| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Endocrine Care |
Department of Vascular Medicine, University Medical Center Utrecht, 3508 GA Utrecht, The Netherlands
Address all correspondence and requests for reprints to: M. Castro Cabezas, M.D., Ph.D., Department of Vascular Medicine, F02.126, University Medical Center Utrecht, P.O Box 85500, 3508 GA Utrecht, The Netherlands. E-mail: . m.castrocabezas{at}azu.nl
Abstract
One of the best studied aspects of the insulin resistance syndrome in familial combined hyperlipidemia (FCHL) is impaired insulin-mediated suppression of FFA by diminished inhibition of hormone-sensitive lipase (HSL). In vitro experiments have shown that stimulation of HSL activity by catecholamines is decreased in FCHL. The aim of this study was to investigate HSL inhibition by insulin and stimulation by endogenous catecholamines in vivo in FCHL patients. Twelve FCHL subjects using lipid-lowering medication and 12 controls underwent a mental stress test after random ingestion of either 50 g glucose or placebo. After ingestion of glucose, insulin concentrations increased from 76.8 ± 21.5 pM to a maximum of 520.2 ± 118.4 pM (P < 0.01) in FCHL and from 38.0 ± 5.0 to 221.7 ± 25.1 pM (P < 0.01) in controls. The percent decreases in plasma FFA during the first hour after glucose ingestion were similar in FCHL and controls (67 ± 5% vs. 72 ± 3%, respectively), suggesting a comparable inhibition of HSL in both. During the placebo test, FFA increased similarly in FCHL (56 ± 9%) and controls (57 ± 19%). In contrast, FFA concentrations did not change during mental stress after ingestion of glucose (from 0.17 ± 0.02 to 0.15 ± 0.02 mmol/liter in FCHL and from 0.11 ± 0.02 to 0.12 ± 0.02 mmol/liter in controls).
In conclusion, the present study provides in vivo evidence for intact insulin-mediated suppression of FFA in FCHL, although this inhibition of HSL was achieved by higher insulin levels, suggesting insulin resistance at the level of HSL. Secondly, the induction of HSL activity by endogenous catecholamines in vivo is not decreased in FCHL, in contrast to earlier in vitro findings. Finally, catecholamine-induced HSL activation can be inhibited by insulin in a similar manner in both FCHL and controls.
FAMILIAL COMBINED hyperlipidemia (FCHL) is the most frequent dominantly inherited dyslipidemia (1, 2, 3, 4). The major characteristic, consistently found by different researchers, is VLDL overproduction, probably mediated by defective uptake and esterification of FFA by peripheral cells (5, 6). In addition, insulin resistance is frequently found in FCHL subjects (7, 8, 9), especially when fasting hypertriglyceridemia is present (10). One of the best studied aspects of insulin resistance in FCHL is impaired insulin-mediated suppression of FFA by diminished inhibition of hormone-sensitive lipase (HSL) (9, 10, 11, 12). It has been postulated that this defect may be located in the adipose tissue of FCHL subjects (10). In addition, elegant in vitro studies by Arner’s group have clearly shown that HSL activity measured in isolated fat cells was decreased in FCHL subjects (13), and in vitro activation was decreased compared with that in controls (14). In addition to HSL activity, many different mechanisms influence plasma FFA concentrations. For example, high plasma VLDL are usually accompanied by higher FFA levels due to the larger amount of substrate available for extracellular lipolysis. Moreover, plasma FFA concentrations are determined by the balance between intravascular lipolysis of triglycerides by lipoprotein lipase and the delivery of these VLDL by blood flow velocity, on the one hand, and the cellular uptake of FFA, on the other hand. In this latter process, several factors play a role: the acylation-stimulating protein (ASP)/C3 pathway, fatty acid transproter/CD36, and other transmembrane transporters (6, 15, 16).
The aim of the present study was to evaluate whether in vivo regulation of HSL is impaired in FCHL subjects. For this purpose a noninvasive test was used that provides information on HSL-mediated FFA changes during activation by endogenous catecholamines and physiologically elevated insulin concentrations (17).
Subjects and Methods
Subjects
The study protocol was approved by the human investigations review committee of University Hospital Utrecht. All participants gave informed consent. Twelve unrelated FCHL patients using lipid-lowering medication were recruited from the Lipid Clinic of Utrecht University Hospital. These subjects met the following criteria: before treatment had been initiated they were known to have primary hyperlipidemia with varying phenotypic expression and elevated plasma apolipoprotein B (apoB) concentrations (>1.2 g/liter); at least one first degree relative had a different hyperlipidemic phenotype; and there was a positive family history of premature coronary heart disease, defined as myocardial infarction or cerebrovascular disease before the age of 60 yr in at least one blood-related relative. In addition, the patients fulfilled the following inclusion criteria: absence of xanthomas, absence of secondary factors associated with hyperlipidemia, body mass index below 30 kg/m2, absence of apoE2/E2 genotype, and consumption of no more than 3 U alcohol/d.
Twelve normolipidemic healthy controls without a family history of cardiovascular disease, with a normal apoB concentration (<1.2 g/liter) and the absence of apoE2/E2 genotype, and not using drugs known to affect lipid metabolism were recruited by advertisement.
Mental stress tests
Two separate mental stress tests were performed at random with at least a 1-wk interval. All participants visited the metabolic ward of our laboratory after a 12-h overnight fast, where an iv cannula was placed in the left brachial vein. The cannula was kept open by continuous 0.9% saline infusion. All peripheral blood samples were obtained from the cannula in tubes containing sodium EDTA (2 mg/ml), placed on ice, and centrifuged immediately for 15 min at 3000 rpm at 4 C. An inhibitor of lipoprotein lipase (tetrahydrolipstatin, Roche, Basel, Switzerland) was added to the plasma immediately after centrifugation (18). The volunteers ingested randomly either a 200-ml placebo solution containing sodium saccharide 8% (wt/vol) or 200 ml of a 20% (wt/vol) glucose solution. The two solutions were identical in color, taste, and viscosity. The subjects were blinded to the type of solution. During the first 60 min of the test the volunteers remained supine in a room without disturbing stimuli. The next 20 min, the participants were subjected to two types of mental stress tests, consisting of letters and figures, as previously described (17). They were asked to subtract two letters in the alphabet from the given letters; for example, CD would give AB. When numbers were given, subjects had to reverse the order; for example, 1234567 would give 7654321. After the mental stress period, the subjects remained supine for 40 min. Peripheral blood samples were obtained before the mental stress test (-60 to 0 min) at 10-min intervals. During the 20 min of stress (0–20 min), blood was collected with an interval of 5 min, and after the stress period (30–60 min), blood was collected every 10 min. During the test, heart rate was also recorded as an estimate of sympathetic activity.
Analytical methods
Plasma samples were stored at -20 C immediately after centrifugation. Triglycerides and cholesterol were measured in duplicate by commercial colorimetric assay (GPO-PAP and Monotest Cholesterol kit, Roche Molecular Biochemicals, Mannheim, Germany). FFA concentrations were measured in plasma samples by an enzymatic colorimetric method (Wako Chemicals GmbH, Neuss, Germany). The quantitative assays of apoB have been described in detail previously (19). Insulin was measured by commercial ELISA (Mercodia, Uppsala, Sweden). For estimation of insulin sensitivity, the homeostasis model assessment (glucose x insulin/22.5) was calculated (20).
Statistics
All values are expressed as the mean ± SEM. Correlations among variables were tested by linear regression analysis (Pearson’s correlation coefficient). For statistical analysis of changes in FFA concentrations and heart rate, repeated measures ANOVA was used with a least significant difference test as post hoc analysis of the curves (time-effect relationship per group).
The relative changes in FFA and heart rate were calculated by correcting for basal values at rest (-60 min) or at the beginning of the mental stress period (0 min). For estimation of the total amount of FFA released by mental stress, the area under the FFA curve (AUC) and the incremental area under the FFA curve (dAUC) were calculated from 0–20 min after correction for the FFA concentration at 0 min.
Statistical significance was reached at P < 0.05 (two-tailed). Calculations were performed using SPSS/PC+ 9.0 (SPSS, Inc., Chicago, IL).
Results
General characteristics (Tables 1
and 2)
Twelve FCHL and 12 control subjects participated in this study. All FCHL patients were using lipid-lowering medication (11 taking statins and 1 taking fibrates). FCHL patients tended to have an increased waist and body mass index and were slightly older than control subject (Table 1). Fasting plasma triglycerides, insulin, and glucose concentrations were higher in FCHL patients. Plasma cholesterol, FFA, and apoB concentrations were not different between the groups (Table 2).
|
|
Heart rate was similar in FCHL and controls before placebo (64 ± 2 vs. 64 ± 3 beats/min) and glucose (63 ± 2 vs. 65 ± 2 beats/min; P = NS). After 5 min of mental stress, an increase of 18% was found in the placebo test in FCHL, and an increase of 14% was found in controls. After glucose treatment, the heart rate increase was 10% in FCHL and 14% in controls. There were no significant differences in heart rate between FCHL and controls.
Insulin concentrations (Fig. 1)
In FCHL, insulin concentrations increased after ingestion of glucose from 76.8 ± 21.5 pM to a maximum of 520.2 ± 118.4 pM (P < 0.01); in controls insulin concentrations increased from 38.0 ± 5.0 to 221.7 ± 25.1 pM (P < 0.01; Fig. 1A). In both groups, maximal insulin concentrations were found at 0 min (the beginning of the mental stress period). Ingestion of placebo resulted in a significant rise in insulin concentrations in both FCHL (from 61.0 ± 16.5 to 93.3 ± 2.8 pM at -40 min; P < 0.05) and controls (from 40.0 ± 5.74 to 73.9 ± 10.0 pM at -40 min; P < 0.01; Fig. 1B).
|
and 3)FCHL patients started with slightly higher FFA concentrations (0.60 ± 0.08 mmol/liter) than controls (0.44 ± 0.08 mmol/liter; P = NS) in the glucose test (-60 min). This was also the case at the beginning of the placebo test (0.66 ± 0.13 mmol/liter in FCHL and 0.37 ± 0.07 mmol/liter in controls; P = NS).
|
|
Neither the glucose test nor the placebo test resulted in statistical significantly different FFA patterns between the groups, as analyzed by ANOVA. During the first 60 min of the glucose test, FFA concentrations decreased significantly in both FCHL (from 0.60 ± 0.08 at -60 min to 0.17 ± 0.02 mmol/liter at 0 min; P < 0.01) and controls (from 0.44 ± 0.08 to 0.11 ± 0.02 mmol/liter; P < 0.05). The percent decrease in FFA during the first hour was not different between FCHL (67 ± 5%) and controls (72 ± 3%; Fig. 2A
). During the first 60 min of the placebo test, a less pronounced significant decrease in FFA was found in FCHL (to 0.40 ± 0.06 mmol/liter, representing a 34 ± 6% decrease; P < 0.05 compared with the fasting value) and controls (to 0.28 ± 0.08 mmol/liter, representing a 28 ± 8% decrease; P < 0.05; Fig. 2B). This FFA decrease was not different between FCHL and controls.
Catecholamine-induced HSL activation
In the placebo test, an increase in FFA was seen after the 20 min of mental stress in FCHL (from 0.40 ± 0.06 at 0 min to 0.57 ± 0.06 mmol/liter; P < 0.01) and in controls (from 0.28 ± 0.08 to 0.43 ± 0.13; P < 0.05; Fig. 2B). The percent increase from 0–20 min in FCHL and controls (56 ± 9% and 57 ± 19%, respectively) as well as the FFA AUC from 0–20 min (9.9 ± 1.1 vs. 8.0 ± 2.4 mmol/h·liter) and FFA dAUC (2.0 ± 0.3 and 2.5 ± 0.9 mmol/h·liter, respectively) were not significantly different (Fig. 3). At the end of the test, at 60 min, FFA concentrations were higher than at the beginning of the mental stress period (0 min) in the FCHL patients (60 min, 0.47 ± 0.06 mmol/liter; P < 0.05 compared with 0 min), whereas in controls FFA concentrations returned to basal levels at the end of the test (0.24 ± 0.03 mmol/liter; P = NS compared with 0 min; Fig. 2B).
Insulin-mediated suppression of catecholamine-induced HSL activation
During the glucose test, FFA concentrations did not change significantly during mental stress in either group (from 0.17 ± 0.02 to 0.15 ± 0.02 mmol/liter at 20 min in FCHL and from 0.11 ± 0.02 to 0.12 ± 0.02 mmol/liter in controls; Fig. 2A).
Discussion
Impaired FFA metabolism is one of the key metabolic characteristics of FCHL subjects (7, 10, 11, 12). Different studies have led to the conclusion that impaired peripheral fatty acid uptake, most likely in combination with decreased HSL activity and decreased HSL suppression by insulin, may result in an enhanced flux of FFA to the liver, thereby increasing very low density lipoprotein production (6, 21). However, most data concerning, HSL activity in FCHL were obtained under unphysiological conditions (10, 11, 12) or were derived from ex vivo experiments (13, 14). In addition, contradictory findings on HSL activity in FCHL have been reported recently. Although it has been suggested that in Swedish FCHL subjects HSL activity and stimulation were impaired (13, 14), these findings were not reproduced in Finnish FCHL patients using the same in vitro assay (22). Our in vivo data for FFA changes representing modulation of HSL activity are in line with normal regulation of HSL activity in FCHL. It should be stressed that our patients were receiving lipid-lowering medication and that this may have influenced the results. However, we are not aware of studies demonstrating an effect of lipid-lowering medication on the regulation of HSL activity. Furthermore, the earlier mentioned in vitro studies (13, 14, 22) were performed with adipocytes from FCHL subjects, using similar medication. Therefore, we do not believe that this can explain the difference between our study and those by Arner et al. (13, 14). Another feature that could have influenced the present data is a different amount of stress induced in FCHL patients and controls. However, as both groups had similar social backgrounds, and the increase in heart rate (representing sympathetic activation) was not different, we do not believe that this has influenced the results.
The FCHL patients were slightly older and more obese than the controls. Theoretically, these differences could lead to decreased HSL stimulation and suppression in the FCHL patients. As there were no differences in HSL modulation, we do not believe that this has influenced the results significantly.
After ingestion of glucose, a decline of FFA concentration was found in both FCHL and controls. In previous reports using hyperinsulinemic clamps, impaired FFA suppression by insulin has been demonstrated in FCHL patients (10, 11, 12), suggesting decreased suppression of HSL by insulin in FCHL. The difference from the present study is that insulin concentrations after ingestion of glucose reached physiological levels. Another explanation for the decrease in FFA concentration during hyperinsulinemia could be increased cellular FFA uptake. Boden et al. (23) have shown, using stable isotopes, that insulin profoundly suppresses FFA release by inhibiting intracellular lipolysis, which theoretically could result in enhanced cellular uptake. We cannot rule out that this may have been one of the mechanisms behind the FFA decrease during the glucose load. Although the relative increases in insulin were similar in controls and FCHL patients, the latter reached higher levels, which is in line with the presence of insulin resistance (7, 8, 9). The same observation of higher fasting FFA concentrations but similar FFA suppression after an oral glucose tolerance test has been reported by Vakkilainen et al. (9) in relatives of FCHL subjects. As at lower insulin concentrations the same FFA suppression was seen in controls and FCHL patients, our results are in agreement with the view that HSL suppression in FCHL is resistant to the action of insulin. Our data are in line with those findings.
HSL is the rate-limiting enzyme of adipose tissue lipolysis (24). Lipolysis is stimulated by catecholamines through phosphorylation (25). To induce elevation of endogenous catecholamines, all subjects were stressed mentally for 20 min. During this period a similar increase in FFA was seen in controls and FCHL patients. Our data are in contrast to the in vitro studies by Arner’s group (13, 14) that showed decreased HSL activation in adipocytes of FCHL patients, but our results are in agreement with the report from Taskinen’s group (22). Furthermore, it has been shown that there were no significant mutations in the HSL gene in Finnish FCHL patients (26). These data suggest that HSL is probably not a major determinant of the FCHL phenotype.
After the 20 min of mental stress in the placebo test, FFA concentrations returned to basal levels only in controls, probably due to FFA uptake by peripheral cells which may be medicated in part by the ASP (27, 28, 29). In FCHL patients, impaired ASP action has been held responsible for decreased uptake of postprandial FFA and consequently increased postprandial FFA flux to liver and VLDL overproduction, as seen in FCHL (30, 31). Diminished ASP-mediated uptake of FFA may explain in part the persistently elevated FFA concentrations after the stress period in FCHL compared with controls. The slightly higher fasting C3 concentrations, the precursor of ASP, in FCHL patients supports the theory of impaired ASP action (31). As all FCHL patients were using lipid-lowering medication, and it has been shown that statins lower plasma C3 (32), this could explain the lack of statistical significance. A different explanation for the persistently elevated FFA concentrations in FCHL at the end of the test (60 min) could be that dephosphorylation of HSL (i.e. inactivation) is impaired in FCHL. As far as we know, there are no reports of in vitro measurements of dephosphorylation of HSL in FCHL. Alternatively, the insulin levels in FCHL patients after placebo may not have been high enough to suppress HSL, resulting in persistently elevated FFA concentrations.
A different aspect of HSL activation in FCHL studied here was the insulin-mediated inhibition of catecholamine-induced HSL, represented by FFA levels during the stress period after ingestion of glucose. The reduced antilipolytic effect of insulin may be part of the insulin resistance syndrome in FCHL, and it was not predictable whether the catecholamine-induced activation of HSL would overcome the suppression by insulin. FFA concentrations did not increase in either group during stress after ingestion of glucose, suggesting that under these conditions insulin-mediated suppression is more potent than catecholamine induction. Similar results have been found in healthy volunteers in our laboratory (17).
The present study shows in vivo that the insulin-mediated suppression of FFA by inhibition of HSL is not decreased in FCHL, albeit higher insulin concentrations are necessary to reach similar FFA suppression as in controls. These data are in agreement with insulin resistance at the level of HSL in FCHL subjects. Secondly, the induction of HSL activity by endogenous catecholamines is not decreased in FCHL patients in vivo. Finally, insulin-mediated inhibition of HSL in vivo overrides the catecholamine stimulation in FCHL and controls.
Acknowledgments
Footnotes
Abbreviations: apo, Apolipoprotein; AUC, area under the curve; ASP, acylation-stimulating protein; dAUC, incremental area under the curve; FCHL, familial combined hyperlipidemia; HSL, hormone-sensitive lipase; IRS, insulin resistance syndrome.
Received June 11, 2001.
Accepted January 9, 2002.
References
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
). Data are the mean ± SEM. Note that the scale of the y-axis is different in B. *, P < 0.01; +, P < 0.05 (compared with the fasting value).
, P < 0.01; 
, P < 0.05 (compared with 0 min).
) and 12 controls (
). Data are the mean ± SEM.