Atherosclerosis,
97
82 (1990) 97-103
Elsevier Scientific Publishers Ireland, Ltd. ATHERO 04469
Metformin-induced changes in serum lipids, lipoproteins, and apoproteins in non-insulin-dependent diabetes mellitus Jiirgen Schneider, Thomas Erren, Peter ZGfel and Hans Kaffamik Department of Internal Medicine, Division Endocrinology and Metabolism,
Philipps-University
Marburg, Marburg (F.R.G.)
(Received 28 September, 1989) (Revised, received 19 January, 1990) (Accepted 26 January, 1990)
Forty patients with NIDDM and hyperlipoproteinemia were selected for a 12-week double-blind placebo-controlled trial to study the effects of metformin on lipoprotein concentration and composition. A significant decrease occurred in VLDL-apo B and all lipid components of VLDL, indicating a decreased number of circulating VLDL, while LDL-apo B was unchanged. Moreover in VLDL the relative TG content increased, the cholesterol content decreased, while in LDL the TG content decreased and the cholesterol content increased, indicating a change in the particle distribution over the spectrum VLDLIDL-LDL. The initially enhanced TG-content in HDL was reduced. While a reduction in VLDL is observed together with improving glucose control irrespective of the applied method, the observed compositional changes in VLDL and LDL have not been described before and seem to be metforminspecific.
Key words: Diabetes type II; Lipoproteins;
Metformin
Introduction Diabetics are predisposed to degenerative arterial vascular diseases (‘diabetic macroangiopathy’): diabetes is one of the main risk factors for atherosclerosis [l-5]. Macroangiopathy is responsible for about 75% of deaths in patients with known diabetes [6]. Whilst microangiopathy largely determines the prognosis in type I diabetes, general atherosclerosis of the large arteries
Correspondence to: Prof. Dr. J. Schneider, Department of Internal Medicine, Division Endocrinology and Metabolism, Khnikum Lahnberge, D-3550 Marburg F.R.G.
0021-9150/90/$03.50
is the major secondary disease of type II diabetes. The high frequency of hyper- and dyslipoproteinemia in type II diabetes is also well known and normalization of hyperlipoproteinemia has become one of the criteria of good metabolic control in type II diabetes. The effect of almost all treatment methods (diet, sulphonylurea drugs and insulin) on hyperlipoproteinemia is known and has been studied during the last decade in view of more recent knowledge of the different, sometimes opposing, effects of various lipoprotein classes and subfractions on atherogenesis. Differentiated lipoprotein analyses are lacking for biguanide therapy, presumably because these substances have been of minor therapeutic importance for the last
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
98 10 years in some countries, especially in the U.S.A. Earlier results, which show a clear reduction in elevated triglyceride values in response to biguanides have recently been confirmed in a large number of patients [7]. The aim of the present study was to investigate the effect of metformin on serum lipids, apoproteins and lipoproteins, and their composition in non-insulin-dependent type II diabetics with hyperlipoproteinemia. Patients and methods
Patients The study was performed as a randomized placebo-controlled two-arm-trial in 40 patients with NIDDM and hyperlipoproteinemia. Patients with unstable angina pectoris, myocardial infarction within 1 year, myocardial and respiratory insufficiency, impaired renal function, hepatic disease besides fatty infiltration, gastrointestinal disorders, malignancies and psychic abnormalities were not included, as well as women of childbearing potency. Inclusion was independent from the quality of glucose control. Diagnosis of type II diabetes was clinical. In doubtful cases, it was confirmed by positive insulin and C-peptide values after glucagon stimulation. All patients were treated effectively with diet and sulfonylurea derivatives for at least 6 months. Hyperlipoproteinemia was defined as total cholesterol > 250 mg/dl and/or triglycerides > 200 mg/dl. The patients were aged between 37 and 77 years, all of them were overweight, with a body mass index (weight (kg)/height (m2)) from 25 to 32. The majority of patients in both groups were treated for hypertension., Diastolic blood pressure at entrance was higher than 95 mm Hg in one case in. the metformin group and in one case in the placebo group. Diagnosis of hypertension and coronary heart disease was distributed evenly between the groups. Further characterization of the patients completing the protocol in the two treatment groups is given in Table 1. Study protocol ) After giving informed consent, the patients were randomized by ,order of entrance to the study. They reported as out-patients at four-weekly inter-
TABLE 1 CHARACTERISTICS TWO TREATMENT
OF THE PARTICIPANTS GROUPS
IN THE
Mean f SD. placebo (n =16)
Male/female Age in years BMI HbAlc (0) HbAlc (12) AHbAlc (12-O)
9/7 61.5 i-12.3 27.1 + 3.0 6.48!c 0.87 6.34f 1.11 -0.13+ 0.69
metformin (n =18)
P
8/10 60.4 f9.5 26.1 k3.1 6.7651.05 6.08 f 0.98 -0.69 f 0.87
n.s. n.s. ns. n.s. n.s. 0.054
vals for checks on diabetes and hyperlipoproteinemia. The drug was given in gradually increasing doses: initially the patients received one, after 4 weeks (when tolerated) 2 capsules containing 0.85 g metformin or placebo, respectively. Drug treatment of accompanying disease was kept constant. As metformin treatment is permitted only in combination with sulphonylurea derivatives in the F.R.G., all patients received at least low doses of such drugs, mainly glibenclamide (minimum 1.75 mg). Method At 0, 4, 8 and 12 weeks, fasting blood samples were drawn and VLDL, LDL, HDL, and HDL isolated by preparative ultracentrifugation in 50-3 Ti rotors in L50 centrifuges ([8,9] Beckman Palo Alto). Cholesterol [lo-121, triglycerides [13], and phospholipids [14] in serum and in the density fractions were determined using enzymatic standard methods; HDL-cholesterol was also determined after polyanion precipitation of apo Bcontaining lipoprotein ([15] Boehringer, Mannheim). Apo A-I, A-II and B were determined in serum and density fractions using radial immunodiffusion on standard commercial plates ([16] Immuno, Heidelberg). HbAl was determined using a microcolumn method ([17,18] Biorad, Munich). Statistical comparison of the data of the patients who completed the 12-week trial (n = 18 in the metformin group, n = 16 in the placebo group) was made by Wilcoxon matched-pairs signed-rank test (comparisons within groups) and Mann-Whitney test (comparisons between groups).
99 TABLE
2
LIPIDS
AND APOPROTEINS
IN SERUM
AND
LIPOPROTEIN
CLASSES
IN THE METFORMIN
GROUP
Values are mean + SD in mg/dl. Weeks: Serum
Chol TG
0
4
8
12
PL AI A II B
242* 340 + 305* 140+ 55+ 104+
43 207 59 27 10 16
232& 43 * 250*161 ** 284+ 59 142+ 29 55+ 10 107+ 20
232+ 44 ** 259+210 ** 284+ 39 151* 29 56+ 10 105& 17
233? 51 * 242k156 *** 290+ 45 151* 40 53* 10 98+ 20
VLDL
Chol TG PL B
56* 243 + 68+ 15*
43 195 60 10
38+ 29 * 173+156 *** 40+ 32 9+ 5
35* 29 *** 186*205 ** 40+ 28 * 13+ 9
34+ 27 *** 170*144 *** 38+ 28 * lo* 4%
LDL
Chol TG PL B
133+ 56+ 96* 72+
42 24 23 23
140* 51* 96+ 72i
37 15 27 27
145* 49* 106+ 76+
41 17 30 21
147* 45+ 107+ 70*
HDL,
Chol TG PL AI A II
24* 11* 41* 30* 14*
15 4 28 20 5
22+ lo+ 40* 27+ 17+
15 3 22 22 5
23* 9& 38i 35k 14,
14 3 12 24 6
22* lo+ 37* 37i 14+
HDL
Chol TG PL AI A II
47+ 24+ 89* 98f 39+
22 8 35 24 9
46k 21+ 81k 98+ 38+
19 5 30 37 12
48+ 19* 83* 106+ 42k
19 5 19 35 8
497t 19+ 84+ lIO+ 44+
HDL-Chol.
(precip.)
38f17
40*
18
41&
18
42+
* P 0.05; * * P 0.01 and
* * * P 0.001 compared
39 16 27 * 20 15 3 21 17 5 21 5 28 29 * 7* 20
to initial value.
RWlltS
Six patients left the study, 2 in the metformin group because of gastrointestinal discomfort from which they recovered immediately, 4 in the placebo group because of lacking effect on blood glucose values, which increased over 200 mg/dl. When compared for sex, age, BMI, initial and final value and change of HbAlc, the treatment groups differed in the change of HbAlc which was higher in the metformin group (Table 1). Lipid and apoprotein concentrations In the group of patients treated with metformin there was a significant reduction in triglycerides in serum and in the density fractions VLDL and LDL. There was a simultaneous, highly significant
fall in cholesterol in VLDL, which produced a significant fall in total cholesterol even though LDL-cholesterol rose by 12% (n.s.). Cholesterol concentrations in the HDL- and HDL,-UC-fractions were unchanged; the increase in precipitated HDL-cholesterol was not significant. The following changes in apoproteins were significant: an increase in apo A-I and apo A-II in the HDL-fraction after 12 weeks, and a fall in apo B in the VLDL fraction after 12 weeks. By contrast, apart from a slight increase in A-I in the HDL, fraction and in HDL phospholipids, no significant changes were observed in the control group (Tables 2 and 3). The P values of all parameters in the treatment groups are summarized in Table 4.
100 TABLE 3 LIPIDS AND APOPROTEINS
IN SERUM AND LIPOPROTEIN
DENSITY
CLASSES IN THE PLACEBO
GROUP
Values are mean f SD in mg/dl. Weeks: 0
4
8
12
Serum
Chol TG PL AI A II B
245* 51 309 f 168 276* 49 125rt 26 51* 9 106+ 28
240* 44 276& 95 275+ 58 131* 22 53& 8 1lOf 12
238* 50 280 * 155 282* 45 127* 20 52f 7 103* 22
239f53 269f85 282 f 41 128*18 52* 8 103 * 19
VLDL
Chol TG PL B
50* 45 219 + 160 54+ 43 11* 9
45* 198f 39* lo+
22 98 25 4
45* 34 197 * 145 44* 47 11+ 8
39*17 186 f 74 41+23 11* 7
LDL
Chol TG PL B
147* 53* 96* 79+
51 16 26 24
149* 58f 104* 76+
41 19 27 20
145* 55* lOl+ 77+
152+45 47*13 109*30 76+15
HDL,
Chol TG PL AI A II
18& lo* 29k 21* 12+
9 4 13 14 4
19* lo* 33f 231t 13f
9 3 13 10 4
17+ 6 lo* 5 32&- 13 22* 9 12* 3
18+ 6 10* 3 33*15 26*12 * 13f 5
HDL
Chol TG PL AI A II
38+ 21* 68* 90+ 36+
13 7 19 23 8
41* 19* 16* 89* 40*
14 6 29 17 5
41* 21* 84* 98+ 39+
11 7 38 14 5
43*10 19+ 5 84*27 * 99* 13 41* 7
HDL-Chol.
(p=ip.)
35+
9
37*
12
34*
10
35*
47 19 28 18
9
* P 0.05; * * P 0.01 and * * * P 0.001 compared to initial value.
Lipoprotein composition The relative content of cholesterol and phospholipids in the VLDL fell by 14 and 21% and triglycerides increased by 9%. In the LDL fraction there was an increase in the proportion of cholesterol by 8% and a fall in triglycerides by 23%, the latter highly significant (Table 5). There were no changes in composition in the placebo group. Relationship with improvement of diabetic control In the group treated with metformin there was a highly significant fall in HbAlc from 6.76 to 6.08, in the control group the value fell from 6.48 to 6.34 (n.s.). Relations between AHbAlc and changes in all lipoprotein parameters were computed and ex-
pressed as Spearman correlation coefficients. Out of the parameters with significantly different changes between the groups, only the increase in the relative content of cholesterol in VLDL revealed a significant correlation to the improvement in HbAlc (see Table 6). Discussion The result that the fall in triglycerides in the VLDL fraction was accompanied by a reduction in VLDL-apo B, the only non-exchangeable and therefore uninterrupted constituent of the individual particle in the lipolytic cascade, leads to the conclusion of a reduction in circulating VLDL particles. Monitoring the composition of the VLDL fraction, however, shows that the relative
101 TABLE
4
TABLE
COMPARISON OF CHANGES VERSUS INITIAL VALUE BETWEEN THE METFORMIN GROUP AND THE PLACEBO GROUP BY MANN-WHITNEY TEST weeks: O/4 Serum TG Serum PL VLDL-TG VLDL-Chol HDL PL HDL, A-I
0.016 0.03 0.03 0.001 ns. 0.03
O/8
o/12
0.009 n.s. 0.04 0.003 ns. ns.
0.009 0.04 0.04 0.01 0.02 n.s.
Only significant
correlations
Apo A-I is. PL i.s. TG in HDL, Chol. % in VLDL
amount of apo B in the VLDL total mass increased in the course of treatment, i.e. this is not a purely numerical reduction in an unchanged particle spectrum. This is particularly clear if one considers the changes in the composition of lipids in the VLDL-fraction. Because the composition of newly secreted VLDL in type II diabetes is normal, irrespective of the existence of hypertriglyceridemia [19], the pathological initial state (elevated cholesterol, reduced triglycerides) must reflect abnormal distribution in the VLDL fraction. This finding has been reported elsewhere [20-221. The relative increase in triglycerides and the relative reductions in cholesterol and phospholipids in the course of treatment in our study suggest a shift away from the lipoproteins of the catabolic final stage in this density fraction towards particles richer in triglycerides and hence normalization.
TABLE
6
RELATIONSHIP BETWEEN CHANGES TEIN PARAMETERS AND IMPROVEMENT
IN
LIPGPROOF HbAlc
are shown. r
P
0.52 0.44 0.48 0.72
0.016 0.037 0.025 0.015
By contrast to VLDL in the LDL fraction the proportion of triglycerides fell and cholesterol rose, with a constant apo B content. An increased proportion of triglycerides in the LDL fraction has been reported in type II diabetes with a certain amount of agreement [23-261. Multidispersity of LDL and equivocal changes in composition of VLDL and LDL as a characteristic of NIDDM have been described recently independent from the VLDL-TG levels [27]. As VLDL and LDL in circulation constitute a continuous spectrum of precursor-product particles with a constant apo-B content but with varying amounts of other apoprotein components and lipid composition (large triglyceride-rich VLDL, remnants, IDL, LDL) we observed a dilution of the ‘middle population’, this means a reduction of the NIDDMprone multidispersity. As for HDL the elevated proportion of triglycerides in HDL affecting both HDL, and HDL,, seems to arise through the free exchange of components between triglyceride-rich
5
EFFECT OF TREATMENT WITH METFORMIN LIPOPROTEINEMIC NIDDM (n =18) Values are expressed
FOR
12 WEEKS
ON COMPOSITION
OF VLDL
AND
LDL IN HYPER-
as mean f SD in % 4
8
12
VLDL
Chol TG PL Apo B
14.7f2.1 62.9 f 6.5 18.7k6.1 3.7* 1.5
16.1+ 2.8 64.4+ 7.8 15.5 *5.4 * 4.Ok1.8
12.7 + 3.4 66.8 f 2.7 15.2k4.1 5.2k5.5
12.7 68.3 14.7 4.3
LDL
Chol TG PL Apo B
36.6k5.1 16.6 f 6.9 27.2k4.0 20.0 + 4.6
38.9k5.1 14.7 f 5.0 26.7 f 4.1 19.7 + 4.4
38.4k3.1 13.4*4.7 28.0 + 2.2 20.2f3.7
39.5k3.5 ** 12.754.9 *** 28.8 + 2.1 19.0 f 3.6 *
week: 0
* P 5 0.05; **p
so.01;*** P 5 0.001 compared
to initial value.
+ + f f
3.6 5.2 2.5 2.5
102 lipoproteins and HDL, and recedes with the reduction of hypertriglyceridemia. The question arises whether the changes in lipoprotein particle numbers and compositions observed in this study are biguanide specific. Hypocaloric diet treatment of type II diabetes also reduces hypertriglyceridemia and increases HDLcholesterol if lo-12% weight loss is achieved [28]. Under sulphonylurea therapy, low HDL values were more likely to be observed, or HDL-related parameters (HDL-cholesterol, apo A-I) did not increase [29-331. Under insulin treatment, both reductions in triglycerides and increases in HDLcholesterol were observed, IDL-cholesterol was unchanged [34]. In the only available report on insulin-induced changes with detailed data on lipoprotein composition in all density classes, a 60% reduction in VLDL-triglycerides was reported, and in contrast to our results, a fall in the proportion of triglycerides and an increase in cholesterol and phosphohpids in VLDL. IDL and LDL were isolated separately: their ratio to one another did not change substantially [35]. The differences between insulin- and metformin-mediated changes are in favour of a metformin-specific effect on VLDL and LDL, which is confirmed by our finding that the changes in VLDL and LDL composition were not significantly correlated with the improvement in HbAlc. This view is further strengthened by the fact that the triglyceridelowering effect of biguanides has been observed in non-diabetic subjects as well as in diabetics [36]. Irrespective of specificity, the practical importance of the observed changes has been underlined recently in the Paris Prospective Study in which hypertriglyceridemia proved to be the only factor positively and significantly associated with coronary death in an 11-year follow up of subjects with impaired glucose tolerance or diabetes [37]. References Panzram, G., Mortality and survival in type I (non-insulindependent) diabetes mellitus, Diabetologia, 30 (1987) 123. Reunanen, A., Mortality in type II diabetes. Ann. Clin. Res., 15 (Suppl. 37), (1983) 26. PyiirBla, K. and Laakso, M., Macrovascular disease in diabetes mellitus. In: Mann, Y.I., Pyijr&ti, K. and Teuscher, A. (Eds.), Diabetes in Epidemiological Perspective, Churchill Livingstone, Edinburgh, 1983, pp. 183.
4 Kannel, W.B. and McGee, D.L., Diabetes and cardiovascular disease. The Framingham study, JAMA, 241 (1983) 2035. 5 Garcia, M.J., McNamara, P.M., Gordon, T. and Kannel, W.B., Morbidity and mortality in diabetics in the Framingham population. A sixteen-year follow study, Diabetes, 23 (1974) 105. 6 Ganda, O.P. Pathogenesis of macrovascular disease in the human diabetic. Diabetes, 29 (1980) 931. 7 Haupt, E., Knick, G., Koschinsky, T., Liebermeister, H., Schneider, J. and Hirche, H., Orale antidiabetische Kombinationstherapie mit Sulfonylharnstoffen und Metformin. Ergebnisse einer dreimonatigen Praxisstudie. Medwelt, 40 (1989) 118. 8 Havel, R.J., Eder, H.A. and Bragdon, J.H., The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum, J. Clin. Invest., 34 (1955) 1345. 9 Radding, C.M. and Steinberg, D., Studies on the synthesis and secretion of serum lipoproteins by rat liver slices, J. Clin. Invest., 39 (1960) 1560. 10 Staehler, F., Munz, E. and Kattermann, R., Enzymatische Bestimmung von Gesamtcholesterin im Serum, Dt. Med. Wschr., 100 (1975) 876. 11 Siedel, J.H., Schlumberger, S., Klose, S., Ziegenhorn, J. and Wahlefeld, A.W. Improved reagent for the enzymatic determination of serum cholesterol., J. Clin. Chem. Clin. B&hem., 19 (1981) 838. 12 Kattermann, R., Kupke, I.R. and Bomer, K., Vorllufig ausgew8hlte Methode fbr die enzymatische Bestimmung des Gesamtcholesterins im Serum. J. Clin. Chem. Clin. B&hem., 21 (1983) 347. 13 Wahlefeld, A.W., In: H.K. Bergmeyer (Ed.), Methoden der enzymatischen Analyse. 3rd edition, Verlag Chemie, Weinheim, 1974, p. 1878. 14 Takayama, M., Itoh, S., Nagasaki, T. and Tanimuri, I., A new enzymatic method for determination of choline-containing phospholipids, Clin. Chim. Acta 79 (1977) 93. 15 Lopes-Virella, M., Stone, P. and Ellis, S., Cholesterol determination in high-density lipoproteins separated by three different methods, Clin. Chem., 23 (1977) 882. 16 Mancini, G., Carbonara, A.D. and Hereman, J.F., Immunochemical quantification of antigen by single radial immuno diffusion, Immunochemistry, 2 (1965) 235. 17 Baynes, J.W., Bunn, H.F. and Goldstein, D.E., National Diabetes Data Group: Report of the Expert Commitee on Glycosylated Hemoglobin, Diabetes Care, 7 (1984) 602. 18 Masuda, Y., Kawata, Y., Fujita, S., Katayama, Y. and ltol, K., Determination of Hemoglobin Ale by a minicolumn method which eliminates Schiff base without preincubation. Translated from Igaku no Ayuma, 30 (1984) 8. 19 Kissebah, A.H., Alfarsi, S., Evans, D.J. and Adams, P.W., Integrated regulation of very-low-density lipoprotein triglyceride and apolipoprotein B kinetics in non-insulin-dependent diabetes mellitus. Diabetes, 31 (1982) 217. 20 Weisweiler, P. and Schwandt, P., Type I (insulin-dependent) versus type II (non-insulin-dependent) diabetes mellitus: Characterization of serum lipoprotein alterations. Eur. J. Clin. Invest., 17 (1987) 87.
103 21 Fielding, C.J., Reaven, G.M., Liu, G. and Fielding, P.E., Increased free cholesterol in plasma low- and very-low-density lipoproteins in non-insulin-dependent mellitus: Its role in the inhibition of cholesteryl ester transfer, Proc. Natl. Acad. Sci. USA, 76 (1984) 2311. 22 Howard, B.V., Abbott, W.G.H., Beltz, W.F., Harper, J.T., Fields, R.M., Grundy, S.M. and Taskinen, M.R., Integrated study of low-density lipoprotein metabolism and very-lowdensity lipoprotein metabolism in non-insulin-dependent diabetes, Metabolism, 36 (1987) 870. 23 Taskinen, M.R., Nikkila, E.A., Kuusi, T. and Hamo, K., Lipoprotein lipase activity and serum lipoproteins in untreated type II (insulin-dependent) diabetes associated with obesity, Diabetologia, 22 (1982) 46. 24 Howard, B.V., Knowler, W.C., Vasquez, B., Kennedy, A.L., Pettitt, D.J. and Bennett, P.H., Plasma and lipoprotein cholesterol and triglyceride in the Pima Indian population. Comparison of diabetics and non-diabetics, Arteriosclerosis, 4 (1984), 462. 25 Schonfeld, G., Birge, C., Miller, J.P., Kessler, G. and Santiago, J., Apolipoprotein B levels and altered lipoprotein composition in diabetes, Diabetes, 23 (1974) 827. 26 Kramer, F.B., Chen, Y.-D.I., Cheung, R.M.C. and Reaven. G.M., Are the binding and degradation of low-density lipoprotein altered in type II (non-insulin-dependent) diabetes mellitus? Diabetologia, 23 (1982) 28. 27 Suzuki, N., Oikawa, S., Hori, S., Fujii, Y., Sakuma, E., Kotake, H., Namai, K. Yoshie, K. and Goto, Y., Appearance of multidisperse low density lipoprotein and altered lipoprotein composition in non-insulin-dependent diabetes with type Ha hyperlipoproteinaemia. Metabolism, 38 (1989) 224. 28 Kennedy, L., Walshe, K., Hadden, D.R., Weaver, J.A. and Buchanan, K.D., The effect of intensity dietary therapy on serum high-density lipoprotein cholesterol in patients with type II (non-insulin-dependent) diabetes mellitus: A prospective study. Diabetologia, 23 (1982) 24.
29 Bar-On, H., Landau, D. and Berry, E., Serum high-density lipoprotein and university group diabetes program results. Lancet, i (1977) 761. 30 Calvert, G.D., Graham, J.J., Mannik, T., Wiese, P.H. and Yeaters, R.A., Effects of therapy on plasma high-density lipoprotein cholesterol concentration in diabetes mellitus, Lancet, ii (1978) 66. 31 Kennedy, A.L., Lappin, T.R.J., Lavery, T.D., Hadden, D.R., Weaver, J.R. and Montgomery, D.A.D., Relation of highdensity lipoprotein cholesterol concentration to type of diabetes and its control, Br. Med. J., 2 (1978) 1191. 32 Wingerd, J., and Petitti, D.B., HDL-cholesterol and diabetes. Lancet, ii (1978) 676. 33 Howard, B.V., Xiaoren, P., Harper, I., Foley, J.E., Cheung, M.C. and Taskinen, M.R., Effect of sulfonylurea therapy on plasma lipids and high-density lipoprotein composition in non-insulin-dependent diabetes mellitus, Am. J. Med. 79 (Suppl. 3 B) (1985) 78. 34 Hughes, T.A., Clements, R.S., Fairclough, P.K., Bell, D.S.H. and Segrest, J.P., Effect of insulin therapy on lipoproteins in non-insulin-dependent diabetes mellitus (NIDDM), Atherosclerosis, 67 (1987) 105. 35 Taskinen, M.R., Kuusi, T., Helve, E., N&kill, E.A., and Yki-Jlrvinen, H., Insulin therapy induces antiatherogenic changes of serum lipoproteins in non-insulin-dependent diabetes, Arteriosclerosis, 8 (1988) 168. 36 Sirtori, C.R., Tremoli, E., Sirtori, M., Conti, F. and Paoletti, R., Treatment of hypertriglyceridemia with metformin, Atherosclerosis, 26 (1977) 583. 37 Fontbonne, A., Eschwege, E., Cambien, F., Richard, J.L., Ducimetiere, P., Thibult, N., Warnet, J.M., Claude, J.R. and Rosselin, G.E., Hypertriglyceridaemia as risk factor of coronary heart disease mortality in subjects with impaired glucose tolerance or diabetes. Results from ll-year followup of the Paris Prospective Study, Diabetologia. 32 (1989). 300.
97
82 (1990) 97-103
Elsevier Scientific Publishers Ireland, Ltd. ATHERO 04469
Metformin-induced changes in serum lipids, lipoproteins, and apoproteins in non-insulin-dependent diabetes mellitus Jiirgen Schneider, Thomas Erren, Peter ZGfel and Hans Kaffamik Department of Internal Medicine, Division Endocrinology and Metabolism,
Philipps-University
Marburg, Marburg (F.R.G.)
(Received 28 September, 1989) (Revised, received 19 January, 1990) (Accepted 26 January, 1990)
Forty patients with NIDDM and hyperlipoproteinemia were selected for a 12-week double-blind placebo-controlled trial to study the effects of metformin on lipoprotein concentration and composition. A significant decrease occurred in VLDL-apo B and all lipid components of VLDL, indicating a decreased number of circulating VLDL, while LDL-apo B was unchanged. Moreover in VLDL the relative TG content increased, the cholesterol content decreased, while in LDL the TG content decreased and the cholesterol content increased, indicating a change in the particle distribution over the spectrum VLDLIDL-LDL. The initially enhanced TG-content in HDL was reduced. While a reduction in VLDL is observed together with improving glucose control irrespective of the applied method, the observed compositional changes in VLDL and LDL have not been described before and seem to be metforminspecific.
Key words: Diabetes type II; Lipoproteins;
Metformin
Introduction Diabetics are predisposed to degenerative arterial vascular diseases (‘diabetic macroangiopathy’): diabetes is one of the main risk factors for atherosclerosis [l-5]. Macroangiopathy is responsible for about 75% of deaths in patients with known diabetes [6]. Whilst microangiopathy largely determines the prognosis in type I diabetes, general atherosclerosis of the large arteries
Correspondence to: Prof. Dr. J. Schneider, Department of Internal Medicine, Division Endocrinology and Metabolism, Khnikum Lahnberge, D-3550 Marburg F.R.G.
0021-9150/90/$03.50
is the major secondary disease of type II diabetes. The high frequency of hyper- and dyslipoproteinemia in type II diabetes is also well known and normalization of hyperlipoproteinemia has become one of the criteria of good metabolic control in type II diabetes. The effect of almost all treatment methods (diet, sulphonylurea drugs and insulin) on hyperlipoproteinemia is known and has been studied during the last decade in view of more recent knowledge of the different, sometimes opposing, effects of various lipoprotein classes and subfractions on atherogenesis. Differentiated lipoprotein analyses are lacking for biguanide therapy, presumably because these substances have been of minor therapeutic importance for the last
0 1990 Elsevier Scientific Publishers Ireland, Ltd.
98 10 years in some countries, especially in the U.S.A. Earlier results, which show a clear reduction in elevated triglyceride values in response to biguanides have recently been confirmed in a large number of patients [7]. The aim of the present study was to investigate the effect of metformin on serum lipids, apoproteins and lipoproteins, and their composition in non-insulin-dependent type II diabetics with hyperlipoproteinemia. Patients and methods
Patients The study was performed as a randomized placebo-controlled two-arm-trial in 40 patients with NIDDM and hyperlipoproteinemia. Patients with unstable angina pectoris, myocardial infarction within 1 year, myocardial and respiratory insufficiency, impaired renal function, hepatic disease besides fatty infiltration, gastrointestinal disorders, malignancies and psychic abnormalities were not included, as well as women of childbearing potency. Inclusion was independent from the quality of glucose control. Diagnosis of type II diabetes was clinical. In doubtful cases, it was confirmed by positive insulin and C-peptide values after glucagon stimulation. All patients were treated effectively with diet and sulfonylurea derivatives for at least 6 months. Hyperlipoproteinemia was defined as total cholesterol > 250 mg/dl and/or triglycerides > 200 mg/dl. The patients were aged between 37 and 77 years, all of them were overweight, with a body mass index (weight (kg)/height (m2)) from 25 to 32. The majority of patients in both groups were treated for hypertension., Diastolic blood pressure at entrance was higher than 95 mm Hg in one case in. the metformin group and in one case in the placebo group. Diagnosis of hypertension and coronary heart disease was distributed evenly between the groups. Further characterization of the patients completing the protocol in the two treatment groups is given in Table 1. Study protocol ) After giving informed consent, the patients were randomized by ,order of entrance to the study. They reported as out-patients at four-weekly inter-
TABLE 1 CHARACTERISTICS TWO TREATMENT
OF THE PARTICIPANTS GROUPS
IN THE
Mean f SD. placebo (n =16)
Male/female Age in years BMI HbAlc (0) HbAlc (12) AHbAlc (12-O)
9/7 61.5 i-12.3 27.1 + 3.0 6.48!c 0.87 6.34f 1.11 -0.13+ 0.69
metformin (n =18)
P
8/10 60.4 f9.5 26.1 k3.1 6.7651.05 6.08 f 0.98 -0.69 f 0.87
n.s. n.s. ns. n.s. n.s. 0.054
vals for checks on diabetes and hyperlipoproteinemia. The drug was given in gradually increasing doses: initially the patients received one, after 4 weeks (when tolerated) 2 capsules containing 0.85 g metformin or placebo, respectively. Drug treatment of accompanying disease was kept constant. As metformin treatment is permitted only in combination with sulphonylurea derivatives in the F.R.G., all patients received at least low doses of such drugs, mainly glibenclamide (minimum 1.75 mg). Method At 0, 4, 8 and 12 weeks, fasting blood samples were drawn and VLDL, LDL, HDL, and HDL isolated by preparative ultracentrifugation in 50-3 Ti rotors in L50 centrifuges ([8,9] Beckman Palo Alto). Cholesterol [lo-121, triglycerides [13], and phospholipids [14] in serum and in the density fractions were determined using enzymatic standard methods; HDL-cholesterol was also determined after polyanion precipitation of apo Bcontaining lipoprotein ([15] Boehringer, Mannheim). Apo A-I, A-II and B were determined in serum and density fractions using radial immunodiffusion on standard commercial plates ([16] Immuno, Heidelberg). HbAl was determined using a microcolumn method ([17,18] Biorad, Munich). Statistical comparison of the data of the patients who completed the 12-week trial (n = 18 in the metformin group, n = 16 in the placebo group) was made by Wilcoxon matched-pairs signed-rank test (comparisons within groups) and Mann-Whitney test (comparisons between groups).
99 TABLE
2
LIPIDS
AND APOPROTEINS
IN SERUM
AND
LIPOPROTEIN
CLASSES
IN THE METFORMIN
GROUP
Values are mean + SD in mg/dl. Weeks: Serum
Chol TG
0
4
8
12
PL AI A II B
242* 340 + 305* 140+ 55+ 104+
43 207 59 27 10 16
232& 43 * 250*161 ** 284+ 59 142+ 29 55+ 10 107+ 20
232+ 44 ** 259+210 ** 284+ 39 151* 29 56+ 10 105& 17
233? 51 * 242k156 *** 290+ 45 151* 40 53* 10 98+ 20
VLDL
Chol TG PL B
56* 243 + 68+ 15*
43 195 60 10
38+ 29 * 173+156 *** 40+ 32 9+ 5
35* 29 *** 186*205 ** 40+ 28 * 13+ 9
34+ 27 *** 170*144 *** 38+ 28 * lo* 4%
LDL
Chol TG PL B
133+ 56+ 96* 72+
42 24 23 23
140* 51* 96+ 72i
37 15 27 27
145* 49* 106+ 76+
41 17 30 21
147* 45+ 107+ 70*
HDL,
Chol TG PL AI A II
24* 11* 41* 30* 14*
15 4 28 20 5
22+ lo+ 40* 27+ 17+
15 3 22 22 5
23* 9& 38i 35k 14,
14 3 12 24 6
22* lo+ 37* 37i 14+
HDL
Chol TG PL AI A II
47+ 24+ 89* 98f 39+
22 8 35 24 9
46k 21+ 81k 98+ 38+
19 5 30 37 12
48+ 19* 83* 106+ 42k
19 5 19 35 8
497t 19+ 84+ lIO+ 44+
HDL-Chol.
(precip.)
38f17
40*
18
41&
18
42+
* P 0.05; * * P 0.01 and
* * * P 0.001 compared
39 16 27 * 20 15 3 21 17 5 21 5 28 29 * 7* 20
to initial value.
RWlltS
Six patients left the study, 2 in the metformin group because of gastrointestinal discomfort from which they recovered immediately, 4 in the placebo group because of lacking effect on blood glucose values, which increased over 200 mg/dl. When compared for sex, age, BMI, initial and final value and change of HbAlc, the treatment groups differed in the change of HbAlc which was higher in the metformin group (Table 1). Lipid and apoprotein concentrations In the group of patients treated with metformin there was a significant reduction in triglycerides in serum and in the density fractions VLDL and LDL. There was a simultaneous, highly significant
fall in cholesterol in VLDL, which produced a significant fall in total cholesterol even though LDL-cholesterol rose by 12% (n.s.). Cholesterol concentrations in the HDL- and HDL,-UC-fractions were unchanged; the increase in precipitated HDL-cholesterol was not significant. The following changes in apoproteins were significant: an increase in apo A-I and apo A-II in the HDL-fraction after 12 weeks, and a fall in apo B in the VLDL fraction after 12 weeks. By contrast, apart from a slight increase in A-I in the HDL, fraction and in HDL phospholipids, no significant changes were observed in the control group (Tables 2 and 3). The P values of all parameters in the treatment groups are summarized in Table 4.
100 TABLE 3 LIPIDS AND APOPROTEINS
IN SERUM AND LIPOPROTEIN
DENSITY
CLASSES IN THE PLACEBO
GROUP
Values are mean f SD in mg/dl. Weeks: 0
4
8
12
Serum
Chol TG PL AI A II B
245* 51 309 f 168 276* 49 125rt 26 51* 9 106+ 28
240* 44 276& 95 275+ 58 131* 22 53& 8 1lOf 12
238* 50 280 * 155 282* 45 127* 20 52f 7 103* 22
239f53 269f85 282 f 41 128*18 52* 8 103 * 19
VLDL
Chol TG PL B
50* 45 219 + 160 54+ 43 11* 9
45* 198f 39* lo+
22 98 25 4
45* 34 197 * 145 44* 47 11+ 8
39*17 186 f 74 41+23 11* 7
LDL
Chol TG PL B
147* 53* 96* 79+
51 16 26 24
149* 58f 104* 76+
41 19 27 20
145* 55* lOl+ 77+
152+45 47*13 109*30 76+15
HDL,
Chol TG PL AI A II
18& lo* 29k 21* 12+
9 4 13 14 4
19* lo* 33f 231t 13f
9 3 13 10 4
17+ 6 lo* 5 32&- 13 22* 9 12* 3
18+ 6 10* 3 33*15 26*12 * 13f 5
HDL
Chol TG PL AI A II
38+ 21* 68* 90+ 36+
13 7 19 23 8
41* 19* 16* 89* 40*
14 6 29 17 5
41* 21* 84* 98+ 39+
11 7 38 14 5
43*10 19+ 5 84*27 * 99* 13 41* 7
HDL-Chol.
(p=ip.)
35+
9
37*
12
34*
10
35*
47 19 28 18
9
* P 0.05; * * P 0.01 and * * * P 0.001 compared to initial value.
Lipoprotein composition The relative content of cholesterol and phospholipids in the VLDL fell by 14 and 21% and triglycerides increased by 9%. In the LDL fraction there was an increase in the proportion of cholesterol by 8% and a fall in triglycerides by 23%, the latter highly significant (Table 5). There were no changes in composition in the placebo group. Relationship with improvement of diabetic control In the group treated with metformin there was a highly significant fall in HbAlc from 6.76 to 6.08, in the control group the value fell from 6.48 to 6.34 (n.s.). Relations between AHbAlc and changes in all lipoprotein parameters were computed and ex-
pressed as Spearman correlation coefficients. Out of the parameters with significantly different changes between the groups, only the increase in the relative content of cholesterol in VLDL revealed a significant correlation to the improvement in HbAlc (see Table 6). Discussion The result that the fall in triglycerides in the VLDL fraction was accompanied by a reduction in VLDL-apo B, the only non-exchangeable and therefore uninterrupted constituent of the individual particle in the lipolytic cascade, leads to the conclusion of a reduction in circulating VLDL particles. Monitoring the composition of the VLDL fraction, however, shows that the relative
101 TABLE
4
TABLE
COMPARISON OF CHANGES VERSUS INITIAL VALUE BETWEEN THE METFORMIN GROUP AND THE PLACEBO GROUP BY MANN-WHITNEY TEST weeks: O/4 Serum TG Serum PL VLDL-TG VLDL-Chol HDL PL HDL, A-I
0.016 0.03 0.03 0.001 ns. 0.03
O/8
o/12
0.009 n.s. 0.04 0.003 ns. ns.
0.009 0.04 0.04 0.01 0.02 n.s.
Only significant
correlations
Apo A-I is. PL i.s. TG in HDL, Chol. % in VLDL
amount of apo B in the VLDL total mass increased in the course of treatment, i.e. this is not a purely numerical reduction in an unchanged particle spectrum. This is particularly clear if one considers the changes in the composition of lipids in the VLDL-fraction. Because the composition of newly secreted VLDL in type II diabetes is normal, irrespective of the existence of hypertriglyceridemia [19], the pathological initial state (elevated cholesterol, reduced triglycerides) must reflect abnormal distribution in the VLDL fraction. This finding has been reported elsewhere [20-221. The relative increase in triglycerides and the relative reductions in cholesterol and phospholipids in the course of treatment in our study suggest a shift away from the lipoproteins of the catabolic final stage in this density fraction towards particles richer in triglycerides and hence normalization.
TABLE
6
RELATIONSHIP BETWEEN CHANGES TEIN PARAMETERS AND IMPROVEMENT
IN
LIPGPROOF HbAlc
are shown. r
P
0.52 0.44 0.48 0.72
0.016 0.037 0.025 0.015
By contrast to VLDL in the LDL fraction the proportion of triglycerides fell and cholesterol rose, with a constant apo B content. An increased proportion of triglycerides in the LDL fraction has been reported in type II diabetes with a certain amount of agreement [23-261. Multidispersity of LDL and equivocal changes in composition of VLDL and LDL as a characteristic of NIDDM have been described recently independent from the VLDL-TG levels [27]. As VLDL and LDL in circulation constitute a continuous spectrum of precursor-product particles with a constant apo-B content but with varying amounts of other apoprotein components and lipid composition (large triglyceride-rich VLDL, remnants, IDL, LDL) we observed a dilution of the ‘middle population’, this means a reduction of the NIDDMprone multidispersity. As for HDL the elevated proportion of triglycerides in HDL affecting both HDL, and HDL,, seems to arise through the free exchange of components between triglyceride-rich
5
EFFECT OF TREATMENT WITH METFORMIN LIPOPROTEINEMIC NIDDM (n =18) Values are expressed
FOR
12 WEEKS
ON COMPOSITION
OF VLDL
AND
LDL IN HYPER-
as mean f SD in % 4
8
12
VLDL
Chol TG PL Apo B
14.7f2.1 62.9 f 6.5 18.7k6.1 3.7* 1.5
16.1+ 2.8 64.4+ 7.8 15.5 *5.4 * 4.Ok1.8
12.7 + 3.4 66.8 f 2.7 15.2k4.1 5.2k5.5
12.7 68.3 14.7 4.3
LDL
Chol TG PL Apo B
36.6k5.1 16.6 f 6.9 27.2k4.0 20.0 + 4.6
38.9k5.1 14.7 f 5.0 26.7 f 4.1 19.7 + 4.4
38.4k3.1 13.4*4.7 28.0 + 2.2 20.2f3.7
39.5k3.5 ** 12.754.9 *** 28.8 + 2.1 19.0 f 3.6 *
week: 0
* P 5 0.05; **p
so.01;*** P 5 0.001 compared
to initial value.
+ + f f
3.6 5.2 2.5 2.5
102 lipoproteins and HDL, and recedes with the reduction of hypertriglyceridemia. The question arises whether the changes in lipoprotein particle numbers and compositions observed in this study are biguanide specific. Hypocaloric diet treatment of type II diabetes also reduces hypertriglyceridemia and increases HDLcholesterol if lo-12% weight loss is achieved [28]. Under sulphonylurea therapy, low HDL values were more likely to be observed, or HDL-related parameters (HDL-cholesterol, apo A-I) did not increase [29-331. Under insulin treatment, both reductions in triglycerides and increases in HDLcholesterol were observed, IDL-cholesterol was unchanged [34]. In the only available report on insulin-induced changes with detailed data on lipoprotein composition in all density classes, a 60% reduction in VLDL-triglycerides was reported, and in contrast to our results, a fall in the proportion of triglycerides and an increase in cholesterol and phosphohpids in VLDL. IDL and LDL were isolated separately: their ratio to one another did not change substantially [35]. The differences between insulin- and metformin-mediated changes are in favour of a metformin-specific effect on VLDL and LDL, which is confirmed by our finding that the changes in VLDL and LDL composition were not significantly correlated with the improvement in HbAlc. This view is further strengthened by the fact that the triglyceridelowering effect of biguanides has been observed in non-diabetic subjects as well as in diabetics [36]. Irrespective of specificity, the practical importance of the observed changes has been underlined recently in the Paris Prospective Study in which hypertriglyceridemia proved to be the only factor positively and significantly associated with coronary death in an 11-year follow up of subjects with impaired glucose tolerance or diabetes [37]. References Panzram, G., Mortality and survival in type I (non-insulindependent) diabetes mellitus, Diabetologia, 30 (1987) 123. Reunanen, A., Mortality in type II diabetes. Ann. Clin. Res., 15 (Suppl. 37), (1983) 26. PyiirBla, K. and Laakso, M., Macrovascular disease in diabetes mellitus. In: Mann, Y.I., Pyijr&ti, K. and Teuscher, A. (Eds.), Diabetes in Epidemiological Perspective, Churchill Livingstone, Edinburgh, 1983, pp. 183.
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29 Bar-On, H., Landau, D. and Berry, E., Serum high-density lipoprotein and university group diabetes program results. Lancet, i (1977) 761. 30 Calvert, G.D., Graham, J.J., Mannik, T., Wiese, P.H. and Yeaters, R.A., Effects of therapy on plasma high-density lipoprotein cholesterol concentration in diabetes mellitus, Lancet, ii (1978) 66. 31 Kennedy, A.L., Lappin, T.R.J., Lavery, T.D., Hadden, D.R., Weaver, J.R. and Montgomery, D.A.D., Relation of highdensity lipoprotein cholesterol concentration to type of diabetes and its control, Br. Med. J., 2 (1978) 1191. 32 Wingerd, J., and Petitti, D.B., HDL-cholesterol and diabetes. Lancet, ii (1978) 676. 33 Howard, B.V., Xiaoren, P., Harper, I., Foley, J.E., Cheung, M.C. and Taskinen, M.R., Effect of sulfonylurea therapy on plasma lipids and high-density lipoprotein composition in non-insulin-dependent diabetes mellitus, Am. J. Med. 79 (Suppl. 3 B) (1985) 78. 34 Hughes, T.A., Clements, R.S., Fairclough, P.K., Bell, D.S.H. and Segrest, J.P., Effect of insulin therapy on lipoproteins in non-insulin-dependent diabetes mellitus (NIDDM), Atherosclerosis, 67 (1987) 105. 35 Taskinen, M.R., Kuusi, T., Helve, E., N&kill, E.A., and Yki-Jlrvinen, H., Insulin therapy induces antiatherogenic changes of serum lipoproteins in non-insulin-dependent diabetes, Arteriosclerosis, 8 (1988) 168. 36 Sirtori, C.R., Tremoli, E., Sirtori, M., Conti, F. and Paoletti, R., Treatment of hypertriglyceridemia with metformin, Atherosclerosis, 26 (1977) 583. 37 Fontbonne, A., Eschwege, E., Cambien, F., Richard, J.L., Ducimetiere, P., Thibult, N., Warnet, J.M., Claude, J.R. and Rosselin, G.E., Hypertriglyceridaemia as risk factor of coronary heart disease mortality in subjects with impaired glucose tolerance or diabetes. Results from ll-year followup of the Paris Prospective Study, Diabetologia. 32 (1989). 300.
