Evidence That Chromium Modulates Cellular Cholesterol Homeostasis and ABCA1 Functionality Impaired By Hyperinsulinemia

Abstract

Objective

Trivalent chromium (Cr³⁺) is an essential micronutrient. Findings since the 1950s suggest that Cr³⁺ might benefit cholesterol homeostasis. Here we present mechanistic evidence in support of this role of Cr³⁺.

Method and Results

High-density lipoprotein cholesterol generation in 3T3-L1 adipocytes, rendered ineffective by hyperinsulinemia, known to accompany disorders of lipid metabolism was corrected by Cr³⁺. Mechanistically, Cr³⁺ reversed hyperinsulinemia-induced cellular cholesterol accrual and associated defects in cholesterol transporter ABCA1 trafficking and apolipoprotein A1-mediated cholesterol efflux. Moreover, direct activation of AMP-activated protein kinase (AMPK), known to be activated by Cr³⁺, and/or inhibition of hexosamine biosynthesis pathway (HBP) activity, known to be elevated by hyperinsulinemia, mimics Cr³⁺ action.

Conclusion

These findings suggest a mechanism of Cr³⁺ action that fits with long-standing claims of its role in cholesterol homeostasis. Furthermore, these data implicate a mechanistic basis for the coexistence of dyslipidemia with hyperinsulinemia.

Trivalent chromium (Cr³⁺) is classified as an essential micronutrient for optimal carbohydrate and lipid metabolism. Although evidence relating Cr³⁺ deficiency and cardiovascular disease (CVD) is fragmentary, deficiency has been linked to reduced high-density lipoprotein cholesterol (HDL-C)¹. A rate-limiting step in HDL-C generation entails cholesterol transporter ABCA1-mediated cholesterol efflux to lipid-poor apolipoprotein A1 (ApoA1). The HDL-C particle formed is preβ-1 HDL-C, a subclass which removes cholesterol from macrophages², a cardioprotective event. These findings raise the question of whether an essential mechanism of Cr³⁺ action involves ABCA1/ApoA1-mediated preβ-1 HDL-C generation. Importantly, ABCA1/ApoA1 dysregulation may represent an unappreciated basis of low HDL-C coexisting with metabolic derangements (e.g., hyperinsulinemia).

Methods

Insulin-sensitive and hyperinsulinemia-induced insulin-resistant 3T3-L1 adipocytes and Cr³⁺ in the picolinate form (CrPic) at a 1 μM dose were used as previously described^3-4. Detailed methods and information on Cr³⁺ doses/forms tested are provided in online material (http://www.atcb.ahahournals.org).

Results

Examination of ABCA1 trafficking revealed that plasma membrane (PM) ABCA1 was diminished by hyperinsulinemic conditions relevant to disease³, yet in the presence of Cr³⁺ this was prevented (Fig. 1A). Endosomal membrane (EM) ABCA1 was elevated by hyperinsulinemia and normalized by Cr³⁺ (Fig. 1B). Total ABCA1 protein was not changed (Fig. S1A). Mechanistically, ABCA1 is regulated by the EM-to-cytosol (Cyto) cycling of the GTPase Rab8. Hyperinsulinemia increased and decreased EM- and Cyto-Rab8, respectively, and these changes were normalized by Cr³⁺ (Figs. 1C, D).

Fig. 1.

Control (Cont.) or hyperinsulinemic (12h Ins.) cells were treated without or with Cr³⁺. (A) Plasma membrane (PM) ABCA1, (B) Endosomal membrane (EM) ABCA1, (C) EM Rab8, and (D) Cytosolic Rab8. n = 4-11. *P < 0.05 versus untreated control.

Cholesterol accumulation has been implicated in EM ABCA1 sequestration in Niemann-Pick disease, type C (NPC)⁵. Like NPC, a substantial increase in EM cholesterol was found in cells cultured under hyperinsulinemic conditions that Cr³⁺ prevented (Fig. 2A). Interestingly, exercise is recognized to increase HDL-C levels, and like exercise, Cr³⁺ increases AMP-activated protein kinase (AMPK) activity⁴, known to suppress cholesterol synthesis⁶. AICAR (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside, an AMPK activator) and Cr³⁺ stimulated AMPK (Fig. 2B), and similarly to Cr³⁺, AICAR lowered EM cholesterol (Fig. 2C) and corrected membrane Rab8/ABCA1 levels (Figs. S1B-E); however, a gain in Cyto-Rab8 was not seen; likely due to a shorter AICAR treatment duration not permitting a detectable level of Rab8 to accumulate in the dilute cytosol fraction. Importantly, Cr³⁺ and AICAR both prevented hyperinsulinemia-impaired ApoA1-mediated cholesterol efflux (Fig. 2D).

Fig. 2.

Control (Cont.) or hyperinsulinemic (12h Ins.) cells were treated without or with Cr³⁺, AICAR, or DON. (A, C) EM cholesterol, (B) pAMPK, and (D) ApoA1-mediated cholesterol efflux. n = 4-11. *P < 0.05 versus untreated control. (E) Model of Cr³⁺ protection against (Blue) hyperinsulinemia-induced cholesterol-associated impairment in (Red) Rab8/ABCA1 trafficking and ApoA1-mediated cholesterol efflux.

Contrasting AMPK, increased hexosamine biosynthesis pathway (HBP) activity has been implicated in cholesterol accrual induced by hyperinsulinemia⁷. Testing the effect of the HBP inhibitor 6-Diazo-5-Oxo-L-Norleucine (DON) revealed Cr³⁺- and AICAR-like effects (Fig. 2D). Neither agent nor Cr³⁺ displayed any effect on control cells. Also, cholesterol lowering with methyl-β-cyclodextrin mimicked the protective effect on ApoA1-mediated cholesterol efflux (Fig. S1F).

Discussion

The role of Cr³⁺ in health and disease is complex. While patients with diabetes on Cr³⁺ supplementation see improvement in hyperglycemia, benefits on raising HDL-C remain unclear⁸. An emerging appreciation is that total HDL-C measurements are misleading in understanding its cardioprotective actions, as the ABCA1-generated preβ-1 HDL-C particle likely represents the “functional” subfraction². Therefore, study demonstrating that Cr³⁺ enhances this ABCA1-mediated event in cells cultured in a diabetic milieu is significant.

As the serum concentration of the preβ-1 HDL-C accounts for only a small fraction of total HDL-C, trials designed to assess the benefits of Cr³⁺ on total HDL-C may have had an inherent flaw in understanding Cr³⁺’s effect. In addition, Cr³⁺ deficiency in humans is expected to be slight, if any⁹, thus measurement of a supplemental effect may be negligible. Nevertheless, analyses reveal popular weight loss diets provide Cr³⁺ at suboptimal levels¹⁰.

Mechanistically, observation that AMPK stimulation ramps up ABCA1/ApoA1 functionality is interesting, given the appreciated benefits of exercise, a known stimulant of AMPK activity, on the prevention of metabolic syndrome and its consequences. In this regard, skeletal muscle and adipose tissue contain more cholesterol than any other organ¹¹. In fact, a new importance of adipose tissue cholesterol in the generation of HDL-C has recently been recognized^12-13. In particular, the generation of preβ-1 HDL-C appears critical in mediating cholesterol efflux from cholesterol-laden macrophages. The idea Cr³⁺ could have an indirect effect on cholesterol handling by macrophages is of interest. Testing this possibility as well as characterizing any direct effect Cr³⁺ may have on macrophage cholesterol metabolism is warranted.

In closing, these data suggest low circulating HDL-C, resulting from metabolic disorder, may arise from hyperinsulinemia/HBP-mediated peripheral tissue cholesterol accrual (Fig. 2E). This is associated with an EM sequestration of Rab8/ABCA1, and low preβ-1 HDL-C. Data also implicate that Cr³⁺ suppresses cholesterol synthesis/accrual via AMPK and this improves Rab8/ABCA1 functionality and HDL-C generation. Whether this cell-based model explains the benefits of Cr³⁺ and/or exercise in humans with diabetes remains to be validated.