BAIBA attenuates insulin resistance and inflammation induced by palmitate or a high fat diet via an AMPK–PPARδ-dependent pathway in mice
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We explored the effects of β-aminoisobutyric acid (BAIBA) on hyperlipidaemic-condition-induced insulin resistance and inflammation as mediated through a signalling pathway involving AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor δ (PPARδ).
Mouse skeletal muscle C2C12 cells and C57BL/6J mice were treated with palmitate or a high-fat diet (HFD) and BAIBA. Inflammation and the expression of genes associated with insulin signalling were determined by western blot and quantitative real-time PCR. Selected genes from candidate pathways were evaluated by small interfering (si)RNA knockdown and specific inhibitors.
BAIBA treatment ameliorated impairment of insulin receptor substrate (IRS)-1/Akt-mediated insulin signalling in palmitate-treated C2C12 myocytes and in skeletal muscle of HFD-fed mice. In addition, BAIBA treatment reversed HFD-induced increases in body weight and improved impaired glucose tolerance in mice. In vitro and in vivo, inhibitory κBα (IκBα) phosphorylation, nuclear factor κB (NFκB) nuclear translocation and downstream inflammatory cytokines were significantly suppressed by BAIBA. Furthermore, BAIBA treatment significantly induced AMPK phosphorylation and expression of PPARδ in C2C12 myocytes and in skeletal muscle of mice. Both compound C, an AMPK inhibitor, and Pparδ (also known as Ppard) siRNA abrogated the inhibitory effects of BAIBA on palmitate-induced inflammation and insulin resistance. BAIBA significantly induced the expression of genes associated with fatty acid oxidation, such as carnitine palmitoyltransferase 1 (Cpt1), acyl-CoA oxidase (Aco; also known as Acox1) and fatty acid binding protein 3 (Fabp3); this effect of BAIBA was significantly reduced by compound C and Pparδ siRNA.
These results are the first to demonstrate that BAIBA attenuates insulin resistance, suppresses inflammation and induces fatty acid oxidation via the AMPK–PPARδ pathway in skeletal muscle.
KeywordsAMP-activated protein kinase Inflammation Insulin resistance β-Aminoisobutyric acid
AMP-activated protein kinase
Dulbecco’s modified eagle medium
Intraperitoneal glucose tolerance test
Insulin receptor substrate-1
Insulin tolerance test
Monocyte chemotactic factor-1
Nuclear factor κB
Peroxisome proliferator-activated receptor-gamma coactivator-1α
Peroxisome proliferator-activated receptor δ
Small interfering RNA
Although regular exercise increases insulin sensitivity in humans , the underlying mechanisms are not completely understood. Begriche et al demonstrated that β-aminoisobutyric acid (BAIBA), a natural catabolite of thymine, reduced body fat percentage through increased fatty acid oxidation and decreased de novo lipogenesis in mice . Recently, Roberts et al identified BAIBA as a small molecule myokine secreted from myocytes with forced expression of peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). BAIBA enhances the browning of white adipose tissue and β-oxidation in the liver through a mechanism mediated by peroxisome proliferator-activated receptor α (PPARα) .
Elevated plasma NEFA levels are observed in individuals with insulin resistance . In particular, saturated NEFA has been reported to induce insulin resistance . Moreover, elevated levels of saturated NEFA induce inflammation, which results in insulin resistance via several pathways involving diacylglycerol-mediated protein kinase C activation  or Toll-like receptors . These pathways lead to activation of nuclear factor-κB (NFκB), a proinflammatory transcription factor, which impairs insulin signalling in skeletal muscle . Under inflammatory conditions, NFκB induces the expression of various proinflammatory cytokines, such as TNF-α and IL-6, which are associated with insulin resistance and type 2 diabetes .
The current study investigated the influence of the novel myokine BAIBA, which exerts ameliorating effects on inflammation and insulin resistance. Furthermore, to elucidate the cellular mechanisms through which BAIBA influences inflammation and insulin resistance, we explored a downstream signal transduction pathway involving AMP-activated protein kinase (AMPK) and PPARδ in C2C12 myocytes and in mouse skeletal muscle.
Cell cultures, reagents and antibodies
The mouse skeletal muscle cell line C2C12 (ATCC, Manassas, VA, USA) was cultured in Dulbecco’s modified eagle medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen), 100 units ml−1 penicillin and 100 μg ml−1 streptomycin (Invitrogen). Cells were incubated in a humidified atmosphere of 5% CO2 at 37°C. Cells were supplemented with 2% horse serum to induce differentiation. Mycoplasma contamination was not detected in C2C12 cells. BAIBA (Sigma, St Louis, MO, USA) was dissolved in distilled water. Compound C (Sigma) was dissolved in DMSO and added to the culture medium. Sodium palmitate (Sigma) was conjugated to 2% BSA (fatty acid free; Sigma) dissolved in DMEM. The final concentration of DMSO did not exceed 0.1%, which did not affect cell viability. In all experiments, cells were treated with palmitate-BSA for 24 h and 2% BSA was used as a control. Anti-phospho Akt (Ser473; 1:1000), anti-Akt (1:2500), anti-insulin receptor substrate (IRS)-1 (1:2500), anti-phospho AMPK (Thr172; 1:1000), anti-AMPK (1:2500), anti-NFκBp65 (1:2500), anti-phospho IκBα (Ser32; 1:1000), anti-IL-6 (1:2500) and anti-PPARδ (1:2500) antibodies were purchased from Cell Signaling (Beverly, MA, USA). Anti-phospho IRS-1 (Tyr 632; 1:1000) and anti-beta-actin (1:5000) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Animals, feeding, treatment, in vivo insulin signalling, intraperitoneal glucose tolerance test and insulin tolerance test
This study was approved by the institutional animal review board (Institutional Animal Care and Use Committee) of Korea University, Seoul, Korea. A control group and two experimental groups of 8-week-old male C57BL/6J (B6) mice were given a normal diet (ND; 11.92 kJ g−1; 12.6% energy from fat, 26.7% from protein and 60.7% from carbohydrate; Brogaarden, Gentofte, Denmark) and a high-fat diet (HFD; 21.92 kJ g−1; 60% energy from fat, 20% from protein and 20% from carbohydrate; Research Diets, New Brunswick, NJ, USA), respectively, for 8 weeks. The HFD-BAIBA group received 150 mg kg−1 day−1 of BAIBA dissolved in drinking water given ad libitum for 8 weeks. Soleus muscle samples were harvested 10 min after mice were given an intraperitoneal injection of human insulin (Novo Nordisk, Princeton, NJ, USA; 10 U kg−1 body weight). For the intraperitoneal glucose tolerance test (IPGTT), mice fasted for overnight (12 h) were given an intraperitoneal injection of glucose (2 g kg−1 body weight). Blood samples were harvested from the tail vein before glucose challenge, as well as 30, 60, 90 and 120 min thereafter. For the insulin tolerance test (ITT), mice fasted for 6 h were given an intraperitoneal injection of human insulin (1 U kg−1 body weight). Blood samples were harvested from the tail vein before glucose challenge, as well as 15, 30, 45 and 60 min thereafter. Serum glucose levels were measured using Accu-Chek III glucose analyser. After an 8 week study period, mice of all groups were killed under anaesthesia after fasting overnight (12 h).
RNA isolation and quantitative real-time PCR
Each complementary DNA (cDNA) sample was analysed for gene expression by quantitative real-time PCR using the fluorescent TaqMan 5′-nuclease assay on an Applied Biosystems 7000 sequence detection system (Foster City, CA, USA). The TaqMan real-time PCR was performed using 2× TaqMan Master Mix and the 20× premade TaqMan gene expression assays (Applied Biosystems). The following PCR conditions were used: 95°C for 10 min, 95°C for 15 s and 60°C for 1 min for 45 cycles. The levels of expression of mouse carnitine palmitoyltransferase 1 (Cpt1) (Mm00463960_m1; Applied Biosystems), acyl-CoA oxidase (Aco) (Mm00801417_m1) and fatty acid binding protein 3 (Fabp3; Mm02342495_m1) messenger RNAs (mRNAs) were normalised to mouse beta-actin (Mn00607939_sl; Applied Biosystems).
C2C12 cells were harvested and proteins were extracted with lysis buffer (PRO-PREP™; Intron Biotechnology, Seoul, Korea) for 60 min at 4°C. Protein samples (30 μg) were subjected to 10% SDS-PAGE, transferred to a nitrocellulose membrane (Amersham Bioscience, Westborough, MA, USA) and probed with primary antibody followed by secondary antibody conjugated with horseradish peroxidase (Amersham Bioscience). The samples were detected with chemiluminescence kits (Amersham Bioscience).
Transfections using siRNAs
0–20 nmol/l small interfering (si)RNA oligonucleotides for PPARδ (SC-36305; Santa Cruz Biotechnology) were used to suppress gene expression. Transfection was performed with Lipofectamine 2000 (Invitrogen), in accordance with the manufacturer’s directions.
Measurement of NFκB nuclear translocation
Cells were fractionated using a Nuclear/Cytosol Fractionation Kit, as described in the manufacturer’s instructions (Biovision, Milpitas, CA, USA).
Glucose uptake, acetyl-CoA and ATP measurement
Glucose uptake levels, intracellular acetyl-CoA and ATP in cells or tissues were measured using assay kits, as described in the manufacturer’s directions (Abcam, Cambridge, MA, USA).
All analyses were performed using the SPSS/PC statistical program (version 12.0 for Windows; SPSS, Chicago, IL, USA). Results are presented as the fold difference relative to control values (mean ± SEM). All of the in vitro experiments were conducted a minimum of three times. Student’s t test or two-way ANOVA were used in the statistical analysis.
BAIBA treatment prevents hyperlipidaemia-induced insulin resistance in C2C12 myocytes and skeletal muscle of HFD-fed mice
BAIBA ameliorates HFD-induced insulin resistance in mice
BAIBA alleviates palmitate-induced inflammation in C2C12 cells and soleus skeletal muscle of HFD-fed mice
BAIBA prevents palmitate-induced inflammation, which leads to improvements in insulin resistance through activation of AMPK
The PPARδ-mediated pathway is involved in the inhibitory effects of BAIBA on palmitate-induced inflammation and insulin resistance
BAIBA induces fatty acid oxidation through AMPK-mediated pathways in C2C12 myocytes and in skeletal muscle of HFD-fed mice
Exercise is known to be an effective intervention for the treatment of insulin resistance and type 2 diabetes . During exercise, secretion of BAIBA from skeletal muscle to the blood is enhanced through activation of PGC1α . BAIBA can convert white adipose tissue to brown adipose tissue, which plays beneficial roles in the regulation of glucose metabolism and insulin sensitivity [3, 23]. Moreover, plasma BAIBA levels are inversely associated with metabolic risk variables and are increased with exercise . Therefore, Roberts et al suggested that BAIBA may contribute to exercise-induced protection from metabolic disease . However, the mechanisms by which BAIBA may improve insulin resistance in skeletal muscle remain unknown.
AMPK is the master regulator of energy homeostasis and has been reported to inhibit inflammation through inhibition of NFκB signalling . AMPK plays a pivotal role in metabolic diseases such as obesity and type 2 diabetes. Obesity-induced increases in serum NEFA concentration can suppress AMPK activity  and impair insulin signalling . High blood glucose levels in patients with type 2 diabetes also induce oxidative stress and activate NFκB. Conversely, AMPK activation can suppress reactive oxygen species production  and can augment the expression of thioredoxin, an inhibitor of the inflammatory response [28, 29]. Increased AMPK activity also attenuates endoplasmic reticulum stress, which results in inhibition of inflammation [30, 31]. AMPK is activated in the muscle of patients with type 2 diabetes during exercise . The present study showed that BAIBA significantly induces AMPK phosphorylation. Furthermore, suppression of AMPK abrogated the suppressive effects of BAIBA on inflammation and insulin resistance. These results reveal one possible mechanism through which the beneficial effects of exercise may be mediated through BAIBA.
PPARδ has emerged as an important metabolic regulator that may mediate the favourable effects of exercise in diverse tissues, such as fat, skeletal muscle, liver and heart. PPARδ receptor activation by a ligand mitigates the macrophage-mediated inflammatory response and regulates lipoprotein metabolism to decrease triacylglycerol and increase HDL-cholesterol levels. Furthermore, PPARδ activation in the liver suppresses hepatic glucose output, leading to attenuation of hyperglycaemia and amelioration of lipopolysaccharide-induced inflammation through inhibition of NFκB activity and TNF-α expression in cardiomyocytes . Fatty acid oxidation and energy dissipation in skeletal muscle and adipose tissue by PPARδ activation exerts many beneficial effects, including reducing body weight, increasing skeletal muscle metabolic rate and exercise endurance, improving insulin sensitivity and lipid profiles and suppressing atherogenic inflammation . Therefore, PPARδ is an exciting new target for the treatment of the metabolic syndrome . The present study is the first to show that BAIBA treatment can increase the expression of PPARδ, resulting in alleviation of inflammation through suppression of the NFκB pathway and reduction of proinflammatory cytokines.
By administration of BAIBA to palmitate-treated myocytes, we demonstrated that BAIBA-induced activation of AMPK and PPARδ was responsible for a reduction in inflammation. Previous studies have shown that NEFAs can induce inflammation via NFκB activation and upregulation of additional mediators, such as TNF-α and IL-6 [34, 35]. Consistent with previous reports, the present study confirmed that palmitate increased IκBα phosphorylation, NFκB nuclear translocation and IL-6 expression, which together result in impaired insulin-stimulated IRS-1 and Akt phosphorylation in C2C12 myocytes. Furthermore, suppression of both AMPK and PPARδ significantly abrogated the suppressive effects of BAIBA on palmitate-induced inflammation, suggesting that induction of AMPK and PPARδ by BAIBA contributes to the attenuation of palmitate-induced inflammation. In agreement with the changes in inflammation markers, BAIBA attenuated palmitate-induced elevation of insulin-stimulated IRS-1 and Akt phosphorylation. In vivo experiments using HFD-fed mice also yielded similar results in that BAIBA inhibited the NFκB pathway and the downstream proinflammatory cytokines, such as TNF-α and MCP-1. Overall, these findings suggest that BAIBA alleviates insulin resistance induced by palmitate or an HFD through attenuation of inflammation via an AMPK–PPARδ-dependent pathway in skeletal muscle. Furthermore, BAIBA improved glucose tolerance and insulin tolerance in mice. In this study, elevated basal glucose levels were detected in experimental mice by performing IPGTTs and ITTs after hyperglycaemia developed as a result of an HFD. Owing to a difference in basal plasma glucose levels, a graph of percentage of basal glycaemia was used to estimate insulin sensitivity in the present study. Furthermore, our study showed that HFD-induced basal plasma glucose levels were suppressed by BAIBA administration. This suppressive effect of BAIBA on basal serum glucose levels may be associated with hepatic gluconeogenesis regulated by PPARδ  or other pathways.
Interestingly, BAIBA administration decreased the body weight of experimental mice, although it did not affect energy intake. These results suggest that loss of body weight is not due to a change in energy intake, but internal mechanisms including fat burning by fatty acid oxidation. Begriche et al indicated no change in body weight but decrease in fat mass by BAIBA treatment in ob/ob mice . This discrepancy may be due to differences in study protocol and experimental animal models.
In agreement with the findings reported by Roberts et al demonstrating that BAIBA induces hepatic fatty acid oxidation through a PPARα-dependent pathway , we also confirmed the role of BAIBA-induced β-oxidation in mouse skeletal muscle cells. Interestingly, the expression levels of PPARδ and β-oxidation-associated genes were increased in the skeletal muscle of HFD-fed mice in the present study. Although the reason for this result is not clear, differences in the stimuli might be involved. This increase in PPARδ expression might be mediated by other HFD-induced molecules. Furthermore, the induction of PPARδ by the HFD may play a role in the compensatory effects needed to maintain lipid homeostasis. The mechanism of PPARδ regulation by an HFD will need to be elucidated in future studies. However, in agreement with our current in vitro experiments, BAIBA administration actively upregulated AMPK phosphorylation and PPARδ expression levels in skeletal muscle of HFD-fed mice.
In summary, the present study is the first to demonstrate that BAIBA attenuates insulin resistance and inflammation induced by palmitate or an HFD through a pathway mediated by AMPK–PPARδ in skeletal muscle. These results may provide insight into the novel mechanisms contributing to the beneficial effects of exercise and may suggest a therapeutic approach to insulin resistance and type 2 diabetes.
This study was supported by a grant from the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C0133).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
TWJ, HJH, HCH, HJY, SHB and KMC provided a substantial contribution to the conception and design of the article, and drafted or revised the article. TWJ and KMC were involved in the acquisition of data and the analysis and interpretation of data. All authors approved the final version of the manuscript. TWJ and KMC are responsible for the integrity of the work as a whole.