Lipodystrophic diabetes mellitus. Investigations of lipoprotein metabolism and the effects of omega-3 fatty acid administration in two patients

Lipodystrophic Diabetes Mellitus. Investigations of Lipoprotein Metabolism and the Effects of Omega-3 Fatty Acid Administration in Two Patients Peter W. Stacpoole,

Janice Alig, Laura L. Kilgore, Clifford M. Ayala,

Peter N. Herbert,

Loren A. Zech, and Waldo

R. Fisher

We investigated the metabolic effects of omega-6 (safflower oil) and omega-3 (fish oil) fatty acid-enriched diets (65% carbohydrate, 20% fat) in two patients with a syndrome of diabetes mellitus, lipodystrophy, acanthosis nigricans, chylomicronemia. and abdominal pain. 3H-glycerol was used to evaluate triglyceride-rich lipoprotein-triglyceride (TRLP-TG) metabolism, and changes in glucose and insulin dynamics were also studied. On the omega-6 diet, both subjects demonstrated four- to five-times normal rates of TRLP-TG production and glycerol biosynthesis, and striking decrements in the fractional catabolic rate (FCR) for TRLP-TG and TRLP-particles. Both subjects had elevations in nonesterified fatty acid (NEFA) concentrations. In one patient, the omega-3 diet markedly decreased serum triglycerides and newly synthesized triglyceride glycerol production, in association with a fall in NEFA. In both subjects, plasma glycerol reutilization for triglyceride synthesis, normal on the omega-6 diet, was abolished on the omega-3 regimen. Plasma postheparin lipolytic activity was normal on both diets. On the omega-3 diet, xanthomas and hepatomegaly decreased and, in the patient who had no reduction in serum triglycerides, pancreatitis attacks virtually ceased. Mean 24-hour serum glucose levels were higher, and both basal and peak C-peptide responses to a carbohydrate meal were blunted on the omgea-3 diet. One patient became ketonuric. We conclude the cause of hypertriglyceridemia in these patients was due to increased lipid synthesis and hypothesize that this is secondary to high plasma concentrations of NEFA. In addition, an omega-3 diet in these subjects inhibited insulin secretion and worsened glucose tolerance. o 1988 by Grune & Stratton, Inc.

P

ATIENTS with lipodystrophic diabetes mellitus constitute an uncommon group of diabetics whose clinical findings include insulin resistance, acanthosis nigricans, hyperlipidemia, and various maturational and endocrine disorders.‘” Insulin resistance in this condition is usually accompanied by fasting hyperinsulinemia and has been ascribed to defects proximal to, at, or distal to the level of the is the most freinsulin receptor. 4-l’ Hypertriglyceridemia quent lipid abnormality in subjects with lipodystrophic diabetes, and may lead to chylomicronemia, eruptive xanthomata, and abdominal pain due to hepatosplenomegaly or pancreatitis (chylomicronemia syndrome, cf I’). The etiology of the hyperlipidemia in lipodystrophic diabetes is uncertain. Studies in three patients by Kissebah,‘**” Franklin,14 and coworkers suggest that both overproduction and underutilization of triglyceride-rich (chylomicron and very low density) lipoprotein triglyceride (TRLP-TG) may contribute to the hyperlipidemia. Although treatment with low-fat diets14 or medium chain triglyceride preparations” may reduce circulating lipid levels in such patients, the mechanism for this effect is unknown.

In recent years, considerable attention has focused on the lipid-lowering properties of omega-3 fatty acids, present in fish and other marine food. M*‘~In patients with phenotypes IV or V hyperlipoproteinemia, administration of a fish-oil enriched diet may lead to rapid and striking reductions in circulating triglycerides.‘*-*’ Although a few patients with hyperlipidemia treated with omega-3 fatty acids also had diabetes mellitus,” the effects of increased dietary omega-3 fatty acids on carbohydrate metabolism in normal or diabetic subjects have not been explored. In the present investigation, radiolabeled glycerol was used as a precursor for TRLP-TG and the kinetics were determined in two subjects with lipodystrophic diabetes and chylomicronemia. Heparin-releasable hepatic triglyceride hydrolase and lipoprotein lipase activities were also measured. Studies were conducted under two steady-state conditions, while patients received diets enriched in either omega6 (safflower oil) or omega-3 (fish oil) fatty acids. In addition, the effects of dietary fat substitution on diabetes control and pancreatic islet cell function were evaluated. MATERIALS

AND METHODS

Patients From the Departments of Medicine (Division of Endocrinology and Metabolism), Pharmacology, and Biochemistry, and the Clinical Research Center. University of Florida, College of Medicine, Gainesville. FL; the Department of Medicine (Division of Nutrition and Metabolism), The Miriam Hospital, Brown University, Providence, RI; and the Laboratory of Theoretical Biology. (Division of Cancer Biology and Diagnosis), National Cancer Institute, National Institutes of Health, Bethesda, MD. Supported by grants HL-32550 and HL-29394from the National Institutes of Health and by a gift from R.P. Scherer, Co. Address reprint requests to Peter W. Stacpoole, PhD, MD, Division of Endocrinology and Metabolism. Box J-226, University of Florida, College of Medicine, Gainesville, FL 32610. o 1988 by Grune & Stratton, Inc. 0026-0495/88/3710-0008603.00/O

944

Both subjects were studied on the Clinical Research Center ward of Shands Hospital, University of Florida. Informed consent was obtained prior to investigation. Patient no. 1 (weight, 41.5 kg; height, 160 cm) was a 21-year-old white male. He had been treated since infancy with diphenylhydantion for a generalized seizure disorder and received Dilantin (1 g/d) throughout the study. Progressive loss of subcutaneous fat began at about age 7 years. Total lipoatrophy, non-insulin-dependent diabetes mellitus, insulin resistance, acanthosis nigricans, eruptive xanthoma, chylomicronemia, and massive hepatosplenomegaly were found at age 17 years. A detailed clinical description of this patient has been published.” Substitution of medium-chain triglycerides for longchain fatty acids markedly decreased both hyperinsulinemia and serum lipid levels, but outpatient compliance was poor and chylomicronemia recurred. Upon referral to the Lipid Clinic at Shands Metabolism, Vol37, No 10 (October), 1988: pp 944-951

MARINE LIPID EFFECTS IN LIPODYSTROPHIC DIABETES

Hospital in February, 1986, the patient had maintained serum triglycerides between 3,000 and 7,000 mg/dL for several years and had sustained repeated attacks of abdominal pain thought to be due to hepatic enlargement. Pancreatitis was never documented. Acanthosis nigricans was confined to the axillae. Eruptive xanthomas were present extensively over the trunk and limbs. No family member had diabetes or hyperlipidemia. The patient had never been treated chronically with insulin or oral glucose-lowering drugs. Patient no. 2 (weight, 52.4 kg; height, 168 cm) was a 20-year-old white female college student. Her past history was notable for chickenpox at age 3 years, followed shortly thereafter by the onset of acanthosis nigricans and lipodystrophy. At age 15 years, a glucose tolerance test disclosed normal fasting and postprandial blood sugar levels, but a fasting insulin level was elevated at 22 pU/mL and increased to 238 pU/mL two hours after glucose ingestion. Additional findings at that time included oligomenorrhea since menarche 2 years earlier, normal intellectual and physical growth and development, normal secondary sex characteristics, mild anemia of unknown cause, mild elevation of SGPT activity, normal serum thyroxine, testosterone, androstenedione, and dehydroepiandrosterone sulfate levels, and normal ovaries by ultrasonography. At age 16 years she experienced her first attack of abdominal pain due to pancreatitis. Chylomicronemia and insulin-dependent diabetes mellitus were diagnosed at that time. Physical examination also revealed atrophy of the subcutaneous fat of the face and limbs, with prominent sparing of a collar of fatty tissue around the neck. Acanthosis nigricans involved the axillae, arms, trunk, legs, and feet. Eruptive xanthomata soon developed over the elbows, lower legs, ankles, and the sole of one foot. She subsequently sustained 14 more bouts of pancreatitis associated with serum triglyceride levels between 2,000 and 5,000 mg/dL. When referred to the Shands Lipid Clinic in July, 1984, she was receiving 17 units of NPH insulin and maintaining near-normoglycemia between attacks of pancreatitis. Further evaluation at that time disclosed normal serum prolactin, LH, and FH levels.

Study Design One month prior to admission, each subject was instructed on a weight-maintaining diet having a caloric distribution of 20% total fat, 65% carbohydrate, and 15% protein. The sources and quantities of fat included vegetable oils and margarines (19.3 g/ 1,000 kcal on the omega-6 diet and 3.3 g/ 1,000 kcal on the omega-3 diet) and fish oil (I 6.0 g/ 1,000 kcal on the omega-3 diet). The fish oil was provided as MaxEPA capsules (R.P. Scherer Co, Troy, MI) in divided doses. The range of daily cholesterol intake was 40 to 47 mg/ 1,000 kcal on the omega-6 and 114 to 147 mg/ 1,000 kcal on the omega-3 regimen, respectively. Each l-g MaxEPA capsule contained 6 mg cholesterol and accounted for the higher daily cholesterol consumption on the omega-3 diet, Following admission to the Clinical Research Center, pediatric nasogastric feeding tubes were inserted into both patients, and a constant infusion of a weight-maintaining liquid diet was maintained throughout a 2-week hospitalization. During this period, the weight of each patient was maintained within 0.5 kg. The caloric distribution was 20% fat (22 g/L), 65% carbohydrate (163 g/L) and 15% protein (38 g/L), with approximately 116 mg cholesterol per liter and a caloric content of about 1,000 kcal/L. During the first admission, a formula was administered consisting of nonfat dry milk, glucose polymers (Polycose, Ross Labortories, Columbus, OH), cooked egg yolk, egg substitute (Eggbeaters, Nabisco Brands, Inc, East Hanover, NJ) and safflower oil (Microlipid, ChesebroughNutricia, Inc. Greenwich, CT). During the second admission, most of the safflower oil emulsion was substituted by a fish oil preparation (Dale Alexander Emulsified Super MaxEPA, Twinlabs, Inc, Ronkonkoma, NY). Each formula matched its respective preadmission

945

Table 1. Fatty Acid Composition Omega-&Supplemented

of Omega-6

Omega-6

Composition Total fat (per L or 1,000 kcal)

and

Liquid Diets

ISafflower Oil)

Omega-3

(FishOil1

21.7 (100)

22.2 (100)

Saturated fat

2.9 (13.8)

5.1 (23.0)

Omega-9

3.9 (18.0)

5.5 (24.8)

Omega-6 (1 B:2, 20:4) Total omega-3 Linolenic (1 B:3)

14.1 (85.0)

5.8 (26.1)

0.2 (1 .O)

4.8 (21.5)

0.2 (0.9)

0.1 (0.3)

EPA (20:5)


DHA (22%)

to. 1 (10.1)

(
2.8 (12.8) 1.8 (8.4)

Values are given in grams, with fat percentage in parentheses.

diet in terms of omega-3 fatty acid content. The fatty acid composition of each liquid formula diet is summarized in Table 1. Patient no. 1 received 2,700 kcal/d, for a total omega-3 fatty acid intake of 12.7 g/d. Patient no. 2 received 1,300 kcal/d, or 6.4 g/d of omega-3 fatty acids. For each subject, 15% of total daily calories was provided by fish oils which, in turn, provided 4.2% of total daily calories as omega-3 fatty acids. During the period of tube feedings, but at least four days prior to injection of ‘H-glycerol, blood was obtained immediately before and ten minutes after intravenous (IV) injection of 75 IU heparin per kilogram of body weight for measurement of plasma nonesterified fatty acids (NEFA) and lipolytic activity. After the final blood was drawn for the kinetic studies, the tube was withdrawn and the respective omega-6 or omega-3 preadmission diet was reinstituted as three daytime feedings and a bedtime snack. The evening insulin dose was withheld for patient no. 2 on the second day of solid food intake. On the morning of the third day, each subject drank 7 mL/kg of a mixed meal (Sustacal, Mead Johnson, Evansville, IN) and blood was obtained for determination of serum glucose, insulin, and C-peptide levels. The caloric distribution of Sustacal is 24% protein (soy protein and sodium caseinate), 21% fat (comprised of linoleic, oleic, and palmitic acids providing a polyunsatured:saturated fat ratio of 2.5) and 55% carbohydrate (primarily sucrose, glucose, and maltose polymers).

Analysis of Samples Serum glucose was measured by a Beckman Glucose Analyzer. Triglycerides were assayed in plasma samples using the Boehringer Manheim enzymatic triglyceride assay kit?’ After five to seven days of stable weight and serum triglycerides, 150 &i ‘H-glycerol (New England Nuclear, Boston, MA) was injected IV and lo-mL blood samples were obtained for glycerol triglyceride specific radioactivity at %, 1,2, 3,4,6, 9, 12, 16, 21, 27, 34, 45, 57, and 69 hours following isotope injection. Blood was collected in the presence of merthiolate, sodium EDTA, and sodium azide (0.1% of each) and was chilled immediately. Centrifugally separated plasma was stored briefly under N, at 4OC. overlayered with 1.006 g/mL KBr solution and then ultracentrifuged at 40,000 rpm for 18 hours, in a Ti50 rotor to separate TRLP. Measured aliquots of these were assayed for triglyceride, and lipids were extracted using the method of Bligh and Dyer.‘* Phospholipids were precipitated from diethyl ether with silicic acid, and the supernatants were transferred to counting vials and evaporated. The glyceride-containing lipids were resolubilized in a toluene-based scintillation fluid and dpm were measured. Nonesterified fatty acids (NEFA) were measured colorimetricalIY,*~using the NEFA Kit K supplied by Shoji Kaisha, Ltd (Osaka, Japan). Postheparin lipases were assayed in plasma obtained ten minutes after the IV injection of heparin, 75 IU per kilogram of body weight. The method for quantifying lipoprotein lipase and hepatic

946

STACPOOLE ET AL

Fig 1. Compartmental model for triglyceride-glycerol metabolism showing three subsystems: plasma glycerol in equilibrium with nonplasma glycerol sources, glycerol conversion pathways by which glycerol is esterified to form cellular triglyceride, and the plasma TRLP-TG delipidation pathwey. Nonplasma derived unlabeled glycerol (endogenously synthesized) enters the TRLP-TO subsystem independently. Glycerol is recycled from TRLP-TG to plasma glycerol kf?.

triglyceride hydrolase was identical to that used previously,*’ except that plasma was diluted 1:2 (v/v) with 0.15 mol/L NaCl before a 20-pL aliquot was taken for analysis. Dilution before assay yields significantly higher values for both lipoprotein lipase and hepatic triglyceride hydrolase activities. Control values for this assay were obtained from a study of 12 normal men.25Total plasma concentrations of apolipoproteins A-lz6 and B2’were measured by radioimmunoassay. Analysis of Data The kinetic data were analyzed using a linear, first-order multicompartmental model of triglyceride metabolism (Fig 1) described by Zech et a12*and the Simulation, Analysis and Modeling (SAAM) computer program.r9 The model was developed for analyzing VLDL triglyceride kinetics, and it proved to fit the data from our subjects equally well. The model includes three subsystems (Fig 1). The first describes the metabolism of free glycerol in plasma and comprises compartments 4 and 5 of Fig 1. The second subsystem comprises the conversion of plasma glycerol to plasma TRLP-TG via fast (compartments 10 and 14) and slow (compartment 24) pathways. The final subsystem includes a multicompartmental delipidation chain for TRLP-TG. The steady-state kinetic analysis permits the calculation of the rate of production or utilization of TRLP-TG and VLDL-glycerol. Since the glycerol tracer is injected into plasma, plasma glycerol utilization for TRLP-TG production is directly measured. By difference, the utilization of nonplasma-derived glycerol for triglyceride synthesis may be calculated; this is designated as newly synthesized

Fig 2. Plot of measured triglyceride specific activity for subject no. 1 on safflower oil diet (Cl) and fish oil diet t 0 1. The curves are computer-generated solutions to the model shown in Fig 1.

Each compartment in the TRLP-TG system is defined by two parameters, eg, L(6,l) and L(4,1), the sum of which represents the fractional turnover rate of compartment 1. The fraction of triglyceride that irreversibly leaves the system at each step in this chain is represented by L(4,1)/L(6,1) and L(4,l) and is the fractional catabolic rate (FCR) for triglyceride; its reciprocal is the residence time for triglyceride (the average time a molecule of TRLP-TG remains in plasma).** Similar parameters are calculated for plasma metabolism of the triglyceride-containing lipoprotein particles metabolized along the pathway from compartments 1 to 8 and out. In the original development of the glycerol model, the utilization of plasma glycerol for triglyceride synthesis had to be modeled by two parallel pathways that convey glycerol from compartment 4, plasma glycerol, to compartment 1, newly secreted TRLP-TG. The justification for this model has been presented; in normal subjects the pathway via the chain, compartments 10 to 14, has the faster turnover.28 RESULTS

Lipid and Lipoprotein Metabolism Figures 2 and 3 present the measured plasma TRLP-TG specific radioactivity, expressed as dpm per milliliter of

plasma v time, for patients no. 1 and no. 2, respectively, while receiving safflower- and fish-oil-supplemented diets. (The measured values of dpm per milligram of triglyceride were multiplied by plasma triglyceride concentration, milligram per milliliter, for convenience in analyzing the data.) Excellent fits of the data from each study were found, using the

33

40

TIRE

(ht.1

947

MARINE LIPID EFFECTS IN LIPODYSTROPHIC DIABETES

d P II / ID

1 Fig 3. Plot of measured triglyceride specific activity for subject no. 2 on safflower oil diet (0) and fish oil diet f 0 1. The curves are computer-generated solutions to the model shown in Fig 1.

I ,,,,

20

TIME

of VLDL Metabolism

in Two Patients With Lipodystrophic

Parsmater Triglyceride (mg/dL) Mass TRLP-TG (mg) Total TRLP-TG production

2,835

k 1 lo*

53.300

,,,,

40

50

I...~

so

70

(hr)

Diabetes Mellitus and in 13 Normal Controls

Patient No. 1 SsfflOWer Oil

,..I

by a modest fall in the FCR of TRLP-TG. In contrast, a fish oil diet in patient no. 2 produced no significant change in plasma triglyceride concentration, TRLP-TG production, glycerol biosynthesis for secretion into triglyceride, or the TRLP-TG FCR. The rate of plasma glycerol utilization, which was normal in each patient on the safflower oil diet, was virtually abolished while receiving fish oil, indicating a major decrease in secretion of triglycerides derived from reesterification of glycerol. As shown in Table 3, the hypertriglyceridemia of both patients was associated with severalfoId elevations in the plasma concentration of NEFA. Institution of a fish oil diet decreased the NEFA level 57% in patient no. 1, and 26% in patient no. 2. The heparin-stimulated activities of hepatic

model for TRLP-TG described above. Table 2 displays data of the model parameters for both subjects on each diet, compared with mean values obtained in 13 normolipidemic males, previously described by Zech et a1.28Under steadystate conditions on a safflower oil diet, both patients demonstrated persistent chylomicronemia and four- to five-fold increases in TRLP-TG production; most of this occurred via glycerol biosynthesis. At the same time, the FCR for both TRLP-TG and TRLP-particles decreased to about one third of normal. Upon changing to a fish oil-supplemented regimen, patient no. 1 demonstrated a 45% decrease in circulating triglycerides. This was attended by similar decrements in TRLP-TG production and newly synthesized triglyceride glycerol, and Table 2. Parameters

3J

PatientNo. 2 Fish oil 1,576

+ 30

26,000

S&lower Oil 2,724

+ 350

Fish Oil 3.057

? 289

Normal 130

69,900

70.400

4,200

3,449

1,674

4.392

4.248

806

TRLP-glycerol production

371

180

472

457

87

(mg/h) Newly synthesized triglyceride

352

180

457

72

(mg/h)

glycerol (mg/h) Plasma glycerol utilized for

19

tl

15

<1

triglyceride synthesis 0.19

FCR TRLP-TG (per h)

0.0647

0.0642

0.0630

0.0604

Residence time TRLP

15.4

15.8

15.9

16.6

0.0588

0.0467

0.0328

0.0350

0.12

21.4

30.4

26.6

8.3

5.3

triglyceride (hi FCR TRLP-particles (per h) Residence time TRLP particles

17.0

(hi L(24.41 (per hi

3.0&1t

5.3E-5

3.3E-1

7.7E-5

4.7E-2

L(10,4) (per hi Ratio L(24,4)/L(10,4)

1.4E- 1

6.4E-5

1.8E- 1

5.2E-5

1.2E-1

2.2

0.84

1.9

1.5

0.39

*Mean + SE for 9 to 11 determinations obtained throughout each hospitalization. TEquivalent to 3.0 x 10-l.

948

STACPOOLE ET AL

Table 3. Effect of Dietary Omega-6 and Omega-3 Fatty Acids on the Plasma Concentrations of Triglycerides, Nonesterified Fatty Acids, and Apoliproteins A-l and 6 and on the Plasma Activities of Hepatic Triglyceride Hydrolase and Lipoprotein Lipase in Two Patients With Lipodystrophic Diabetes Mellitus

Patient/Diet

Plasma Triglycerides lme/dLI

PlasmaNEFA (pEq/LI Preheparin

Postheparin

Hepatic Triglyceride Hybolese Activity (HEq NEFA/mL/hl

Lipoprotein Lipase Activity (NEqNEFA/mL/h)

Apolipoprotein A-l (mg/dL)

Apolipoprotein B (mg/dL)

1 Omega-8

2,835

* 110

4,050

15,322

13.17

20.91

89

112

Omega-3

1,576

? 30

1,734

8,828

12.27

18.51

109

101

2 Omega-6

2,724

+ 350

2,732

9,726

6.18

11.94

79

82

Omega-3

3.057

f 289

2,025

8,385

11.13

17.58

61

354 r 145

13.3 ? 3.2

17.0 + 5.3

101 i 24

97 + 23

48

50

25

49

49

Normal ? SD Reference

89 + 37 47

75

Patients were studied after five weeks of isocaloric diets enriched in either omega-6 or omega-3 fatty acids. Blood was obtained immediately before and ten minutes after IV injection of heparin (75lU/kg).

Samples were analyzed as described in Materials and Methods.

triglyceride hydrolase and lipoprotein lipase in both subjects were normal on the safflower oil diet and did not change appreciably on the fish oil regimen. Carbohydrate Metabolism

An unexpected finding during the course of these studies was an apparent worsening of postprandial glucose tolerance in both patients while treated with fish oils. During the final 2 weeks of constant nasogastric administration of the safflower oil diet, patient no. 1 had a mean *SEM serum glucose of 252 + 12 mg/dL (n = 10) and was not treated with insulin or oral glucose-lowering agents. Patient no. 2 had a mean glucose level of 169 f 16 mg/dL (n = 8) and had no ketonuria while receiving a split dose of regular and NPH insulin that totaled 47 U/24 h. However, during the final 2 weeks of fish oil administration (a total of 6 weeks on a fish oil diet), the mean serum glucose concentration of patient no. 1 was 363 f 12 mg/dL (n = 9) and that of patient no. 2 was 308 + 13 mg/dL (n = 29). During the fish oil regimen, patient no. 2 frequently registered small to moderate ketonuria on “dipstick” and required steadily increasing doses of insulin to a maximum of 62 U/24 h. Neither subject became acidotic or hyperkalemic. To assess the effects of the two diets on pancreatic B cell function, blood was obtained before and after consumption of 7 mL/kg of Sustacal for serum glucose, insulin, and Cpeptide determinations. Each test was conducted three days after discontinuing tube feedings but while maintaining an isocaloric diet supplemented with either safflower or fish oil. As shown in Table 4, fasting serum glucose levels in both patients were higher on the fish oil than on the safflower oil regimen. Following Sustacal administration to patient no. 1, serum glucose on the safflower oil diet increased to a maximum of 248 mg/dL, or 48% above basal, and increased to a maximum of 362 mg/dL, or 92% above basal, on the fish oil diet. The peak glucose excursion following Sustacal in patient no. 2 was similar on both diets, although the incremental change from basal glucose levels was 88 mg/dL on the omega-6 diet and only 48 mg/dL on the omega-3 regimen. As noted in other subjects with lipodystrophic diabetes mellitus,‘0.‘4 basal serum C-peptide levels in both subjects on the safflower oil diet were high, relative to that of healthy controls or patients with diabetes but without lipo-

dystrophy (J. Silverstein and N. MacLaren, personal communication). However, basal C-peptide concentrations were decreased 37% in patient no. 1 and 55% in patient no. 2 while receiving fish oils, and the C-peptide response to Sustacal was blunted in both individuals. Similarly, basal and stimulated insulin secretion were also markedly diminished in patient no. 1 on the fish oil diet. DISCUSSION

Lipid Metabolism in Lipodystrophic Diabetes Mellitus

The results of these investigations are noteworthy for several reasons. First, they demonstrate a marked increase in TRLP-TG production in these patients with lipodystrophic diabetes mellitus. Similar findings have been reported by Franklin and coworkersI in a 2-year-old child with diabetes and complete lipoatrophy. In this child, studies with 13’1VLDL and ‘H-glycerol showed increased synthesis of both the apoB and triglyceride components of VLDL. Treatment with a low-fat diet led to a reduction in VLDL-TG synthesis but no change in VLDL-apoB production. Both of our subjects showed rates of triglyceride synthesis on a low-fat safflower oil diet that were fourfold to fivefold greater than Table 4. Effect of Dietary Omega-6 and Omega-3 Fatty Acids on Serum Glucose, C-Peptide, and Insulin Concentrations Following Sustacal Administration in Two Patients With Lipodystrophic Diabetes Mellitus C-Peptide(pg/mL) Insulin(HU/mL) Glucose(mg/dL) Time Patient hnin) Omega-6 Omega-3 Omega-6 Omega-3 Omega-6 Omega-3 1

2

0

167

189

3.98

2.51

159

30

203

240

4.80

3.53

280

70 95

60

248

330

5.85

4.08

464

156

SO

240

362

7.43

2.85

333

136

120

225

334

7.00

3.60

332

142

0

156

232

1.12

0.50

30

208

258

1.24

0.52

60

242

280

1.25

0.51

90

244

272

1.28

0.53

120

231

246

1.28

0.54

Patients were studied after 8 weeks of isocaloric diets enriched in either omega-6 or omega-3 fatty acids. On the morning after an overnight fast, each subject received a liquid mixed meal of Sustacal (7 mL/kg). Blood was obtained for analysis of serum glucose. C-peptide, and insulin levels as described in Materials and Methods.

MARINE LIPID EFFECTS IN LIPODYSTROPHIC

949

DIABETES

in normals, and this was associated with a fivefold to sixfold increase in glycerol secretion, arising from endogenously synthesized glycerol, rather than from plasma glycerol. As reported for a few other patients with lipodystrophy and hyperlipemia,“*3@3’ our patients also had a striking increase in plasma NEFA, reflecting either deficient clearance of plasma NEFA by fat cells or enhanced adipose tissue lipolysis. For many years it has been recognized that hepatic triglyceride synthesis is stimulated by the availability of NEFA in the hepatic perfusate.“37 In the present setting, one may presume that the accelerated hepatic use of nonplasma sources of glycerol is stimulated by the glycerol requirement for enhanced triglyceride synthesis. Also contributing to the hyperlipemia, as demonstrated in this and other studies,‘* was a decrease in the FCR of TRLP-TG and of TRLP-particles. The reason for this is uncertain. Although plasma lipolytic activity has been reported to be decreased in some patients with lipodystrophic diabetes,30 data presented here and elsewhere38 reveal that this does not always occur. We initially observed apparently low postheparin lipases in our patients but observed that this was due to competition of plasma triglycerides with our assay substrate. Dilution of postheparin plasma or clarification by ultracentrifugation revealed normal postheparin lipolytic activity. It is possible that the increased plasma residence time of TRLP-TG in lipodystrophic diabetes is due to partial saturation of the lipolytic pathway by hypersecretion of large TRLP particles that require greater time for completion of their delipidation. Effects of Fish Oils on Lipid Metabolism A second major finding of this study is that the composition of dietary fat may have important metabolic consequences in patients with lipodystrophic diabetes. An isocaloric substitution of fish for safflower oil led to a substantial fall in circulating triglycerides in patient no. 1 but no change in triglycerides in patient no. 2. The greater effect observed in patient no. 1 may have been due to his higher daily absolute consumption of omega-3 fatty acids (12.7 g), compared with that of patient no. 2 (6.1 g). The lipid-lowering response in patient no. 1 was also associated with a proportionate decrease in both VLDL and triglyceride glycerol production, while these parameters remained constant in subject no. 2. Patient no. 1 also had a marked fall in NEFA but the decrease in NEFA in patient no. 2 was modest. If enhanced availability of NEFA to the liver is the driving force for triglyceride overproduction, then the observed differences in response may be related to NEFA availability. Reduced VLDL production during administration of omega3 fatty acids in subject no. 1 was not accompanied by a change in FCR or residence time of TRLP-TG. Thus no appreciable change in the kinetics of the lipolysis reaction occurred as a result of fish oil administration. These results are similar to data obtained by others20*39 using labeled glycerol and indicate that the major effect of omega-3 fatty acids on triglyceride-rich lipoproteins is on production, rather than on removal, mechanisms. While use of a glycerol tracer provides only limited insight into mechanisms of triglyceride biosynthesis, it is neverthe-

less interesting to examine the rate constants for the two pathways by which plasma glycerol is utilized for triglyceride synthesis. For both subjects on safflower oil the rate constant for the fast pathway, L(10,4), was very similar to normals; however the slow pathway, L(24,4), was accelerated and became the predominant route for glycerol reutilization, thus reversing the ratio of L(24,4): L(10,4). When the subjects were receiving fish oil the utilization of plasma glycerol, phosphorylated by glycerol kinase, was almost abolished. Since the kinase is a predominantly hepatic enzyme, the observation may account for the subjects’ persisting chylomicronemia. Dietary fish oil appears to have a major impact on hepatic glycerol metabolism. Effects of Fish Oils on Carbohydrate Metabolism Epidemiologic investigations have indicated that populations chronically consuming large amounts of marine oils may be at decreased risk for developing diabetes mellitus.“OA In addition, recent investigations by Storlien and coworkers’* demonstrate that partial substitution of omega-6 by omega-3 fatty acids in a high-fat diet improves both peripheral and hepatic insulin sensitivity in nondiabetic rats. It was surprising, therefore, to note that substitution of fish for safflower oil in our patients resulted in deterioration of glucose tolerance. This conclusion is substantiated by increases in both fasting and postprandial hyperglycemia in our subjects and the development of ketonuria and increased insulin requirements in patient no. 2. The underlying mechanisms for these changes are uncertain, but may include inhibition of endogenous insulin secretion under both basal conditions and in response to a mixed meal. It is known that the consumption of omega-3 fatty acids results in their accumulation in the membranes of platelets and cells,43-46with relative depletion of omega-6 fatty acids at these sites. This has led to the postulate that fish oils may induce changes in the structure and fluidity of cell membranes.16 It is conceivable, therefore, that such alterations in membrane lipid composition could modify the signal for secretion of insulin from islet cells, influence the action of insulin on target tissues, or impair transport of glucose across cell membranes. These possibilities are not mutually exclusive, but will remain speculative until tested directly. It might be argued that the deterioration of carbohydrate tolerance by fish oils may reIlect a response peculiar to patients with lipodystrophy and/or extreme insulin resistance that will not occur in the general diabetic population. However, we are aware of preliminary reports, published in abstract form, from three laboratories that relate to potential effects of omega-3 fatty acids on carbohydrate metabolism. Studies by Lardinois and coworkers49 indicate that dietary omega-3 fatty acids are incorporated into the phospholipid membranes of rat pancreatic islet cells, and this effect might modify beta cell responsiveness to normal stimuli. Additional experiments” have been conducted by these investigators in which normal subjects underwent four fat tolerance tests derived from fish, cocoa butter, corn, and olive oil sources. Plasma excursions of glucose, insulin, and gastric inhibitory polypeptide (GIP) were determined following fat ingestion. Compared with the other fat challenges, the fish-oil test

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Other Effects of Fish Oils Two other interesting observations were made in patient no. 2 during her course of study. She had experienced persistent chylomicronemia and 14 hospitalizations for abdominal pain and pancreatitis, usually with hyperamylasemia, in the 5 years before fish-oil administration. She was subsequently treated for 5 months with a diet containing 10 to 20 gm of fish oils (MaxEPA). Despite persistence of chylomicronemia and with triglycerides as high as 7,700 mg/dL on fish oils, she was free of abdominal pain. Furthermore, her hyperpigmentation also decreased in intensity and her xanthomata regressed. She was then placed on an outpatient 20% fat diet containing safflower oil in preparation for her initial kinetic study. Within 3 weeks, she sustained two bouts of nausea, vomiting, and abdominal pain, and one episode required hospitalization. She has now

been maintained on 15% of calories as fish oils for an additional 11 months and has experienced only three mild cases of abdominal pain without associated vomiting. All were treated conservatively at home. During this interval, her serum triglycerides averaged about 2,100 mg/dL, her xanthomata nearly resolved, and her acanthosis also improved. Blood glucose control was excellent, but her insulin requirement increased to 74U/24h. Because of the apparent symptomatic improvement in her chylomicronemia syndrome, we have elected to maintain this patient indefinitely on a fish oil-supplemented diet and to make adjustments in insulin dose appropriate to maintain tight glycemic control. The reasons for these metabolic effects in patient no. 2 are unknown. Particularly intriguing, however, is the improvement in her chylomicronemia syndrome. Why extreme hypertriglyceridemia predisposes toward pancreatitis is unknown, but under most circumstances the persistent presence of circulating chylomicrons increases the risk of pancratic inflammation and abdominal pain.” Is it reasonable to postulate that omega-3 fatty acids may decrease the inflammatory response associated with chylomicrons by altering neutrophil function, as has been demonstrated in humans at sites of synovial inflammation?# Or does a change in pancreatic cell membrane composition enhance their resistance to the actions of neutrophils or other mediators of inflammation? Finally, is it possible that the omega-3 diet altered the size or composition of our patient’s chylomicrons, making them less injurious to the pancreas? Regardless of the actual mechanism, the observations in our patient suggest that it is not the presence of chylomicronemia per se that is the immediate cause of pancreatic injury. Controlled studies employing fish oil supplementation in other patients with pancreatitis and hypertriglyceridemia appear to be warranted. ACKNOWLEDGMENT We are indebted to Dr George W. Moore, Tulsa, OK, for referring patient no. 1 and Dr James A. Hotz, Albany, GA, for referring patient no. 2. We thank the nursing and dietary staff of the Clinical Research Center at Shands Hospital, University of Florida for their assistance in conducting these studies and Penny Moeller for typing the manuscript.

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