Ablation of Iqgap2 protects from diet-induced hepatic steatosis due to impaired fatty acid uptake

Abstract

Long-chain fatty acids (LCFA) serve as structural components for membrane biogenesis and as primary energy sources during mitochondrial β-oxidation reactions. Hepatic LCFA uptake is complex, with characteristics suggestive of a dual-kinetic model manifested by rapid (carrier-assisted/facilitated) and delayed (passive diffusional) phases. Our previous work using mice deficient of the Iqgap2 gene established a highly novel link between IQGAP2, a putative GTPase-activating protein, and hepatocarcinogenesis. Now we report that Iqgap2 deficiency also results in selective loss of the facilitated phase of hepatocyte LCFA uptake with preservation of the diffusional component. This molecular defect was seen in Iqgap2(-/-) hepatocytes of all ages studied (1-, 4-, 8-months). The loss of facilitated LCFA uptake protected against development of hepatic triglyceride accumulation in Iqgap2-deficient mice fed high-fat diet, consistent with a fundamental role in physiological fat partitioning. These phenotypic changes could not be explained by genetic loss of fatty acid processing proteins known to regulate lipid uptake or metabolic processing pathways. Iqgap2-deficient livers also displayed enhanced insulin sensitivity.

Conclusion: These observations identify a novel property of the putative GTPase-activating protein IQGAP2 in LCFA uptake in vitro and in vivo, and implicate IQGAP2 in an intracellular signaling pathway necessary for functional fatty acid uptake, lipid processing, and, possibly, glucose homeostasis.

Figure 1

Defective fatty acid partitioning in diet-modified Iqgap2^-/- mice. (A) Weight change and alterations in body composition for high-fat diet; asterisk indicates statistically significant differences (p < 0.05). % Fat was calculated as a percent of total body fat weight measured by NMR from total body weight. (B) Fresh-frozen liver sections stained with Oil Red O and counterstained with hematoxylin demonstrate protection against hepatic steatosis in Iqgap2^-/- mice. A size bar denotes 200 micron. Hepatic (C) and serum (D) triglyceride content is expressed as the mean ± SEM (n=6 per genotype), hepatic triglyceride levels are expressed in mg per mg of total protein. Asterisk indicates statistically significant differences (p<0.05).

Figure 2

Selective loss of facilitated fatty acid uptake in Iqgap2^-/- hepatocytes. (A) LCFA uptake by isolated hepatocytes from Iqgap2^-/- and wild-type mice was measured in real time using BODIPY-FA/Q-Red.1 [32]. Data plots reflect those of a complete set of paired experiments representative of six identical experiments. All data points are mean relative fluorescence units (RFU) per 10⁵ cells/well from triplicate wells (SEM not shown to minimize plot complexity). Three empirical phases of uptake (A, B, C) are shown. (B) [³H]palmitate uptake by Iqgap2^-/- and wild-type hepatocytes. Uptake is expressed as mean decay per minute (DPM) per 2 × 10⁵ cells ± SEM from triplicate wells. p < 0.05 for 60 and 120 seconds time points. Plot is representative of four independent experiments. (C) Viability of isolated hepatocytes was assessed by MTT assay. Data are expressed as mean relative absorbance at 540 nm per 2 x 10⁵ cells ± SEM from triplicate wells. Difference in absorbance between Iqgap2^-/- and wild-type is not statistically significant (p = 0.099). (D) Apoptosis in isolated hepatocytes was quantified by ApoStrand ELISA and expressed as mean relative absorbance at 405 nm per 10⁴ cells ± SEM from triplicate wells. Asterisk indicates p < 0.05 compared with all other groups.

Figure 3

Effect of BAPTA-AM on LCFA uptake by HepG2 cells. HepG2 cells were pre-incubated with 125 M, 500 M and 1 mM BAPTA-AM for 30 minutes at 25°C prior to real time LCFA uptake quantification using BODIPY-FA/Q-Red.1. All plots represent the mean ± SEM from triplicate wells from one representative set of complete experiments (repeated once). DMSO treatment was used as a positive control. The results are expressed as relative fluorescent units (RFU) per 10⁵ cells per second.

Figure 4

Hepatic expression of genes involved in FA processing in Iqgap2^-/- and wild-type mice. Quantitative RT-PCR was completed using oligonucleotide primers specific for: fatty acid transport proteins (fatp1-5), fatty acid binding proteins (fabp 1-5), caveolin-1 (cave1), FAT/CD36, acyl-Coenzyme A oxidase 1 (Acox1), carnitine palmitoyltransferase 1a (Cpt1a) and sterol regulatory element-binding protein SREBP-1c and SREBP-2 genes (see Table 1). The relative transcript expression was normalized to actin expression, calculated using the comparative threshold cycle number (Δ-Ct method) and expressed as relative transcript abundance in ng mRNA per 1 ng of β-actin mRNA. Mean expression levels from triplicate wells and standard deviation were log₁₀-transformed and plotted on a logarithmic scale.

Figure 5

Expression levels of lipogenesis-related genes in IQGAP2-deficient livers. (A) Hepatic FAS, PPARα and PEPCK RNA transcript levels measured by qRT-PCR. RNA transcript levels of fatty acid synthase (FAS), peroxisome proliferator-activated receptor α (PPARα) and phosphoenolpyruvate carboxykinase (PEPCK) were measured in response to a single intraperitoneal insulin injection (5U/kg) by qRT-PCR. The relative transcript expression was calculated as described for Figure 4. (B) Immunoblot confirming protein expression of FAS, FABP1 and caveolin-1 in wild-type, Iqgap2^-/- and Iqgap1^-/-/Iqgap2^-/- livers. The blots are representative of three independent experiments analyzing three different groups of mice. (C)Densitometric quantification of hepatic FAS protein expression normalized to GAPDH levels.

Figure 6

Enhanced insulin sensitivity of IQGAP2-deficient liver. (A) Glucose tolerance test (GTT) using 6-month-old male wild-type, Iqgap2^-/- and Iqgap1^-/-/Iqgap2^-/- mice fed regular diet and fasted overnight. Glucose (2 g/kg) was administered as a single intraperitoneal injection and blood samples were collected from a tail vein at the specified times. Data are presented as mean SEM, n = 5 in each group. The graph is representative of two independent experiments using different mice. (B) GTT using 6-month-old male wild-type and Iqgap2^-/- mice fed high fat diet (HFD) for 8 weeks and fasted overnight prior test. Data presented as mean SEM, n = 5 in each group. The graph is representative of three independent experiments using different mice. (C) Immunoblot analysis of hepatic total levels and the phosphorylated forms of Akt (Ser473) and GSK3β (Ser9) in response to insulin stimulation in 6-month-old male wild-type, Iqgap2^-/- and Iqgap1^-/-/Iqgap2^-/- mice. The blot is representative of four independent studies. (D) Densitometric quantification of hepatic levels of phospho-Akt and phospho- GSK3β in response to insulin stimulation normalized to GAPDH levels.

Figure 7

Schema describing the proposed IQGAP2-mediated mechanism of facilitated FFA uptake by hepatocytes. Conformational change of the IQGAP2 protein in response to a signaling cue (possibly Ca²⁺ or phosphorylation) is shown.