Insulin resistance and action in hypertriglyceridemia

Diabetes Research and Clinical Practice, 14 (1991) Q 1991 Elsevier

Science Publishers

55-62

55

B.V. All rights reserved 016%8227/91/%03.50

DIABET 00541

Insulin resistance and action in hypertriglyceridemia S. Zuniga-Guarjardo, The Division of Endocrinology

G. Steiner, S. Shumak

and B. Zinman

and Metabolism. Toronto General Hospital, Mount Sinai Hospital, and The Department of Medicine, University of Toronto, Ontario, Canada

(Received 11 February 1991) (Revision accepted 11 June 1991)

Summary To define further the characteristics of insulin insensitivity associated with hypertriglyceridemia, the metabolic responses to the euglycemic insulin clamp were evaluated in 6 hypertriglyceridemic male patients and compared to 5 normal male controls. At baseline, the hypertriglyceridemic patients had elevated triglycerides (687 & 172 vs 78 t 7 mg/dl, P < 0.005) and free fatty acids (702 + 36 vs 444 + 42 PM/~, P -c 0.005) concentrations. During the euglycemic insulin clamp, steady-state plasma glucose concentrations were similar in both groups (90.2 + 1.5 vs 88.8 & 2.3 mg/dl*, ns) as were steady state insulin levels (142 2 10.1 vs 132.2 ? 6.8 pU/ml**). The amount of glucose metabolized during the last hour of the clamp (M) was significantly reduced in the hypertriglyceridemic patient (2.9 k 0.4 vs 6.2 & 0.7 mgemin-‘*kg‘, P -c 0.001). Changes in free fatty acid, glycerol, B-hydroxybutyrate, lactate and pyruvate during the euglycemic insulin clamp were similar indicating a preserved antilipolytic, antiketogenic and glycolytic intermediate (lactate + pyruvate) response to insulin and glucose infusion in the hypertriglyceridemic patients. In summary, hypertriglyceridemia is associated with insulin resistance as it relates to muscle glucose utilization. However, this is not universal, as a number of other insulin responsive pathways appear to be unaffected. Key words: Hypertriglyceridemia;

Insulin resistance;

Introduction Hypertriglyceridemia has been associated with insulin resistance and glucose intolerance [l-9]. Interestingly, the insulin insensitivity in hyper-

Correspondence

to: B. Zinman, Mount Sinai Hospital, 600 University Avenue, Suite 782, Toronto, Ontario M5G 1X5, Canada. * To convert glucose to mM/l multiply x 0.056. ** To convert IRI to pM/l multiply x 7.18.

Euglycemic insulin clamp

triglyceridemic patients has been shown to be independent of obesity or diabetes [2,4,7]. That the insulin insensitivity may be specific for both the organ and the metabolic pathway was suggested in hypertriglyceridemic patients by studies demonstrating impaired insulin action peripherally with normal suppression of hepatic glucose production [ 3,4 1. Similar dissociations between the peripheral effects of insulin on glucoregulation and its antilipolytic, antiketogenic and suppressive effects on hepatic glucose production have

56 Methods

been shown in obesity [ lo-131 and in hypertensives [ 141. Other studies [ 15-161 have demonstrated that the infusion of Intralipid to increase circulating triglyceride and FFA concentrations resulted in impaired insulin action on peripheral glucose uptake as assessed by the euglycemic insulin clamp. Since muscle is the major site of glucose disposal during the hyperinsulinemic glucose clamp, abnormalities in muscle glucose uptake, oxidation and storage have been implicated as the primary defects responsible for the observed insulin insensitivity. Of interest, in these studies the insulin-induced suppression of hepatic glucose production was unaffected by Intralipid infusion. The previous demonstration that hypertriglyceridemic humans were resistant and the suggestions that, in other insulin-resistant states, the resistance may be organ-specific led to the present study. It was undertaken to confirm, by another method, that lean hypertriglyceridemics are insulin-resistant and to examine whether the insulinresistance associated with hypertriglyceridemia is general or specific. The action of insulin was assessed on several metabolic pathways including (muscle) glucose utilization, antilipolysis and antiketogenesis in lean patients with hypertriglyceridemia using the euglycemic insulin clamp. Metabolic responses were compared to normal controls.

TABLE

Subjects Six non-diabetic males with primary hypertriglyceridemia consented to participate in the study. Pertinent clinical and anthropometric data are shown in Table 1. Five lean, male, normal subjects served as controls. None of the controls and hypertriglyceridemic subjects had hepatic, renal or heart disease, or were taking any medications that could interfere with glucose or lipid metabolism. All subjects were consuming a normal diet containing at least 250 gm of carbohydrate per day for 3 days prior to the study. All participants were fully informed about the purpose, nature and potential risks of the study and had given informed, written consent in accordance with the Human Subjects Review Committee of the University of Toronto. They were studied in the Clinical Investigation Unit of the Toronto General Hospital after an overnight fast. Euglycemic insulin clamp protocol A euglycemic insulin clamp study was performed in each hypertriglyceridemic and control subject, as previously described [ 16,171. After an overnight fast, an indwelling catheter was inserted into an antecubital vein for glucose and insulin administration. A second catheter was placed in a retrograde fashion into a superficial hand vein in the contralateral arm for blood sampling. The hand was kept in a warming chamber at 69” C to ensure arterialization of the venous blood [ 191. The insulin infusion was initiated with a priming dose of crystalline pork insulin (Iletin II, Eli Lilly

1

Clinical data

Age

Sex

(yr)

Ht (cm)

wt

IBW

BMI

(kg)

(%)

(kg/m* )

Normal controls (5)

33.8 k 2.1

5 Male

114.6 * 4.2

77.8 f 4.4

109.3 + 3.1

25.4 f 0.6

Hypertriglyceridemic subjects (6)

45.8 k 3.5**

6 Male

170.1 * 2.2

82.6 + 2.7

120.6 i 2.P

28.4 i_ 0.5*

* P < 0.005 as compared

with normal controls;

** P < 0.05 as compared

with normal controls.

57

Co., IN) for ten min, followed by a continuous insulin infusion of 40 mu/m2 for a subsequent 1 lo-min period to achieve steady-state hyperinsulinemia of approximately 100 pU/ml above basal level. The plasma glucose concentrations were maintained within 5 y0 of the fasting level by measuring plasma glucose concentrations from arterialized venous blood samples every five min with subsequent adjustment of the 20% glucose infusion rate. The glucose metabolized (M) during the second hour of the glucose clamp was calculated on the basis of the amount of glucose infused with a correction made for changes in glucose pool size [

171.

The validity of these measurements assumes that endogenous glucose production has been entirely suppressed. This has been previously documented in normal subjects, hypertensives and hypertriglyceridemic patients with the insulin and glucose concentrations used in these studies [3,4,14-161. The metabolic clearance rate of insulin (MCR,) was calculated by dividing the insulin infusion rate by the mean insulin concentrations during the last 60 min of the clamp and was corrected for changes in the endogenous insulin secretion as measured by C-peptide concentrations. Plasma immunoreactive insulin (IRI), glucagon (IRG), lactate, pyruvate, B-hydroxybutyrate (B-OHB), glycerol and free fatty acids (FFA) were determined at fasting and at timed intervals after the initiation of the glucose clamp. Analytical methods Plasma glucose was measured by the glucose oxidase method (Beckman Glucose Analyzer II, Beckman Instruments Co., Fullerton, CA). Samples for IRI and IRG were placed into prechilled tubes containing 0.5 ml of Trasylol (10,000 ml KIU, Miles Pharmaceutical, Rexdale, ONT) then centrifuged with minimal delay at 4 oC and supernatants frozen at - 20’ C until assayed. IRI and IRG were determined by standard radioimmunoassay as previously described [ 2 11. Pyruvate, lactate, B-hydroxybutyrate, glycerol

were determined using a microfluorometric adaptation of standard enzymatic methods [ 201. Free fatty acids were estimated by the radiochemical microtechnique of Ho [22]. Plasma fasting triglycerides (TG) were assayed using a commercial enzymatic kit (Boehringer Mannheim, Montreal,

PQ). Statistical analyses To compare differences between groups, Student’s unpaired t-test was employed while Student’s paired t-test was used for comparisons within the same group. Linear regression analyses were done by conventional methods. Data is given as mean & standard error of the mean in the text, tables and figures.

Results Baseline studies Apart from elevations in triglyceride concentrations (687 + 172 vs 78 ? 7 mg/dl, P < 0.005) the hypertriglyceridemic and control subjects had similar and normal baseline concentrations of glucose, IRI, IRG, lactate and pyruvate (Table 2). Fasting free fatty acid concentrations were elevated in the hypertriglyceridemic patients (702 k 36 vs 444 + 42 PM/~, P -C 0.005) as were the B-hydroxybutyrate concentrations (153 2 45 vs 42 + 11, P < 0.05). Although somewhat higher in the hypertriglyceridemic patients, glycerol concentrations (72 + 10 vs 66 f 14 PM/~) were not significantly different (Fig. 3). Euglycemic insulin clamp studies Steady-state plasma glucose concentrations were similar in both groups (hypertriglyceridemic vs controls 90.2 _c 1.5 vs 88.8 ~tr2.3 mg/dl, P = ns). The amount of glucose metabolized during the last hour of the clamp (M) was significantly reduced in the hypertriglyceridemic patients vs (2.9 * 0.4 6.2 f. 0.7mg.min’.kgg’, P < 0.001; Fig. 1). The steady state plasma insulin concentrations and the metabolic clearance rate of insulin were similar for hypertri-

TABLE 2 Fasting plasma concentrations

Norma1 controls Hypertriglyceridemic

of glucose, insulin, glucagon and triglyceride

Glucose

IRI

IRG

Triglyceride

Pyruvate

Lactate

(mg/dl)

(pub-4

bdml)

@WV

(PM/U

(PM/O

88.6 _+2.7 90.2 + 1.5

14.3 + 0.5 17.2 t 2.9

284.5 t 33.3 216.1 + 20.3

78 + 7 687 f 172*

96.9 f 9.1 82.3 f 8.1

851.8 + 98.4 723.9 + 85.1

* P < 0.005 as compared with normal controls. To convert: Glucose to mM/l multiply by 0.056; IRI to pM/l multiply by 7.18.

S.S.P.G

M

lOOr

1’0

80

a

60

6

40

4

7

5

IE

73

z

2 _’

20

E p

2

2

. -. .c E

6.423

zr

4.337

75.

I

2.250 1 *p
Fig. 1. Steady-state plasma glucose (SSPG) concentrations and glucose metabolized (M) during the last hour of the euglycemic clamp in normal controls (hatched bars) and hypertriglyceridemic (open bars) patients. All data are expressed as mean + SE. To convert glucose to mM/l multiply x 0.056.

control (IRI normal and glyceridemic 142.9 + 10.1 vs 137.2 k 6.8 pIJIm and MCR, 7.8 + 0.7 vs 8.5 of:0.6 ml - min ’ * kg- ‘). When combining both groups, a significant negative correlation between FFA and M was observed as illustrated in Fig. 2 (r = 0.77, P < 0.01). Other metabolic responses

Glycerol, FFA and B-hydroxybutyrate responses to the euglycemic clamp are shown in Fig. 3. Similar responses were observed in normal controls and hypertriglyceridemic patients.

Discussion These studies using the euglycemic hyperinsulinemic insulin clamp demonstrated that hyper-

FFA

uMIL

Fig. 2. Correlation of baseline free fatty acid (FFA) concentrations and glucose metabolized (M) in normal controls and hypertriglyceridemic subjects combined. (r = 0.775, P < 0.01).

triglyceridemic humans, had an M value for glucose of less than half that of the normal controls (Fig. 1). Hence, in these terms they were insulin resistant. It is important to note that the hypertriglyceridemic patients were 120.6 + 2.6% IBW compared to the controls (109.3 + 3.1% IBW, Table 1). However, this was unlikely to account for the reduction in M as patients with greater degrees of obesity (136 + 6% IBW) demonstrated normal insulin stimulated glucose utilization during similar euglycemic clamp testing [ 231. Another variable that can affect insulin sensitivity is age. The hypertriglyceridemic patients were older than the controls (45.8 k 3.5 vs 33.8 _+2.7 years). Studies of insulin action and aging [24-261 documented a significant dilference in insulin sensitivity in young subjects compared to the elderly (ie. greater than 60 years of

59

%

FFA

%

I 60

I 120

Time min

Fig. 3. Glycerol, FFA and B-hydroxybutyrate at baseline and during the euglycemic clamp ( yO change from baseline) in normal controls (NC) and hypertriglyceridemic (HTG) subjects. Baseline absolute values are given in the insert in this figure. All data expressed as mean f SE.

age), but not in the 35 to 45 year age group that we studied. Furthermore, these studies are in accord with previous tracer kinetic studies in which we demonstrated that lean, age-matched hypertriglyceridemics were resistant to insulin’s effect on glucose utilization [4]. It therefore seems likely that some aspect of the metabolic abnormality present in the hypertriglyceridemic patients was responsible for the decrease in glucose utilization during the euglycemic clamp. Muscle would appear to be the target organ affected since the major portion of the glucose infused during the clamp is taken up by this tissue with proportionally smaller amounts

being consumed by adipose, kidney and red cells [27]. However, without tissue specific measurements, it is not possible to draw more precise conclusions regarding the site of insulin resistance to glucose metabolism. Triglycerides, free fatty acids and B-hydroxybutyrate were clearly elevated at baseline in the hypertriglyceridemic patients. With chronic adaptation of muscle to increased fatty acid oxidation, irrespective of whether the fatty acids are derived from plasma FFA or from local lipolysis of the triglycerides, one would predict that muscle glucose uptake and oxidation would decrease consistent with glucose fatty acid cycle [28,29]. This hypothesis is supported by the observed decrease in muscle glucose metabolism in response to experimental elevations in circulation triglyceride and FFA [ 15,161. Increased fatty acid oxidation produced a decrease in glucose transport, oxidation and storage [ 151. Our studies provide further evidence in support of this hypothesis since we found a significant negative correlation between FFA concentrations and muscle glucose utilization (M), (Fig. 2). It is also possible that the elevated B-hydroxybutyrate concentrations in the hypertriglyceridemic patients contributed to a decrease in muscle glucose uptake. To examine the effect of insulin on other metabolic pathways, the concentrations of B-hydroxybutyrate, free fatty acids and glycerol were measured throughout the euglycemic clamp. The normal control subjects responded as has been previously. described [ 181. The concentrations of glycerol, FFA, and B-hydroxybutyrate (expressed as percent change) decreased similarly in the hypertriglyceridemics and the control subjects. Thus the antilipolytic and antiketogenic responses to insulin and glucose infusion were preserved in these insulin resistant hypertriglyceridemic patients. These studies confirm that hypertriglyceridemia is associated with significant insulin resistance. More importantly they demonstrate that the presence of insulin resistance in muscle cannot be extrapolated to other tissues and other metabolic pathways.

60 Acknowledgements We would like to acknowledge the important contribution of the staff of the Clinical Investigation Unit of the Toronto General Hospital in the performance of these studies. The excellent technical assistance of Betty Noble and the staffs of the Banting and Best Diabetes Centre Core Laboratory and the Atherosclerosis and Lipid Research Program is recognized. The secretarial assistance of Susan Desveaux and Yvonne Bidney in preparation of the manuscript is greatly appreciated. The studies were supported by the Medical Research Council of Canada and the Canadian Diabetes Association.

II

12

13

I4

15

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