Reversible Abnormalities in Postheparin Lipolytic Activity During the Late Phase of Release in Diabetes M ellitus (Postheparin Lipolytic Activity in Diabetes) John D. Brunzell,
Daniel
Porte, Jr., and Edwin L. Biermon
To test whether abnormalities in multiphasic release of lipoprotein lipase are associated with hypertriglyceridemia in diabetes mellitus, postheparin lipolytic activity (PHLA) was measured during a high-dose, constant heparin infusion in 20 diabetic subjects with hypertriglyceridemia, 25 nondiabetic hypertriglyceridemic subiects and 7 normal subiects. The standard low heparin dose PHLA and the PHLA during the early phase of the hep arin infusion were the same in all groups. In contrast, the PHLA during the late phase of the heparin infusion was lower in the 12 untreated diabetic subjects than in the 25 nondiabetic hypertriglyceridemic and the 7 normal subjects ( p < 0.001). An abnormality in late phase PHLA in the untreated diabetic subjects was more apparent when it was compared to the level of PHLA attained during the early phase infusion (Equilibrium of the heparin PHlA/60 min PHLA). The relative PHLA in the late phase of the infusion was lower in the untreated diabetic subjects (0.671 f 0.147) than in the nondiabetic hypertriglyceridemic subiects (0.847 + 0.019, p < O.OOl), or in the chronically treated diabetic subjects (0.823 + 0.108, p < 0.05). Among the untreated diabetic
L
IPOPROTEIN
subjects, increasing fasting glucose levels were associated with both decreasing absolute PHLA levels at the late phase of the infusion ( r = -0.61, p < 0.02) and greater decreases in relative PHLA during the infusion ( r = -0.80, p < 0.001). Treatment of the diabetes with long-term oral sulfonylurea or insulin therapy corrected the abnormality in the late phase PHLA with an associated decrease in plasma triglyceride levels ( p < 0.001). In five subjects with a deficient PHLA response to a standard, low dose of heparin, the PHLA response was low throughout the heparin infusion. With treatment, the PHLA response to the low heparin dose corrected rapidly toward normal in those two diabetic subjects with PHLA deficiency, and the early PHLA response during the heparin infusion increased. However, the late phase abnormality in all untreated diabetic subiects did not correct to normal until after several months of antihyperglycemic therapy. In the untreated diabetic subjects the degree of elevation of the plasma triglyceride level appeared to result from the interaction of the abnormality in PHLA with the presence or absence of an inherited familial lipid disorder.
LIPASE, present
major role in plasma hepatic lipolysis accounts
in a variety of tissues, appears to play a triglyceride removal in man; and in animals extrafor the removal of up to 90% of chylomicron tri-
From the Department of Medicine, The University of Washington School of Medicine and the Seattle Veterans Administration Hospital. Received for publication December 18.1974. Dr. Brunzell was a V.A. Research Associate during the course of these studies. Part of this work was performed at the University Hospital Clinical Research Center (FR-37) and part at the Harborview Medical Center Clinical Research Center (RR1333). Supported by NIH grant AMG6670. Reprint requests should be addressed to Dr. John D. Brunzell, Veterans Administration Hospital, 4435 Beacon Avenue South, Seattle, Washington 98108. Presented in part at the 32nd Annual Meeting of the American Diabetes Association, Washington, D.C.. June 25.1972. o 1975 by Grune & Stratton, Inc. Metabolism,Vol. 24, No. 10 (October), 1975
1123
BRUNZELL,
1124
PORTE,
AND BIERMAN
glyceride (TG) from blood.’ It has been indirectly estimated as postheparin lipolytic activity (PHLA),* a measurement of lipolytic activity on triglyceride substrate after a small dose of intravenous heparin. A familial disorder with low PHLA and severe hypertriglyceridemia has been described3 and is associated with decreased adipose tissue levels of lipoprotein lipase.4 A reduction of this enzyme activity associated with hypertriglyceridemia has been described in hypothyroidism,5 in uremia,6 and in dysgammaglobulinemic conditions.’ Adults with diabetes mellitus are known to have a disproportionate prevalence of hypertriglyceridemiasv9 with elevated very low density lipoprotein and chylomicron levels. Severe uncontrolled diabetes is almost always associated with some degree of hypertriglyceridemia and treatment of the diabetes has been shown to be associated with a decrease in plasma TG levels.‘O~” Low PHLA levels have been reported in several patients with untreated overt diabetes and hypertriglyceridemia.” Both the elevated plasma TG levels and the low PHLA levels rapidly returned toward normal during insulin therapy. Similarly, insulin-requiring juvenile diabetics developed an increase in TG levels and a fall in PHLA when insulin was withdrawn for a 48-hr period. Impaired clearance of intravenously injected labeled very low density lipoproteins from the plasma following insulin withdrawal in these patients suggests that PHLA is functionally significant in TG removal.13 Although a few untreated diabetic patients have low PHLA, by far the majority have normal PHLA associated with hypertriglyceridemia.‘4 Just as in diabetic patients with PHLA deficiency, plasma TG levels fall when these untreated diabetic patients with normal PHLA are treated with insulin. This suggests that hypertriglyceridemia in these diabetics may in part be related to insulin deficiency and, perhaps, to a subtle defect in lipoprotein lipase not detected by the standard PHLA assay. Recently, two new approaches have been developed to search for more specific abnormalities in lipoprotein lipase. The first relates to purification of the enzyme. Postheparin lipolytic activity is now known to consist of multiple enzymatic activities that emanate from different tissue sources.‘5-‘s The subfractionation of the enzymatic activity in plasma I9920 and the specific tissue sites from which these enzymes arise2’-23needs further examination. The second approach relates to assessment of dynamic changes in enzymatic activity. Lipoprotein lipase release has been demonstrated to be multiphasic in rat hearts,24 as has PHLA release following heparin in man.2526 In earlier studies it was found that despite normal low dose PHLA responses, PHLA levels during the late phase of a high dose, prolonged (3-6 hr) heparin infusion were reduced in a few diabetic subjects. 27In the present study, this observation has been systematically explored in an attempt to relate this finding to the hypertriglyceridemia associated with the diabetic state. MATERIALS
AND METHODS
Forty-five subjects, both nondiabetic (Table 1) and diabetic (Table 2) with hypertriglyceridemia and normal postheparin lipolytic activity * were the subjects of this study. They were referred from the University of Washington Affiliated Hospital system or the local community because of hypertriglyceridemia and were studied while hospitalized on a metabolic ward. An elevated plasma triglyceride concentration was defined as a value greater than the age and sex
is c)I
45M
48M
58F
55M
5OM
37M
34M
39M
34M
57M
58M
58M
48M
64M
53M
AR
JCr
ABr
WEs
WR
BY
MMi
88
OB
WF
WM
EB
GM
VI
FK
108
69.4
77.7
62.9
98.4 111
115
143
155
137
84.9 133
133
101.0
83.3
118
83.0
90.6
142
94.6
122
104
72.0 86.0
126
108
88.0 53.8
121
120
08.3
77.3
142
88.1
248 206
362 374 420
a7 a7
9453
8EOt
97
243
34
71
+88
0.162
0.562
0.370
0.349
0.752
0.626
0.671
0.689
0.476
0.179
0.581
0.298
1.028
0.830
0.521
0.478
0.245
0.662
0.622
0.600
0.733
0.562
0.434
0.409
0.522
0.613
0.650
0.479
0.603
0.628
0.514
0.476
0.932
0.656
0.379
0.697
~P.rcwntcban9ein f&in9 TG leveb from basal to fat-free dii.
$ Historyof chybmicmnrin fastingpbrmo by 3% polyvinylpyrrolidone technique.
t percenfof meanideal body weighton h’atropolitonlife Inwronce Tables.
‘Relotim with hyperlipidemia:+ press”+;- abknt, nonfamiliol hy@pidemio;
0.173
0.516
0.163
0.466
0.236
0.854
0.405
0.369
0.654
0.221
0.634
0.477
0.365
0.55 1
0.583
0.565
0.390
0.381
0.331
0.416
0.331
0.522
0.398
0.481
0.250
0.749
0.412
0.368
0.697
0.577
0.6
+74
+74
+73
+74
56
+56
-5
+2
+49
+11
+41
+23
+72
-14
+36
+lD3
+181
+57
-20
+53
+40
+19
+a7
+30
+122
+91
+75
+145
-29
+153
S.D.
43
169
124
94 61
121 233
31
216
155
172
160
140
285
208
182
398
198
165
293
300
872
316
218
177
240
356
160
416
339
242
208
297
0 not studied,or no relatinr w&able.
8.3
88
104
95
66
136
55
84 a7
61
62
759
703
798f 11771
374
367x
3ast
1145
428
82
81
7.8
95
112
106
105
103
103
102
102
3820:
193
97 98
255
542
s59t
555r
97
96
95
95
1858$
92 95
333
188
516t
92
90
90
88
84
316
216
271
288
214
84
261
80
Mean
TOTAL
8.6
13.0
55M
Do
112
112
83.2
70.3
S.D.
31M
JB 109
31M
FS
122
92.4
93
111
45M
IT
54.5
77.4
19F
BR
108 108
71.7 SO.0
89.5
36M
Mwn
46M
EM
RCO
Normolinidemic
14
49M
DT
104
66.2
123
52M
JCn
98.0
11.3
58M
WF
120 140
Ea.4
83.2
39M
118
86.4
S.D.
46M
BH
RE
114
68.2 127
121
91.5
105
89.4
% l&W,
(K) 72.3
D
W.ipht
f
30M
51M
41M
LC
VD
59M
RCU
AT
42M
CD
hmilia,
Hypwlipidunia’
Table 1. Nondiabetic Subjects
0.492
0.751 0.775
0.317
I.100
0.801 0.318
0.167
0.495
0.151
0.499
0.105
0.856
0.088
0.890
0.859
0.951
0.602 0.332
0.810
1.051
0.900
0.859
0.109
0.847
0.758
0.980
0.860
0.507
0.705
0.620
0.409
0.175
0.494
0.226
1007
0.714
0.712
0.787 0.371
0.376
0.809 0.934
0.230
0.875 0.503
O.wO
0.810 0.554
0.540
0.594
0.618
0.710
0.869 0.392 0.308
0.889
0.555 0.378 0.533
0.920 0.789
0.553
0.570
0.714 0.88 1
0.367
0.7w 0.376
0.810
1.130 0.750
0.428 0.755
0.861
0.6XKl
43F
RCO
+
+
0
220
117
71.5
122.8
119
105
82.4
62.9
TG
163
137
122
108
83
200
189
110
103
316
304
300
288
269
234
212
200
185
404
207
364
339%
800% 952%
610%
432% 36701
4605%
669
4770%
u7
wJW)% 2105%
830% 6510t
(9530)%
1177 (258011
173
597
w/d1
162
160
w/d1
F.ltilVd Gl”cO*
306
178
170
97
208
362
353
816
245
465
Ul
-19
+211
~61
-35
+43
-54
-15
-36
+3
-91
-21
-25 -71
194
-70
-89
-89
-15
-86
-18
-27
+69
502
387
(728)
849
336
(952)
(512)
228
313
wifdl
Chofeskrol
0.609
0.536
0.579 0.160
S.D.
0.789
0.416
0.380
0.660
0.456
0.7U
Mean
0.299
0.523
0.788
0.485
0.225
0.347
0.232
0.716
0.244
0.231
0.271
0.305
0.352
0.260
0.392
0.488
0.604
0.479 0.255
0.441
0.490
0.869
0.671 0.147
0.133
0.409
0.510
0.468
0.761
0.680
0.624
0.639
0.719
0.790
0.900
0.790
0.759
0.388
0.323
0.212
0.178
0.502
0.310
0.464
0.312
0.438
0.477
0.379
0.387
0.660
No Diabetic Therapy 60 EQ EQwJ
0.467
0.408
0.468
Standard PHtA wEq FFA/ml/ mi”
Diabetic Subjects
% AT-G.6 on Fat Frw Diet
Table 2.
initially studied when had defkiency of PHLA and untreated diabetes (Table 3).
)Measured on on ad lib diet.
*‘MM0
(
11Drug therapy for diabetm NPH: NPH insulin; CPM: chlorpropamide; ACET: acetoheximids; TOL: tolbutamide.
#Percent change in fasting TG levels from basal to fat free diet.
tHistory of chylomicrons in fasting plasma by 3% polyvinyl pyrrolidone technique.
TPercent of mean ideal body weight based on Metropolitan life Insurance Tables.
*Relatives with hyperlipidemio: + present; - absent, nonfamilial hyperlipidemia; 0 not studied, or no relatives available.
63M
Cw
111
70.7
+
62M
UM
lit
ARe
122
67.5
+
43F
RI
+
121
17F
64.0
we
+
77.9
55F
127
148
95.0
MM0**
+
55M
159
118
54F
84.4
53.7
156
146
91.3
93.4
132
109
73.2
70.4
89
67.8
EA
+
0
+
+
0
+
161
96.3
127
135
73.2
135
77.8
82.7
125
% wwt
DPr
Intially treated
UF
SS
33M
RCO
45F
56M
TC
NB
23M
RS
42M
47M
JS
47M
41M
WK
CG
+
45M
GH
CR
+
+
43M
OL
+
+
93.4
weight
(Kg)
45M
Ag*fSsx
F.Xliliql tfyperlipidemia’
RB
Subject
lnitiolly Untrwred
NPH
NPH
CPM
TOL
Tot
Nffl
NPH
NPH
ACE,
NPH
NPH
NPH
NPH
NPH
CPM
NPH
Dw
0.345 0.146
0.163
0.477
0.152
0.379
0.250
0.176
0.230
0.246
0.431
0.343
0.527
0.579
Tharopy EQ
0.570
0.734
0.411
0.716
0.431
0.415
0.438
0.358
0.674
0.597
0.642
0.839
Short-Term 60
0.126
0.388
0.650
0.370
0.529
0.580
0.424
0.525
0.668
0.639
0.574
0.821
0.690
EQ/M)
0.190
0.704
0.714
0.692
1.104
0.719
0.411
0.652
0.642
0.697
60
0.223
0.588
0.517
0.567
1.072
0.705
0.345
a.463
0.462
0.568
Eq
0.710
0.720
0.815
Eql60
O.lOR
0.823
0.724
0.819
0.976
0.98 1
0.839
Chronic Therapy
PtftA During Heporin Infusion (pEq FFA/ml/min)
POSTHEPARIN
LIPOLYTIC
ACTIVITY
1127
corrected 94th percentile of values obtained in a control population of 950 adults in the same community,28 (spouses of the families of the subjects in this and another study% from this laboratory). Excluded were hypertriglyceridemic subjects with broad beta disease (type III hyperlipoproteinemia), hypothyroidism, uremia, nephrotic syndrome, auto-immune disease, or a recent history of therapy with a drug known to affect plasma triglyceride metabolism. Twenty-five of the 45 subjects were nondiabetic as determined by a fasting plasma glucose level less than 115 mg/lOO ml. This plasma glucose level is the level above which an acute insulin response (3-5 min) to a glucose injection is absent.29 Of the twenty diabetic subjects, 12 had untreated overt diabetes with fasting glucose levels above 150 mg/lOO ml, three had recently been started on insulin or oral sulfonylurea therapy, and five had been on long-term insulin or oral sulfonylurea therapy. Seven of the untreated diabetic subjects were restudied after several weeks of insulin or oral sulfonylurea therapy and three after long-term therapy as well (Table 2). Five subjects with overt de$iency of postheparin lipolytic activity (see the following), two and three with idiopathic lipoprotein lipase deficiency, were related to untreated diabetes studied for purposes of comparison (Table 3). Seven subjects with normal glucose and lipid levels were also studied (Table 1): Three on the formula diets described in the following, and four on ad libitum diets with assurances made that their weight was stable and that their diets were of normal comp.osition. The subjects were fed constant composition liquid formula diets with the calories calculated to maintain constant weight. Most subjects were sequentially fed a basal diet for 2 wk (40% calories as fat, 45% as carbohydrate, and 15% as protein) and a fat-free, high carbohydrate diet for 2 wk (85% carbohydrate, 15% protein).” Plasma triglyceride, cholesterol, and glucose levels were measured three times weekly on both the basal and fat-free, high carbohydrate diets. Some subjects with a history of recurrent abdominal pain could not be maintained on fat containing basal diet because of severe hypertriglyceridemia, and some normal subjects were studied on the fat containing diet only (Table 1). The effect of the diets on plasma triglyceride levels was estimated by comparison of the mean of the last two triglyceride levels on the fat containing diet to the mean of the plasma triglyceride level on days 8 and 10 of the fat-free diet. Plasma triglyceride,” cholesteroL3’ and the presence or absence of chylomicrons32 were determined by previously reported techniques. Plasma glucose was measured by the autoanalyzer ferricyanide method. Plasma postheparin lipolytic activity was measured with Ediole substrate both after a low dose of hepn (380 U/m”), and on a separate day during a prolonged (3-5 hr) maximal heparin infusion. ’ The mfuston was begun with a loading dose of heparin (2280 U/m2 or approximately 60 U/kg body weight) which appears to be maximal since no more PHLA is released with larger doses (Fig. 1). The half-life of lipolytic activity was estimated to be 30 min for the purposes of infusing heparin by constant delivery pump to maintain constant circulating levels2’(actual measured infusion rate: 1984 U/m’/hr). PHLA was measured at 30 min intervals during the infusion. Normal values for the low dose standard PHLA with Ediole2 were based on mean values f 2 SD. (0.460 f 130 uEq FFA/ml/min) obtained in 26 normal individuals of both sexes, 19-54 yr old, on ad libitum diets (mean of 6, 8, and 10 min samples after 380 U/m2 of heparin or approximately lOU/kg for subjects of normal weight). Thus, 0.200 uEq FFA/ml/min was used as the lower limit of normal. In this study the PHLA response to low dose heparin was measured after an overnight fast on the basal diet. Except for several normal subjects, the maximal PHLA during the heparin infusion was measured while on the fat-free diet. The presence or absence of a familial form of hypertriglyceridemia was determined by examination of the cholesterol and triglyceride levels among all living first degree relatives and most second degree relatives. A genetic form of hypertriglyceridemia was determined to be present if a subject had one or more first degree relatives with plasma triglyceride levels equal to or greater than the ninety-ninth percentile of the control population.28 If a subject had four or more first degree relatives with normal plasma triglyceride and cholesterol levels and no abnormal relativea, a diagnosis of nonfamilial hypertriglyceridemia was made. Results are reported as mean f standard deviation.
5OM
WE1
Familial
84
113
86.7
+
present;
82
93
TG
0.803 0.629
0.112
on fat-free
+
weight
diet only.
TG from
in fasting
body
in fasting
change
)Measured
percent
(
IHistory
ideal
of mean
of chylomicrons
tPercent
with
*Relatives
hyperlipidemio:
based
Patient
basal
plasma diet.
polyvinyl
of Dr. R. Havel,
to fat-free
by 3%
San
Francisco,
pyrrolidone
Calif.
technique.
Tables.
hyperlipidemia;
Life Insurance
non-familial
on Metropolitan
-absent,
0 not studied
or no relatives
available.
0.213
0.736
0.121
198
336$
0.044
966
4220$
0.172
278
0.060
268
S.D.
82.9
-
108
Mean
57.2
+ 163
0.420
LB
0.950
55F
41F
MM0
0.102
0.178
0.126
0.192
0.098
0.978
0.072 0.082 0.178
0.243
-59
0.123
EC?/60
equilibrium
60 min
0.202
264
8451
-54
PHLA
pEq FFAjmljmin
Standard
0.162
(167)
% ATG on Fat Free Diet@
0.106
1075
3371
mg/dl
Cholesterol
(255)
mg/dU
-79
to diabetes
81
116
50.0
(w/d4
Fasting Glucose
75.0
% tewt
0
(Kg)
Weight
+
Hyperlipidemia*
Table 3. Subjects with Deficiency of Posthcparin Lipolytic Activity
+30
secondaw
3OF
PHLA deficiency
32M
AF
Age/Sex
PHLA deficiency
PG
Subject
Idiopathic
POSTHEPARIN
LIPOLYTIC
1129
ACTIVITY
Y
4 q
l.O-
8 0.8?I 0.6-
Fig. 1. Mean (*SD) peak posthsparin lipolytic activity in normal subiects after varying doses of intmvenout heparin.
$
0.4-
;
0.2-
s
L
Maximal I,‘PHLA
I
1
I
Standard
T 0
PHLA
n=3 0
I 50 HEPARIN
I
100 PULSE
I
150
I
200
I
250
(UNITS/Kg)
RESULTS
Characterization of Subjects
The fasting plasma glucose levels in the nondiabetic hypertriglyceridemic subjects ranged from 80-l 12 mg/lOO ml (Table 1) and in the untreated diabetics from 160-316 mg/lOO ml (Table 2). Among the subjects with hypertriglyceridemia, the plasma triglyceride levels in the untreated diabetic subjects (3156 f 2832 mg/lOO ml) were higher on the basal diet than those of the nondiabetic subjects (703 f 759 mg/lOO ml; p < O.OOl), and were frequently associated with the presence of chylomicrons in plasma on the basal diet after an overnight fast (A). The untreated diabetic subjects as a group had a fall in plasma triglyceride levels on the fat free diet (-44 f 46%; p < 0.001) which suggests that a large component of their circulating plasma triglyceride was of dietary origin. In contrast, the group of nondiabetic hypertriglyceridemic subjects had a rise in TG on the fat free, high carbohydrate diet (+56 f 56%; p < 0.001). The standard low dose PHLA was in the normal range in both groups of hypertriglyceridemic subjects. It appeared to be lower in the untreated diabetic subjects (0.369 f 0.104 uEq FFA/ml/min) than in the nondiabetic hypertriglyceridemic subjects (0.466 f 0.163) but the difference was not significant. PHLA During the Heparin Infusion Normal and nondiabetic hypertriglyceridemic subjects. The peak PHLA during the heparin infusion was reached at 60 min in both the normal and the nondiabetic hypertriglyceridemic subjects, and was similar in both groups of subjects (0.562 f 0.162 and 0.581 f 0.179 crEqFFA/ml/min, respectively). The equilibrium PHLA, defined as the mean of three values during the last hour of heparin infusion, (late phase of release) was significantly lower than the 60 min value in both groups of subjects (p < O.OOl), but again the two groups were similar (normal: 0.499 f 0.151; hypertriglyceridemic: 0.494 f 0.175, pEqFFA/ml/min). The absolute value of PHLA among individuals in both groups was highly variable, but the equilibrium PHLA appeared to be a function of the 60-min value (r = 0.94, y = 0.910X + 0.0297, Fig. 2). The relationship of the equilibrium PHLA to the 60 min PHLA was fairly constant and similar in both groups (equilibrium/60 min; normal 0.890 + 0.088, hypertriglyceridemic 0.847 rt 0.109). Thus, no differences in PHLA during the heparin infusion could be demonstrated between these two groups.
BRUNZELL, PORTE, AND
3 IL2
n = 32
t
p ( ,001
.s
s
I
r
BIERMAN
. . . /A .
5.94
A’ $ dv” , , , , , dkLk;~~~~i~~~~ 0%
.
0.4 PHLA
AT
60
0.6 MINUTES
pEq
1.2 FFA/min/ml
and at equilibrium infusion.
of hoparin
Untreated diabetic subjects. The 60 min PHLA in the untreated diabetic subjects (0.579 f 0.160, pEqFFA/ml/min) was no different from that in the nondiabetic subjects (0.577 i 0.173). However, the equilibrium PHLA was lower in the untreated diabetic subjects (0.388 f 0.133) than the nondiabetic subjects (0.495 f 0.167, p < 0.05). This difference is amplified when the ratio of the equilibrium PHLA to the 60-min PHLA (EQ/60) is examined. This ratio in the untreated diabetic subjects (0.671 i 0.147) was much lower than in the nondiabetic subjects (0.856 f 0.105, p < 0.001). Although the equilibrium PHLA was lower in the untreated diabetic subjects, it was a function of the 60-min value (r = 0.76, y = 0.630x-0.0234, p < 0.005, Fig. 3). While there was no relationship between the 60-min PHLA and the fasting glucose level, the degree of hyperglycemia in the untreated diabetic subjects was inversely related to the level of the equilibrium PHLA (r = 0.61, p < 0.02, Fig. 4). Thus, those subjects with the highest fasting glucose levels had the lowest
Fii. 3. Relationship between portheparin lipolytic activity at 60 min and at equilibrium of heparin infusion in untroatod diabetic subjects with fasting comhyp4ycemia ( -I parod to relationship in nondbbotic rubjcts (-----) from Fig. 2.
g 3 Q
1 0.4
0 PHLA
AT
60
1 0.0 MINUTES
pEq
1 1.2 FFA/min/ml
POSTHEPARIN
LIPOLYTIC
ACTIVITY
1131
n=iz r : -.6l
P (.02
Fig. 4. Relationship between postheparin lipolytic activity at equilibrium of heparin infusion and fasting plasma glucose Ievelr in untreated diabetic rubjects with elevated fasting plasma glucose levels.
0%
I
I
I
250
200
I50
FASTING
PLASMA
GLUCOSE
300 mg/dl
PHLA levels during the late phase of the heparin infusion. The EQ/60 was also inversely related to the fasting glucose level (r = 0.80, p < O.OOl,~Fig.5). Recently treated diabetic subjects. Twelve diabetic subjects were studied within 2 mo of receiving insulin or oral sulfonylurea therapy. Those subjects who did not respond to oral sulfonylurea therapy with a decrease in fasting glucose levels and glycosuria were put on insulin therapy. The fasting plasma glucose levels for these recently treated diabetics (151 f 51 mg/dl) were lower than those in the untreated diabetic subjects (234 f 59 mg/dl, p < 0.02). The 60-min PHLA was again essentially the same as in the untreated diabetic subjects and the nondiabetic subjects. However, despite the treatment of hyperglycemia in these subjects, the defect in PHLA during the late phase of the heparin infusion persisted. The equilibrium PHLA (0.345 rEqFFA/ml/min) was lower than that in the nondiabetic subjects (0.495 f 0.167, p < 0.02) as
n = 12
r =-.80 p < ,001 .
FASTING
PLASMA
GLUCOSE
mg/dl
Fig. 5. Relationship between the relative postheparin lipolytic activity at equilibrium (RO/60) of the heparin infusion with fasting plasma glucose levels in untreated diabetic subjects.
1132
BRUNZELL,
PORTE,
AND
BIERMAN
was the EQ/60 ratio (recently treated diabetics 0.588 f 126, nondiabetics 0.856 + 0.105, p < 0.001). None of these parameters in the recently treated diabetics were different from that found in the untreated diabetic subjects. Paired plasma triglyceride levels did not change during this short period of treatment (see the following). In contrast, when eight diabetic subjects who had been on insulin or sulfonylurea therapy for longer than 6 mo without an intervening episode of ketoacidosis were studied, their PHLA release appeared to be the same as in nondiabetic subjects. The lower equilibrium PHLA was now not detectable (chronically treated diabetics 0.588 + 0.223 rEqFFA/ml/min; nondiabetic 0.495 f 0.167), nor was the EQ/60 ratio (chronically treated diabetics: 0.823 f 0.108; nondiabetic: 0.856 f 0.105). Thus, both of these parameters were significantly improved when compared to the untreated diabetics (equilibrium PHLA: p < 0.05, EQ/60 ratio: p < 0.05) and the recently treated diabetics (equilibrium PHLA: p < 0.01, EQ/60 ratio: p < 0.001). The mean fasting plasma glucose for these eight chronically treated diabetic subjects was 154 i 75 mg/dl. In the chronically treated subjects there was no correlation between glucose levels and PHLA levels. Seven subjects studied in Subjects studied serially during course of therapy. the untreated diabetic state were restudied after a short course of insulin or oral sulfonylurea therapy. The findings in these paired studies were the same as when the groups were examined as a whole. Thus, short-term treatment decreased fasting glucose levels, without changing the equilibrium PHLA levels. This group allows comparison of the effects of short term therapy on plasma triglyceride levels. On the fat free diet plasma triglyceride levels were no different (untreated diabetic state: 647 f 230 mg/dl; after a short course on therapy: 569 f 284 mg/dl). PHLA during the heparin infusion was studied in the untreated and/or recently treated diabetic state and again after chronic treatment with insulin or oral sulfonylurea in three subjects. The defect in the equilibrium PHLA corrected in each of these subjects in the chronically treated state when studied serially. Chronic therapy with insulin or oral sulfonylureas was associated with a decrease (89%) in plasma triglyceride levels from (33 10 f 2850 mg/dl) in the untreated state to (373 f 304 mg/dl, p < 0.001) in the chronically treated diabetic state. The three subjects with Subjects with postheparin lipolytic activity de$ciency. idiopathic PHLA deficiency and the two subjects with PHLA deficiency secondary to untreated diabetics had low levels of PHLA throughout the heparin infusion and were, thus, different from the untreated diabetics who had a normal low dose PHLA and a defect only in the late phase (equilibrium) PHLA (Table 3). One of the untreated diabetic subjects with PHLA deficiency (MMo) was treated for 2 wk with insulin with return of the 60-min PHLA to normal but with persistence of the low equilibrium PHLA (see MMO, Table 2) such that she now appeared to resemble other recently treated diabetic subjects.
POSTHEPARIN
LIPOLYTIC
1133
ACTIVITY
Role of Familial and Secondary Causes of Hypertriglyceridemia First degree relatives with significant hypertriglyceridemia were found in 13 of the 15 nondiabetic hypertriglyceridemic subjects who had large enough families for evaluation. Similarly, nine of 11 of the untreated diabetic subjects had nondiabetic relatives with significant hypertriglyceridemia. The untreated diabetic subjects with nonfamilial hypertriglyceridemia had lower plasma triglyceride levels (538 f 259 mg/dl) than those untreated diabetic subjects with a familial form of hypertriglyceridemia and the defect in PHLA (4234 f 2703 mg/dl, p < 0.02) (Table 2). All but one of the eight subjects with a concomitant familial form of hypertriglyceridemia remained hypertriglyceridemic after treatment of diabetes. In contrast, in all subjects who appeared to have a nonfamilial form of hypertriglyceridemia, triglyceride levels returned to normal with chronic insulin or oral sulfonylurea therapy. DISCUSSION
overt Bagdade et a1.12noted low PHLA levels in a few hypertriglyceridemic, diabetic patients that rapidly returned to normal after a course of insulin therapy. However, those patients seem to be rare, since most diabetic patients have been found to have a normal PHLA following a low dose of intravenous heparin (10 U/kg).14 In a previous report three diabetic subjects were noted to be unable to sustain PHLA levels during the late phase of a 3-5 hr heparin infusion.27 This decrease in PHLA appeared to be unrelated to the state of treatment of the diabetes, however, the possible differences between short and long term insulin therapy on PHLA were not appreciated. In the present study, differences in PHLA during a maximal, prolonged heparin infusion have been demonstrated between nondiabetic subjects (either with or without concomitant hypertriglyceridemia) and untreated diabetic subjects. The factor(s), apparently independent of the hypertriglyceridemia or the diabetic state, that account for the wide variation in the PHLA in the early phase of the heparin infusion (60 min) in all groups of subjects are unknown. It does not appear to be due to the range of circulating levels of insulin in the basal state, since there is no relationship between obesity (a major determinant of basal insulin levels”) and the 60 min PHLA value, or for that matter between obesity and any other parameter of PHLA measured in this study. Shafrir suggested that the PHLA released shortly after a low dose heparin injection was a function of the TG level,% but there was no relationship in this study between the 60-min PHLA value and the TG concentration. Whatever the factors are that control the levels of PHLA during the early phase of the heparin infusion, the equilibrium PHLA levels appear to be a function of these early levels in the nondiabetic subjects (Fig. 2). The decrease in equilibrium PHLA in the untreated diabetic subjects was even more notable when considered in relation to the 60-min level, presumably due to the variance introduced by factors in the two groups of subjects which relate primarily to the early phase of PHLA release. That the decline in late phase PHLA is related to abnormal carbohydrate metabolism or insulin deficiency, is supported by the fact that those diabetic
1134
BRUNZELL.
PORTE,
AND BIERMAN
subjects with the highest fasting plasma glucose levels had the lowest PHLA levels during the late phase of the infusion (Figs. 4 and 5). The return of PHLA and triglyceride levels to or toward normal with long term insulin or oral sulfonylurea therapy supports the relationship between the diabetic state and abnormal PHLA release. The mechanism for the decrease in PHLA during the late phase of the heparin infusion is speculative at this time. Decreased protein synthesis that is known to occur secondary to insulin deficiency may be a factor. The early phase may be related to the storage of lipoprotein lipase, perhaps in capillary endothelial cells 35,36that remains normal until the defect in the later phase becomes severe enough to cause abnormalities in the early phase of release as well. The later phase may relate to adipose tissue storage or synthesis of the enzyme or enzyme precursors 37and, thus, could be the site of the abnormality produced by insulin deficiency. Alternatively, in untreated diabetes the enzyme may be removed from the plasma more rapidly thereby causing lower levels during the prolonged heparin infusion. This seems unlikely since the half-life of the enzyme is the same in nondiabetics as in untreated diabetic subjects.12 Beta-adrenergic stimulation has been shown to suppress release of lipoprotein lipase from adipose tissue in vitro.38,39 Untreated diabetic subjects have elevated catecholamine levels? and this may be an inhibiting factor in the release of PHLA. However, attempts to induce a defect in PHLA during a heparin infusion in normal subjects with a simultaneous epinephrine infusion were unsuccessful, and the defect in PHLA in an untreated diabetic subject was not reversed by 4 mo of propranolol therapy (unpublished observations). Glucagon is also increased in untreated diabetes4’ and this hormone has been shown to decrease adipose tissue lipoprotein lipase.4’ Further studies are necessary to clarify the role of these factors on abnormalities of PHLA in diabetes. The reason for the delay in the return to normal of PHLA in the late phase of the heparin infusion in the diabetic subjects restudied after a short period of oral sulfonylurea or insulin therapy, or following a recent episode of ketoacidosis, is unknown. A low synthesis rate of enzyme may be present. A similar delay in the return of PHLA to normal has been reported in hypothyroidism since it was found that > 6 wk was required to reverse abnormalities of TG metabolism to normal.5S43 Studies of lipoproteins lipase in adipose tissue of insulin deficient rats indicate that insulin plays an important role in the regulation of this enzyme. Lipoprotein lipase decreases in insulin deficiency and the decrease is reciprocally related to the severity of the diabetes.“*45 Furthermore, there is in vitro evidence that insulin is important for the synthesis and release of the enzyme in adipose tissue, since the amount of enzyme released increases when adipose tissue is incubated in the presence of insulin. 44.46Low levels of lipoprotein lipase activity have been documented in adipose tissue from patients with familial lipoprotein lipase deficiency and preliminary studies reveal a similar decrease in adipose tissue lipoprotein lipase in humans with untreated diabetes4’ To date, heart lipase is the only other tissue lipoprotein lipase to be studied in experimental diabetes. Borensztajn, et al. 4s found lipoprotein lipase in rat hearts to be independent of insulin levels. However, Atkin and Meng24 have
POSTHEPARIN
LIPOLYTIC
1135
ACTIVITY
demonstrated a biphasic release of PHLA from the perfused hearts of normal rats and also examined the in vivo pattern of release in alloxan treated rats. Following alloxan treatment the early phase of release was virtually identical to that in the normal rat, but there was a significant decrease in the amount of PHLA released during the later phase. In vivo insulin treatment reversed the abnormality in the later phase to normal just as it did in the present study. They also demonstrated that the enzyme activity of rat hearts could be depleted by repetitive exposure to heparin and that the rate of repletion was deficient in the alloxan diabetic rats. They postulated that their data were consistent with two tissue pools of lipoprotein lipase the early phase being a small storage pool and the later phase a pool related to the synthesis of lipoprotein lipase. The data in the human subjects in the present study are consistent with this concept. A diagnosis of a concomitant familial form of hypertriglyceridemia was possible in many of the patients in this study by evaluation of the distribution of plasma TG and cholesterol levels among their relatives. Recent evidence obtained from family studies suggests that the familial forms of hypertriglyceridemia segregate independently from diabetes.“’ However, when these two disorders coexist in the subjects in this study the level of plasma triglyceride was markedly elevated and, thus, appeared to be related to an interaction between a familial form of hypertriglyceridemia and overt diabetes with its PHLArelated TG removal defect. The abnormality in PHLA in untreated diabetes in patients with familial lipid disorders appears to cause an additional increment in plasma TG levels. After reversal of the PHLA abnormality with insulin or oral sulfonylurea therapy, plasma TG levels in those diabetics with a concomitant familial form of hypertriglyceridemia returned to levels found in subjects with monogenic hypertriglyceridemia2* free of diabetes. On the other hand in diabetics with nonfamilial hypertriglyceridemia plasma TG levels were restored to normal following several months of insulin or oral sulfonylurea therapy. Further support for the pathophysiological role of the abnormality in PHLA in plasma TG removal in diabetes,49 stems from the correlation between maximal plasma lipolytic rate, (maximal TG removal rate) during the heparin infusion” and the late phase PHLA. ACKNOWLEDGMENTS The authors would like to express their gratitude to Ms. Martha Campbell, Shirley Corey, and Diane Stevens for their assistance.
Kimura,
Martha
Pleasant,
Ellen
REFERENCES I. Bergman EN, Have1 RJ, Wolfe, BM, Bohmer T: Quantitative studies of the metabolism of chylomicron triglyceride and cholesterol by liver and extrahepatic tissues of sheep and dogs. J Clin Invest 50: 183, 1971 2. Fredrickson DA, Ono K, Davis LL: Lipolytic activity of postheparin plasma in hyperglyceridemia. J Lipid Res 4:24-33, 1963 3. Have1 RJ, Gordon RS Jr: Idiopathic hyperlipemia: Metabolic studies in an affected family. J Clin Invest 39: 1777-l 790, 1960
4. Harlan WR, Winesett PS, Wasserman AJ: Tissue lipoprotein lipase in normal individuals and in individuals with exogenous hypertriglyceridemia and the relationship of this enzyme to assimilation of fat. J Clin Invest 46: 239-247, 1967 5. Porte D Jr, O’Hara DD, Williams RH: The relation between postheparin lipoytic activity and plasma triglyceride in myxedema. Metabolism 15:107-l 13, 1966 6. Bagdade JD, Porte D Jr, Bierman EL:
1136
Hypertriglyceridemia: A metabolic of chronic renal failure. N Engl
RRIJNZELL,
consequence J Med 279:
181-185, 1968 7. Glueck CJ, Kaplan AP, Levy RI, Greten H, Gralnick H, Fredrickson DS: A new mechanism of exogenous hyperglyceridemia. Ann Intern Med 71: 1051-1062, 1969 8. Man EB, Peters JP: Serum lipids in diabetes. J Clin Invest 14:579, 1935 9. New MI, Roberts TN, Bierman EL, Reader GG: The significance of blood lipid alterations in diabetes mellitus. Diabetes 12: 208-2 12, 1963 IO. Thannhauser SJ: Hyperlipidemia in severe untreated diabetes with secondary eruptive xanthoma, in: Lipidoses: Disease of the Intracellular Lipid Metabolism (3rd ed). Grune St Stratton, New York, 1958 296 pp 11. Chance GW, Albutt EC, Edkins SM: Serum lipids and lipoproteins in untreated diabetic children. Lancet I:1 126, 1969 12. Bagdade JD, Porte Jr D, Bierman EL: Diabetic lipemia. A form of acquired fatinduced lipemia. N Engl J Med 276:427-433, 1967 13. Bagdade JD, Porte D Jr, Bierman EL: Acute insulin withdrawal and the regulation of plasma triglyceride removal in diabetic subjects. Diabetes 17:127-132.1968 14. Wilson DE, Schreibman PH, Arky RA: Postheparin lipolytic activity in diabetic patients with a history of mixed hyperlipemia. Relative rates against artificial substrates and human chylomicrons. Diabetes 18:562-566, 1969 15. Biale Y, Shafrir E: Lipolytic activity toward tri- and monoglycerides in postheparin plasma. Clin Chim Acta 23:413-419, 1969 16. Greten H, Levy RI, Fredrickson DS: Evidence for separate monoglyceride hydrolase and triglyceride lipase in postheparin human plasma. J Lipid Res l&326-330, 1969 17. Vogel WC, Brunzell JD, Bierman EL: A comparison of triglyceride, momoglyceride, and phospholipid substrates for postheparin lipolytic activities from normal and hypertriglyceridemic subjects. Lipids 6805-814. 1971 18. Zieve FJ, Zieve L: Postheparin phospholipase and postheparin lipase have different tissue origins. Biochem Biophys Res Commun 47:148&1485, 1972 19. Krauss RM, Windmueller HG, Levy RI, Fredrickson DS: Selective measurement of two different triglyceride lipase activities in rat postheparin plasma. J Lipid Res 14286-295, 1973
PORTE,
AND BIERMAN
20. Fielding CJ: Further characterisation of lipoprotein lipase and hepatic postheparin lipase from rat plasma. Biochim Biophys Acta 280:569-578, 1972 21. Egelrud T, Olivecrona T: The purification of a lipoprotein lipase from bovine skim milk. J Biol Chem 247:6212-6217, 1972 22. Ehnholm C, Shaw W, Harlan W, Brown V: Separation of two types of triglyceride lipase from human postheparin plasma. Circulation (Suppl IV) 48:112, 1973 23. Bensadoun A, Ehnholm C, Steinberg D, Brown WV: Purification and characterization of lipoprotein lipase from pig adipose tissue. J Biol Chem 249:2220-2227, 1974 24. Aktin E, Meng HC: Release of clearing factor lipase (lipoprotein lipase) in vivo and from isolated perfused hearts of alloxan diabetic rats. Diabetes 21:149-l 56, 1972 25. Boberg J: Heparin-released blood plasma lipoprotein lipase activity in patients with hyperlipoproteinemia. Acta Med Stand 191:97102, 1972 26. Brunzell JD, Smith ND, Porte D Jr, Bierman EL: Evidence for multiphasic release of postheparin lipolytic activity (PHLA). J Ciin Invest 51:16a. 1972 27. Porte Jr. D, Bierman EL: The effect of heparin infusion of plasma triglyceride in vivo and in vitro with a method for calculating triglyceride turnover. J Lab Clin Med 73:631648, 1969 28. Goldstein JL, Hazzard WR, Schrott HG, Bierman EL, Motulsky AC: Hyperlipidemia in coronary heart disease. II. Genetic analysis of lipid levels in 176 families and delineation of a new inherited disorder: combined hyperlipidemia. J Clin Invest 52: 1544-1568, 1973 29. Lerner RL, Porte D Jr, Acute and steadystate insulin responses to glucose in nonobese diabetic subjects. J Clin Invest 51:1624-1631, 1972 30. Brunzell JD, Lerner RL, Hazzard WR, Porte Jr. D, Bierman EL: Improved glucose tolerance with high carbohydrate feeding in mild diabetes. N Eng J Med 284521-524, 1971 31. Bierman EL, Porte D Jr, O’Hara DD, Schwartz M, Wood FC Jr: Characterization of fat particles in plasma of hyperlipemic subjects maintained on fat-free high carbohydrate diets. J Clin Inves 44:261-270, 1965 32. O’Hara DD, Porte Jr. D. Williams RH: The use of constant composition polyvinylpyrollidone (PVP) columns to study the interaction of fat particles with plasma. J Lipid Res 7~264, 1966
POSTHEPARIN
LIPOLYTIC
ACTIVITY
33. Bagdade JD, Bierman EL, Porte D Jr: The significance of basal insulin levels in the evaluation of the insulin response to glucose in diabetic and nondiabetic subjects. J Clin Invest 46:1549-1557, 1967 34. Shafrir E, Biale Y: Effect of experimental hypertriglyceridaemia on tissue and serum lipoprotein lipase activity. Eur J Clin invest 1: 19-24, 1970 35. Robinson DS: Assimilation, Distribution and Storage: The function of the plasma triglycerides in fatty acid transport, in M. Florkin and E. M. Stotz (eds) Comprehensive Biochemistry Amsterdam, Elsevier, 18:51-l 16, 1970 36. Blanchette-Mackie EJ, Scow RO: Site of lipoprotein lipase activity in adipose tissue perfused with chylomicrons. Electron microscope cytochemical study. J Cell Bio15 1:I-25, 1971 37. Schotz MC, Garfinkel AS: Effect of nutrition of species of lipoprotein lipase. Biochim Biophys Acta 270~472-478, 1972 38. Wing PR, Salaman MR, Robinson PS: Clearing factor lipase in adipose tissue. Factors influencing the increase in enzyme activity produced on incubation of tissue from starved rats in vitro. Biochem J 99:648-656, 1966 39. NikkilP EA, and Pykalistb 0: Regulation of adipose tissue lipoprotein lipase synthesis by intracellular free fatty acid. Life Sciences 7: 1303-1309, 1968 40. Christensen NJ: Plasma norepinephrine and apinephrine in untreated diabetes, during fasting and after insulin administration. Diabetes 23:1-8; 1974 41. Unger RH: Glucagon and diabetes mellitis. Adv Metab Disord 6:73-98, 1974 42. Nestel PJ, Austin W: Relation between
1137
adipose tissue lipoprotein lipase activity and compounds which affect intracellular lipolysis. Life Sciences 8:157-164, 1969 43. Baum D, Guthrie R, Brunzell JD, Vogel WC, Bierman EL: Triglyceride abnormality in infantile hypothyroidism. Am J Dis Child 125: 612-613, 1973 44. Pykllisto OJ: Regulation of adipose tissue lipoprotein lipase by free fatty acids. Thesis, University of Helsinki, Finland, 1970 45. Borensztajn J, Samols DR. Rubenstein AH: Effects of insulin on lipoprotein lipase activity in the rat heart and adipose tissue. Am J Physiol223:1271-1275, 1972 46. Wing PR, Salaman MR, Robinson DS: Clearing factor lipase in adipose tissue. A medium in which the enzyme activity of tissue from starved rats increased in vitro. Biochem J 99640647, 1966 47. Pykalisto OJ, Smith PH. Bierman EL, Brunzell JD: Decreased adipose tissue lipoprotein lipase in untreated diabetic man. Diabetes, (Suppl) 1:348, 1974 48. Brunzell JD, Hazzard WR, Motulsky AG, Bierman EL: Evidence for diabetes mellitus and genetic forms of hypertriglyceridemia as independent entities. Metabolism 24:00&000, 1975. 49. Brunzell JD, Porte D Jr, Bierman EL: PHLA Depletion: A defect in plasma triglyceride removal related to hyperglycemia. Diabetes Zl(Supp1 1):342, 1972 50. Brunzell JD, Hazzard WR, Porte D Jr, Bierman EL: Evidence for a common, saturable, triglyceride removal mechanism for chylomicrons and very low density lipoproteins in man. J Clin Invest 52:1578-1585, 1973
Daniel
Porte, Jr., and Edwin L. Biermon
To test whether abnormalities in multiphasic release of lipoprotein lipase are associated with hypertriglyceridemia in diabetes mellitus, postheparin lipolytic activity (PHLA) was measured during a high-dose, constant heparin infusion in 20 diabetic subjects with hypertriglyceridemia, 25 nondiabetic hypertriglyceridemic subiects and 7 normal subiects. The standard low heparin dose PHLA and the PHLA during the early phase of the hep arin infusion were the same in all groups. In contrast, the PHLA during the late phase of the heparin infusion was lower in the 12 untreated diabetic subjects than in the 25 nondiabetic hypertriglyceridemic and the 7 normal subjects ( p < 0.001). An abnormality in late phase PHLA in the untreated diabetic subjects was more apparent when it was compared to the level of PHLA attained during the early phase infusion (Equilibrium of the heparin PHlA/60 min PHLA). The relative PHLA in the late phase of the infusion was lower in the untreated diabetic subjects (0.671 f 0.147) than in the nondiabetic hypertriglyceridemic subiects (0.847 + 0.019, p < O.OOl), or in the chronically treated diabetic subjects (0.823 + 0.108, p < 0.05). Among the untreated diabetic
L
IPOPROTEIN
subjects, increasing fasting glucose levels were associated with both decreasing absolute PHLA levels at the late phase of the infusion ( r = -0.61, p < 0.02) and greater decreases in relative PHLA during the infusion ( r = -0.80, p < 0.001). Treatment of the diabetes with long-term oral sulfonylurea or insulin therapy corrected the abnormality in the late phase PHLA with an associated decrease in plasma triglyceride levels ( p < 0.001). In five subjects with a deficient PHLA response to a standard, low dose of heparin, the PHLA response was low throughout the heparin infusion. With treatment, the PHLA response to the low heparin dose corrected rapidly toward normal in those two diabetic subjects with PHLA deficiency, and the early PHLA response during the heparin infusion increased. However, the late phase abnormality in all untreated diabetic subiects did not correct to normal until after several months of antihyperglycemic therapy. In the untreated diabetic subjects the degree of elevation of the plasma triglyceride level appeared to result from the interaction of the abnormality in PHLA with the presence or absence of an inherited familial lipid disorder.
LIPASE, present
major role in plasma hepatic lipolysis accounts
in a variety of tissues, appears to play a triglyceride removal in man; and in animals extrafor the removal of up to 90% of chylomicron tri-
From the Department of Medicine, The University of Washington School of Medicine and the Seattle Veterans Administration Hospital. Received for publication December 18.1974. Dr. Brunzell was a V.A. Research Associate during the course of these studies. Part of this work was performed at the University Hospital Clinical Research Center (FR-37) and part at the Harborview Medical Center Clinical Research Center (RR1333). Supported by NIH grant AMG6670. Reprint requests should be addressed to Dr. John D. Brunzell, Veterans Administration Hospital, 4435 Beacon Avenue South, Seattle, Washington 98108. Presented in part at the 32nd Annual Meeting of the American Diabetes Association, Washington, D.C.. June 25.1972. o 1975 by Grune & Stratton, Inc. Metabolism,Vol. 24, No. 10 (October), 1975
1123
BRUNZELL,
1124
PORTE,
AND BIERMAN
glyceride (TG) from blood.’ It has been indirectly estimated as postheparin lipolytic activity (PHLA),* a measurement of lipolytic activity on triglyceride substrate after a small dose of intravenous heparin. A familial disorder with low PHLA and severe hypertriglyceridemia has been described3 and is associated with decreased adipose tissue levels of lipoprotein lipase.4 A reduction of this enzyme activity associated with hypertriglyceridemia has been described in hypothyroidism,5 in uremia,6 and in dysgammaglobulinemic conditions.’ Adults with diabetes mellitus are known to have a disproportionate prevalence of hypertriglyceridemiasv9 with elevated very low density lipoprotein and chylomicron levels. Severe uncontrolled diabetes is almost always associated with some degree of hypertriglyceridemia and treatment of the diabetes has been shown to be associated with a decrease in plasma TG levels.‘O~” Low PHLA levels have been reported in several patients with untreated overt diabetes and hypertriglyceridemia.” Both the elevated plasma TG levels and the low PHLA levels rapidly returned toward normal during insulin therapy. Similarly, insulin-requiring juvenile diabetics developed an increase in TG levels and a fall in PHLA when insulin was withdrawn for a 48-hr period. Impaired clearance of intravenously injected labeled very low density lipoproteins from the plasma following insulin withdrawal in these patients suggests that PHLA is functionally significant in TG removal.13 Although a few untreated diabetic patients have low PHLA, by far the majority have normal PHLA associated with hypertriglyceridemia.‘4 Just as in diabetic patients with PHLA deficiency, plasma TG levels fall when these untreated diabetic patients with normal PHLA are treated with insulin. This suggests that hypertriglyceridemia in these diabetics may in part be related to insulin deficiency and, perhaps, to a subtle defect in lipoprotein lipase not detected by the standard PHLA assay. Recently, two new approaches have been developed to search for more specific abnormalities in lipoprotein lipase. The first relates to purification of the enzyme. Postheparin lipolytic activity is now known to consist of multiple enzymatic activities that emanate from different tissue sources.‘5-‘s The subfractionation of the enzymatic activity in plasma I9920 and the specific tissue sites from which these enzymes arise2’-23needs further examination. The second approach relates to assessment of dynamic changes in enzymatic activity. Lipoprotein lipase release has been demonstrated to be multiphasic in rat hearts,24 as has PHLA release following heparin in man.2526 In earlier studies it was found that despite normal low dose PHLA responses, PHLA levels during the late phase of a high dose, prolonged (3-6 hr) heparin infusion were reduced in a few diabetic subjects. 27In the present study, this observation has been systematically explored in an attempt to relate this finding to the hypertriglyceridemia associated with the diabetic state. MATERIALS
AND METHODS
Forty-five subjects, both nondiabetic (Table 1) and diabetic (Table 2) with hypertriglyceridemia and normal postheparin lipolytic activity * were the subjects of this study. They were referred from the University of Washington Affiliated Hospital system or the local community because of hypertriglyceridemia and were studied while hospitalized on a metabolic ward. An elevated plasma triglyceride concentration was defined as a value greater than the age and sex
is c)I
45M
48M
58F
55M
5OM
37M
34M
39M
34M
57M
58M
58M
48M
64M
53M
AR
JCr
ABr
WEs
WR
BY
MMi
88
OB
WF
WM
EB
GM
VI
FK
108
69.4
77.7
62.9
98.4 111
115
143
155
137
84.9 133
133
101.0
83.3
118
83.0
90.6
142
94.6
122
104
72.0 86.0
126
108
88.0 53.8
121
120
08.3
77.3
142
88.1
248 206
362 374 420
a7 a7
9453
8EOt
97
243
34
71
+88
0.162
0.562
0.370
0.349
0.752
0.626
0.671
0.689
0.476
0.179
0.581
0.298
1.028
0.830
0.521
0.478
0.245
0.662
0.622
0.600
0.733
0.562
0.434
0.409
0.522
0.613
0.650
0.479
0.603
0.628
0.514
0.476
0.932
0.656
0.379
0.697
~P.rcwntcban9ein f&in9 TG leveb from basal to fat-free dii.
$ Historyof chybmicmnrin fastingpbrmo by 3% polyvinylpyrrolidone technique.
t percenfof meanideal body weighton h’atropolitonlife Inwronce Tables.
‘Relotim with hyperlipidemia:+ press”+;- abknt, nonfamiliol hy@pidemio;
0.173
0.516
0.163
0.466
0.236
0.854
0.405
0.369
0.654
0.221
0.634
0.477
0.365
0.55 1
0.583
0.565
0.390
0.381
0.331
0.416
0.331
0.522
0.398
0.481
0.250
0.749
0.412
0.368
0.697
0.577
0.6
+74
+74
+73
+74
56
+56
-5
+2
+49
+11
+41
+23
+72
-14
+36
+lD3
+181
+57
-20
+53
+40
+19
+a7
+30
+122
+91
+75
+145
-29
+153
S.D.
43
169
124
94 61
121 233
31
216
155
172
160
140
285
208
182
398
198
165
293
300
872
316
218
177
240
356
160
416
339
242
208
297
0 not studied,or no relatinr w&able.
8.3
88
104
95
66
136
55
84 a7
61
62
759
703
798f 11771
374
367x
3ast
1145
428
82
81
7.8
95
112
106
105
103
103
102
102
3820:
193
97 98
255
542
s59t
555r
97
96
95
95
1858$
92 95
333
188
516t
92
90
90
88
84
316
216
271
288
214
84
261
80
Mean
TOTAL
8.6
13.0
55M
Do
112
112
83.2
70.3
S.D.
31M
JB 109
31M
FS
122
92.4
93
111
45M
IT
54.5
77.4
19F
BR
108 108
71.7 SO.0
89.5
36M
Mwn
46M
EM
RCO
Normolinidemic
14
49M
DT
104
66.2
123
52M
JCn
98.0
11.3
58M
WF
120 140
Ea.4
83.2
39M
118
86.4
S.D.
46M
BH
RE
114
68.2 127
121
91.5
105
89.4
% l&W,
(K) 72.3
D
W.ipht
f
30M
51M
41M
LC
VD
59M
RCU
AT
42M
CD
hmilia,
Hypwlipidunia’
Table 1. Nondiabetic Subjects
0.492
0.751 0.775
0.317
I.100
0.801 0.318
0.167
0.495
0.151
0.499
0.105
0.856
0.088
0.890
0.859
0.951
0.602 0.332
0.810
1.051
0.900
0.859
0.109
0.847
0.758
0.980
0.860
0.507
0.705
0.620
0.409
0.175
0.494
0.226
1007
0.714
0.712
0.787 0.371
0.376
0.809 0.934
0.230
0.875 0.503
O.wO
0.810 0.554
0.540
0.594
0.618
0.710
0.869 0.392 0.308
0.889
0.555 0.378 0.533
0.920 0.789
0.553
0.570
0.714 0.88 1
0.367
0.7w 0.376
0.810
1.130 0.750
0.428 0.755
0.861
0.6XKl
43F
RCO
+
+
0
220
117
71.5
122.8
119
105
82.4
62.9
TG
163
137
122
108
83
200
189
110
103
316
304
300
288
269
234
212
200
185
404
207
364
339%
800% 952%
610%
432% 36701
4605%
669
4770%
u7
wJW)% 2105%
830% 6510t
(9530)%
1177 (258011
173
597
w/d1
162
160
w/d1
F.ltilVd Gl”cO*
306
178
170
97
208
362
353
816
245
465
Ul
-19
+211
~61
-35
+43
-54
-15
-36
+3
-91
-21
-25 -71
194
-70
-89
-89
-15
-86
-18
-27
+69
502
387
(728)
849
336
(952)
(512)
228
313
wifdl
Chofeskrol
0.609
0.536
0.579 0.160
S.D.
0.789
0.416
0.380
0.660
0.456
0.7U
Mean
0.299
0.523
0.788
0.485
0.225
0.347
0.232
0.716
0.244
0.231
0.271
0.305
0.352
0.260
0.392
0.488
0.604
0.479 0.255
0.441
0.490
0.869
0.671 0.147
0.133
0.409
0.510
0.468
0.761
0.680
0.624
0.639
0.719
0.790
0.900
0.790
0.759
0.388
0.323
0.212
0.178
0.502
0.310
0.464
0.312
0.438
0.477
0.379
0.387
0.660
No Diabetic Therapy 60 EQ EQwJ
0.467
0.408
0.468
Standard PHtA wEq FFA/ml/ mi”
Diabetic Subjects
% AT-G.6 on Fat Frw Diet
Table 2.
initially studied when had defkiency of PHLA and untreated diabetes (Table 3).
)Measured on on ad lib diet.
*‘MM0
(
11Drug therapy for diabetm NPH: NPH insulin; CPM: chlorpropamide; ACET: acetoheximids; TOL: tolbutamide.
#Percent change in fasting TG levels from basal to fat free diet.
tHistory of chylomicrons in fasting plasma by 3% polyvinyl pyrrolidone technique.
TPercent of mean ideal body weight based on Metropolitan life Insurance Tables.
*Relatives with hyperlipidemio: + present; - absent, nonfamilial hyperlipidemia; 0 not studied, or no relatives available.
63M
Cw
111
70.7
+
62M
UM
lit
ARe
122
67.5
+
43F
RI
+
121
17F
64.0
we
+
77.9
55F
127
148
95.0
MM0**
+
55M
159
118
54F
84.4
53.7
156
146
91.3
93.4
132
109
73.2
70.4
89
67.8
EA
+
0
+
+
0
+
161
96.3
127
135
73.2
135
77.8
82.7
125
% wwt
DPr
Intially treated
UF
SS
33M
RCO
45F
56M
TC
NB
23M
RS
42M
47M
JS
47M
41M
WK
CG
+
45M
GH
CR
+
+
43M
OL
+
+
93.4
weight
(Kg)
45M
Ag*fSsx
F.Xliliql tfyperlipidemia’
RB
Subject
lnitiolly Untrwred
NPH
NPH
CPM
TOL
Tot
Nffl
NPH
NPH
ACE,
NPH
NPH
NPH
NPH
NPH
CPM
NPH
Dw
0.345 0.146
0.163
0.477
0.152
0.379
0.250
0.176
0.230
0.246
0.431
0.343
0.527
0.579
Tharopy EQ
0.570
0.734
0.411
0.716
0.431
0.415
0.438
0.358
0.674
0.597
0.642
0.839
Short-Term 60
0.126
0.388
0.650
0.370
0.529
0.580
0.424
0.525
0.668
0.639
0.574
0.821
0.690
EQ/M)
0.190
0.704
0.714
0.692
1.104
0.719
0.411
0.652
0.642
0.697
60
0.223
0.588
0.517
0.567
1.072
0.705
0.345
a.463
0.462
0.568
Eq
0.710
0.720
0.815
Eql60
O.lOR
0.823
0.724
0.819
0.976
0.98 1
0.839
Chronic Therapy
PtftA During Heporin Infusion (pEq FFA/ml/min)
POSTHEPARIN
LIPOLYTIC
ACTIVITY
1127
corrected 94th percentile of values obtained in a control population of 950 adults in the same community,28 (spouses of the families of the subjects in this and another study% from this laboratory). Excluded were hypertriglyceridemic subjects with broad beta disease (type III hyperlipoproteinemia), hypothyroidism, uremia, nephrotic syndrome, auto-immune disease, or a recent history of therapy with a drug known to affect plasma triglyceride metabolism. Twenty-five of the 45 subjects were nondiabetic as determined by a fasting plasma glucose level less than 115 mg/lOO ml. This plasma glucose level is the level above which an acute insulin response (3-5 min) to a glucose injection is absent.29 Of the twenty diabetic subjects, 12 had untreated overt diabetes with fasting glucose levels above 150 mg/lOO ml, three had recently been started on insulin or oral sulfonylurea therapy, and five had been on long-term insulin or oral sulfonylurea therapy. Seven of the untreated diabetic subjects were restudied after several weeks of insulin or oral sulfonylurea therapy and three after long-term therapy as well (Table 2). Five subjects with overt de$iency of postheparin lipolytic activity (see the following), two and three with idiopathic lipoprotein lipase deficiency, were related to untreated diabetes studied for purposes of comparison (Table 3). Seven subjects with normal glucose and lipid levels were also studied (Table 1): Three on the formula diets described in the following, and four on ad libitum diets with assurances made that their weight was stable and that their diets were of normal comp.osition. The subjects were fed constant composition liquid formula diets with the calories calculated to maintain constant weight. Most subjects were sequentially fed a basal diet for 2 wk (40% calories as fat, 45% as carbohydrate, and 15% as protein) and a fat-free, high carbohydrate diet for 2 wk (85% carbohydrate, 15% protein).” Plasma triglyceride, cholesterol, and glucose levels were measured three times weekly on both the basal and fat-free, high carbohydrate diets. Some subjects with a history of recurrent abdominal pain could not be maintained on fat containing basal diet because of severe hypertriglyceridemia, and some normal subjects were studied on the fat containing diet only (Table 1). The effect of the diets on plasma triglyceride levels was estimated by comparison of the mean of the last two triglyceride levels on the fat containing diet to the mean of the plasma triglyceride level on days 8 and 10 of the fat-free diet. Plasma triglyceride,” cholesteroL3’ and the presence or absence of chylomicrons32 were determined by previously reported techniques. Plasma glucose was measured by the autoanalyzer ferricyanide method. Plasma postheparin lipolytic activity was measured with Ediole substrate both after a low dose of hepn (380 U/m”), and on a separate day during a prolonged (3-5 hr) maximal heparin infusion. ’ The mfuston was begun with a loading dose of heparin (2280 U/m2 or approximately 60 U/kg body weight) which appears to be maximal since no more PHLA is released with larger doses (Fig. 1). The half-life of lipolytic activity was estimated to be 30 min for the purposes of infusing heparin by constant delivery pump to maintain constant circulating levels2’(actual measured infusion rate: 1984 U/m’/hr). PHLA was measured at 30 min intervals during the infusion. Normal values for the low dose standard PHLA with Ediole2 were based on mean values f 2 SD. (0.460 f 130 uEq FFA/ml/min) obtained in 26 normal individuals of both sexes, 19-54 yr old, on ad libitum diets (mean of 6, 8, and 10 min samples after 380 U/m2 of heparin or approximately lOU/kg for subjects of normal weight). Thus, 0.200 uEq FFA/ml/min was used as the lower limit of normal. In this study the PHLA response to low dose heparin was measured after an overnight fast on the basal diet. Except for several normal subjects, the maximal PHLA during the heparin infusion was measured while on the fat-free diet. The presence or absence of a familial form of hypertriglyceridemia was determined by examination of the cholesterol and triglyceride levels among all living first degree relatives and most second degree relatives. A genetic form of hypertriglyceridemia was determined to be present if a subject had one or more first degree relatives with plasma triglyceride levels equal to or greater than the ninety-ninth percentile of the control population.28 If a subject had four or more first degree relatives with normal plasma triglyceride and cholesterol levels and no abnormal relativea, a diagnosis of nonfamilial hypertriglyceridemia was made. Results are reported as mean f standard deviation.
5OM
WE1
Familial
84
113
86.7
+
present;
82
93
TG
0.803 0.629
0.112
on fat-free
+
weight
diet only.
TG from
in fasting
body
in fasting
change
)Measured
percent
(
IHistory
ideal
of mean
of chylomicrons
tPercent
with
*Relatives
hyperlipidemio:
based
Patient
basal
plasma diet.
polyvinyl
of Dr. R. Havel,
to fat-free
by 3%
San
Francisco,
pyrrolidone
Calif.
technique.
Tables.
hyperlipidemia;
Life Insurance
non-familial
on Metropolitan
-absent,
0 not studied
or no relatives
available.
0.213
0.736
0.121
198
336$
0.044
966
4220$
0.172
278
0.060
268
S.D.
82.9
-
108
Mean
57.2
+ 163
0.420
LB
0.950
55F
41F
MM0
0.102
0.178
0.126
0.192
0.098
0.978
0.072 0.082 0.178
0.243
-59
0.123
EC?/60
equilibrium
60 min
0.202
264
8451
-54
PHLA
pEq FFAjmljmin
Standard
0.162
(167)
% ATG on Fat Free Diet@
0.106
1075
3371
mg/dl
Cholesterol
(255)
mg/dU
-79
to diabetes
81
116
50.0
(w/d4
Fasting Glucose
75.0
% tewt
0
(Kg)
Weight
+
Hyperlipidemia*
Table 3. Subjects with Deficiency of Posthcparin Lipolytic Activity
+30
secondaw
3OF
PHLA deficiency
32M
AF
Age/Sex
PHLA deficiency
PG
Subject
Idiopathic
POSTHEPARIN
LIPOLYTIC
1129
ACTIVITY
Y
4 q
l.O-
8 0.8?I 0.6-
Fig. 1. Mean (*SD) peak posthsparin lipolytic activity in normal subiects after varying doses of intmvenout heparin.
$
0.4-
;
0.2-
s
L
Maximal I,‘PHLA
I
1
I
Standard
T 0
PHLA
n=3 0
I 50 HEPARIN
I
100 PULSE
I
150
I
200
I
250
(UNITS/Kg)
RESULTS
Characterization of Subjects
The fasting plasma glucose levels in the nondiabetic hypertriglyceridemic subjects ranged from 80-l 12 mg/lOO ml (Table 1) and in the untreated diabetics from 160-316 mg/lOO ml (Table 2). Among the subjects with hypertriglyceridemia, the plasma triglyceride levels in the untreated diabetic subjects (3156 f 2832 mg/lOO ml) were higher on the basal diet than those of the nondiabetic subjects (703 f 759 mg/lOO ml; p < O.OOl), and were frequently associated with the presence of chylomicrons in plasma on the basal diet after an overnight fast (A). The untreated diabetic subjects as a group had a fall in plasma triglyceride levels on the fat free diet (-44 f 46%; p < 0.001) which suggests that a large component of their circulating plasma triglyceride was of dietary origin. In contrast, the group of nondiabetic hypertriglyceridemic subjects had a rise in TG on the fat free, high carbohydrate diet (+56 f 56%; p < 0.001). The standard low dose PHLA was in the normal range in both groups of hypertriglyceridemic subjects. It appeared to be lower in the untreated diabetic subjects (0.369 f 0.104 uEq FFA/ml/min) than in the nondiabetic hypertriglyceridemic subjects (0.466 f 0.163) but the difference was not significant. PHLA During the Heparin Infusion Normal and nondiabetic hypertriglyceridemic subjects. The peak PHLA during the heparin infusion was reached at 60 min in both the normal and the nondiabetic hypertriglyceridemic subjects, and was similar in both groups of subjects (0.562 f 0.162 and 0.581 f 0.179 crEqFFA/ml/min, respectively). The equilibrium PHLA, defined as the mean of three values during the last hour of heparin infusion, (late phase of release) was significantly lower than the 60 min value in both groups of subjects (p < O.OOl), but again the two groups were similar (normal: 0.499 f 0.151; hypertriglyceridemic: 0.494 f 0.175, pEqFFA/ml/min). The absolute value of PHLA among individuals in both groups was highly variable, but the equilibrium PHLA appeared to be a function of the 60-min value (r = 0.94, y = 0.910X + 0.0297, Fig. 2). The relationship of the equilibrium PHLA to the 60 min PHLA was fairly constant and similar in both groups (equilibrium/60 min; normal 0.890 + 0.088, hypertriglyceridemic 0.847 rt 0.109). Thus, no differences in PHLA during the heparin infusion could be demonstrated between these two groups.
BRUNZELL, PORTE, AND
3 IL2
n = 32
t
p ( ,001
.s
s
I
r
BIERMAN
. . . /A .
5.94
A’ $ dv” , , , , , dkLk;~~~~i~~~~ 0%
.
0.4 PHLA
AT
60
0.6 MINUTES
pEq
1.2 FFA/min/ml
and at equilibrium infusion.
of hoparin
Untreated diabetic subjects. The 60 min PHLA in the untreated diabetic subjects (0.579 f 0.160, pEqFFA/ml/min) was no different from that in the nondiabetic subjects (0.577 i 0.173). However, the equilibrium PHLA was lower in the untreated diabetic subjects (0.388 f 0.133) than the nondiabetic subjects (0.495 f 0.167, p < 0.05). This difference is amplified when the ratio of the equilibrium PHLA to the 60-min PHLA (EQ/60) is examined. This ratio in the untreated diabetic subjects (0.671 i 0.147) was much lower than in the nondiabetic subjects (0.856 f 0.105, p < 0.001). Although the equilibrium PHLA was lower in the untreated diabetic subjects, it was a function of the 60-min value (r = 0.76, y = 0.630x-0.0234, p < 0.005, Fig. 3). While there was no relationship between the 60-min PHLA and the fasting glucose level, the degree of hyperglycemia in the untreated diabetic subjects was inversely related to the level of the equilibrium PHLA (r = 0.61, p < 0.02, Fig. 4). Thus, those subjects with the highest fasting glucose levels had the lowest
Fii. 3. Relationship between portheparin lipolytic activity at 60 min and at equilibrium of heparin infusion in untroatod diabetic subjects with fasting comhyp4ycemia ( -I parod to relationship in nondbbotic rubjcts (-----) from Fig. 2.
g 3 Q
1 0.4
0 PHLA
AT
60
1 0.0 MINUTES
pEq
1 1.2 FFA/min/ml
POSTHEPARIN
LIPOLYTIC
ACTIVITY
1131
n=iz r : -.6l
P (.02
Fig. 4. Relationship between postheparin lipolytic activity at equilibrium of heparin infusion and fasting plasma glucose Ievelr in untreated diabetic rubjects with elevated fasting plasma glucose levels.
0%
I
I
I
250
200
I50
FASTING
PLASMA
GLUCOSE
300 mg/dl
PHLA levels during the late phase of the heparin infusion. The EQ/60 was also inversely related to the fasting glucose level (r = 0.80, p < O.OOl,~Fig.5). Recently treated diabetic subjects. Twelve diabetic subjects were studied within 2 mo of receiving insulin or oral sulfonylurea therapy. Those subjects who did not respond to oral sulfonylurea therapy with a decrease in fasting glucose levels and glycosuria were put on insulin therapy. The fasting plasma glucose levels for these recently treated diabetics (151 f 51 mg/dl) were lower than those in the untreated diabetic subjects (234 f 59 mg/dl, p < 0.02). The 60-min PHLA was again essentially the same as in the untreated diabetic subjects and the nondiabetic subjects. However, despite the treatment of hyperglycemia in these subjects, the defect in PHLA during the late phase of the heparin infusion persisted. The equilibrium PHLA (0.345 rEqFFA/ml/min) was lower than that in the nondiabetic subjects (0.495 f 0.167, p < 0.02) as
n = 12
r =-.80 p < ,001 .
FASTING
PLASMA
GLUCOSE
mg/dl
Fig. 5. Relationship between the relative postheparin lipolytic activity at equilibrium (RO/60) of the heparin infusion with fasting plasma glucose levels in untreated diabetic subjects.
1132
BRUNZELL,
PORTE,
AND
BIERMAN
was the EQ/60 ratio (recently treated diabetics 0.588 f 126, nondiabetics 0.856 + 0.105, p < 0.001). None of these parameters in the recently treated diabetics were different from that found in the untreated diabetic subjects. Paired plasma triglyceride levels did not change during this short period of treatment (see the following). In contrast, when eight diabetic subjects who had been on insulin or sulfonylurea therapy for longer than 6 mo without an intervening episode of ketoacidosis were studied, their PHLA release appeared to be the same as in nondiabetic subjects. The lower equilibrium PHLA was now not detectable (chronically treated diabetics 0.588 + 0.223 rEqFFA/ml/min; nondiabetic 0.495 f 0.167), nor was the EQ/60 ratio (chronically treated diabetics: 0.823 f 0.108; nondiabetic: 0.856 f 0.105). Thus, both of these parameters were significantly improved when compared to the untreated diabetics (equilibrium PHLA: p < 0.05, EQ/60 ratio: p < 0.05) and the recently treated diabetics (equilibrium PHLA: p < 0.01, EQ/60 ratio: p < 0.001). The mean fasting plasma glucose for these eight chronically treated diabetic subjects was 154 i 75 mg/dl. In the chronically treated subjects there was no correlation between glucose levels and PHLA levels. Seven subjects studied in Subjects studied serially during course of therapy. the untreated diabetic state were restudied after a short course of insulin or oral sulfonylurea therapy. The findings in these paired studies were the same as when the groups were examined as a whole. Thus, short-term treatment decreased fasting glucose levels, without changing the equilibrium PHLA levels. This group allows comparison of the effects of short term therapy on plasma triglyceride levels. On the fat free diet plasma triglyceride levels were no different (untreated diabetic state: 647 f 230 mg/dl; after a short course on therapy: 569 f 284 mg/dl). PHLA during the heparin infusion was studied in the untreated and/or recently treated diabetic state and again after chronic treatment with insulin or oral sulfonylurea in three subjects. The defect in the equilibrium PHLA corrected in each of these subjects in the chronically treated state when studied serially. Chronic therapy with insulin or oral sulfonylureas was associated with a decrease (89%) in plasma triglyceride levels from (33 10 f 2850 mg/dl) in the untreated state to (373 f 304 mg/dl, p < 0.001) in the chronically treated diabetic state. The three subjects with Subjects with postheparin lipolytic activity de$ciency. idiopathic PHLA deficiency and the two subjects with PHLA deficiency secondary to untreated diabetics had low levels of PHLA throughout the heparin infusion and were, thus, different from the untreated diabetics who had a normal low dose PHLA and a defect only in the late phase (equilibrium) PHLA (Table 3). One of the untreated diabetic subjects with PHLA deficiency (MMo) was treated for 2 wk with insulin with return of the 60-min PHLA to normal but with persistence of the low equilibrium PHLA (see MMO, Table 2) such that she now appeared to resemble other recently treated diabetic subjects.
POSTHEPARIN
LIPOLYTIC
1133
ACTIVITY
Role of Familial and Secondary Causes of Hypertriglyceridemia First degree relatives with significant hypertriglyceridemia were found in 13 of the 15 nondiabetic hypertriglyceridemic subjects who had large enough families for evaluation. Similarly, nine of 11 of the untreated diabetic subjects had nondiabetic relatives with significant hypertriglyceridemia. The untreated diabetic subjects with nonfamilial hypertriglyceridemia had lower plasma triglyceride levels (538 f 259 mg/dl) than those untreated diabetic subjects with a familial form of hypertriglyceridemia and the defect in PHLA (4234 f 2703 mg/dl, p < 0.02) (Table 2). All but one of the eight subjects with a concomitant familial form of hypertriglyceridemia remained hypertriglyceridemic after treatment of diabetes. In contrast, in all subjects who appeared to have a nonfamilial form of hypertriglyceridemia, triglyceride levels returned to normal with chronic insulin or oral sulfonylurea therapy. DISCUSSION
overt Bagdade et a1.12noted low PHLA levels in a few hypertriglyceridemic, diabetic patients that rapidly returned to normal after a course of insulin therapy. However, those patients seem to be rare, since most diabetic patients have been found to have a normal PHLA following a low dose of intravenous heparin (10 U/kg).14 In a previous report three diabetic subjects were noted to be unable to sustain PHLA levels during the late phase of a 3-5 hr heparin infusion.27 This decrease in PHLA appeared to be unrelated to the state of treatment of the diabetes, however, the possible differences between short and long term insulin therapy on PHLA were not appreciated. In the present study, differences in PHLA during a maximal, prolonged heparin infusion have been demonstrated between nondiabetic subjects (either with or without concomitant hypertriglyceridemia) and untreated diabetic subjects. The factor(s), apparently independent of the hypertriglyceridemia or the diabetic state, that account for the wide variation in the PHLA in the early phase of the heparin infusion (60 min) in all groups of subjects are unknown. It does not appear to be due to the range of circulating levels of insulin in the basal state, since there is no relationship between obesity (a major determinant of basal insulin levels”) and the 60 min PHLA value, or for that matter between obesity and any other parameter of PHLA measured in this study. Shafrir suggested that the PHLA released shortly after a low dose heparin injection was a function of the TG level,% but there was no relationship in this study between the 60-min PHLA value and the TG concentration. Whatever the factors are that control the levels of PHLA during the early phase of the heparin infusion, the equilibrium PHLA levels appear to be a function of these early levels in the nondiabetic subjects (Fig. 2). The decrease in equilibrium PHLA in the untreated diabetic subjects was even more notable when considered in relation to the 60-min level, presumably due to the variance introduced by factors in the two groups of subjects which relate primarily to the early phase of PHLA release. That the decline in late phase PHLA is related to abnormal carbohydrate metabolism or insulin deficiency, is supported by the fact that those diabetic
1134
BRUNZELL.
PORTE,
AND BIERMAN
subjects with the highest fasting plasma glucose levels had the lowest PHLA levels during the late phase of the infusion (Figs. 4 and 5). The return of PHLA and triglyceride levels to or toward normal with long term insulin or oral sulfonylurea therapy supports the relationship between the diabetic state and abnormal PHLA release. The mechanism for the decrease in PHLA during the late phase of the heparin infusion is speculative at this time. Decreased protein synthesis that is known to occur secondary to insulin deficiency may be a factor. The early phase may be related to the storage of lipoprotein lipase, perhaps in capillary endothelial cells 35,36that remains normal until the defect in the later phase becomes severe enough to cause abnormalities in the early phase of release as well. The later phase may relate to adipose tissue storage or synthesis of the enzyme or enzyme precursors 37and, thus, could be the site of the abnormality produced by insulin deficiency. Alternatively, in untreated diabetes the enzyme may be removed from the plasma more rapidly thereby causing lower levels during the prolonged heparin infusion. This seems unlikely since the half-life of the enzyme is the same in nondiabetics as in untreated diabetic subjects.12 Beta-adrenergic stimulation has been shown to suppress release of lipoprotein lipase from adipose tissue in vitro.38,39 Untreated diabetic subjects have elevated catecholamine levels? and this may be an inhibiting factor in the release of PHLA. However, attempts to induce a defect in PHLA during a heparin infusion in normal subjects with a simultaneous epinephrine infusion were unsuccessful, and the defect in PHLA in an untreated diabetic subject was not reversed by 4 mo of propranolol therapy (unpublished observations). Glucagon is also increased in untreated diabetes4’ and this hormone has been shown to decrease adipose tissue lipoprotein lipase.4’ Further studies are necessary to clarify the role of these factors on abnormalities of PHLA in diabetes. The reason for the delay in the return to normal of PHLA in the late phase of the heparin infusion in the diabetic subjects restudied after a short period of oral sulfonylurea or insulin therapy, or following a recent episode of ketoacidosis, is unknown. A low synthesis rate of enzyme may be present. A similar delay in the return of PHLA to normal has been reported in hypothyroidism since it was found that > 6 wk was required to reverse abnormalities of TG metabolism to normal.5S43 Studies of lipoproteins lipase in adipose tissue of insulin deficient rats indicate that insulin plays an important role in the regulation of this enzyme. Lipoprotein lipase decreases in insulin deficiency and the decrease is reciprocally related to the severity of the diabetes.“*45 Furthermore, there is in vitro evidence that insulin is important for the synthesis and release of the enzyme in adipose tissue, since the amount of enzyme released increases when adipose tissue is incubated in the presence of insulin. 44.46Low levels of lipoprotein lipase activity have been documented in adipose tissue from patients with familial lipoprotein lipase deficiency and preliminary studies reveal a similar decrease in adipose tissue lipoprotein lipase in humans with untreated diabetes4’ To date, heart lipase is the only other tissue lipoprotein lipase to be studied in experimental diabetes. Borensztajn, et al. 4s found lipoprotein lipase in rat hearts to be independent of insulin levels. However, Atkin and Meng24 have
POSTHEPARIN
LIPOLYTIC
1135
ACTIVITY
demonstrated a biphasic release of PHLA from the perfused hearts of normal rats and also examined the in vivo pattern of release in alloxan treated rats. Following alloxan treatment the early phase of release was virtually identical to that in the normal rat, but there was a significant decrease in the amount of PHLA released during the later phase. In vivo insulin treatment reversed the abnormality in the later phase to normal just as it did in the present study. They also demonstrated that the enzyme activity of rat hearts could be depleted by repetitive exposure to heparin and that the rate of repletion was deficient in the alloxan diabetic rats. They postulated that their data were consistent with two tissue pools of lipoprotein lipase the early phase being a small storage pool and the later phase a pool related to the synthesis of lipoprotein lipase. The data in the human subjects in the present study are consistent with this concept. A diagnosis of a concomitant familial form of hypertriglyceridemia was possible in many of the patients in this study by evaluation of the distribution of plasma TG and cholesterol levels among their relatives. Recent evidence obtained from family studies suggests that the familial forms of hypertriglyceridemia segregate independently from diabetes.“’ However, when these two disorders coexist in the subjects in this study the level of plasma triglyceride was markedly elevated and, thus, appeared to be related to an interaction between a familial form of hypertriglyceridemia and overt diabetes with its PHLArelated TG removal defect. The abnormality in PHLA in untreated diabetes in patients with familial lipid disorders appears to cause an additional increment in plasma TG levels. After reversal of the PHLA abnormality with insulin or oral sulfonylurea therapy, plasma TG levels in those diabetics with a concomitant familial form of hypertriglyceridemia returned to levels found in subjects with monogenic hypertriglyceridemia2* free of diabetes. On the other hand in diabetics with nonfamilial hypertriglyceridemia plasma TG levels were restored to normal following several months of insulin or oral sulfonylurea therapy. Further support for the pathophysiological role of the abnormality in PHLA in plasma TG removal in diabetes,49 stems from the correlation between maximal plasma lipolytic rate, (maximal TG removal rate) during the heparin infusion” and the late phase PHLA. ACKNOWLEDGMENTS The authors would like to express their gratitude to Ms. Martha Campbell, Shirley Corey, and Diane Stevens for their assistance.
Kimura,
Martha
Pleasant,
Ellen
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