intervention | 0.19 percentage of total free fatty acid | Standard Error 0.02 |
| Omega-3 | EPA and DHA Concentrations in Adipose Tissue | DHA Baseline | 0.14 percentage of total free fatty acid | Standard Error 0.01 |
| Omega-3 | EPA and DHA Concentrations in Adipose Tissue | DHA Post-Intervention | 0.28 percentage of total free fatty acid | Standard Error 0.02 |
| Placebo | EPA and DHA Concentrations in Adipose Tissue | DHA Post-Intervention | 0.16 percentage of total free fatty acid | Standard Error 0.02 |
| Placebo | EPA and DHA Concentrations in Adipose Tissue | EPA baseline | 0.07 percentage of total free fatty acid | Standard Error 0.01 |
| Placebo | EPA and DHA Concentrations in Adipose Tissue | DHA Baseline | 0.15 percentage of total free fatty acid | Standard Error 0.01 |
| Placebo | EPA and DHA Concentrations in Adipose Tissue | EPA Post-Intervention | 0.07 percentage of total free fatty acid | Standard Error 0.01 |
Post Hoc
EPA and DHA Concentrations in Plasma
Post hoc analyses were conducted to test whether EPA and DHA concentrations in plasma in response to intervention explained variation in outcome measures of adipose tissue lipolysis insulin sensitivity and inflammatory markers post-intervention.
Time frame: approximately after 6 months of treatment
| Arm | Measure | Group | Value (MEAN) | Dispersion |
|---|---|---|---|---|
| Omega-3 | EPA and DHA Concentrations in Plasma | EPA Baseline | 0.95 percentage of total free fatty acid | Standard Error 0.22 |
| Omega-3 | EPA and DHA Concentrations in Plasma | EPA Post-Intervention | 6.0 percentage of total free fatty acid | Standard Error 0.92 |
| Omega-3 | EPA and DHA Concentrations in Plasma | DHA Baseline | 0.89 percentage of total free fatty acid | Standard Error 0.23 |
| Omega-3 | EPA and DHA Concentrations in Plasma | DHA Post-Intervention | 3.5 percentage of total free fatty acid | Standard Error 0.84 |
| Placebo | EPA and DHA Concentrations in Plasma | DHA Post-Intervention | 0.90 percentage of total free fatty acid | Standard Error 0.18 |
| Placebo | EPA and DHA Concentrations in Plasma | EPA Baseline | 1.2 percentage of total free fatty acid | Standard Error 0.27 |
| Placebo | EPA and DHA Concentrations in Plasma | DHA Baseline | 1.2 percentage of total free fatty acid | Standard Error 0.39 |
| Placebo | EPA and DHA Concentrations in Plasma | EPA Post-Intervention | 1.1 percentage of total free fatty acid | Standard Error 0.19 |