Atherosclerosis, 91 (1991) 25-34 C 1991 Elsevier Scientific Publishers

ATHERO

25 Ireland,

Ltd. All rights

reserved

0021-91SO/91/$03.50

04712

Apolipoprotein A-IV polymorphism, and its role in determining variation in lipoprotein-lipid, glucose and insulin levels in normal and non-insulin-dependent diabetic individuals M. Ilyas Kamboh

‘, R.F. Hamman

2, S. Iyengar

I, C.E. Aston

’ and

R.E. Ferrell

’

’Depurtment of Human Genetics, Graduate School of Public Health, lJnic,ersity of Pittsburgh, Pittsburgh, PA I.5261 (U.S.A.). and ’ Department of Prec,entir,e Medicine and Biometrics, Unit,ersity of Colorudo School o,f Medicine, Dencer, CO 80262 (U.S.A.) (Received 15 April, 1991) (Revised, received 5 July, 1991) (Accepted 29 July, 1991)

Apolipoprotein A-IV (ape A-IV) is a major component of several lipoprotein particles and may, therefore, play an important role in lipid metabolism. Genetic polymorphism of apo A-IV has been reported in humans and several other animal species. The presence of two common alleles, apo A-IV * 1 and apo A-IV * 2 has been documented in several human populations. In this investigation, we have determined apo A-IV polymorphism by isoelectric focusing-immunoblotting in 82 non-insulin-dependent diabetic and 204 control non-Hispanic Whites from the San Luis Valley, Colorado. We have also estimated the impact of apo A-IV polymorphism on eight quantitative traits: total cholesterol, HDLcholesterol, HDL3 and HDL2-cholesterol, LDL-cholesterol, triglycerides, fasting glucose and fasting insulin. No statistically significant difference was seen in apo A-IV allele frequencies between the control and diabetics, and these frequencies were comparable with those reported for U.S. White and European populations. Among controls, the impact of the apo A-IV polymorphism was significant on LDLcholesterol (P = 0.04) in females and on fasting insulin levels (P = 0.06) in males. In diabetics, the effect was significant on insulin (P = 0.03) levels in males only. Furthermore, our data suggest that when making comparison of lipid profiles between the controls and diabetics the presence or absence of common apo A-IV phenotypes should be taken into account as these appear to effect these comparisons.

Key words: Apolipoprotein betes

A-IV; Polymorphism;

Correspondence to: Dr. M.I. Kamboh, Department of Human Genetics. Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA 15261, U.S.A. Phone: (412) 624-301X: Fax: (412) 624-3020.

Lipoprotein-lipid

levels; Non-insulin-dependent

dia-

Introduction

Apolipoprotein A-IV (ape A-IV) is a 46000 Da glycoprotein containing 6% carbohydrate by

26 weight and 376 amino acid residues [l--5]. Apo A-IV is synthesized in enterocytes of the small intestine during fat absorption as preapo A-IV and the mature protein is secreted into the lymph associated with chylomicrons [6,7]. The average level of plasma apo A-IV varies from 14 to 37 mg/dl depending upon the immunological methods of determination [4,5,7,81. Early reports about its plasma distribution indicated that although apo A-IV was associated with chylomicrons, very low density lipoproteins [2-41, and high density lipoproteins (HDL) [8,9], apo A-IV was primarily found in the lipoprotein free fraction after ultracentrifugation [5,10]. Recently, Lagrost et al [ll] re-evaluated the distribution of apo A-IV and showed, using high performance gel filtration, that apo A-IV in fasting serum is mainly associated with HDL particles and is not in the free form. Lagrost et al. [ll] further showed that this association with HDL did not result from the known in vitro redistribution of apo A-IV induced by lecithin : cholesterol acyltransferase (LCAT) [12-141 since apo A-IV was observed in sera in the presence of LCAT inhibitors. Apo A-IV has been reported to play a role in the activation of the LCAT enzyme [15], in the efflux of cholesterol from cells [16,17], in modulating the activation of lipoprotein lipase [181, and as an activator or potentiator of a plasma HDL conversion factor [19]. However, the precise role of apo’ A-IV in lipid metabolism is yet to be delineated. Recently, a deletion of the apo A-IV gene has been suggested to be associated with fat malabsorption, which points out the role of apo A-IV on intestinal lipoprotein synthesis and secretion [20]. The structural gene for apo A-IV is located on human chromosome llq in a tightly linked cluster with apolipoproteins A-I and C-III, and has been characterized in detail [21] Genetic polymorphism of apo A-IV has been reported in humans [22-281 and other species [29,30). The role of apo A-IV polymorphism in determining plasma lipoprotein-lipid levels has been investigated in several general population studies [26-28,31-331. In this investigation we have determined the apo A-IV phenotype and allele frequencies in non-insulin-dependent diabetic (NIDDM) and control subjects from the

San Luis Valley in Southern Colorado. We have estimated the impact of apo A-IV polymorphism on eight quantitative traits: total cholesterol, high density lipoprotein cholesterol, HDL,-cholester01, HDL,-cholesterol, low density lipoprotein cholesterol, triglycerides, glucose and insulin. Population, subjects and methods Population

The San Luis Valley Diabetes Study was designed as a geographically based case-control study of NIDDM in Alamosa and Conejos counties of southern Colorado. The study area is predominantly biethnic with non-Hispanic Whites (Angles), comprising 54.9% of the total population of the two counties. The non-Hispanic White (NHW) families came to the valley from a variety of northern European countries. Settlement by these groups began in earnest in the mid-1800s considerably postdating the intermittant occupation of the area by native Americans and Spanish explorers, Historical patterns of intermarriage between the NHW and American Indian population or the Hispanic population is not well documented from an anthropological perspective. Estimates of Amerindian admixture for this population indicate that this Anglo group has approximately 3% American Indian genes. This is similar to admixture levels reported for Angles living in the San Antonio, TX area. Overall the area is considered rural, with one major population center in the city of Alamosa. The area’s primary economic activities are farming and tourism, with the median income, as of 1980, for both counties ranked below the median for the state. All subjects in this report are non-Hispanic White and were classified based on their response to the 1980 census question: “Are you of Spanish/ Hispanic origin or descent?” [34]. Subjects

A total of 204 non-diabetics aged 26-75 years (mean k SE, 53.53 f 0.85 yrs) and 82 diabetics aged 28-75 yrs (59.58 f 1.03 yrs) were studied. Among the non-diabetic controls, there were 106 males with a mean age of 53.34 f 1.13 yrs and 98 females with a mean age of 53.73 k 1.28 yrs. The diabetic group consisted of 46 males with mean

27

However, the formula was used only if triglyceride values were less than 400 mg/dl. Individuals with triglycerides level above 400 mg/dl were not included in the analysis. Fasting serum insulin level was measured by a double antibody radioimmunoassay [40] and fasting glucose levels were obtained using glucose oxidase [41].

age 58.34 f 1.46 and 36 females with mean age 61.17 + 1.38 yrs. A summary of the clinical features of both the controls and diabetics is given in Table 1. Unpaired Student’s t-tests indicated that all variables are significantly different between the diabetics and controls with the exception of total cholesterol in males and females, and HDL,-cholesterol and age in males only. In all analyses, males and females were tested separately. Lipoprotein-lipid, men ts

glucose

and

Apo A-IV phenotyping

Apo A-IV phenotyping was determined on native plasma collected at the time of a standard oral glucose tolerance test, using polyacrylamide isoelectric-focusing (IEF) gels followed by immunoblotting as described previously [24]. Briefly, plasma samples absorbed on 5 x 4 mm Whatman 3 MM wicks were applied 1 cm from the cathode and IEF was performed on polyacrylamide gels at 2500 V, 30 W, and 250 mA for 3 h. Following the completion of IEF, proteins were transferred from the gel onto a 0.20~pm pore size nitrocellulose filter by simple diffusion for 10 min at room temperature. The filter was first probed with rabbit anti-human apo A-IV (provided by Dr.

insulin measure-

Total serum cholesterol was measured by the esterase-oxidase method [35], total HDL-cholesterol and HDL,-cholesterol were determined enzymatically following magnesium precipitation 136,371 and serum triglycerides by the enzymatic procedure of Stavropoulous and Crouch [38]. HDL,-cholesterol measurement was obtained by subtracting HDL,-cholesterol from total HDLcholesterol. LDL-cholesterol value was calculated according to the formula of Friedewald et al. [39].

TABLE

I

BIOMETRIC SUBJECTS,

AND QUANTITATIVE DATA (MEAN&SE) SAN LUIS VALLEY, CO, 1984-1987

FOR

THE

NON-HISPANIC

WHITE

CONTROL

Variable

Gender

Control

NIDDM

Count

M F M F M F M F M F M F M F M F M F M F M F

106 98 53.34* 1.13 53.73 + 1.28 26.36+ 0.37 25.23 f 4.94 215.98 f 4.43 221.39* 5.07 44.90 If- 0.98 58.49 + 1.49 38.12+ 0.72 42.30 k 0.86 6.78+ 0.46 13.19i_ 0.85 138.21 f 4.26 135.06 + 4.07 158.26 + 14.00 139.17* 6.51 97.28 + 0.79 92.40 + 0.80 11.39+ 0.62 10.99 f 0.99

46 36 58.34 + 1.46 61.17+ 1.38 29.14+ 0.66 32.02* 0.97 204.85 f 6.20 211.22+ 8.02 38.1 I + 1.70 46.14k 2.15 32.65+ I.41 37.67 * 1.32 5.46? 0.68 8.47 + I.30 113.61 f 8.64 111.44+ 7.78 223.76 + 26.83 214.19+ 19.72 193.15_+ 11.55 178.69+ 11.48 16.67 + 2.30 21.92+ 5.37

Age (yrs) BMI

(kg/m’)

Total cholesterol

(mg/dl)

HDL-cholesterol

(mg/dl)

HDL,-cholesterol

(mg/dl)

HDL,-cholesterol

(mg/dl)

LDL-cholesterol Triglycerides Glucose

(mg/dl) (mg/dl)

fasting

(mg/dl)

Insulin fasting (FIU/ml)

AND

NIDDM

28 K.H. Weisgraber, Gladstone Foundation Laboratories) followed by goat anti-rabbit IgG conjugated with alkaline phosphatase. After extensive washing, apo A-IV isoproteins were visualized by histochemical staining.

package BMDP-IV. Initially, all dependent variables, with the exception of LDL-cholesterol, were normalized by taking natural logs to ensure the distribution of the dependent variable was Gaussian. Covariates considered were body mass index and age. Body mass index (BMI) was calculated as weight/ height2 (kg/m’). Significant covariates for each dependent variable were identified using stepwise regression. In control males, BMI was identified as a significant covariate for HDL-cholesterol, HDL,- and HDL,-cholesterol, triglyceride, glucose and insulin variables. BMI had the same effect among control females and, in addition, the effect of age was a significant covariate for HDL-cholesterol, HDL,-cholesterol, LDL-cholesterol and total cholesterol. Those covariates found to be significant for the control group were used for the diabetic group. In addition, a dichotomous variable classifying females into pre- and post-menopause was considered as a covariate but was found not to be significant. The dependent variables were adjusted for the effects of age and BMI as indicated.

Statistical methods Apo A-IV allele frequencies were estimated by allele counting. Hardy-Weinberg equilibrium was tested by a chi-square goodness of fit test. Apo A-IV l-l and 2-1 phenotypes were used to estimate the impact of apo A-IV polymorphism on the 8 quantitative traits (the dependent variables): triglyceride, fasting glucose, fasting insulin, total cholesterol, HDL-cholesterol, HDL,- and HDL,cholesterol and LDL-cholesterol levels. The number of apo A-IV 3-l phenotypes was too small to be included in the statistical analysis. To test the null hypothesis of equality of mean levels of quantitative variables between the l-l and 2-l phenotypes, an analysis of covariance was performed separately for males and females for each of the 8 quantitative traits using the computer

TABLE

2

DISTRIBUTION OF HISPANIC WHITES Gender

APO

n

A-IV

PHENOTYPE

Female

Combined

ALLELE

A-IV * 1

A-IV * 2

106 % 98 %

86 81.1 86 87.8

20 18.9 12 12.2

0.906

0.094

0.939

0.061

204 %

172 84.3

32 15.7

0.922

0.078

l-l

Female

Combined

IN CONTROL

2-1

AND

NIDDM

Allele frequencies

Phenotypes

(b) NIDDM Male

FREQUENCIES

Allele frequencies

Phenotypes l-l

(a) Control Male

AND

2-1

3-l

A-IV * 1

A-IV * 2

A-IV * 3

46 % 36 %

40 87.0 28 77.8

6 13.0 7 19.4

0 0.0 1 2.8

0.935

0.065

0.000

0.889

0.097

0.014

82 %

68 82.9

13 15.9

1 1.2

0.915

0.079

0.006

NON-

29

Apo A-IV phenotype and allele frequencies

Apo A-IV phenotypes and plasma lipoprotein-lipid, glucose and insulin levels between controls and diabetics

Two common apo A-IV phenotypes, l-l and 2-1, were observed in both the NIDDM and control groups. The rare phenotype, 3-1, was seen in one diabetic individual. No 2-2 phenotype was observed in either group. The distribution of apo A-IV phenotypes and allele frequencies in male, female and gender pooled, control and diabetic Anglos are presented in Tables 2a and 2b. The distribution of apo A-IV phenotypes was in Hardy-Weinberg equilibrium in normal males (P > 0.70) and females (P > O.SO), and in diabetic males (P > 0.90) and females (P > 0.60). HardyWeinberg equilibrium also still held when genders were pooled in controls (P > 0.25) and diabetics (P > 0.25), and when pooled controls and diabetics were compared (P > 0.50). Apo A-IV allele frequencies were similar between genders within each group and the combined values were similar between controls and diabetic groups. Although the apo A-IV * 2 allele frequency is slightly higher in control males and lower in diabetic males than in the corresponding female samples, the difference is not statistically significant (P > 0.25). A comparison of apo A-IV allele frequencies between the Anglos from Colorado and other Caucasian populations is given in Table 3. Apo A-IV allele frequencies in Anglo residents of the San Luis Valley are indistinguishable from those in other U.S. and European Caucasian populations.

Before making a comparison among the two apo A-IV phenotypes (1-l and 2-l), in the control and diabetic groups separately (Tables 5 and 6), we first made a comparison between the corresponding A-IV phenotypes between the controls and diabetics to determine whether the eight quantitative traits differ significantly within each of the two phenotypes between these two groups (Table 4). Irrespective of the gender and A-IV phenotype, only the fasting glucose level is significantly higher in diabetics than controls. For the remaining seven quantitative traits, the difference between controls and diabetics depends either on gender or A-IV phenotype. In females, but not in males, total cholesterol was significantly lower in diabetics whether the A-IV phenotype was l-l (P = 0.005) or 2-1 (P = 0.025). In diabetics, the following traits were significantly lower than in controls: HDL-cholesterol in females carrying the A-IV l-l phenotype (P = O.OOO>, and males carrying the A-IV 2-l phenotype (P = 0.02); HDL,cholesterol in both the males (P = 0.05) and females (P = 0.000) of the A-IV l-l phenotype: HDL,-cholesterol only in females (P = 0.01) of the A-IV l-l phenotypes; and LDL-cholesterol both in the males (P = 0.056) and females (P = 0.005) of the A-IV l-l phenotype. Similarly, diabetic females, but not males, of the A-IV 1-l phenotype have significantly (P = 0.003) higher triglycerides. In contrast, diabetic males, but not females, of the A-IV 2-l phenotype have signifi-

Results

TABLE

3

APO A-IV ALLELE

Population Non-Hispanic control diabetic U.S. Whites U.S. Whites Germans Dutch Austrians

FREQUENCIES

n

IN SELECTED

GROUPS

A-IV * 1

A-IV + 2

Other

Reference

204 82 127

0.922 0.915 0.090

0.078 0.078 0.088

0 0.006 0.003

453 1,000 1,393 473

0.927 0.923 0.901 0.918

0.067 0.075 0.079 0.077

0.004 0.002 0.020 0.004

Present study Present study Kamboh and Ferrell (1987) [24] Eichner et al. (1989) Menzel et al. (1982) deKniff et al. (1988) Menzel et al. (1988)

White

[32] [22] [31] [26]

30 TABLE 4 COMPARISON OF MEAN ADJUSTED VALUES OF PLASMA LIPOPROTEIN-LIPID, GLUCOSE AND INSULIN LEVELS (+ SE) BETWEEN CONTROL AND DIABETIC WITHIN THE APO A-IV I-I AND 2-l PHENOTYPES IN NON-HISPANIC WHITES Variable

Gender

A-IV l-l

A-IV 2-l

Control Phenotype count

M F

Total cholesterol

M F

(mg/dlI HDL-cholesterol (mg/dlI HDL,-cholesterol (mg/dl) HDL,-cholesterol (mg/dl) LDL-cholesterol (mg/dl) Triglycerides (mg/dl) Glucose fasting (mg/dl) Insulin fasting (~IU/ml)

Diabetic

86 86

F-value

P

Control

40 28

216.52+ 5.07 223.05 + 4.02 1.10 1.54

20 12

201.01* 197.71 f

Diabetic

F-value

P

6 7

7.64 7.46

2.69 8.77

0.10 0.005

215.97+ 9.27 293.51+ 28.55

222.75 + 17.30 133.02 + 43.63

0.11 8.23

0.74 0.025

40.61+ 1.65 46.06 + 2.86

1.76 14.22

0.19 0.000

47.08 + 1.92 60.38 f 6.61

36.90 f 3.58 42.34 + 10.10

6.08 1.47

0.02 0.24

34.16+ 37.78 +

1.28 1.70

3.87 15.10

0.05 0.000

38.68 k 1.70 43.10+ 3.44

32.75 f 3.17 39.25 + 5.26

2.64 0.25

0.12 0.63

2.07 6.82

3.18 1.99

0.09 0.18

M F

43.32+ 58.78 +

M F

37.29 + 0.85 45.57 + 0.91

M F

6.03+ 13.21+

0.49 0.86

6.45+ 8.28+

0.73 1.60

0.21 6.82

0.65 0.01

8.40+ 1.11 17.28 + 4.47

M F

136.97+ 135.83 f

5.37 3.77

117.59+ 111.89+

8.10 6.99

3.74 8.40

0.056 0.005

140.10+ 11.85 157.82 f 23.58

98.63k22.12 69.80 + 36.03

2.65 2.75

0.12 0.12

M F

170.27 + 16.40 141.93 f 7.33

182.49 + 24.72 191.15+ 13.26

0.16 9.44

0.69 0.003

146.06 + 37.83 236.56& 53.16

367.45 f 70.64 103.48k81.23

7.42 1.23

0.01 0.28

M F

97.36 k 4.71 92.50 f 3.74

191.45 + 7.10 180.89 f 6.95

114.43 115.84

0.000 0.000

94.92 + 11.30 78.73 f 20.09

211.25+21.10 198.47 + 30.69

22.96 7.01

0.000 0.02

M F

11.77k $89 11.36rt 1.40

1.34 0.92

0.25 0.34

13.36 + 3.43 16.92+ 15.00

24.48 f 6.41 37.13 + 22.91

2.27 0.36

0.15 0.56

13.69 f 14.32 f

1.34 2.61

4.15+ 3.09+

TABLE 5 MEAN ADJUSTED VALUES OF PLASMA LIPOPROTEIN-LIPID, GLUCOSE AND INSULIN LEVELS (+ SE) BETWEEN APO A-IV PHENOTYPES IN NON-HISPANIC WHITES WITH NORMAL GLUCOSE TOLERANCE Variable

Gender

Phenotype count a

M F M F M F M F M F M F M F M F M F

Total cholesterol (mg/dl) HDL-cholesterol

(mg/dlI

HDL,-cholesterol

(mg/dl)

HDL,-cholesterol

(mg/dlI

LDL-cholesterol

(mg/dlI

Triglycerides (mg/dl) Glucose fasting (mg/dl) Insulin fasting (~IU/mlJ

A-IV phenotypes l-l

2-l

86 86 209.37 + 4.58 213.90+ 3.97 43.12kO.95 56.89 + 1.48 37.10+ 0.76 44.58 + 0.86 4.98 + 0.40 10.86 + 0.82 140.91 f 4.36 132.315 3.76 131.19k6.45 125.44+6.29 97.25 f 0.88 92.09 + 0.84 9.56 f 0.48 9.06 + 0.41

20 12 213.58 + 218.16k 46.91+ 55.54 + 38.67+ 44.07 f 6.76+ 9.33* 140.71 k 154.81 f 130.58+ 124.80 + 95.65 f 91.92k 11.85 f 9.04*

a Includes cases with missing data or data beyond limits.

9.63 11.34 2.14 3.87 1.64 2.29 1.12 1.90 8.93 10.06 13.23 16.79 1.80 2.25 1.22 1.10

F-value

P

0.16 0.13 2.75 0.12 0.77 0.05 2.75 0.50 0.00 4.39 0.00 0.00 0.63 0.00 3.51 0.00

0.69 0.72 0.10 0.73 0.38 0.82 0.10 0.48 0.98 0.04 0.97 0.99 0.43 0.97 0.06 0.97

31 TABLE

6

MEAN ADJUSTED VALUES OF PLASMA LIPOPROTEIN-LIPID, APO A-IV PHENOTYPES IN NON-HISPANIC WHITES WITH Gender

Variable

Phenotype

count

”

Total cholesterol

(mg/dl)

HDL-cholesterol

tmg/dl)

HDL,-cholesterol

(mg/dl)

HDL+holesterol

(mg/dl)

LDL-cholesterol Triglycerides

(mg/dl) tmg/dl)

Glucose

fasting (mg/dll

Insulin

fasting (~IU,/mll

a Includes

cases with missing

M F M F M F M F M F M F M F M F M F data or data beyond

cantly (P = 0.01) higher triglycerides trols.

GLUCOSE NIDDM

AND INSULIN

A-IV phenotypes l-l

2-1

39 28 197.89 f 207.62 + 37.32+ 44.70 + 31.85k 37.41 k 4.58k 6.65& 129.46 + 126.71+ 165.95 f 188.70+ 177.74+ 167.94+ 15.00+ 19.82+

6 7 21557k 19.00 215.4Oi 17.46 34.48 + 3.76 45.97 + 5.46 28.81 + 3.59 36.16+ 3.31 3.13+ 1.16 6.71 + 2.10 144.90 + 20.87 107.78 + 14.64 266.35 + 65.15 175.69k37.03 187.71 k31.68 164.84 f 24.94 32.01 k 9.99 30.57 & 9.01

6.84 8.30 1.60 2.56 1.56 1.65 0.62 1.05 7.06 6.14 15.91 19.63 11.76 12.55 1.65 3.17

LEVELS

t + SE) BETWEEN

F-value

P

0.82 0.16 0.46 0.04 0.56 0.11 0.93 0.00 0.49 1.40 3.24 0.09 0.09 0.01 5.24 1.67

0.37 0.69 0.50 0.84 0.46 0.74 0.34 0.98 0.49 0.25 0.08 0.77 0.77 0.91 0.03 0.21

limits.

than con-

Apo A-IV polymorphism and plasma lipoproteinlipid, glucose, and insulin levels in the diabetic group

Apo A-IV polymorphism and plasma lipoproteinlipid, glucose, and insulin levels in the control group

The adjusted mean values of eight quantitative traits between the two A-IV phenotypes in the control group of males and females are shown in Table 5. Males showed no significant difference in seven quantitative traits between the l-l and 2-1 phenotypes. The fasting insulin level was marginally higher (P = 0.06) in the 2-l phenotype (11.85 _t 1.22 mg/dl) as compared to the l-l phenotype (9.56 k 0.48 mg/dl) in males. In females, only LDL-cholesterol (P = 0.04) showed statistically significant differences between the two A-IV phenotypes. The 2-l phenotype was associated with higher LDL-cholesterol (154.81 + 10.06 vs 132.31 -+ 3.76 mg/dl) as compared to the l-l phenotype. The remaining six traits were comparable between the two phenotypes in females.

Table 6 presents the comparison of quantitative levels of eight traits between the two A-IV phenotypes in diabetic subjects stratified by gender. Only the fasting insulin level was significant (P = 0.03) between the l-l and 2-l phenotypes in males. The triglycerides were higher in the 2-1 phenotype than the l-l phenotype but this difference did not reach a significant level at the 5% level. Discussion

Genetically determined structural polymorphism in the apo A-IV polypeptide has been reported in a number of populations [22-28,421. However, only a few studies have attempted to analyze the impact of this structural polymorphism on lipoprotein-lipid levels [26-28,31-331. In this study we have, for the first time, compared

32 the apo A-IV phenotype and allele frequencies and estimated their effects on eight quantitative traits, which are risk factors for cardiovascular disease, in NIDDM and normoglycemic subjects in a non-Hispanic White population residing in the San Luis Valley of Southern Colorado. Historically, non-Hispanic White families came to this area from a variety of northern European countries and recent genetic data indicate that they have approximately 3% Amerindian genes in their gene pool (Iyengar et al., unpublished data). Despite this admixture, the apo A-IV allele frequencies in non-Hispanic White were very similar to those reported for several European and U.S. White populations (see Table 3). In this study, we found no difference in apo A-IV allele frequencies between the diabetic and control subjects. The reported studies regarding the impact of apo A-IV polymorphism on lipoprotein-lipid levels either looked only at men [31] or women [32] or with gender combined [26-28,331. To our knowledge, no study has considered males and females from the same population separately while analyzing the impact of allelic variation at the apo A-IV locus on quantitative traits. Due to very different lipid profiles of males and females, and their different susceptibility to cardiovascular disease, it is important to consider them separately: the present study has demonstrated the importance of this point by identifying sex-specific effects of the A-IV polymorphism on quantitative traits in both the control and the diabetic subjects. Previously reported effects in non-diabetics of the apo A-IV polymorphism on triglycerides [27,31] total HDL-cholesterol [25,26] and on glucose [28] were not apparent in the non-diabetic population considered here. However, we observed marginal effects on fasting insulin (P = 0.061, HDL- and HDL,-cholesterol (P = 0.10) in males, and a significant effect on LDL-cholesterol (P = 0.04) in non-diabetic females. Atherosclerosis accounts for about 25% more deaths in diabetics than non-diabetics [43], and the atherosclerotic process is similar in its pathophysiology and anatomical distribution in both groups. For this reason, it seems probable that the well established relationship between elevated lipoprotein lipids and cardiovascular disease in the non-diabetic may also apply to the

diabetic. The increased prevalence of atherosclerosis in NIDDM may be due to adverse effects of diabetes on plasma lipoprotein-lipid levels [44]. Therefore, examination of the role of genes in determining these quantitative traits in both the normals and diabetics is important. To our knowledge, there is no prior study of the relationship between apo A-IV polymorphism and plasma lipoprotein-lipid levels in NIDDM, and for this reason, we have investigated this relationship in the NIDDM subjects from the San Luis Valley. On the average, diabetics had higher triglycerides and fasting glucose levels and lower LDL and HDL-cholesterol levels than non-diabetic controls. Interestingly, however, this variation was dependent on both the gender and apo A-IV phenotype (see Table 4). These data suggest that when making comparison of lipid profile between diabetics and non-diabetics, the apo A-IV phenotypes should be taken into account as this appears to effect these comparisons. In both the control and diabetic males, the apo A-IV polymorphism has an effect on the fasting insulin level (Tables 5 and 6) in the same direction. Although in both groups the insulin fasting level is higher in the 2-l phenotype than the l-l phenotype, the difference is signifcant in diabetics (P = 0.03) and marginal in controls (P = 0.06). In contrast to the diabetic group, where the apo A-IV polymorphism did not show a significant effect on the LDL-cholesterol level, the apo A-IV effect was significant in controls (P = 0.04). A significant age difference was found between the control group and the diabetic group. However, we expect that this difference does not affect our results since the effect of age was removed as a covariate in the analyses. To confirm this expectation, a stratified resampling scheme was used to generate a random subsample of the control group with the same mean age and age by decade distribution as the diabetic group. Analysis of the lipoprotein-lipid, glucose and insulin profiles of the whole control group and the subgroup found no significant differences between these groups. In conclusion, the present data indicate that the apo A-IV polymorphism has a small effect on LDL-cholesterol, fasting insulin and possibly the HDL- and HDL,-cholesterol levels in non-di-

33

abetics, and a moderate effect on the insulin and triglycerides level in the non-Hispanic White diabetics. However, considering the high number of comparisons performed, the odds of finding spurious significant association, just by chance, are relatively high. Furthermore, when making comparison of lipid profiles between the control and diabetics, the apo A-IV polymorphism should be taken into consideration as it effects these comparisons. In the absence of knowledge of the precise role of apo A-IV in lipid metabolism, the physiologic effect of the apo A-IV polymorphism on the quantitative traits in NIDDM is open to speculation. Elevated triglyceride levels are characteristic of the diabetic state and apo A-IV is found in triglyceride-rich particles. In this study, the apo A-IV phenotype has a marginal impact on triglyceride levels in diabetic males. The lack of an effect in females may reflect the smaller sample size. The elevated triglyceride levels observed in diabetics may arise through the effect of insulin on lipoprotein lipase, the enzyme which catalyzes the hydrolysis of plasma triglycerides; and the genotype specific elevation of insulin levels in NIDDM perhaps reflect an interaction between insulin levels and apo A-IV genotype in determining diabetic hypertriglyceridemia. Recently, the mutation leading to the amino acid sequence difference in the apo A-IV * 2 allele was determined [45] and structure-function studies coupled with genotype specific metabolic studies will define the role of apo A-IV in the normal and diabetic state. Acknowledgements

This research project was funded by the National Dairy Board and administered in cooperation with the National Dairy Council and by N.I.H. Grant HL44672. Core funding for the San Luis Valley Diabetes Study was from N.1.H Grant DKC-30747 and CRC core grant CRC-RR0005. We would like to thank Lori Kelly for technical assistance and Debbie Biernesser for preparing the manuscript. We thank the residents of Alamosa and Canejos counties for their participation in this study and the many collaborators from the San Luis Valley Diabetes Study who provided advice and assistance.

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