Circulating serum fatty acid synthase is elevated in patients with diabetes and carotid artery stenosis and is LDL-associated

Abstract

Background and aims: Diabetes is an independent risk factor for carotid artery stenosis (CAS). Fatty acid synthase (FAS), an essential de novo lipogenesis enzyme, has increased activity in the setting of diabetes that leads to altered lipid metabolism. Circulating FAS (cFAS) was recently observed in the blood of patients with hyperinsulinemia and cancer. We thought to evaluate the origin of cFAS and its role in diabetes-associated CAS.

Methods: Patients with diabetes and no diabetes, undergoing carotid endarterectomy (CEA) for CAS, were prospectively enrolled for collection of plaque and fasting serum. FPLC was used to purify lipoprotein fractions, and ELISA was used to quantify cFAS content and activity. Immunoprecipitation (IP) was used to evaluate the affinity of cFAS to LDL-ApoB.

Results: Patients with CAS had higher cFAS activity (p < 0.01), and patients with diabetes had higher cFAS activity than patients with no diabetes (p < 0.05). cFAS activity correlated with serum glucose (p = 0.03, r² = 0.35), and cFAS content trended with plaque FAS content (p = 0.06, r² = 0.69). cFAS was predominantly in LDL cholesterol fractions of patients with CAS (p < 0.001), and IP of cFAS demonstrated pulldown of ApoB. Similar to patients with diabetes, db/db mice had highest levels of serum cFAS (p < 0.01), and fasL^-/- (tissue-specific liver knockdown of FAS) mice had the lowest levels of cFAS (p < 0.001).

Conclusions: Serum cFAS is higher in patients with diabetes and CAS, appears to originate from the liver, and is LDL cholesterol associated. We postulate that LDL may be serving as a carrier for cFAS that contributes to atheroprogression in carotid arteries of patients with diabetes.

Keywords: Carotid artery stenosis; Diabetes; Fatty acid synthase; Lipoprotein; Serum biomarker.

Figure 1:

Serum cFAS is elevated in patients with CAS and diabetes. (A) cFAS content (ng/μL) in the fasting serum of patients diagnosed with high-grade CAS and scheduled for CEA (n=26) was compared to fasting serum of patients who do not have CAS or other major cardiovascular co-morbidities (n=13). Patients with high-grade CAS demonstrated significantly higher cFAS content in their fasting serum compared to control patients. Control patients consistently demonstrated low serum cFAS content. ***p<0.001, Mann-Whitney. Error bars represent the SEM. (B) cFAS enzyme activity (arbitrary units, AU) was also evaluated between patients with high-grade CAS (n=26) and control patients (n=13). Patients with high-grade CAS demonstrated significantly higher cFAS enzyme activity compared to control patients **p<0.01, Mann-Whitney. Error bars represent the SEM. (C) Patients with diabetes and CAS (n=13) had the highest levels of cFAS enzyme activity compared to patients with no diabetes and CAS (n=13), and control patients (n=13). *p<0.05, **p<0.01, Kruskal-Wallis. Error bars represent the SEM.

Figure 2:

Serum cFAS correlates with serum glucose and carotid plaque FAS and saturated fatty acid content. (A) Serum cFAS activity and content were evaluated in patients with diabetes and CAS (n=13), patients with no diabetes and CAS (n=13), and controls with no diabetes and no CAS (n=13). Patients with diabetes and CAS have significantly higher serum glucose compared to patients with no diabetes and CAS. *p<0.05, Kruskal-Wallis. Error bars represent the SEM. cFAS activity (B) but not content (C) has a significant correlation with serum glucose (patient with diabetes and CAS, triangle; patient with no diabetes and CAS, square; patient control with no CAS, circle). r², Spearman correlation. (D) No significant difference was observed in serum LDL between patients with and without diabetes, and control patients. (E and F) No correlation was observed between cFAS activity/content and LDL. (G) Open exposure of the right carotid artery bifurcation with the head towards the left (cranial) and the feet towards the right (caudal). Visible within the clamped carotid bifurcation is irregular ulcerated plaque. Extracted CEA plaque demonstrates a minimally (Min) diseased portion at the distal edge, and a maximally (Max) diseased portion at the carotid bifurcation region. (H) fasn RNA transcript between Max and Min diseased CEA portions was evaluated using RT-PCR. No differences were observed in fasn and between patients with and without diabetes. (I) FAS protein content in CEA plaque homogenates were evaluated by Western blot analysis. (J) Higher levels of FAS were observed in Max diseased plaque compared to Min diseased plaque (n=6 patients). *p<0.05, Mann-Whitney U test. Error bars represent the SEM. (K) FAS protein content in CEA plaque homogenates was evaluated using ELISA. Compared to patients with no diabetes, there was a 39% increase in FAS in the Max and Min CEA plaques of patients with diabetes. Error bars represent the SEM. (L) FAS protein content in CEA plaque demonstrated a moderate correlation with serum cFAS activity, but this difference was not statistically significant. r², Spearman correlation; p=0.06. (M) The ratio of the relative content of total saturated fatty acid (SFA) to total unsaturated fatty acid (USFA) was significantly higher in the Max CEA plaques of patients with diabetes. **p<0.001, Kruskal-Wallis. Error bars represent the SEM.

Figure 3:

Serum cFAS content and activity is elevated in serum LDL cholesterol isolates and co-immunoprecipitates with ApoB. (A) Fasting serum from patients with diabetes and CAS (n=13), no diabetes and CAS (n=13), and control patients (n=13). Serum was added to an FPLC column and cholesterol isolate concentrations were evaluated to determine VLDL, LDL, and HDL concentrations. (B) Although control patients demonstrated a mild non-significant increase in serum LDL, no differences in VLDL and HDL were observed between the study groups. (C) Area under the curve (AUC) analysis demonstrated an overall increase in cholesterol isolates in control patients relative to patients with and without diabetes and CAS. *p<0.05, Kruskal-Wallis. Error bars represent the SEM. (D) cFAS content in peak VLDL, LDL, and HDL isolates was evaluated with ELISA from patients with diabetes (n=8) and patients with no diabetes (n=7). The LDL peak isolate demonstrated the highest levels of cFAS. ***p<0.001, Kruskal-Wallis. Error bars represent the SEM. (E) cFAS activity in LDL peak isolates from patients with diabetes (n=5) and patients with no diabetes (n=5) was evaluated by FAS activity assay. This demonstrated significantly higher FAS activity levels (arbitrary units; AU) in peak LDL isolates of patients with diabetes. *p<0.05, Mann-Whitney. Error bars represent the SEM. (F and G) Serum LDL fractions from a patient with diabetes was collected and ApoB immunoprecipitated. Wash, eluent, and immunoprecipitation (IP) fractions were collected. Subsequent Western blots were performed for ApoB (F) and FAS (G). An FPLC purified LDL fraction (30 μg of protein) was used as positive control for the ApoB blot, and a murine liver homogenate (10 μg of protein) was used as positive control for the FAS blot. (H) Similarly, serum LDL fractions from a patient with diabetes (1) and a patient with no diabetes (2) were collected and FAS was immunoprecipitated. The procedure wash, eluent, and IP fractions were collected and Western blots performed for ApoB (top blot), and FAS (bottom blot). Whole serum (30 μg of protein), murine liver homogenate (2.5 μg of protein) was used as positive control for ApoB and FAS blots, respectively.

Figure 4:

Differential expression of FAS and cFAS in murine tissue-specific liver and skeletal muscle FAS knockdowns. (A) FAS protein content from liver homogenates of +/+, db/db, fasL^−/−, and fasM^−/− mice were evaluated using Western blotting. (A and B) fasL^−/− mice demonstrated significantly less FAS in the liver, while db/db mice demonstrated significantly higher FAS in the liver. *p<0.05. Error bars represent the SEM. (C) FAS protein content from anterior tibialis muscle homogenates from the same mouse groups was also evaluated using Western blotting. (C and D) fasM^−/− mice showed significantly less FAS in the muscle tissue, and db/db mice also showed significantly higher FAS in the muscle. *p<0.05. Error bars represent the SEM. (E) Whole serum was collected from all mouse groups, and cFAS was then evaluated using Western blot. fasL^−/− mice had significantly less cFAS compared to +/+ and fasM^−/−. db/db mice had significantly higher cFAS compared to all other mouse groups. **p<0.01. Error bars represent the SEM. GAPDH blot was used as loading control for all Western blots.

Figure 5:

cFAS originates from the liver and may be transported by LDL to the peripheral vasculature to influence plaque formation. cFAS produced by the liver is extruded into the blood stream in semi-soluble form conjugated to ApoB in non-HDL cholesterol particles such as VLDL and LDL. Intravascular LDL particles traverse the endothelium along with their cFAS cargo, which is then delivered to the sub-endothelium. Accumulation of cFAS in the sub-endothelium is thought to contribute to SFA plaque content and peripheral arterial atheroprogression.