The effects of propionate and valerate on insulin responsiveness for glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes via G protein-coupled receptor 41

Abstract

Since insulin resistance can lead to hyperglycemia, improving glucose uptake into target tissues is critical for regulating blood glucose levels. Among the free fatty acid receptor (FFAR) family of G protein-coupled receptors, GPR41 is known to be the Gαi/o-coupled receptor for short-chain fatty acids (SCFAs) such as propionic acid (C3) and valeric acid (C5). This study aimed to investigate the role of GPR41 in modulating basal and insulin-stimulated glucose uptake in insulin-sensitive cells including adipocytes and skeletal muscle cells. Expression of GPR41 mRNA and protein was increased with maximal expression at differentiation day 8 for 3T3-L1 adipocytes and day 6 for C2C12 myotubes. GPR41 protein was also expressed in adipose tissues and skeletal muscle. After analyzing dose-response relationship, 300 µM propionic acid or 500 µM valeric acid for 30 min incubation was used for the measurement of glucose uptake. Both propionic acid and valeric acid increased insulin-stimulated glucose uptake in 3T3-L1 adipocyte, which did not occur in cells transfected with siRNA for GPR41 (siGPR41). In C2C12 myotubes, these SCFAs increased basal glucose uptake, but did not potentiate insulin-stimulated glucose uptake, and siGPR41 treatment reduced valerate-stimulated basal glucose uptake. Therefore, these findings indicate that GPR41 plays a role in insulin responsiveness enhanced by both propionic and valeric acids on glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and in valerate-induced increase in basal glucose uptake in C2C12 myotubes.

Figure 1. GPR41 expression.

GPR41 mRNA (A) and protein (B) levels were assayed in 3T3-L1 preadipocytes, differentiated adipocytes on day 8, C2C12 myoblasts, and differentiated myotubes on day 6 by quantitative real-time PCR and immunoblotting, as described in the Methods. Bar graph expresses relative values to 3T3-L1 preadipocytes or C2C12 myoblasts. **P<0.01, vs. 3T3-L1 preadipocytes or C2C12 myoblasts. GPR41 protein levels were measured during differentiation of 3T3-L1 adipocytes (C) and C2C12 myotubes (D). Day 0 was set for adipocyte differentiation by growing 3T3-L1 preadipocytes to 2 days post-confluence in growth medium (GM) and for myotubes by growing C2C12 myoblasts to confluence in GM. **P<0.01 and *P<0.05, vs. GM. Gel images are representative of the three experiments. All values expressed in the bar graphs are means ± SEM and the average of three similar, independent experiments, each performed in triplicate. The expression level of GPR41 protein was measured in five different tissues of nine-week-old male C57BL/6 mice (E) as described in the Methods. Gel images shown are representative of four similar experiments with different protein extracts of five tissues from four mice (n = 4). Total GPR41 protein was normalized to the β-actin level in cell lines and GAPDH level in tissues.

Figure 2. Cytotoxicity of propionic acid and valeric acid in both non-differentiated and differentiated 3T3-L1 and C2C12 cells.

3T3-L1 preadipocytes (A), C2C12 myoblasts (B), 3T3-L1 adipocytes (C) and C2C12 myotubes (D) were exposed to the indicated concentrations of propionic acid or valeric acid for 24 h, and cell viability was measured at 570 nm using the MTT assay as described in the Methods. Digitonin (100 µg/mL) was used as the positive control. Results are the means ± SEM of three similar independent experiments, each performed in triplicate.

Figure 3. Dose-response relationship between SCFAs and glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes, and time-course analysis.

For the determination of dose-response relationship, 3T3-L1 adipocytes (A) or C2C12 myotubes (C) were starved and treated with various concentrations of propionic acid or valeric acid for 30 min in the absence or presence of insulin (100 nM) in KRPH buffer. For the analysis of time-course, 3T3-L1 adipocytes (B) and C2C12 myotubes (D) were treated with 300 µM propionic acid or 500 µM valeric acid for the indicated time in the absence or presence of insulin in KRPH buffer. After adding 2-deoxy-[³H]-glucose for 10 min, glucose uptake was measured in the cell lysates as described in the Methods. Results are the means ± SEM of three similar independent experiments, each performed in quadruplicate. *P<0.05 and **P<0.01 for propionic acid, and ^# P<0.05 and ^## P<0.01 for valeric acid, vs. insulin-stimulated glucose uptake with no SCFA treatment. ^$ P<0.05 for propionic acid, ⁺ P<0.05 and ⁺⁺ P<0.01 for valeric acid, vs. basal glucose uptake with no SCFA treatment.

Figure 4. Effect of SCFAs on basal and insulin-stimulated glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes.

The activity of 300 µM propionic acid and 500 µM valeric acid to increase basal and insulin-stimulated glucose uptake was measured in 3T3-L1 adipocytes (A) and C2C12 myotubes (B) as described in the Methods. These cells were treated with rosiglitazone (10 µM), as a positive control, for 48 h, and glucose uptake was measured in the cell lysates. Results are the means ± SEM of three similar independent experiments, each performed in quadruplicate. *P<0.05, **P<0.01, ***P<0.001, vs. basal glucose uptake (no insulin stimulation), ^## P<0.01, vs. insulin-stimulated glucose uptake with no SCFA treatment.

Figure 5. Effects of siRNA for GPR41 on SCFA-induced rise in insulin-stimulated glucose uptake in 3T3-L1 adipocytes and C2C12 myotubes.

After confirming the expression of GPR41 protein expression by transfecting with siRNA for GPR41 (siGPR41,100 nM) using Lipofectamine RNAiMAX for 48 h in 3T3-L1 adipocytes (A) or C2C12 myotubes (C), cells were treated with 300 µM propionic acid or 500 µM valeric acid for 30 min in the absence or presence of insulin (100 nM) in KRPH buffer. Glucose uptake was measured in the lysates of 3T3-L1 adipocytes (B) or C2C12 myotubes (D) as described in the Methods. Results are the means ± SEM of three similar independent experiments, each performed in quadruplicate. **P<0.01 and ***P<0.001, vs. basal glucose uptake with siControl (negative control siRNA). ⁺⁺ P<0.01 and ⁺⁺⁺ P<0.001 vs. basal glucose uptake with siGPR41, ^## P<0.01 vs. insulin-stimulated glucose uptake with SCFAs and siControl.