Elevated Plasma Glucose and Lowered Triglyceride ... - Diabetes Care

Elevated Plasma Glucose and Lowered Triglyceride Levels From Omega-3 Fatty Acid Supplementation in Type II Diabetes

We studied the effect of omega-3 fatty acids (co3FA) on glucose homeostasis and lipoprotein levels in eight type II (non-insulin-dependent) diabetic subjects ingesting 8 g/day to3FA for 8 wk as marine-lipid concentrate capsules. After w3FA supplementation, fasting plasma glucose levels increased 22% (P = .005) and meal-stimulated glucose increased 35% (P = .036). The percentage of glucose elevation correlated with percentage ideal body weight (r = .73, P = .04). No significant changes were seen in fasting or meal-stimulated plasma insulin, glucose disposal, or insulin-to-glucagon ratios. Very-low-density lipoprotein cholesterol and triglyceride (TG) levels showed consistent reductions of 56% (P < .001) and 42% (P < .001), respectively, after o>3FA supplementation. Total cholesterol levels decreased 7% (P < .05) without alteration in low- or high-density lipoprotein cholesterol. Thus, o>3FA supplementation at a dose of 8 g/day significantly improves plasma TG levels but increases fasting and meal-stimulated glucose concentrations in the type II diabetic patient not treated with insulin or sulfonylurea agents. Marine-lipid concentrate capsules supplying large amounts of to3FAs should be used cautiously in the type II diabetic patient. Diabetes Care 12:276-81, 1989

mega-3 fatty acids (a>3FAs) derived from fish oils have been shown to lower plasma lipid levels when given to normal (1-4) and hypertriglyceridemic (4-6) subjects. These fatty acids appear to reduce triglyceride (TG) synthesis, producing a marked decrease in very-low-density lipopro-

O

From the Division of Metabolism, Endocrinology and Nutrition, Department of Medicine, University of Washington, Seattle, Washington. Address correspondence and reprint requests to John W. Ensinck, MD, Department of Medicine, RC-14, University of Washington, Seattle, WA 98195.

276

Karen E. Friday, MD Marian T. Childs, PhD Christine H. Tsunehara, RD Wilfred Y. Fujimoto, MD Edwin L. Bierman, MD John W. Ensinck, MD

tein cholesterol (VDL-chol) and TG levels (1-8). Smaller reductions are seen in low-density lipoprotein (LDL) cholesterol levels in normal subjects (1,2), whereas variable LDL changes have been noted in hyperlipidemic subjects (4,6-8), including a paradoxical increase in LDL-chol in subjects with familial combined hyperlipidemia (7). Coronary heart disease is the major cause of morbidity and mortality in the type II (non-insulin-dependent) diabetic patient. For this reason, the use of a>3FA to reduce the risk of coronary heart disease by lowering TG and cholesterol levels (1-6) and platelet aggregation (3,9,10) would seem justified, provided diabetic individuals respond the same as normal subjects. Theoretically, o>3FA-rich diets could change membrane composition in a manner that alters insulin action. A recent study suggests that in type II diabetic subjects, insulin sensitivity is increased after addition of co3FA to the diet (11). Epidemiologic studies showing a low incidence of diabetes in Eskimo populations (12-14), who consume large amounts offish (15), would tend to support this hypothesis. Thus, it appears that oo3FA supplementation could potentially benefit the type II diabetic patient in three ways: 7) improved glucose homeostasis, 2) reduced TG and cholesterol levels, and 3) decreased platelet aggregation. This study was designed to test whether supplemental w3FA derived from fish oils affects lipoprotein levels and glucose homeostasis in type II diabetic subjects. MATERIALS AND METHODS

Subjects. Participants included eight type II diabetic men not treated with insulin or sulfonylurea agents for at least 2 mo before enrollment and during the 8-wk

DIABETES CARE, VOL. 12, NO. 4, APRIL 1989

K.E. FRIDAY AND ASSOCIATES

study (Table 1). Other medications were held constant. Their weights ranged from 95 to 139% (112 ± 6%, mean ± SE) of ideal body weight (IBW) (16) and remained stable throughout the study. The protocol was approved by the Human Subjects Review Committee, and each volunteer gave informed consent before participation. Diet. Diet histories were obtained at the beginning and end of the 8-wk study. In addition, each subject recorded a 3-day diet diary after 4 wk of a>3FA supplementation. Each subject was instructed to decrease his fat intake by 15 g/day to compensate for calories in the marine-lipid concentrate. No other changes were made in the subjects' regular diets. Baseline diet contained 2263 ± 2 1 4 cal/day, 303 ± 78 g cholesterol/day, 32 ± 3% fat, 19 ± 1% protein, and 49 ± 3% carbohydrate (mean ± SE). No significant changes were detected in dietary composition and seafood consumption as assessed by diet diaries and histories at 4 and 8 wk. Fatty acid supplementation. Each subject received ~8 g o)3FA/day for 8 wk provided in 15 marine-lipid concentrate capsules per day marketed as RES-Q1000 (Pharmacaps, Elizabeth, NJ). Each capsule contained 1000 mg methyl ester fatty acids, providing 200 mg docosahexaenoic acid (DHA, 22:6w3), 300 mg eicosapentaenoic acid (EPA, 20:5a>3), and 1 mg cholesterol. Subjects took 5 capsules 3 times daily with meals. Protocols. Metabolic studies were performed on 2 consecutive days in the fasting state before and after 8 wk of w3FA supplementation. Peripheral venous blood samples were drawn 15, 10, 5, and 0 min (baseline) before and 30, 60, 90, 120, 180, and 240 min after ingestion of a liquid mixed meal (Ensure Plus), providing 15 cal/kg body wt not exceeding 1080 cal. Meal composition was 53% carbohydrate, 15% protein, and 32% fat. Plasma insulin, glucose, and glucagon levels were measured in each blood sample. Increments in insulin and glucose over mean baseline values were calculated for 4 h after ingestion of the liquid mixed meal and reported as arbitrary incremental area units. Arterialized venous blood samples were obtained 15, 10, 5, and 0 min before and 10, 12, 14, 16, 19, 22, 25, and 30 min after an intravenous injection over TABLE 1 Subject characteristics

Subjects

Age (yr)

Baseline weight (kg)

Postweight (kg)

Height (cm)

Baseline ideal body weight (%)

1 2 3 4 5 6 7 8

57 58 62 62 65 69 70 70

98.0 73.0 55.3 85.5 56.5 76.2 78.7 63.0

100 71.6 55.5 85.3 57.4 75.1 79.9 63.2

175 173 159 168 158 186

139 104 99 132 102 95

181 156

104 118

DIABETES CARE, VOL. 12, N O . 4, APRIL 1989

1 min of 50% dextrose in water providing 0.3 g glucose/kg body wt. Insulin and glucose levels were measured in each plasma sample. Glucose disposal (Kg) was calculated as Kg = (0.69 -r t1/2)100. Measurements. Enzymatic methods were used for cholesterol and TC quantification of whole plasma and plasma fractions (17). Density (d) ultracentrifugation of whole plasma (d = 1.006 g/ml) was performed at 105,000 x g for 18 h to separate VLDL (d < 1.006) from LDL and high-density lipoprotein (HDL; d > 1.006) (18). HDL was isolated from the d > 1.006 fraction with dextran sulfate-magnesium precipitation (19). If turbidity was detected after dextran sulfate-magnesium precipitation in hypertriglyceridemic plasma, thesupernatant was filtered with a 0.22-|xM filter to remove uncompletely sedimented LDL aggregates before analysis of the supernatant for HDL-chol (19). LDL-chol was calculated as the difference between the d > 1.006 fraction and HDL-chol (18). VLDL-chol was calculated as the difference between total plasma cholesterol and the d > 1.006 fraction (18). Measured VLDL-chol in the d < 1.006 lipoprotein fraction (results not shown) served as quality control for calculated VLDL-chol (18). Plasma and erythrocyte membrane fatty acid composition was determined before and after 8 wk of o>3FA supplementation in each subject. Plasma fatty acids were determined after lipid extraction by the method of Folch et al. (20) and those in erythrocyte membranes after extraction with chloroform isopropanol (21). Fatty acids were transesterified with methanolic-H2SO4 and determined by gas chromatography with a SP2330 capillary column, a Hewlett-Packard chromatograph 5790A with flame ionization detector, and a Hewlett-Packard 3390A integrator. Fatty acid peaks were identified by comparison with standard mixtures of fatty acids. Plasma glucose concentrations were measured by the glucose oxidase method with a YSI model 23A glucose analyzer. Fasting plasma glucose values represent the mean of eight values obtained on 2 days ( - 1 5 , - 1 0 , - 5 , and 0 min of liquid mixed-meal administration and intravenous glucose tolerance test). Plasma insulin was measured by radioimmunoassay with a double-antibody method with guinea pig antiinsulin serum supplying the first antibody and goat antiguinea pig serum as the second antibody (22,23). Glucagon was quantitated with a polyethylene glycol precipitation assay (24). Fasting plasma insulin and glucagon represents the mean of eight values obtained on 2 days ( - 1 5 , - 1 0 , - 5 , and 0 min of liquid mixedmeal administration and intravenous glucose tolerance test). Fasting blood was collected in EDTA tubes, and stable glycosylated hemoglobin was analyzed with a Glycaffin GHB test (Isolab, Akron, OH). Statistics. Data are expressed as means ± SE and analyzed with the two-tailed Student's paired t test. When measurements did not show a normal distribution, data were compared with the nonparametric Wilcoxon's signed-rank test. Correlation coefficients were calculated with the least-squares linear regression method.

277

OMEGA-3 FATTY ACIDS IN TYPE II DIABETES

RESULTS Fatty acid plasma and erythrocyte m e m b r a n e c o m position. Supplementation with w3FA induced significant changes in erythrocyte and plasma fatty acid composition. DHA comprised 2.4 ± 0.4% of the total plasma fatty acid at baseline, increasing to 6.0 ± 0.4% (P < .001) after w3FA supplementation, whereas erythrocyte DHA rose from 3.5 ± 0.9 to 7.0 ± 0.9% (P = .01) of total fatty acids. EPA accounted for 0.7 ± 0.2% of the total plasma fatty acid at baseline and rose to 7.8 ± 1.1% (P < .001) after the addition of w3FAs, whereas erythrocyte EPA increased from 0.5 ± 0.2 to 3.9 ± 0.5% ( P < .001). Plasma lipoprotein composition. Supplementation with co3FA induced a reduction of 56 ± 6% in VLDL cholesterol and 42 ± 8% in total TG levels (Table 2). Total cholesterol levels decreased to 7 ± 3%, suggesting that changes in VLDL-chol accounted for most of the change in total cholesterol. No consistent alterations were noted in LDL- and HDL-chol after the addition of co3FA. Five subjects increased their LDL-chol levels, resulting in a mean increase of 13 ± 9% for the group. Likewise, HDL-chol increased in four subjects with a mean increase of 8 ± 7% for the group. An inverse linear relationship existed between absolute change in LDL-chol and absolute decrease in total TG levels (r = - . 8 9 , P = .003). Changes in lipoprotein concentrations did not correlate with percentage IBW, fasting plasma glucose levels, percentage of glucose change,

or the degree of DHA and EPA enrichment in erythrocytes or plasma. Glucose homeostasis. Fasting plasma glucose rose in all but one subject after 8 wk of w3FA supplementation (22 ± 5%, P = .005). Mean plasma glucose increased from 159 ± 13 to 193 ± 16 mg/dl. The change in plasma or erythrocyte DHA and EPA concentrations did not correlate with the degree of glucose elevation. Percentage glucose change was positively correlated with percentage IBW (r = .73, P = .04; Fig. 1). To assess glucose responses to a mixed meal in this group of diabetic subjects with a wide range in fasting glucose levels (122-222 mg/dl), increments in plasma glucose over fasting levels were measured for 4 h after the ingestion of a liquid mixed meal with results expressed as incremental area units. An increase was observed in six subjects (P = .018, n = 8; Fig. 2), with a mean rise of 35 ± 15% (P = .036). The changes in fasting and postprandial glucose could not be explained by consistent alterations in glucose disposal (/,) with a mean prew3FA KR of 0.73 ± 0.04 compared to 0.81 ± 0.6 after 8 wk of co3FA (NS). Glycosylated hemoglobin values of 10.0 ± 1.0% before co3FA supplementation remained unchanged at 10.2 ± 1.4% (NS, n = 7) after 8 wk of w3FA. Plasma insulin. Fasting plasma insulin (mean of 8 fasting insulin values for each individual) before w3FA averaged 21 ± 2 ixU/ml and did not change after co3FA supplementation (22 ± 3 |xU/ml, NS). Incremental meal-stimulated insulin area units showed no consistent alteration after 8 wk of w3FA supplementation (Fig. 2).

TABLE 2 Lipoprotein alterations with omega-3 fatty acid (a>3FA) supplementation Subjects

Total cholesterol (mg/dl) Pre-u)3FA Post-3FA supplementation in untreated type II diabetic men and did not study individuals well controlled with insulin and/or oral hypoglycemic agents. Further investigation will be necessary to test whether the hyperglycemic effects of a)3FA supplementation may be reversed by aggressive diabetic control with insulin and/or oral hypoglycemic agents.

ACKNOWLEDGMENTS We thank Robin Vogel for expert technical assistance and Janetta Shepard, Jo Casterline, Ann Ferguson, Scott Johnson, and Kristi Mclntyre for preparation of the manuscript. RES-Q1000 capsules were kindly provided by Pharmacaps, Inc., Elizabeth, New Jersey. This study was supported by U.S. Public Health Service Grant ROI-AM34937 from the Diabetes Research and Education Foundation and National Institutes of Health Grants RR-37, 5-P30-AM-17047, 5-P01-HL-30086, 5-T32-HL-07028, and 1-P30-AM-35816. This study was presented in part at the 47th annual meeting of the American Diabetes Association, 7-9 June 1987, Indianapolis, Indiana.

REFERENCES 1. Harris WS, Connor WE, McMurray, MP: The comparative reductions of the plasma lipids and lipoproteins by dietary polyunsaturated fats: salmon oil versus vegetable oils. Metabolism 32:179-84, 1983 2. Illingworth DR, Harris WS, Connor WE: Inhibition of low density lipoprotein synthesis by dietary omega-3 fatty acids in humans. Arteriosclerosis 4:270-75, 1984 3. Bradlow BA, Chetty N, van der Westhuyzen J, Mendelsohn D, Gibson JE: The effects of a mixed fish diet on platelet function, fatty acids and serum lipids. Thromb Res 29:561-68, 1983 4. Nestel PJ, Connor WE, Reardon MF, Connor S, Boston SW: Suppression by diets rich in fish oil of very low density lipoprotein production in man. ) Clin Invest 74:8289, 1984 5. Phillipson BE, Rothrock DW, Connor WE, Harris WS, Illingworth DR: Reduction of plasma lipids, lipoproteins, and apoproteins by dietary fish oils in patients with hypertriglyceridemia. N Engl J Med 312:1210-16, 1985 6. Simons LA, Hickie JB, Balasubramaniam S: On the effects of dietary n-3 fatty acids (maxEPA) on plasma lipids and lipoproteins in patients with hyperlipidemia. Atherosclerosis 54:75-88, 1985 7. Failor RA, Childs MT, Bierman EL: The effect of omega-


K.E. FRIDAY AND ASSOCIATES

8.

9.

10.

11.

12. 13.

14. 15. 16. 17.

18.

19.

20. 21. 22.

3 and omega-6 fatty acid-enriched diets upon plasma lipoproteins and apoproteins in subjects with familial combined hyperlipidemia. Metabolism 37:1021-28, 1988 Sullivan DR, Sanders TAB, Trayner IM, Thompson GR: Paradoxical elevation of LDL apoprotein B levels in hypertriglyceridaemic patients and normal subjects ingesting fish oil. Atherosclerosis 61:129-34, 1986 Lorenz R, Spengler U, Fischer S, Duhm J, Weber P: Platelet function, thromboxane formation and blood pressure control during supplementation of the Western Diet with cod liver oil. Circulation 67:504-11, 1983 Von Schacky C, Fisher S, Weber PC: Long-term effects of dietary marine w-3 fatty acids upon plasma and cellular lipids, platelet function, and eicosanoid formation in humans. I Clin Invest 76:1626-31, 1985 Popp-Snijders C, Schouten JA, Heine RJ, van der Meer J, van der Veen EA: Dietary supplementation of omega-3 polyunsaturated fatty acids improves insulin sensitivity in non-insulin-dependent diabetes. Diabetes Res 4:141-47, 1987 Sagild U, Jorgen LC, Jespersen S, Andersen S: Epidemiological studies in Greenland 1962-1964. I. Diabetes mellitus in Eskimos. Acta Med Scand 179:29-39, 1966 Kromman N, Green A: Epidemiological studies in the Upernavik District, Greenland: incidence of some chronic diseases 1950-1974. Acta Med Scand 208:401406, 1980 Mouratoff GJ, Carroll NV, Scott EM: Diabetes mellitus in Athabaskan Indians in Alaska. Diabetes 18:29-32, 1969 Bang HO, Dyerberg J, Sinclair HM: The composition of the Eskimo food in northwestern Greenland. Am j Clin Nutr 33:2657-71, 1980 New weight standards for men and women. Metrop Life Insur Co Stat Bull 40:1-4, 1959 Warnick GR: Enzymatic methods for quantification of lipoprotein lipids. In Methods in Enzymology. Albers JJ, Segrest JP, Eds. Orlando, FL, Academic, 1986, p. 1 0 1 23 Lipid Research Clinics Program: Manual of Laboratory Operations. Lipid and Lipoprotein Analysis. Vol. 1. Washington, DC, U.S. Govt. Printing Office 1974 (DHEW NIH publ. no. 75-628) Warnick GR, Benderson J, Albers JJ: Dextran sulfate - M g 2 + precipitation procedure for quantification of high-density-lipoprotein cholesterol. In Selected Methods of Clinical Chemistry. Vol. 10. Cooper GR, Ed. Washington, DC, Am. Assoc. Clin. Chem., 1983, p. 91-97 Folch J, Lees M, Stanley GHS: A single method for the isolation and purification of total lipids from animal tissue. I Biol Chem 226:497-507, 1957 Rose HG, Oklander M: Improved procedure for the extraction of lipids from human erythrocytes. / Lipid Res 6:428-31, 1965 Morgan CR, Luzarow A: Immunoassay of insulin: two


antibody system: plasma insulin levels of normal, subdiabetic and diabetic rats. Diabetes 21:115-26, 1963 23. Stentz FB, Wright RK, Kitabchi AE: A rapid means of separating A14-125l-insulin from heterogenous labeled insulin molecules for biologic studies. Diabetes 31:1128-31, 1982 24. Henquin JC, Malvaux P, Lambert AE: Glucagon immunoassay using polyethylene glycol to precipitate antibodybound hormone. Diabetologia 10:61-68, 1974 25. Glauber HS, Wallace P, Brechtel G: Adverse effects of omega-3 fatty acids in non-insulin-dependent diabetes mellitus. Ann Intern Med 108:663-68, 1988 26. Topping DL, Trimble RP, Storer GB: Failure of insulin to stimulate lipogenesis and triglycerol secretion in perfused livers from rats adapted to dietary fish oil. Biochim Biophys Acta 927:423-28, 1987 27. Storlien LH, Kraegen EW, Chisholm DJ, Ford GL, Bruce DG, Pascoe WS: Fish oil prevents insulin resistance induced by high-fat feeding in rats. Science 237:885-87, 1987 28. Felig P, Wahren J, Hendler R, Brundin T: Splanchnic glucose and amino acid metabolism in obesity. J Clin Invest 53:582-90, 1974 29. Cherrington AD, Chiasson JL, Liljenquist JE, Jennings AS, Keller U, Lacy WW: The role of insulin and glucagon in the regulation of basal glucose production in the postabsorptive dog. ) Clin Invest 58:1407-18, 1976 30. Unger RH: Glucagon and the insulin:glucagon ratio in diabetes and other catabolic illnesses. Diabetes 20:83438, 1971 31. Nestel PJ, Connor WE, Reardon MF, Connor S, Wong S, Boston R: Suppression by diets rich in fish oil of very low density lipoprotein production in man. j Clin Invest 74:82-89, 1984 32. Sanders TAB, Sullivan DR, Reeve J, Thompson GR: Triglyceride-lowering effect of marine polyunsaturates in patients with hypertriglyceridemia. Arteriosclerosis 5:45965, 1985 33. Rose HG, Haft GK, Juliano J: Clofibrate-induced low density lipoprotein elevation: therapeutic implications and treatment by colestipol resin. Atherosclerosis 23:413-27, 1976 34. Packard CJ, Munro A, Lorimer AR, Gotto AM, Shepard J: Metabolism of apolipoprotein B in large triglyceride-rich very low density lipoproteins of normal and hypertriglyceridemic subjects. I Clin Invest 74:2178-92, 1984 35. Schectman G, Kaul S, Kissebah A: Effect of fish oil on plasma lipoproteins and platelet function in non-insulindependent diabetes mellitus (NIDDM) (Abstract). Diabetes 36 (Suppl. 1):12A, 1987 36. Walden CE, Coleman J, Retzlaff B, Childs MT, Albers JJ, Knopp RH: Considerations in the therapeutic use of fish oils in hypertriglyceridemia (Abstract). Clin Res 35:371 A, 1987

281