High-Dose Statin Therapy Does Not Induce Insulin Resistance in Patients with Familial Hypercholesterolemia

Abstract

Background:

Insulin resistance is thought to play a pathophysiological role in the development of atherosclerosis. Decreased adiponectin levels are associated with hyperinsulinemia, insulin resistance, and coronary artery disease. Patients with familial hypercholesterolemia (FH) develop premature atherosclerosis and should be insulin resistant and have low adiponectin levels.

Methods:

A total of 51 homozygous FH (HoFH) and 20 heterozygous FH (HeFH) patients were studied before and after statin therapy. Twenty normocholesterolemic subjects were controls. Fasting lipograms, glucose, insulin, proinsulin, adiponectin, and high-sensitivity C-reactive protein (hsCRP) were measured. Insulin resistance was calculated with the homeostasis model assessment (HOMA-IR) formula. Carotid intima media thickness (CIMT) was measured as a subclinical marker of atherosclerosis.

Results:

On multiple regression analysis, the major determinant of insulin resistance measured by HOMA-IR was body mass index (BMI) (r=0.54; P=0.004). On simple linear regression, the highest correlation was with BMI (r=0.39; P=0.0002). Log hsCRP correlated with BMI (r=0.35; P<0.002) and insulin resistance (r=0.22; P=0.05). Low-density lipoprotein cholesterol (LDL-C) and CIMT did not correlate with insulin resistance. Unexpectedly, adiponectin levels were highest in HoFH patients and correlated with LDL-C (r=0.34; P=0.001). No change in the degree of IR was observed with statin therapy.

Conclusions:

FH patients are not insulin resistant and do not have low adiponectin levels. There was no significant change in insulin resistance with high-dose statin therapy.

Introduction

T he elucidation of the molecular basis of familial hypercholesterolemia (FH) by Goldstein and Brown proved a causal relationship between a high concentration of low-density lipoprotein cholesterol (LDL-C) and atherosclerotic cardiovascular disease.¹ FH is an inherited autosomal disorder caused mainly by mutations in the LDL receptor gene. Mutations in three other associated genes, apolipoprotein B, proprotein convertase subtilisin/kexin type 9, and, more rarely, the autosomal recessive hypercholesterolemia adaptor protein, may lead to a similar phenotype with varying severity. The condition is characterized by extremely high LDL-C levels that predispose patients with FH to premature atherosclerosis, predominantly coronary artery disease (CAD). Individuals with the more severe homozygous form of FH (HoFH) develop clinically significant atherosclerosis in early childhood and, if untreated, they rarely survive beyond the age of 30 years, whereas those with the less severe heterozygous form (HeFH) usually present with CAD in the third to fifth decade.¹

The prevalence of overweight and obesity is increasing worldwide. Obesity, particularly visceral obesity, is associated with insulin resistance, and it has been suggested that insulin resistance and the accompanying compensatory hyperinsulinemia promote the development of atherosclerosis.² Meta-analyses of data from prospective studies have shown a significant association between hyperinsulinemia and cardiovascular mortality independently of other risk factors.^3,4 Fasting hyperinsulinemia and insulin resistance also have a positive correlation with atherosclerosis as measured by carotid intima media thickness (CIMT) ultrasonography,⁵ which is regarded as an early subclinical marker for atherosclerosis. The combination of hyperinsulinemia and insulin resistance increases the likelihood of developing type 2 diabetes mellitus (T2DM), hypertension, dyslipidemia, and CAD.⁶

Obesity is increasingly recognized as an inflammatory condition.⁷ However, the link between obesity, inflammation, and CAD is not completely understood. One postulated mechanism is that adipocytes secrete increased levels of adipocytokines into the circulation that may induce an inflammatory response throughout the body.⁸ This inflammatory process in atherosclerotic arteries may lead to an elevated level of the acute-phase reactant C-reactive protein (CRP) in patients with CAD.⁹ Increased CRP is also associated with insulin resistance, and this finding has led to the concept that inflammatory pathways may link CAD with insulin resistance.¹⁰ One of the adipocytokines, a collagen-like protein, adiponectin, has been intensively evaluated as a potential cardiovascular marker.¹¹ Adiponectin is distinct from other adipocytokines in that it is inversely related to obesity¹² and decreased levels are associated with CAD,¹³ hyperinsulinemia, and insulin resistance.¹⁴

More than a decade ago, our research group reported that insulin resistance appeared to play little role in the pathogenesis of atherosclerosis in a small number of patients with HoFH or HeFH.¹⁵ It is important to verify this conclusion, and we are now able to reassess our findings in a larger cohort of patients with this disorder. The aim of the present study was to evaluate the association of atherosclerosis, obesity, inflammation, adiponectin, and insulin resistance in groups of patients with HoFH or HeFH.

Patients and Methods

Patients

Patients with FH were recruited from the lipid clinic at the Charlotte Maxeke Johannesburg Hospital. They comprised 51 HoFH patients (24 males and 27 females) and 20 HeFH patients (10 males and 10 females). Twenty healthy normocholesterolemic subjects (7 males and 13 females) with no history of hypercholesterolemia were used as controls.

Criteria for the diagnosis of HoFH were: (1) Genetic confirmation of two mutant alleles at the LDL receptor gene locus; or (2) an untreated LDL-C level >13.0 mmol/L together with either cutaneous and/or tendinous xanthoma before 10 years of age, and/or evidence of an elevated LDL-C level >4.9 mmol/L before lipid-lowering therapy consistent with HeFH in both parents. Diagnosis of HeFH was based on the presence of a family history of hypercholesterolemia, clinical signs of FH together with an elevated total cholesterol concentration, and confirmation by DNA analysis of FH LDL receptor mutations.

Sixteen HoFH patients and three HeFH patients had evidence of coronary atherosclerosis on coronary angiography. None of the patients were receiving LDL apheresis. The majority of patients were taking lipid-lowering therapy, predominantly a statin at the maximal dose of 40 mg of rosuvastatin, 80 mg of atorvastatin, or 80 mg of simvastatin daily. Seven of the HoFH patients and 6 of the HeFH patients had moderate hypertension (blood pressure ≥130/85 mmHg) and were taking antihypertensive medication. All subjects gave informed consent, and the study was approved by the Committee for Research on Human Subjects of the University of the Witwatersrand.

Methods

Biochemical assays

The patients' most recent untreated (no lipid-lowering therapy) blood samples were compared with their treated samples at the time nearest to the study analysis. Fasting concentrations of plasma glucose, serum total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDL-C) were determined by enzymatic methods. Reagents were supplied by Roche Diagnostics GmbH, Mannheim, Germany. LDL-C levels were calculated using the Friedewald formula.¹⁶ Enzyme-linked immunosorbent assays (ELISA) were used to measure serum concentrations of insulin (Millipore Corporation, Billerica, MA) (reference range, 5–15 mU/L); proinsulin (Linco Research, St Charles, MO) (reference range, 3–20 pmol/L), and adiponectin (R&D Systems, Inc., Minneapolis, MN) (reference range, 0.8–21.5 μg/mL). An immunoturbidometric assay was used to measure high-sensitivity CRP (hsCRP) (Tina-quant, Roche Diagnostics GmbH, Mannheim, Germany) (reference intervals: low risk <1.0 mg/L; average risk 1.0–3.0 mg/L; high risk >3.0 mg/L). Intraassay coefficients of variance were <5.0% for all assays.

Calculation of homeostasis model assessment of insulin resistance

The homeostasis model assessment of insulin resistance (HOMA-IR) equation was used to calculate insulin resistance in the patients and control subjects from their fasting glucose and insulin concentrations as follows¹⁷: [Insulin (mU/L)]×[glucose (mmol/L)]/22.5. A high HOMA-IR score denoted insulin resistance.

Measurement of CIMT

The carotid arteries of patients and control subjects were evaluated with high-resolution B-mode ultrasonography using a previously validated technique.¹⁸ All of the subjects were examined in the supine position. Both common carotid arteries were scanned longitudinally to visualize the intima media complex of the far wall of the artery. The distance between the echo arising from the lumen intima interface and the media adventitia interface was taken as a measure of the intima media complex. CIMT was defined as the average of five measurements randomly selected between 10 mm and 30 mm proximal to the carotid bifurcation. The same observer performed all the measurements and was blinded to the patients' drug therapy or any previous ultrasound findings. Using this technique, the intraobserver variation was 6.7%.

Statistical analysis

Data were analyzed using GB-STAT (Dynamic Microsystems, Inc., Silver Spring, MD), with a P value<0.05 considered significant. Differences between two groups were determined by the unpaired t-test or the Mann–Whitney U-test for parametric and nonparametric data, respectively. One-way analysis of variance (ANOVA) was used to compare differences between more than two groups. Because of the skewed distribution of hsCRP, median values of hsCRP were compared in the study groups using the nonparametric Kruskal–Wallis test. All of the variables, including natural log-transformed hsCRP values, were assessed in multiple linear and simple linear regression analyses to reveal any significant correlations with insulin resistance as measured by HOMA-IR. Results are expressed as mean±standard deviation (SD), median (range), or proportion (%).

Results

Fasting profiles of the study groups

Table 1 summarizes the anthropometric measurements and fasting biochemical variables of the HoFH patients, HeFH patients, and control subjects. There were small but significant differences between untreated groups of HoFH and HeFH patients and the control subjects for age and CIMT (ANOVA both P<0.0001), hsCRP (Kruskal–Wallis P<0.04), glucose and adiponectin (ANOVA both P<0.001), and insulin (ANOVA P<0.01).

Table 1.

Anthropometric Measurements and Fasting Biochemical Variables in Patients with Homozygous and Heterozygous Familial Hypercholesterolemia, and Control Subjects

	HoFH patients (n=51)		HeFH patients (n=20)
	Untreated	Treated	Untreated	Treated	Control subjects (n=20)
Gender (male (M)/female (F))	24 M/27 F	—	10 M/10 F	—	7 M/13 F
Age (years)	23.6±13.8^e	—	41.4±13.6	—	42.4±10.4
BMI (kg/m²)	22.0±5.6^a	—	25.0±4.6	—	23.1±2.9
Cigarette smoker	10 (20%)	—	1(5%)	—	1 (5%)
CIMT (mm)	1.45±0.57^e	—	0.74±0.21^c	—	0.55±0.12
hsCRP (mg/L)	1.84^a	0.86	1.28	0.90	0.86
median (range)	(0.2–9.9)	(0.33–6.6)	(0.35–9.1)	(0.37–6.8)	(0.16–3.1)
Glucose (mmol/L)	4.3±1.1^c,e	5.0±0.6	5.5±0.9^a	5.7±1.0	4.8±1.2
Insulin (mIU/L)	11.6±8.8^d	9.9±6.0	7.9±6.6	10.7±7.3	5.7±3.8
Proinsulin (pmol/L)	2.6±2.9	1.9±2.0	3.4±0.9^a	3.2±0.8	2.9±0.4
Adiponectin (μg/mL)	18.8±11.2^a,c,e	11.4±6.4	8.9±5.1^a	8.6±4.9	14.0±7.8
HOMA	2.3±1.9^b	2.2±1.5	2.0±1.7	2.8±2.2	1.3±1.0

Results are expressed as mean±SD, proportion (%) or median (range).

P<0.05 HoFH untreated patients vs. HeFH untreated patients (BMI), HoFH untreated patients vs. control subjects (hsCRP, adiponectin), and HeFH untreated patients vs. control subjects (glucose, proinsulin, adiponectin).

P<0.01 HoFH untreated patients vs. control subjects.

P<0.002 HoFH untreated patients vs. HoFH treated patients (glucose, adiponectin) and HeFH untreated patients vs. control subjects (CIMT).

P<0.001 HoFH untreated patients vs. control subjects.

P<0.0001 HoFH untreated patients vs. HeFH untreated patients and control subjects (age, CIMT), and HoFH untreated patients vs. HeFH untreated patients (glucose, adiponectin).

HoFH, homozygous familial hypercholesterolemia; HeFH, heterozygous familial hypercholesterolemia; BMI, body mass index; CIMT, carotid intima media thickness; hsCRP, high-sensitivity C-reactive protein; HOMA, homeostasis model assessment of insulin resistance; SD, standard deviation.

Separate pairwise comparisons between these three groups found that HoFH patients were younger than HeFH patients and control subjects (both P<0.0001), and BMI was lower in the HoFH patients than HeFH patients (P<0.05). CIMT values in HoFH patients were higher than HeFH patients and control subjects (both P<0.0001), and CIMT was also higher in HeFH patients than control subjects (P<0.002). HoFH patients had lower glucose levels than HeFH patients (P<0.0001), and glucose was higher in the HeFH patients than control subjects (P<0.05). The concentration of hsCRP was slightly higher in the HoFH patients than the HeFH patients, and both groups were higher than control subjects, significantly so in the HoFH patients (P<0.05). HoFH patients had higher levels than control subjects for insulin (P<0.001) and HOMA-IR (P<0.01); proinsulin was higher in HeFH patients than control subjects (P<0.05). There was little change in HOMA-IR in the FH patients with high-dose statin therapy. Unexpectedly, adiponectin levels were highest in HoFH patients compared with the HeFH patients (P<0.0001) and control subjects (P<0.05), but were lower in the HeFH patients than control subjects (P<0.05). Within the HoFH group, untreated patients had a lower glucose concentration and a higher adiponectin level than treated patients (both P<0.002). There were no significant differences within the HeFH group for any of the variables.

Lipid profiles of the study groups

Lipid profiles of the HoFH patients, HeFH patients and control subjects are presented in Table 2. Significant differences were found between the untreated groups of HoFH and HeFH patients and control subjects for TC, HDL-C, and LDL-C (ANOVA all P<0.0001). As expected, separate pairwise comparisons between these three groups showed that TC and LDL-C concentrations were higher in the HoFH patients than in the HeFH patients and control subjects (both P<0.0001), and they were also higher in the HeFH patients than control subjects (both P<0.0001). TG levels were similar in the untreated and treated patients and control subjects. HDL-C concentration was lower in the HoFH patients than HeFH patients (P<0.01) and control subjects (P<0.0001). Within the HoFH and HeFH groups, untreated patients had higher concentrations of TC and LDL-C than treated patients (both P<0.0001).

Table 2.

Lipid Profiles of Patients with Homozygous and Heterozygous Familial Hypercholesterolemia, and Control Subjects

	HoFH patients (n=51)		HeFH patients (n=20)
	Untreated	Treated	Untreated	Treated	Control subjects (n=20)
Total cholesterol (mmol/L)	16.5±3.5^b,c	12.7±3.6	9.2±1.1^c	5.7±0.9	4.8±0.6
Triglycerides (mmol/L)	1.4±1.1	1.6±0.8	1.7±1.0	1.4±0.8	1.2±0.5
HDL-C (mmol/L)	0.9±0.3^a,c	1.0±0.3	1.4±0.5	1.5±0.5	1.6±0.3
LDL-C (mmol/L)	14.9±3.5^b,c	11.0±3.3	7.0±1.0^c	3.6±0.8	2.6±0.5

Results are expressed as mean±SD.

P<0.05 HoFH untreated vs. HeFH untreated patients (HDL-C).

P<0.001 HoFH untreated vs. HoFH treated patients (TC, LDL-C).

P<0.0001 HoFH untreated vs. HeFH untreated patients and control subjects (TC, LDL-C), HoFH untreated patients vs. control subjects (HDL-C), and HeFH untreated vs. HeFH treated patients and control subjects (TC, LDL-C).

HoFH, homozygous familial hypercholesterolemia; HeFH, heterozygous familial hypercholesterolemia; HDL-C, high-density lipoprotein cholesterol: LDL-C, low-density lipoprotein cholesterol; SD, standard deviation.

Regression analysis

Multiple regression analysis of the combined groups of patients and control subjects in which HOMA-IR was the dependent variable and age, BMI, TG, HDL-C, LDL-C, CIMT, log hsCRP, proinsulin, and adiponectin were the independent variables revealed that the major determinant of insulin resistance was BMI (r=0.54; P=0.004) (Table 3). On simple linear regression, the highest correlation was between HOMA-IR and BMI (r=0.39; P=0.0002) (Fig. 1). Log hsCRP correlated with BMI (r=0.35; P<0.002) and insulin resistance (r=0.22; P=0.05). LDL-C and CIMT did not correlate with insulin resistance. Adiponectin correlated significantly with LDL-C (r=0.34; P=0.001), but not with CIMT (r=0.12; P>0.05).

FIG. 1.

Linear regression plot of combined groups of patients with homozygous or heterozygous familial hypercholesterolemia and control subjects, displaying a significant correlation between homeostasis model assessment (HOMA) and body mass index (BMI).

Table 3.

Multiple Regression Analysis of the Combined Groups of Patients with Homozygous or Heterozygous Familial Hypercholesterolemia and Control Subjects, with HOMA As the Dependent Variable

Independent variables	B coefficient	Standard error (B)	t Value	Probability
Age	−0.006198	0.021626	−0.2866	0.7757
BMI	0.169627	0.056628	2.9955	0.0044
Triglycerides	−0.165556	0.25229	−0.6526	0.5150
HDL-C	−0.55239	0.651131	−0.8484	0.4006
LDL-C	0.048779	0.65552	0.7441	0.4606
CIMT	0.231377	0.560743	0.4126	0.6818
Log hsCRP	−0.133103	0.29033	−0.4585	0.6488
Proinsulin	−0.077765	0.102726	−0.757	0.4529
Adiponectin	−0.030745	0.24222	−1.2693	0.2107

HOMA, homeostasis model assessment; BMI, body mass index; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; CIMT, carotid intima media thickness, hsCRP, high-senstivity C-reactive protein.

Discussion

This study assessed the relationship between atherosclerosis, insulin resistance, and the associated conditions of obesity and inflammation in patients with HoFH or HeFH. We also evaluated the effect of high-dose statin therapy on insulin resistance in this cohort. The main finding was that markers of insulin resistance, i.e., HOMA-IR, insulin, and hsCRP correlated more closely with BMI than with the severity of atherosclerosis. There was no significant difference in HOMA-IR scores between the patient groups, nor was there any significant change in insulin resistance with high-dose statin therapy. Unexpectedly, adiponectin levels were highest in the HoFH patients. Although the target LDL-C level was not achieved in our FH patients, LDL-C levels decreased significantly with lipid-lowering therapy, predominantly statin therapy.

It has been evident for several years that obesity is associated with inflammation that is causally involved in the development of insulin resistance. Numerous studies have documented metabolic abnormalities are interrelated with insulin resistance and obesity, including glucose intolerance, hyperinsulinemia, dyslipidemia, and elevated hsCRP concentration.^19
–21 By contrast, previous studies of patients with FH, including our own report, have found normal resistance to insulin.^15,22,23 This observation has been confirmed in the present study, in which our patients had normal glucose and insulin levels, and they were not insulin resistant as assessed by the HOMA-IR model. HOMA scores were similar in the HoFH and HeFH patients, supporting the view that FH is not an insulin-resistant state.

Research from the past decade has described the relationship between measures of obesity such as BMI and inflammation as determined by hsCRP. In a recent study, Brooks et al.²⁴ demonstrated that hsCRP had a strong correlation with BMI. Khaodhiar et al.²⁵ and Guldiken et al.²⁶ separately showed similar results in their studies. These reports are in keeping with our finding that hsCRP correlated closely with BMI. However, hsCRP concentrations in our untreated HoFH and HeFH patients were within the average risk range of 1.0–3.0 mg/L, and after treatment both groups had levels below 1.0 mg/L, which indicated a low risk of inflammation. Results from a meta-analysis have shown that statin therapy reduces hsCRP in healthy individuals and those with stable vascular disease,²⁷ and it appears to have had the same effect on our FH patients.

Statins are being prescribed increasingly, and recently published data indicate that statin therapy, particularly high-dose therapy, is associated with increased diabetes risk.^28,29 In contrast to these reports, we and other researchers do not agree with this finding.³⁰ Indeed, high-dose statin therapy did not aggravate insulin resistance in our FH patients, and in our experience the occurrence of diabetes is unusual in FH subjects. Statins appear to reduce hsCRP in a largely LDL-C–independent manner, and their antiinflammatory properties have been suggested as a potential mechanism beyond LDL-C reduction for the efficacy of these agents.³¹ Despite not achieving target LDL-C levels, the significant decrease in LDL-C levels seen in our FH patients is in agreement with several clinical trials that have shown that statins lower LDL-C levels substantially in patients with HoFH and HeFH.^32
–34 Statins probably reduce LDL-C by inhibiting hepatic cholesterol synthesis, thereby limiting cholesterol availability for the formation and secretion of apolipoprotein B, and by increasing residual LDL receptor activity.³⁵

High concentrations of very-low-density lipoprotein (VLDL) triglycerides and low levels of HDL-C are associated with insulin resistance,³⁶ and it has been proposed that insulin resistance, through its effects on lipoprotein metabolism, could also increase the risk of atherosclerosis.³⁷ However, our FH patients had normal or only mildly elevated TG, and there was no relationship between LDL-C levels and insulin resistance. Although insulin has the capacity to modify LDL-C levels, this does not necessarily imply that patients with hypercholesterolemia that is caused by genetic mutations would have insulin resistance. Our HoFH and HeFH patients had high LDL-C levels, but they did not have a significant degree of insulin resistance, which indicates that cholesterol metabolism in FH is regulated independently of mechanisms related to insulin resistance.

Contrary to expectations, adiponectin was not decreased in our FH patients. Although adiponectin concentrations were higher in the HoFH patients, this probably reflected their younger age and lower BMI. Adiponectin stimulates fatty acids oxidation, reduces TG, and improves glucose metabolism by increasing insulin sensitivity.³⁸ In addition, adiponectin inhibits the inflammatory process that accompanies atherosclerosis.¹¹ As reported in a recent study by Nawrocki et al.,³⁹ who found no association between adiponectin levels and atherosclerosis in mice, we also noted that in our study of FH patients, adiponectin did not correlate with CIMT, a subclinical marker of atherosclerosis.

Our study should be considered within the context of its limitations. The approximate calculation of insulin resistance using the HOMA-IR model is dependent on the precision of the insulin assay, which may be affected by insulin's short plasma half-life and the biological pulsatility of insulin secretion.¹⁷ While the standard reference method to estimate insulin resistance is the euglycemic hyperinsulinemic clamp,⁴⁰ HOMA-IR has been strongly correlated with insulin resistance in several studies^41,42 and in our own comparison of the clamp technique with surrogate indices in patients with CAD.⁴³ The use of antihypertensive medication in some of our patients, which might have aggravated insulin resistance, was minimal and was unlikely to have influenced the overall results.

The present extension of our earlier study to include a larger cohort of HoFH and HeFH patients confirmed that BMI is the major determinant of insulin resistance. Our FH patients had normal glucose metabolism and they were not insulin resistant. Moreover, there was no worsening of insulin resistance with high-dose statin therapy. Adiponectin was not decreased, neither was it related to atherosclerosis; therefore, adiponectin is more likely to indicate obesity rather than atherosclerosis. Lipid profiles of both groups of FH patients improved significantly with statin therapy; however, there was no relationship between the degree of insulin resistance and LDL-C or markers of inflammation and atherosclerosis. We conclude that in the absence of any known causes of insulin resistance, our findings strengthen the opinion that FH patients are not insulin resistant and high-dose statin therapy does not induce insulin resistance in patients with FH. Atherosclerosis per se is not an insulin-resistant state.

Footnotes

Author Disclosure Statement

All authors have no competing financial interests to report.

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