Hepatocyte medium was purchased from ScienCell (Carlsbad, CA, USA). Dulbecco's modified Eagle's medium (DMEM), Hank's balanced salt solution (HBSS), PBS and collagenase type IV (200 units/mg) were from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). Sodium pyruvate, sodium L-lactate, dexamethasone, bovine serum albumin (BSA), 8-bromo-cyclic adenosine monophosphate (8-bromo-cAMP), acetate, propionate, sodium butyrate, Trichostatin A (TSA), CI994, Entinostat (MS-275), PCI-34051 and tubacin were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). All primers used for reverse-transcription quantitative polymerase chain reaction (RT-qPCR) were synthesized by Shanghai Biological Engineering Technology & Services Co., Ltd. (Shanghai, China). Anti-cAMP response element-binding protein (CREB; cat no. 9197S) and anti-phosphorylated (p)-CREB (Ser133; cat no. 9198) as well as anti-mouse immunoglobulin (Ig)G (cat no. 14709) and anti-rabbit IgG conjugated with horseradish peroxidase (cat no. 7074) were from Cell Signaling Technology Inc. (Beverly, MA, USA). The antibody to GAPDH (cat no. sc25778) was purchased from Santa Cruz Biotechnology, Inc. (Danvers, MA, USA).

A total of 20 Male C57BL/6 mice (age, 6–8 weeks; weight, 16–18 g) were purchased from Shanghai Slack Experimental Center (Shanghai, China). Primary hepatocytes were isolated from C57BL/6 mice by a modified version of the collagenase method. In a biosafety cabinet, mouse livers were perfused with 10 ml calcium-free HBSS through the portal vein under anesthesia with 10% chloral hydrate (Sigma-Aldrich; Merck, KGaA). The liver was excised by careful dissection and transferred to a 10-cm tissue culture dish. Mice were then sacrificed via cervical dislocation. The isolated liver was perfused with 0.05% collagenase IV (Gibco; Thermo Fisher Scientific, Inc.) dissolved in 20 ml calcium-containing HBSS in a recirculating manner for 15 min. Hepatocytes extracted from digested livers were filtered through a 100-µm cell strainer, washed 3 times with PBS and re-suspended in hepatocyte medium containing 100 U/ml penicillin, 100 µg/ml streptomycin, 0.1% BSA and hepatocyte growth factor (all from ScienCell Research Laboratories, Inc., San Diego, CA, USA). The cells were seeded on 6-well or 24-well plates and incubated in a tissue culture incubator at 37°C (95% air and 5% CO₂). After 24 h, the cells had already attached to the plates and the medium was replaced with DMEM containing 5.5 mM glucose and 0.25% BSA, followed by drug treatment. All mice used in the present study for isolation of hepatocytes were fed a normal diet under a regular schedule and were not fasted. All of the experimental procedures involving the use of animals were approved by the Animal Use and Care Committee of Shanghai Jiaotong University School of Medicine (Shanghai, China).

Mouse primary hepatocytes were cultured in DMEM supplemented with 0.25% BSA overnight at 37°C with 5% CO₂. To evaluate the effect of butyrate on hepatic gluconeogenesis, mouse primary hepatocytes were incubated with sodium butyrate for 8 h at various concentrations (0, 0.1, 1, 5, or 10 mmol/l). To investigate the effect of HDAC inhibitors on hepatic gluconeogenesis, cells were incubated in glucose-free DMEM containing gluconeogenic substrates (10 mmol/l sodium lactate and 1 mmol/l sodium pyruvate), 8-bromo-cAMP (100 µmol/l) and with the following HDAC inhibitors: TSA (100 nmol/l), CI994 (10 µmol/l), MS-275 (10 µmol/l), PCI-34051 (10 µmol/l), Tubacin (10 µmol/l) or sodium butyrate (5 mmol/l). Following 8 h incubation at 37°C, cell culture supernatants were collected for measuring the glucose content. Mouse primary hepatocytes were then treated with sodium butyrate (5 mmol/l) and TSA (100 nmol/l) in the presence of 8-bromo-cAMP (100 µmol/l) for 8 h at 37°C, the mRNA expression of gluconeogenic genes were detected using RT-qPCR. To compare the effects of three SCFAs on hepatic glucose production, mouse hepatocytes were treated with acetate (5 mmol/l), propionate (5 mmol/l) or sodium butyrate (5 mmol/l) for 8 h at 37°C in the presence of gluconeogenic substrates (10 mmol/l sodium lactate and 1 mmol/l sodium pyruvate). To test whether SCFAs affect hepatic gluconeogenesis as substrates, mouse hepatocytes were incubated with one of three different SCFAs (acetate, propionate, or sodium butyrate; 5 mmol/l each) and 100 µM 8-bromo-cAMP in the absence of gluconeogenic substrates (10 mmol/l sodium lactate and 1 mmol/l sodium pyruvate) for 8 h at 37°C. To assess the phosphorylation levels of CREB, mouse hepatocytes were treated with butyrate (5 mmol/l) or 8-bromo-cAMP (100 µmol/l) for 1 h at 37°C.

Glucose production was assayed as described previously (16). In brief, hepatocytes were seeded into 24-well plates at 2.5×10⁵ cells/well. After 24 h, these hepatocytes were pre-stimulated with DMEM containing 5.5 mM glucose, 0.25% BSA and 100 nM dexamethasone for 16 h. Cells were then washed three times with PBS and incubated in glucose production buffer (DMEM without glucose, serum or phenol red, and supplemented with 1 mM sodium pyruvate and 10 mM sodium lactate). After 8 h, the cell culture supernatants were collected for measuring the glucose content using a glucose oxidase kit (Applygen Co., Beijing, China).

Total RNA was extracted from mouse primary hepatocytes using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions. Complementary DNA was synthesized by RT using Random Primers (Promega Corp., Madison, WI, USA) according to the manufacturer's protocol. RT-qPCR was performed in a Roche LightCycler 480 system (Roche Diagnostics, Basel, Switzerland) using SYBR Premix EX Taq (Takara, Tokyo, Japan). The PCR conditions were as follows: Denaturation at 95°C for 10 sec, followed by 40 cycles of 95°C for 5 sec. The annealing temperature was then reduced to 60°C for 31 sec with a final elongation step at 72°C for 300 sec. At the end of the amplification, a melting curve was generated in the temperature range of 60–95°C. The sequences of primers used are listed in Table I. Relative gene expression levels were quantified based on the cycle threshold (Cq) values and normalized to the reference gene β-actin. The relative gene expression was calculated using the 2^−ΔΔCq method (17).

Primary hepatocytes were treated with radioimmunoprecipitation lysis buffer containing protease and phosphatase inhibitors (Merck KGaA) and centrifuged at a speed of 6,000 × g for 10 min at 4°C. The supernatant was then used to analyze the expression levels of specific proteins. Protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific, Inc.). A total of 10 µg protein was loaded per lane and separated in 15% SDS-PAGE. Samples were then transferred onto polyvinylidene fluoride membranes (ECL Advance; Cell Signaling Technology, Inc). Prior to western blot analysis, membranes were blocked with 5% skimmed milk for 2 h at 37°C and subsequently incubated at 4°C overnight with rabbit anti-mouse primary antibodies sourced from Cell Signaling Technology Inc. These included cAMP response element-binding protein (CREB; cat. no. 9197S; 1:1,000) and phosphorylated (p)-CREB (Ser133; cat. no. 9198; 1:1,000). GAPDH antibodies (cat. no. sc25778; 1:1,000) were purchased from Santa Cruz Biotechnology, Inc. Membranes were then incubated with mouse anti-rabbit horseradish peroxidase-labeled IgG secondary antibodies (1:2,000; cat. no. 7074; Cell Signaling Technology Inc.,) overnight at 4°C. The results were visualized using an immobilon ECL ultra western HRP substrate (cat. no. WBULS0100; Merck KGaA) and images were captured using an LAS-4000 Super CCD Remote Control Science Imaging System (Fuji, Tokyo, Japan).

The amount of ATP was measured by the luciferin-luciferase method according to the protocol of the ATP detection kit (cat. no. S0026; Beyotime Institute of Biotechnology, Inc., Haimen, China). Primary hepatocytes were seeded onto 24-well plates at 2.5×10⁵ cells/well. After 24 h, these cells received the same dexamethasone pre-stimulation as described in the glucose production assay. The cells were then treated with propionate (5 mmol/l) and different concentrations of sodium butyrate (0, 0.1, 1, 5 and 10 mmol/l) for 1 h prior to lysis with lysis buffer (200 µl/well) from the ATP detection kit. After centrifugation at 12,000 × g for 5 min at 4°C, the supernatant was transferred to a fresh tube for the ATP test. The luminescence of a 20-µl sample was assayed in a luminometer (Perkin Elmer, Inc., Waltham, MA, USA) together with 100 µl ATP detection buffer from the ATP detection kit. The protein concentration was determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc.). The concentration of ATP was normalized to that of protein in the same cell lysate.

All values are expressed as the mean ± standard error of the mean from at least three independent experiments. Two group comparisons were performed using a Student's t-test. All statistical analyses were performed using SPSS 19.0 (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

To determine the effects of butyrate on hepatic gluconeogenesis, mouse primary hepatocytes were incubated with various concentrations of sodium butyrate for 8 h. Unexpectedly, sodium butyrate stimulated hepatic glucose production in a dose-dependent manner, causing a significant increase at the concentration of 1 mM (P<0.01; Fig. 1A). Under the same conditions, the expression of genes associated with hepatic gluconeogenesis, including phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (G6Pase), FoxO1, PGC-1α and hepatocyte nuclear factor 4α (HNF4α) was also increased. Consistent with the results on gluconeogenesis, sodium butyrate dose-dependently increased the mRNA expression of these gluconeogenic genes, with the maximum efficacy at the concentration of 10 mM (Fig. 1B-F).

To investigate whether sodium butyrate-stimulated gluconeogenesis is involved in the inhibition of HDACs, the effects of other HDAC inhibitors, including TSA, CI994, MS-275, PCI-34051 and tubacin, on hepatic glucose production in mouse primary hepatocytes were detected. After mouse hepatocytes were incubated with 100 µM 8-bromo-cAMP for 8 h, glucose production was markedly increased. In the presence of 100 nM TSA, 10 µM CI994 or 10 µM PCI-34051, 8-bromo-cAMP-stimulated gluconeogenesis was significantly decreased. However, sodium butyrate treatment further enhanced glucose production induced by 8-bromo-cAMP. The other two HDAC inhibitors had no significant effect (Fig. 2A). TSA is a potent inhibitor of class I and II HDAC (18). The present study further compared the effects of TSA and sodium butyrate on the expression of the five gluconeogenic genes. As expected, the expression of all of these gluconeogenic genes was strongly induced by 8-bromo-cAMP. Contrary to the action of sodium butyrate, TSA suppressed cAMP-stimulated gluconeogenic gene expression (Fig. 2B-F). It is therefore unlikely that sodium butyrate promotes gluconeogenesis via inhibiting HDAC activity.

To investigate whether sodium butyrate stimulates gluconeogenesis as an energy substrate, the present study compared the effects of three SCFAs on hepatic glucose production. Mouse hepatocytes were treated with 5 mM acetate, propionate or sodium butyrate for 8 h in the presence of gluconeogenic substrates (sodium lactate and sodium pyruvate). Similar to sodium butyrate, propionate also promoted hepatic glucose production (Fig. 3A). Sodium butyrate supplementation led to increases in the expression of all of the selected gluconeogenic genes. Propionate significantly triggered the mRNA expression of PEPCK, G6Pase, FoxO1 and PGC-1α, but not that of HNF4α (Fig. 3B-F). Although acetate had no significant effects on glucose production, it significantly increased the expression of FoxO1 and PGC-1α (Fig. 3A, D and E).

To test whether sodium butyrate induces glucose production as a type of gluconeogenic substrate, mouse primary hepatocytes were incubated with one of three SCFAs in the absence of gluconeogenic substrates. Acetate, propionate and sodium butyrate all increased hepatic gluconeogenesis. Propionate had the most noticeable effect. In the presence of three SCFAs, addition of 8-bromo-cAMP further enhanced the glucose production (Fig. 4A). In agreement with the results on gluconeogenesis, 8-bromo-cAMP significantly enhanced butyrate and propionate-stimulated expression of the five gluconeogenic genes (Fig. 4B-F). Acetate supplementation only led to mild increases in PEPCK and PGC-1α expression (Fig. 4B and E). It is well accepted that propionate is a substrate for hepatic glucose production (19). Therefore, it is reasonable to presume that butyrate also stimulates hepatic gluconeogenesis as a substrate.

It is well-known that cAMP/CREB is an important signaling pathway for hepatic gluconeogenesis (20,21). The present study investigated whether the cAMP/CREB signaling pathway is involved in butyrate-mediated induction of gluconeogenesis. As ATP is the substrate for cAMP production, the present study first detected the intracellular ATP concentration after mouse primary hepatocytes were incubated with various concentrations of sodium butyrate for 1 h. In parallel with glucose production, sodium butyrate treatment resulted in the accumulation of intracellular ATP in a dose-dependent manner, exhibiting a significant effect at the concentration of 1 mM (P<0.05; Fig. 5A). However, gluconeogenic substrates (sodium lactate and sodium pyruvate) as well as propionate did not alter the intracellular ATP concentration (Fig. 5B). Next, western blot analysis was performed to evaluate the phosphorylation state of CREB in butyrate-treated hepatocytes. Similar to 8-bromo-cAMP, sodium butyrate also stimulated the phosphorylation of CREB (Fig. 5C). Overall, these results indicated that butyrate activates cAMP/CREB signaling and stimulates the expression of hepatic gluconeogenic genes, at least in part via increasing the accumulation of intracellular ATP.