Obesity-induced elevated palmitic acid promotes inflammation and glucose metabolism disorders through GPRs/NF-κB/KLF7 pathway

Reagents and materials

In total, 40 mM PA solution: PA (Sigma-Aldrich, St. Louis, USA, 0.0614 g) was added to 3 ml NaOH solution (0.1 mol/l), placed in a 75 °C full saponification water bath for 30 min until the PA particles are completely dissolved and the liquid is colorless and transparent. Then the liquid was added to 3 ml BSA (40%, free of fatty acid) solution immediately with sufficient mixing. In total, 100 mM AH7614 solution: AH7614 (TOCRIS, England, 10 mg) was dissolved in 285 μl DMSO. In total, 100 mM GW1100 solution: 5 mg GW1100 (MCE, America, 5 mg) was dissolved in 960 μl DMSO. In total, 10 mM Bay 11-7082 solution: Bay 11-7082 (MCE, America, 5 mg) was dissolved in 2.41 ml DMSO.

Animal experiment

Animal experiments were approved by the Medical Ethics Committee of the First Affiliated Hospital of Shihezi University School of Medicine (Code: A2019-086-01). Four-weeks-old C57BL/6 male mice were purchased from Slack Jingda Laboratory Animal Company (Hunan) and raised in the animal room of Shihezi University School of Medicine, 3–5 mice/cage were reared. The food and water were changed once a day, the weight and body length were measured weekly. After adaptive feeding for 1 week, mice were divided into normal control diet feeding group (NCD, 10% calories from fat) and high-fat diet feeding group (HFD, 60% calories from fat) randomly. After feeding for 7 weeks, mice in HFD were grouped into four groups: HFD group (DMSO), HFD and intraperitoneal injection of GW1100 (HFD + GW1100 5 mg/kg/day), HFD and intraperitoneal injection of AH7614 (HFD + AH7614, 5 mg/kg/day), HFD and intraperitoneal injection of Bay 11-7082 (HFD + Bay 11-7082, 2.5 mg/kg/day) randomly, the drug injection lasted for 5 weeks.

Glucose tolerance test and insulin tolerance test

Glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at the 12th week. GTT: the feed was removed and water was provided for 12 h, 50% glucose solution (4 μl/g) was injected intraperitoneally, the blood glucose was measured at 0, 0.5, 1, 1.5 and 2 h, respectively. ITT: the feed was removed and the mice were continuously supplied with water for 6 h. The mice were intraperitoneally injected with different doses of insulin (0.5 IU/g), the blood glucose was measured at 0 h, 0.5 h, 1 h, 1.5 h and 2 h, respectively. After GTT and ITT tests, the samples of adipose tissue, liver, and serum were collected for analysis.

Cell culture

The pre-adipocyte 3T3-L1 was cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicilinstreptomycin at 37 °C for 48 h under full of atmosphere of 5% carbon dioxide and 95% air. The source of the cell line was identified by STR profiling and tested for mycoplasma contamination. Differentiation was initiated after 2 days with cell growth coverage rate reaching 100%. First, changing culture medium into induce medium I with 0.5 mmol IBMX, 1 μM dexamethasone, and 1 μg/ml insulin for 2 days, then induce medium I was replaced by induce medium II without IBMX and dexamethasone every 48 h for 6–8 days until mature adipocytes was reached about 90%. HepG2 and HEK293T cells were cultured in DMEM with 10% FBS and 1% penicilinstreptomycin at 37 °C under full of atmosphere of 5% carbon dioxide and 95% air.

Cell treatment

After induction of adipocytes and culturation of HepG2 cells sucessfully in the six-well plate, adding 2 μl DMSO as blank control group, PA group was added with 2 μl DMSO and 10 μl PA storage solution (40 mM) until the final concentration was 200 μM. GW1100 and AH7614 groups were blocked by adding 1, 2, 3 μl GW1100 or AH7614 storage solution to the final concentration of 50, 100, 150 μM, respectively. In total, 1 μl Bay 11-7082 were added to the culture medium until the final concentration was 10 μM to block p-p65.

Quantitative reverse transcription PCR

Total RNA was isolated from HepG2 cells and 3T3-L1 adipocytes using TRIZOL reagent (Life Technologies, 15596-026, USA). Reverse transcription using Thermo Scientific Revert Aid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622, USA), conditions for cDNA amplification were as follows: 25 °C for 5 min, 42 °C for 60 min, and 70 °C for 15 min.

Real-time PCR was used to measure gene expression using SYBR® Select Master Mix (Applied Biosystems, 4472908, USA), PCR reactions were conducted in 10 μl volumes the detailed PCR reaction were as follow: 94 °C for 5 min, the denaturation temperature was 94 °C for 30 s, the annealing temperature was 53 °C for 30 s, and the extension temperature was 72 °C for 30 s, for a total of 40 cycles. Relative expression levels of related genes were determined by qRT-PCR normalized to GAPDH or β-actin. Primer sequences are available in Table 1.

Table 1 Primes sequences used in this study.Western blot

The separated proteins were electroblotted onto nitrocellulose filter membrane and blocked for 2 h at room temperature with Tris-buffered saline containing 5% BSA. Nitrocellulose filter membrane were incubated at 4 °C overnight with antibodies, rabbit anti-KLF7 (Abcam; ab197690), rabbit anti-IL-6 (Abcam, ab214429), rabbit anti-GPR120 (Abcam; ab230869), rabbit anti-GPR40 (Thermo Fisher; PA5-75351), rabbit anti-p-IKKβ (Cell Signaling Technology; 2697S), rabbit anti-T-IKKβ (Abcam; ab124957), rabbit anti-p-IκB (Cell Signaling Technology; 2859S), rabbit anti-T-IκB (Cell Signaling Technology; 4812S), rabbit anti-p-p65 (Cell Signaling Technology; 3033S), rabbit anti-T-p65 (Cell Signaling Technology; 8242S), rat anti-GAPDH (ZSGB-BIO; TA-08), rabbit anti-TBP (Cell Signaling Technology; 44059S), rat anti-β-Actin (ZSGB-BIO; TA-10) were used at a dilution of 1:1000. The secondary antibodies (ZSGB-BIO; ZB2301 and ZB2305) were incubated at 25 °C for 2 h at a working ratio of 1:10,000.

Luciferase reporter gene experiment

Using Eukaryotic Promotor Database software to query the gene sequence of KLF7 promoter region. Jaspar Database predicts the binding site of p65 and KLF7 promoter region. Human KLF7 promoter region core segment luciferase plasmid (2001 bp) and truncated luciferase reporter gene plasmids (1501, 1001, 501 bp) (GenePharma), each luciferase reporter gene plasmid and p65 overexpression plasmid as well as the renilla fluorescent plasmid was co-transfected into HepG2 cells. Testing equipment: full-wavelength scanning multi-functional microplate reader (BioTeK); testing kit: (Promega Dual-Glo® Luciferase Assay System E2920) for sample addition testing in the following order: Add Dual-Glo® Luciferase Assay Reagent to the plate, Incubate at 20–25 °C for 10 min–2 h, Measure firefly luminescence. Add Dual-Glo® Stop & Glo®Reagent to the plate, Incubate at 20–25 °C for 10 min, Measure Renilla luminescence. Calculate ratio of firefly: Renilla luminescence for each well, Normalize the sample well ratio to the ratio from a control (or series of control) wells.

Chromatin immunoprecipitation assay

In total, 2–4 × 106 cells were crosslinked with 1% formaldehyde for 10 min at room temperature, and then DNA was fragmented into 200–500 bp fragments by Ultrasonic instruments (Sonics & Materials Inc, VCX150, USA). Antibodies specific for p65 (CST, 8242S) or unspecific IgG (CST;3900S) were used for ChIP assay. The DNA was then purified and analyzed by qRT-PCR to detect specific DNA sequences of KLF7 promoter. The sequences of primers are shown in Supplementary Table 1.

Glucose metabolism assay

HepG2 cells and 3T3-L1 adipocytes were grown in 6-well plates and treated with PA (200 μM) or PA and blocker (100 μM), culture medium was collected for measurement of glucose concentration using the glucose oxidase method (F006-1-1, Nanjing Jiancheng Bioengineering Institute, China).

Biochemical indicator test

Triglycerides (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by TC assay kit (A110-1-1, Nanjing Jiancheng Bioengineering Institute, China), TC assay kit (A111-1-1, Nanjing Jiancheng Bioengineering Institute, China), HDL assay kit (A112-1-1, Nanjing Jiancheng Bioengineering Institute, China), and LDL assay kit (A113-1-1, Nanjing Jiancheng Bioengineering Institute, China). PA, IL-6, MCP-1, TNF-α in plasma were detected by mice PA ELISA Kit (A185255, Shanghai Fusheng Bioengineering Institute, China), mice serum IL-6 ELISA Kit (F10830, Shanghai XiTang Bioengineering Institute, China), mice serum MCP-1 ELISA Kit (F11130, Shanghai XiTang Bioengineering Institute, China), mice serum TNF-α ELISA Kit (F11630, Shanghai XiTang Bioengineering Institute, China).

Statistical analysis

The statistical software SPSS 18.0 was used for data analysis. For the data conform to the normal distribution and similar variance, Student’s t test were used when two groups of data were compared. If the comparison between three groups of data, one-way ANOVA-LSD analysis was conducted. p < 0.05, the difference is statistically significant. Values are expressed as mean ± SEM (Prism 7; GraphPad Software). In this research, there is no power analysis was performed to determine the sample size. The sample size was based on the previous studies employing in mice. No animals were excluded from the analyses and the blind rule was not used in this study.

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