Sodium Butyrate Improves Liver Glycogen Metabolism in Type 2

Article Cite This: J. Agric. Food Chem. 2019, 67, 7694−7705

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Sodium Butyrate Improves Liver Glycogen Metabolism in Type 2 Diabetes Mellitus Wen-Qian Zhang,† Ting-Ting Zhao,† Ding-Kun Gui,‡ Chen-Lin Gao,†,§ Jun-Ling Gu,† Wen-Jun Gan,† Wei Huang,§ Yong Xu,§ Hua Zhou,†,∥ Wei-Ni Chen,⊥ Zhi-Long Liu,*,⊥ and You-Hua Xu*,†

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Faculty of Chinese Medicine, State Key Laboratory of Quality Research in Chinese Medicine, Macau University of Science and Technology, Avenida Wai Long, Taipa, Macao 999078, People’s Republic of China ‡ Department of Nephrology, Shanghai Jiaotong University Affiliated Sixth People’s Hospital, Shanghai 200080, People’s Republic of China § Department of Endocrinology, Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan 646000, People’s Republic of China ∥ Macau Institute for Applied Research in Medicine and Health, Avenida Wai Long, Taipa, Macao 999078, People’s Republic of China ⊥ Department of Endocrinology, Zhuhai Hospital of Integrated Traditional Chinese and Western Medicine, Zhuhai 519000, People’s Republic of China ABSTRACT: Liver plays a central role in modulating blood glucose level. Our most recent findings suggested that supplementation with microbiota metabolite sodium butyrate (NaB) could ameliorate progression of type 2 diabetes mellitus (T2DM) and decrease blood HbA1c in db/db mice. To further investigate the role of butyrate in homeostasis of blood glucose and glycogen metabolism, we carried out the present study. In db/db mice, we found significant hypertrophy and steatosis in hepatic lobules accompanied by reduced glycogen storage, and expression of GPR43 was significantly decreased by 59.38 ± 3.33%; NaB administration significantly increased NaB receptor G-protein coupled receptor 43 (GPR43) level and increased glycogen storage in both mice and HepG2 cells. Glucose transporter 2 (GLUT2) and sodium-glucose cotransporter 1 (SGLT1) on cell membrane were upregulated by NaB. The activation of intracellular signaling Protein kinase B (PKB), also known as AKT, was inhibited while glycogen synthase kinase 3 (GSK3) was activated by NaB in both in vivo and in vitro studies. The present study demonstrated that microbiota metabolite NaB possessed beneficial effects on preserving blood glucose homeostasis by promoting glycogen metabolism in liver cells, and the GPR43-AKT-GSK3 signaling pathway should contribute to this effect. KEYWORDS: type 2 diabetes mellitus, glycogen, liver, microbiota, sodium butyrate

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T2DM, Qin and colleagues10 found butyrate-producing bacteria within gut lumen are significantly decreased in this population. Previous studies suggest protective effects of butyrate on liver, for example, antiapoptosis,11 anti-inflammation,12 and antioxidant.13 Under physiological conditions, the concentration of butyric acid in the liver is not high, but the supply of butyric acid by feeding can still affect the pathological changes and inflammation of fatty liver caused by high-fat diet feeding.14 Khan and Jena found that sodium butyrate (NaB) administration ameliorated liver vesicular steatosis and inhibited fat deposition in STZ-induced diabetic rats.15 Recently, there is a report that demonstrated that butyrate can modulate mitochondrial function within hepatocytes in T2DM animal model.4 Previously, we found NaB supplementation could ameliorate progression of T2DM and decrease blood HbA1c level in db/db mice.16

INTRODUCTION Insulin resistance (IR) and hyperglycemia are the most two important characteristics of type 2 diabetes mellitus (T2DM). Liver plays a pivotal role in controlling blood glucose level in that it absorbs glucose into hepatocytes and transfers it into glycogen to decrease blood glucose, and degrades the stored glycogen into glucose to elevate blood glucose if needed.1,2 Unfortunately, the function of liver is severely damaged under T2DM settings.3,4 Hence, preserving liver function is very important in ameliorating both the occurrence and the development of T2DM. Gut microbiota is an indispensable organ for preserving physiological functions of the organism,5 and its imbalance will contribute to a series of diseases, including T2DM.6 Besides inhibiting colonization and proliferation of pathogens within gut lumen, there also exist cometabolism phenomena between gut microbiota and the organism.7 It was found that microbiota derived metabolites can be absorbed into blood circulation.8 Short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate, are the main microbiota metabolic products. Although butyrate only accounts for 15% of the total SCFAs,9 its function is very important. By identifying markers for © 2019 American Chemical Society

Received: Revised: Accepted: Published: 7694

April 3, 2019 June 20, 2019 June 22, 2019 June 22, 2019 DOI: 10.1021/acs.jafc.9b02083 J. Agric. Food Chem. 2019, 67, 7694−7705

Article

Journal of Agricultural and Food Chemistry

Figure 1. Sodium butyrate (NaB) ameliorated liver function in db/db mice. (A) Gross observation of liver; (B) H&E and (C) PAS staining of liver (magnification: 100 and 400); and (D) WB detection for GPR43 expression. n = 6. Met: metformin. **p < 0.01.

Evidence from both reports and our previous finding suggest potential effects of NaB on stabilizing blood glucose level and liver function. Yet the underlying mechanism is still not fully understood. To further explore the role and mechanism of NaB on T2DM, specifically, on liver function in stabilizing blood glucose level, we designed the present study. Both in vivo and in vitro experiments were carried out. Our present findings will supply a new idea and strategy toward the treatment of T2DM.

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carbohydrate, and 20% protein). All mice were housed in an animal house with a 12 h daylight cycle at 25 °C. Before drug intervention, all mice were fed adaptively for 7 days. Diabetic animals (db/db mice) were randomly divided into three groups as follows: (1) model group (n = 6); (2) intervention group in which animals were orally treated with NaB (0.5 g/kg/d, n = 6);16−18 and (3) positive control group in which animals were orally administrated with metformin (0.15 g/kg/ day, n = 6). Six C57BL/6 mice were set as normal control. Each day, all animals were administrated with drugs as mentioned above or 0.45 mL of normal saline (for normal and model animals) by gavage for 5 continuous weeks. At the end of the experiment, liver was collected for histological or biochemical study. Hematoxylin-Eosin (H&E) and Periodic Acid-Schiff (PAS) Staining. The liver sample was fixed within 4% paraformaldehyde over 24 h; after being paraffinized, the sections (0.4 μm) were stained with H&E or PAS staining solution according to the standard procedure. Histopathological images were observed under microscope (Olympus, Japan). Cell. Hepatocyte cell line HepG2 cells were purchased from American Type Culture Collection (ATCC) (Manassas, VA). The cells were cultured in high-glucose MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in the incubator. MTT Assay for Cell Viability. Cells were seeded into a 96-well cell culture plate. After being incubated with indicated drugs for 24 or 48 h, fresh MEM containing 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)) was added to the culture system. Culture medium was discarded 4 h later, and dimethyl sulfoxide (DMSO) was applied to dissolve formazan crystals. Cell viability was assayed by a plate reader under 550 nm (Molecular Devices, U.S.). Cell Glucose Uptake Assay. To observe the ability of HepG2 cells to uptake glucose, combined methods including microscopy fluorescence density observation and flow cytometry were applied. A fluorescencent indicator, 2-NBDG, which can mimic glucose uptake in living cells, was used. For this end, cells were deprived from FBS overnight followed by drug treatment for 24 h, and then 2-NBDG (100 μM) was added to the culture system. Thirty minutes later, the cells were washed with cold PBS. Fluorescence density was assayed under a Live Cell Imaging System (Olympus, Japan) or by a flow cytometer (BD FACSAria III).

MATERIALS AND METHODS

Materials. Metformin (Met, lot: A0830A) and sodium butyrate (NaB) were, respectively, provided by GBCBIO Technology (Guangzhou, China) and Meilun Biological Technology (Dalian, China). Lipopolysaccharide (LPS), which is derived from Escherichia coli 055:B5, was purchased from Sigma (St. Louis, MO; product number: L2880). Primary antibodies including GLUT2 (ab54460) and SGLT1 (ab14685) were derived from Abcam (Cambridge, MA). Antibodies or agents for GAPDH (sc-47724), GPR43 (sc-32906), Akt (sc-514032), p-Akt (sc-8312), GSK-3α/β (sc-7291), p-GSK-3α/β (sc-81496), GPR43-siRNA (SC-77339), and control siRNA-A (SC37007) were purchased from Santa Cruz (Dallas, TX). Glycogen detection kit was supplied by Biovision (Milpitas, CA). 2-NBDG was from Life Technologies (Carlsbad, CA). Kits for hexokinase (HK), glucose-6-phosphate dehydrogenase (G6PDH), and pyruvate kinase (PK) were derived from Jiancheng (Nanjing, Jiangsu, China); glycogen synthase (GCS) and glycogenphosphorylase (GPa) were derived from Solarbio Life Sciences (Beijing, China). All other reagents were from commercial sources. Animals. All animal care and investigation conform to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85-23, revised 1996) and were approved by the Macau University of Science and Technology. Male animals, including 7−8 weeks old db/db mice (20−30 g) and C57BL/6 mice (15−20 g), and animal chows were supplied by Cavens Lab Animal Co. Ltd. (Changzhou, China). C57BL/6 mice were fed with regular chow (2 g/10 g, comprised of 5% fat, 53% carbohydrate, and 23% protein), and db/db mice were fed with a high-fat diet (2 g/10 g, comprised of 40% fat, 40% 7695

DOI: 10.1021/acs.jafc.9b02083 J. Agric. Food Chem. 2019, 67, 7694−7705

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Figure 2. Sodium butyrate (NaB) modulated liver AKT-GSK3 signaling proteins activation in db/db mice. (A) WB detection of AKT-GSK3 signaling proteins; (B) ratio of p-AKT toward total AKT; (C) ratio of p-GSK3α toward total GSK3α; and (D) ratio of p-GSK3β toward total GSK3β. n = 6. Met: metformin. *p < 0.05, **p < 0.01.

Figure 3. HepG2 cell viability assay by MTT. (A) LPS, (B) sodium butyrate (NaB), and (C and D) insulin concentrated-dependently inhibited HepG2 cell viability. n = 3. Met: metformin. *p < 0.05, **p < 0.01, vs normal. Knock-Down of GPR43. Expression of GPR43 was knockeddown according to protocol from the manufacturer. Immunofluorescence Assay. Cells at exponential state were incubated with insulin, LPS, NaB, or Metformin (2 mM)19 as indicated. Twenty-four hours later, cells were treated with 4% paraformaldehyde for 30 min. The cells then were further incubated with 5% BSA. After that, cells were incubated with primary antibodies including GLUT2 (1:200), SGLT1 (1:200), p-AKT (1:200), AKT (1:200), p-GSK3 (1:200), GSK3 (1:200), or GPR43 (1:200) at 4 °C overnight. After being washed with PBS, cells were further incubated with FITC- or CY3-conjugated secondary antibody. The nucleus was stained with DAPI. Finally, the cells were observed under a confocal

laser scanning microscopy (Leica TCS SP8, Germany), and the fluorescent density was determined by Image-J software. Western Blot (WB) Analysis. Total proteins from tissue or cells were extracted by RIPA lysis buffer. Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide and then transferred to PVDF membranes (pore size 0.45 μm). Membranes were incubated with primary antibodies including GPR43 (1:800), SGLT1 (1:800), GLUT2 (1:1000), AKT (1:1000), p-AKT (1:1000), GSK3α/β (1:1000), and p-GSK3α/β (1:1000) at 4 °C overnight and then washed with TBST [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20]. Subsequently, membranes were incubated with secondary antibodies at room temperature for 2 h. Finally, membranes were visualized by ECL from Santa Cruz (Dallas, 7696


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Figure 4. Sodium butyrate (NaB) reversed cell glucose uptake ability under high insulin + LPS condition. (A) High-insulin concentrationdependently inhibited cell glucose uptake: (a−f) indicated the concentration of insulin at 0, 10−9, 10−8, 10−7, 10−6, and 10−5 μmol/L, respectively. (B) Flow cytometry of cell glucose uptake, and (C) intracellular density of 2-NBDG fluorescence was compared (*p < 0.05, vs normal). (D) LPS concentration-dependently aggregated insulin resistance as determined by flow cytometry. (E) 2-NBDG fluorescence was analyzed for data (D) (*p < 0.05). n = 3.

there existed fibrosis and steatosis, the staining of glycogen within liver cells was significantly decreased, and NaB preserved the structure of hepatic lobule and level of glycogen within the cells. GPR43 is one of the most important receptor that mediates function of NaB. Here, we also found protein expression of GPR43 in db/db mice was significantly decreased by 59.38 ± 3.33% (p < 0.01, vs normal), while both metformin (Met) and NaB administration could reverse this reduction (p < 0.01, vs db/db) (Figure 1D). AKT-GSK3 signaling pathway participated in both insulin resistance and liver glycogenesis. To this end, we determined their activation status within liver tissue by Western blot. As shown in Figure 2, the phosphorylation of AKT was significantly increased while p-GSK3α/β (inactivation form) was decreased in db/db mice (p < 0.01, vs normal), and both Met and NaB inhibited effects of diabetes on AKT-GSK3 (p < 0.05, vs db/db). It was interesting to observe that the effects of NaB on AKT were more significant than those of Met (p < 0.05) (Figure 2A and B).

TX). GAPDH (1:1000) was applied as control. Relative expression of protein was analyzed by Image-J software. Enzyme Immunoassay (EIA). Levels of intracellular glycogen, GCS, HK, GPa, G6PDH, and PK were determined by kits according to the manufacturers’ protocols. Statistical Analysis. All data were obtained from more than three independent repeated experiments, and were analyzed by SPSS 19.0 software; all data that fit into the normal distribution were expressed as mean ± standard deviation (S.D.), and the differences among groups were analyzed by one-way ANOVA method. p < 0.05 was considered as statistically significant.

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RESULTS Sodium Butyrate (NaB) Improved Liver Glycogen Synthesis in db/db Mice. As shown in Figure 1A, liver in db/db mice was shown with obvious hypertrophy and steatosis, and supplementation with NaB significantly improved the appearance of liver. By H&E (Figure 1B) and PAS (Figure 1C) staining, we can obviously observe in db/db mice that the structure of hepatic lobule was changed in that 7697


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Figure 5. Sodium butyrate (NaB) increased intracellular glycogen under high insulin (Ins)+LPS condition. (A) Flow cytometry of cell glucose uptake in normal, Ins, Ins+LPS, and Ins+LPS+NaB groups, and (B) intracellular density of 2-NBDG fluorescence was compared among groups in (A). (C) Intracellular content of glycogen was determined by kit. n = 3. *p < 0.05, **p < 0.01.

NaB Increased Glycogen Level in HepG2 Cell Line. To elucidate the effects and mechanism of NaB on liver glycogene metabolism, we applied a cell line, HepG2 cells, in the following study. As liver cell possesses strong regenerative ability, we first want to exclude the influence of cell amount on the determination of glycogen within HepG2 cells. As shown in Figure 3A,B, cell viability was inhibited by LPS or NaB in a concentration-dependent manner after 24 h incubation. We then further incubated the cells with insulin at different concentrations for 24 h (Figure 3C) or 48 h (Figure 3D), and found hyperinsulinemia decreased cell viability in a concentration-dependent manner at both time points. Hyperinsulinemia or insulin resistance (IR)-inhibited glucose uptake was further verified via 2-NBDG detection by immunofluence microscopy (Figure 4A) and flow cytometry (Figure 4B,C). Converging with literature reports and our present findings, we applied 1 μg/mL of LPS,20 0.5 mM of NaB,21 and 10−7 mol/L of insulin22 at 24 h in the following study except for otherwise stated. IR and low-grade inflammation are two characteristics of T2DM. To understand the glucose uptake ability of liver cells under this condition, HepG2 cells were cultured with 10−7 mol/L of insulin plus different concentrations of LPS. We found glucose uptake was reduced with concentration increase of LPS (Figure 4D,E). Converging from reports20,22 and our present observation (Figure 3A), we finally applied 10−7 mol/L of insulin plus 1 μg/mL of LPS (Ins+LPS) to stimulate cells in the following study. To investigate the effects of NaB on liver glycogenesis, the cells were first incubated with agents indicated and detected by flow cytometry. As depicted in Figure 5A,B, Ins+LPS treatment significantly decreased glucose uptake by as high as 27.23 ± 11.23% (p < 0.05, vs normal), while NaB supplementation significantly reversed reduction of glucose uptake induced by Ins+LPS (p < 0.05); there was no statistical significance

between normal and Ins+LPS+NaB groups. This was further demonstrated by intracellular glycogen detection in that Ins +LPS significantly reduced intracellular level of glycogen by as high as 49.05 ± 1.22% (p < 0.01, vs normal), and NaB or Met administration significantly promoted glycogenesis (p < 0.01, vs Ins+LPS); there was no statistical significance among normal, Ins+LPS+NaB, and Ins+LPS+Met groups concerning glycogenesis (Figure 5C). NaB Promoted Metabolism of Glycogen within HepG2 Cells. Normal metabolism of glycogen is pivotal for maintaining both liver cell and the organism in a healthy status, and this metabolism relies on a series of enzymes. In the present study, we found Ins+LPS significantly reduced the activity of both anabolic and catabolic enzymes of liver glycogen (Figure 6), and thus inhibited glycogen metabolism in the two ends. Expectedly, NaB incubation increased levels of anabolic enzymes including hexokinase (HK) and glycogen synthase (GCS) by 57.98 ± 23.67% and 42.88 ± 3.28%, respectively (p < 0.05, vs Ins+LPS) (Figure 6A and B), suggesting NaB increased conversion of intracellular glucose into glycogen. On the other hand, we also found NaB elevated level of catabolic enzymes including glycogenphosphorylase (GPa), glucose-6-phosphate dehydrogenase (G6PDH), and pyruvate kinase (PK) by 18.82 ± 6.79%, 58.14 ± 10.91%, and 25.70 ± 7.62%, respectively (p < 0.05, vs Ins+LPS) (Figure 6C−E), which therefore ensured efficient utilization of liver glycogen. Glucose Transport Proteins Were Upregulated by NaB. Glucose transporters play pivotal roles in mediating hepatocyte glucose uptake. By immunofluorescence WB assay, we found the expression of glucose transporter 2 (GLUT2) was significantly decreased under high insulin settings (p < 0.05, vs normal), while NaB inhibited this decrement (Figure 7A, D, and G). 7698


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Figure 6. Sodium butyrate (NaB) increased activity of enzymes participating in liver glycogen metabolism under high insulin (Ins)+LPS condition. Intracellular content of (A) hexokinase (HK), (B) glycogen synthase (GCS), (C) glycogenphosphorylase (GPa), (D) glucose-6-phosphate dehydrogenase (G6PDH), and (E) pyruvate kinase (PK) was determined by kits. n = 3. *p < 0.05, **p < 0.01.

Sodium-dependent glucose cotransporter 1 (SGLT1) is mainly expressed in small intestine and proximal tubule of the nephron to mediate glucose absorption and reabsorption. Recent finding indicates that it is also expressed on liver biliary duct cells.23 In the present study, we observed SGLT1 was also located on HepG2 cell membranes (Figure 7B); moreover, Ins +LPS significantly inhibited its expression (p < 0.01, vs normal), and this reduction was reversed by NaB administration (p < 0.01, vs Ins+LPS) (Figure 7B, E, and H). In line with findings in animal tissue (Figure 1), Ins+LPS reduced expression of GPR43 in HepG2 cells (p < 0.01, vs normal), and administration with either NaB or Met reversed this reduction (p < 0.01, vs Ins+LPS) (Figure 7C, F, and I). To further demonstrate the role of GPR43 in mediating effects of NaB, its protein expression was knocked-down by siRNA. We found siRNA-GPR43 significantly inhibited NaB-induced expression of GPR43 (Figure 7G and K).

No statistical significance was observed for GLUT2, SGLT1, and GPR43 expression between normal and Ins+LPS+NaB groups. AKT-GSK3 Pathway Was Involved in NaB-Mediated Liver Glycogenesis in HepG2 Cells. To further validate involvement of AKT-GSK3 signaling pathway proteins in HepG2 cells, their expression and phosphorylation were determined by both immunofluorescence and WB. For this end, we first incubated the cells with Ins+LPS as indicated for a time-course ranging from 0 to 60 min, and observed the content of AKT and GSK3α/β as well as their phosphorylated proteins by immunofluorescence method. It was observed that the content of intracellular AKT and GSK3α/β was not significantly changed with time, while their phosphorylated proteins varied according to the stimulation of Ins+LPS, and the highest level of p-AKT and p-GSK3α/β was observed at 30 and 5 min, respectively (Figure 8A−D). According to the report,24 AKT is the upstream signaling pathway protein for 7699


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Figure 7. Sodium butyrate (NaB) increased GLUT2, SGLT1, and GPR43 expressions. Immonofluorescence assay for (A) GLUT2, (B) SGLT1, and (C) GPR43 under a laser scanning confocal microscope (magnification: 800); and relative fluorescence intensity (D−F) for them were determined. Western Blot analysis for the above proteins was rechecked (G−I). siRNA-GPR43 was applied to further demonstrate GPR43 in mediating effects of NaB by WB (J and K). n = 3. *p < 0.05, **p < 0.01.

GSK, so we applied “30 min” in the following study. As can be observed in Figure 8B and D, p-GSK3α/β was significantly reduced at 30 min as compared to that at 0 min. In line with

our findings in animal tissue (Figure 2), the level of p-AKT was significantly increased while p-GSK3 was reduced by Ins+LPS, and NaB incubation reversed this variation (Figure 8E−L). 7700


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Figure 8. Sodium butyrate (NaB) modulated AKT and GSK3 activation within HepG2 cells. High insulin (Ins)+LPS activated (A and C) AKT and (B and D) GSK3 in a time-dependent manner as observed under a laser scanning confocal microscope (magnification: 800), and a most significant activation was observed at 30 and 5 min, respectively (**p < 0.01, vs 0 min). Cells were stimulated with agents Ins, Ins+LPS, or Ins+LPS +NaB for 30 min, and activation of (E and G) AKT and (F and H) GSK3 was further determined by immunofluorescence; (I−L) WB was applied for further determination of AKT/GSK3. n = 3. *p < 0.05, **p < 0.01.

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DISCUSSION

chain fatty acids (SCFAs) against Type 2 Diabetes Mellitus (T2DM). Yet its specific role and mechanism still needs to be

The pivotal role of cometabolism between gut microbiota and the organism has been more recognized. Recent understanding of this cometabolism suggests the important function of short-

fully explored. Liver is the pivotal organ on regulating blood glucose level via glocogen metabolism; we found in the present 7701


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Figure 9. Proposed mechanism of sodium butyrate (NaB) on promoting glycogen metabolism under T2DM settings.

ameliorate liver vesicular steatosis and inhibited fat deposition in STZ-induced diabetic rats;15 modulation of liver mitochondrial function might be one of the mechanisms.4 Considering glucose and glycogen utilization, there is a report found in animal skeletal muscle that feeding with glucose plus acetic acid could significantly accelerate glycogen repletion as compared to that without acetic acid.33 In the present study, we demonstrated both in vivo and in vitro that NaB supplementation positively promoted glycogenesis within hepatocytes. Our present findings obviously supplied new and direct evidence that microbiota metabolite NaB plays a key role in modulating the blood glucose level. One important way that mediates function of SCFAs is GPR43. GPR43, also known as free fatty acid receptor 3 (FFAR2), was reported to be distributed on immune cells, gastrointestinal epithelial cells,9 and white adipose tissue.34 Here, we found GPR43 was also distributed in liver tissue; its expression was significantly reduced in db/db mice and Ins +LPS stimulated HepG2 cells, and this reduction was inhibited by NaB supplementation. Study concerning NaB and GPR43 on liver is rare, but there is one report that supports our present finding that an abundance of GPR43 mRNA was modulated according to diet.35 In our previous study,16 oral administration with NaB or metformin significantly ameliorated composition alteration of gut microbiota. GPR43 is the receptor for SCFAs and can be induced by its ligand, for example, NaB. In the present study, either Met or NaB administration increased expression of GPR43. We postulated that Met may upregulate expression of GPR43 via at least two ways: (1) it increased the abundance of butyrate-producing bacteria within gut lumen, which thus increased the level of butyrate; and (2) it inhibited LPS-induced effects on GPR43.9 However, a specific mechanism deserves further investigation.

study that sodium butyrate (NaB), an SCFA, could increase glycogenesis within hepatocytes under T2DM settings. Insulin resistance is the most important characteristic of T2DM. Although the underlying mechanism still needs to be unveiled, recent understanding suggests that gut dysbacteriosis is positively related to the occurrence and development of T2DM.10 Microbiota metabolites (mainly SCFAs) play a protective role against T2DM. Besides serving as the energy source of gut epithelial cells and promoter for glucagon sample peptide 1/2 in intestinal L cells,25 SCFAs have been demonstrated to have anti-inflammation and antioxidative stress damage ability in glomecular mesangial cells.26 Most recently, a study suggested microbiota products correlated with blood glucose control.27 There are mainly three types of SCFAs, acetate, propionate, and butyrate. Although it only shares 15% of all SCFAs, butyrate exhibits the most potential effect against diabetic damage.9,28 This has been verified by Qin and colleagues10 in T2DM population. From this aspect of view, NaB supplementation is believed to be a promising strategy against diabetes.4,29 This was further demonstrated by Vidrine and colleagues that NaB absorption into blood did not influence production of both hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), but local generated butyrate increased these two factors.30 In our previous study, oral supplementation with NaB could decrease blood glucose and HbA1c levels in db/db mice,16 suggesting the protective role of NaB on blood glucose metabolism. Liver is one of the most important organs in modulating blood glucose level via the glycogen metabolism pathway. Unfortunately, as high as 19% cases in T2DM population may concurrently have liver dysfunction.31 Depletion of gut microbiota has been reported to be related to liver disease.32 Also, microbiota metabolic product NaB administration could 7702


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activation of AKT; therefore, its phosphorylated form was found to be increased. We also observed in our in vitro study that phosphorylation of AKT and GSK3 stimulated by Ins +LPS was not synchronized (Figure 8A−D), and at time point “30 min”, level of pGSk3 was even lower than that at “0 min”. This phenomenon was also attributed to the fact that GSK3 could be modulated by more than one upstream signaling pathway protein (e.g., Smad complex and Wnt). However, more work should be carried out to completely uncover the GSK3-related signaling pathway in modulating glycogen metabolism. In conclusion, we found in the present study that NaB supplementation could increase hepatocyte glucose uptake and promote glycogen metabolism, and the GPR43-AKT-GSK3 pathway should contribute to the above effects. Our present findings will supply a new idea and strategy toward T2DM intervention.

The effect of liver on modulating blood glucose relies on glucose transport proteins. There are mainly two types of glucose transport proteins, glucose transporters (GLUTs) and sodium-glucose cotransporters (SGLTs); GLUTs are mostly distributed in insulin-sensitive tissues, while SGLTs are mainly found in intestinal mucosa and renal proximal tubule cells.36 GLUT2 is the major type of GLUTs that is responsible for hepatic glucose utilization.37 Under T2DM settings, expression of GLUT2 in most tissues is reduced.38 This is in line with our present finding that GLUT2 was reduced in Ins+LPS stimulated hepatocytes. Studies concerning influence of SCFAs on GLUT2 are rare and mostly focus on intestinal tissues. Reports suggested that SCFAs supplementation increased GLUT2 abundance in intestinal cells.39,40 Here, we demonstrated in hepatocytes that NaB administration reversed Ins+LPS stimulated GLUT2 expression. This effect should be attributed to glucose uptake increment by liver cells. Besides GLUT2, we also demonstrated distribution of SGLT1 on hepatocyte membrane; our present finding is in line with a report from Sharma and colleagues.42 The role of SGLT1 in glycogenesis is still to be elucidated, but understanding from Sharma and colleagues41 suggests that SGLT1 plays a key role against bacterial infection; we postulated that this should at least in part contribute to the beneficial effects of NaB against diabetic inflammation and hepatocytes dysfunction. Glycogen homeostasis relies on the balance between glycogenesis and glycogenolysis. These two processes are regulated by hormone and nervous stimuli and were strictly controlled by many rate-limiting enzymes (Figure 9). In T2DM population, glycogen synthase (GCS) is inactivated;42 this obviously reduced the glycogen storage ability of hepatocytes in T2DM. On the other hand, glycogen phosphorylase (GPa) controls glycogen degradation, and high glucose will inhibit its activity.43 In our present study, we found NaB supplementation increased the activity of both glycogenesis and glycogenolysis enzymes, and we believe this effect increased the uptake of glucose into cytoplasma, on the one hand, and promoted utilization of glucose (through glycogenolysis), on the other hand; therefore, NaB was shown with significant hypoglycemic effect in hepatocytes. This effect is obviously more protective to organism as it avoids glycogen utilization impairment and thereafter liver cirrhosis or fatal low blood glucose.44 Activation of GCS and GPa is mediated by their upstream signaling pathway proteins. Overexpression/overactivation of glycogen synthase kinase 3 (GSK3) can inhibit activation of GCS.45 When being phosphorylated, activation of GSK3 is inhibited; in this sense, the level of p-GSK3 is positively related to activation of GCS. Both in vivo and in vitro studies in our present work observed that NaB administration significantly increased the level of p-GSK3α/β. It has been well demonstrated that AKT inhibited GSK3.46 In our present study, the relative level of p-AKT was found to be increased, while p-GSK3 was reduced in db/db mice or Ins+LPS stimulated HepG2 cells. This was inconsistent with some previous report that both AKT and GSK3 were reduced.47 This should be attributed to a different disease model. As T2DM has been recognized as a low-grade-inflammatory disease, IR accompanied by inflammation plays a central role in promoting development of T2DM. In our previous report, we confirmed that both LPS and inflammatory cytokines were increased in db/db mice.16 In this sense, we stimulated HepG2 cells by Ins+LPS. LPS stimuli will significantly promote

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected], [email protected]. *E-mail: [email protected]. ORCID

You-Hua Xu: 0000-0002-3258-013X Funding

This work was supported by the Science and Technology Development Fund of Macau (FDCT: 0093/2018/A3). Notes

The authors declare no competing financial interest.

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REFERENCES

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Sodium Butyrate Improves Liver Glycogen Metabolism in Type 2

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