Sodium Pyruvate

Fluoroquinolones suppress gluconeogenesis by inhibiting fructose 1,6bisphosphatase in primary monkey hepatocytes

Takuma Iguchia,⁎, Koichi Gotoa, Kyoko Watanabeb, Kazuyuki Hashimotob, Takami Suzukic, Hiroyuki Kishinoa, Kazunori Fujimotoa, Kazuhiko Moria

Abstract

Dysglycemia is one of the most serious adverse events associated with the clinical use of certain fluoroquinolones. The purpose of this study was to investigate the effects of the representative fluoroquinolones moxifloxacin and gatifloxacin on hepatic gluconeogenesis using primary monkey hepatocytes. Glucose production was induced after the cells were incubated for 4 h with 10 mM sodium lactate and 1 mM sodium pyruvate as gluconeogenic substrates. Under these conditions, moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis at concentrations of 100 μM or higher. Transcriptome analysis of rate-limiting enzymes involved in hepatic gluconeogenesis revealed that moxifloxacin and gatifloxacin at a concentration of 1000 μM did not affect the expression of key gluconeogenic enzymes such as phosphoenolpyruvate carboxykinase, glucose 6-phosphatase, and fructose 1,6-bisphosphatase. Furthermore, metabolome analysis, in vitro glucose production assay using additional gluconeogenic substrates, and fructose 1,6-bisphosphatase assay using the cell extracts showed that fluoroquinolones enzymatically suppressed hepatic gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. These inhibitory effects may involve in the clinically relevant dysglycemia associated with fluoroquinolones in human.

Keywords:
Gluconeogenesis
Fructose 1,6-bisphosphatase
Fluoroquinolones
Hepatocyte
Cynomolgus monkey

1. Introduction

Fluoroquinolones are potent antibacterial agents with a broad spectrum of activity against DNA gyrase and DNA topoisomerase IV in Gram-positive and Gram-negative bacteria (Owens Jr. and Ambrose, 2000). The currently available fluoroquinolones have a favorable safety profile; however, certain fluoroquinolones have been associated with dysglycemia, which is one of the most serious adverse events that can sometimes be life-threatening and result in hospitalization (Mohr et al., 2005; Graumlich et al., 2005; Lewis and Mohr III, 2008). The risk of clinically relevant dysglycemia varies among fluoroquinolones. In an analysis of the U.S. Food and Drug Administration (FDA) database, the risk of severe hypoglycemia and hyperglycemia was greater with gatifloxacin than with other fluoroquinolones (Frothingham, 2005; Aspinall et al., 2009). Gatifloxacin was withdrawn voluntarily from the market because of increased risk of hypoglycemia and hyperglycemia in patients treated with it (Mehlhorn and Brown, 2007).
Direct insulinotropic effect via blocking ATP-sensitive potassium channels of pancreatic β cells was suggested as one of the most likely causes of hypoglycemia induced by common fluoroquinolones (Maeda et al., 1996; Saraya et al., 2004). In addition, the mean pancreas-toplasma ratios of gatifloxacin was higher than that of other fluoroquinolones in animals and humans (Ishiwata et al., 2006; Nagai et al., 2010; Yabe et al., 2019), being associated with high risk of dysglycemia for gatifloxacin. In contrast, it has also been reported that fluoroquinolones like gatifloxacin did not stimulate insulin secretion in the presence of physiological concentrations of glucose in vitro (Ghaly et al., 2009), and they also induced hypoglycemia even in type I diabetes model rats (Absi et al., 2013). The complexity of biological events suggests that fluoroquinolones modulate multiple glycemic control mechanisms other than insulinotropic effects and consequently elicit variable effects.
The liver and, to a lesser extent, the kidneys are the primary organs responsible for endogenous glucose production (Roden and Bernroider, 2003; Gerich et al., 2001). These organs maintain euglycemia by rapid clearance of glucose from the portal vein in the absorptive state after a meal, and by controlled production of glucose in the post-absorptive state at a sufficient rate (Agius, 2007). Drozak et al. (2008) reported that gatifloxacin substantially decreased the glucose synthesis rate by impairing mitochondrial pyruvate uptake. This result strongly suggests that gatifloxacin-induced hypoglycemic episodes are caused by not only facilitating insulin secretion but inhibiting hepatic glucose synthesis. However, the effect of fluoroquinolones on several rate-limiting enzymes involved in gluconeogenesis regulation has not yet been elucidated.
Cynomolgus monkey is one of the most commonly used animals for preclinical toxicity evaluation. Since it is a non-human primate, it is considered to be anatomically, physiologically, and genetically closer to humans than other laboratory animals like rats and mice (Register, 2009; Uchida et al., 2012). Recently, Yoshimatsu et al. (2018) reported that oral administration of gatifloxacin induced dose-dependent hypoglycemia in monkeys that was comparable to that in humans, based on the sensitivity with regard to the effect on blood glucose homeostasis.
The purpose of this study was to investigate the effects of the representative fluoroquinolones moxifloxacin and gatifloxacin on hepatic gluconeogenesis using primary monkey hepatocytes. Moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis to a similar extent in monkey hepatocytes. Furthermore, metabolome analysis, in vitro assay using additional gluconeogenic substrates, and fructose 1,6bisphosphatase assay using the cell extracts suggested that fluoroquinolones suppressed hepatic gluconeogenesis by inhibiting fructose 1,6-bisphosphatase.

2. Materials and methods

2.1. Chemicals

Moxifloxacin was purchased from Sequoia Research Products (Oxford, UK), and gatifloxacin was purchased from LKT laboratories (St. Paul, MN, USA). Both drugs were dissolved in 1 N sodium hydroxide (Kishida Chemical, Osaka, Japan) to prepare 1000 μM stock solution. Insulin solution from bovine pancreas (Sigma-Aldrich Japan, Tokyo, Japan) was used at 10 mg/mL as a stock solution. Other reagents were of analytical grade.

2.2. Cells

Cryopreserved primary hepatocytes isolated from 3 male cynomolgus monkeys were obtained from KAC (Tokyo, Japan). The frozen cells were thawed and centrifuged at 50 ×g for 5 min and resuspended in a culture medium composed of glucose-free Dulbecco’s modified Eagle’s medium supplemented with 5% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 1 μM dexamethasone, and 67 μM N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate. The cells were plated at a density of 15 ± 3 × 104 cells/cm2 in collagencoated tissue culture 24- or 96-well plates and incubated overnight at 37 °C with 5% CO2.

2.3. Fluoroquinolone treatment

After the overnight incubation, the culture medium was changed to a glucose production medium composed of Dulbecco’s phosphate-buffered saline supplemented with 25 mM NaHCO3, 25 mM HEPES, 1% bovine serum albumin, 10 mM sodium lactate and 1 mM sodium pyruvate as gluconeogenic substrates (Chen et al., 2012). The cells were incubated for 4 h in the glucose production medium at various fluoroquinolone concentrations (10, 100, or 1000 μM). These concentrations were based on a preliminary experiment that showed no cytotoxicity for fluoroquinolones concentrations up to 1000 μM. To clarify the effect of fluoroquinolones on fructose 1,6-bisphosphatase, which is involved in the rate-limiting step of fructose-1,6-bisphosphate conversion to fructose-6-phosphate, the cells were exposed to fluoroquinolones at 1000 μM with either 10 mM phosphoenolpyruvate, fructose-1,6-bisphosphate, or 10 mM fructose-6-phosphate in the presence or absence of 10 mM sodium lactate and 1 mM sodium pyruvate.

2.4. Glucose production assay

The glucose level in the medium was quantified by using a commercial assay kit (Glucose CII Test Wako, Fujifilm Wako Pure Chemical Corporation, Osaka, Japan) according to the manufacturer’s instructions. The glucose level of each sample was calculated as the percentage of corresponding vehicle control samples with gluconeogenic substrates that was set as 100% unless otherwise described.

2.5. Gene expression analysis

Total RNA was isolated from hepatocytes on a 24-well plate using the RNeasy mini kit (QIAGEN, Tokyo, Japan) after fluoroquinolone treatment. One microgram of RNA was reverse-transcribed with the SuperScript VILO cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. Gene expression levels of rate-limiting enzymes involved in hepatic gluconeogenesis were measured using the Express SYBR Green qPCR Supermix (Thermo Fisher Scientific). Cycling was performed at 50 °C for 2 min, 95 °C for 2 min, followed by 45 cycles of 95 °C 15 s and 60 °C 1 min. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene. PCR used the following primers synthesized by Thermo Fisher Scientific: fructose 1,6-bisphosphatase (F: 5′-CAGTGCCACCATGCTGGTCCTTG-3′, R: 5′-GCGTAGCCCTCGTTAAGGCTG TAG-3′); glucose 6-phosphatase (F: 5′-GGTGATTGGAGACTGGCTCAAC CTC-3′, R: 5′-CACAGGTGACAGGGAACTGCTTTATCAG-3′); phosphoenolpyruvate carboxykinase 1 (F: 5′-CAGGCGGCTGAGAAGTATGACAA CTG-3′, R: 5′-GGATGGGCACTGTGTCTCTTTGCTC-3′); phosphoenolpyruvate carboxykinase 2 (F: 5′-CTGAGTCCACTGCTGCAGCAG AAC-3′, R: 5′-GGCAGATCCTGGTTGACCTGCTC-3′); pyruvate dehydrogenase 2 (F: 5′-CCGTCTCCAACCAGAACATCCAGTAC-3′, R: 5′-GGG TTGGTGCTGCCGTCAAAGATG-3′); pyruvate kinase (F: 5′-CCGTGAAC CTCCAGAAGCCATCTG-3′, R: 5′-CCTGTCACCACAATCACCAGGT CTC-3′); GAPDH (F: 5′-GGGAAGGTGAAGGTCGGAGTCAAC-3′, R: 5′-GCCATGGGTGGATCATACTGGAACATG-3′).

2.6. Metabolome analysis

In the glucose production assay and gene expression analysis, moxifloxacin and gatifloxacin showed similar inhibitory effects, but moxifloxacin showed apparent effects with less variation. In addition, metabolome analysis was designed to obtain the clues regarding the inhibited metabolic pathway of gluconeogenesis by fluoroquinolones. Therefore, only moxifloxacin was used in the analysis. Cells were harvested into phosphate buffered saline. Metabolites were extracted from the cells according to the method of Bligh and Dyer (1959). Extracted aqueous solution was measured by an LC-MS/MS method. LC separation was performed using an Ultimate 3000 HPLC system (DIONEX, Thermo Fisher Scientific) and Mastro C18 column (150 mm × 2.1 mm, 3 μm, Shimadzu GLC, Tokyo, Japan) with a linear gradient elution using mobile phases of 15 mM tributylamine/10 mM acetic acid (A) and methanol (B) at a flow rate of 300 μL/min at a column temperature of 40 °C. The eluate was analyzed by a TSQ Vantage triple-stage quadrupole mass spectrometer (Thermo Fisher Scientific) with positive/negative ion switching electrospray ionization in multiple-reaction monitoring mode to detect 103 metabolites associated with gluconeogenesis. Data acquisition and processing were performed using TraverseTM MS Version 1.0 (Reifycs, Tokyo, Japan).

2.7. Fructose-1,6-bisphosphatase activity assay

Cytoplasmic extracts from the cells were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) supplemented with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific). The total protein concentration of the extracts was determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Fructose 1,6-bisphosphatase activity in the extracts was assayed using Fructose-1,6-Bisphosphatase Activity Assay Kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s instructions. In the assay, fructose-1,6-bisphosphatase hydrolyzed fructose-1,6-bisphosphate into fructose-6-phosphate. Fructose-6-phosphate was used in an enzyme-coupled reaction system that measured spectrophotometrically at 450 nm. One unit of enzyme activity was defined as that amount which generates 1 μmol of fructose-6-phosphate per min. The enzyme activity of the incubation mixture containing the serial dilutions of fructose-6-phosphate with known concentration (for standard curve), moxifloxacin or gatifloxacin at 1000 μM, or fructose1,6-bisphosphatase-1 inhibitor (Cayman Chemical, Ann Arbor, MI, USA) at 100 μM (as a positive control) was measured.

2.8. Statistics

For the glucose production assay, means and standard deviations were calculated from three independent biological samples in triplicate or quadruplicate. Transcriptome and metabolome analyses were conducted from one biological sample in triplicate or quadruplicate based on the availability of the cells. Statistical analyses between groups were performed with the parametric test one-way analysis of variance (ANOVA) followed by Student’s t-test, Dunnett’s multiple comparisons test, or paired t-test. These analyses were performed using SAS System Release 9.2 (SAS Institute, Cary, NC, USA). A P value of P < .05 was considered statistically significant. 3. Results 3.1. Effects of moxifloxacin and gatifloxacin on gluconeogenesis in monkey hepatocytes Under our experimental conditions, primary monkey hepatocytes produced large amounts of glucose in the presence of the gluconeogenic substrates lactate and pyruvate (Fig. 1). Moxifloxacin and gatifloxacin dose-dependently suppressed gluconeogenesis at concentrations of 100 μM or higher. The IC50 values (95% confidence interval) of moxifloxacin and gatifloxacin on gluconeogenesis were 143 μM (72 μM 320 μM) and 292 μM (137 μM - 920 μM), respectively. Although the IC50 value of moxifloxacin was lower than that of gatifloxacin, no significant difference between moxifloxacin and gatifloxacin was observed at the same concentrations. In addition, moxifloxacin and gatifloxacin increased cellular ATP content involved in the gluconeogenic pathway leading from pyruvate to glucose at similar concentrations (Fig. 2). These results suggested that the inhibitory effects of moxifloxacin and gatifloxacin on gluconeogenesis did not result from a lack of ATP in monkey hepatocytes. Meanwhile, this result also demonstrated that moxifloxacin and gatifloxacin had no cytotoxic potential at concentrations up to 1000 μM in monkey hepatocytes. 3.2. Effects of moxifloxacin and gatifloxacin on gluconeogenic gene expression For a better understanding of the inhibitory effects of moxifloxacin and gatifloxacin on gluconeogenesis, gene expression levels of ratelimiting enzymes involved in hepatic gluconeogenesis were evaluated. Gluconeogenic substrates clearly upregulated the expression of phosphoenolpyruvate carboxykinase 1 and glucose 6-phosphatase. However, these gluconeogenic genes were suppressed by the presence of insulin (Fig. 3), which is consistent with previous reports (Gabbay et al., 1996; Dickens et al., 1998). On the other hand, moxifloxacin and gatifloxacin at a concentration of 1000 μM did not suppress the upregulated phosphoenolpyruvate carboxykinase 1 and glucose 6-phosphatase with gluconeogenic substrates. Instead, it upregulated or tended to upregulate these genes. In addition, fluoroquinolone did not affect the expression of other gluconeogenic genes including mitochondrial isoform phosphoenolpyruvate carboxykinase 2, fructose 1,6-bisphosphatase, and glycolytic genes like pyruvate dehydrogenase 2 or pyruvate kinase. 3.3. Metabolome analysis of monkey hepatocytes exposed to moxifloxacin Gluconeogenic gene expression pattern was not correlated with inhibitory effects of moxifloxacin and gatifloxacin on glucose production from monkey hepatocytes. Therefore, quantitative analysis of the metabolites was conducted to identify the key molecule in gluconeogenesis using cellular extracts from monkey hepatocytes exposed to moxifloxacin at 1000 μM. Metabolome analysis identified that 17 and 13 out of 103 metabolites associated with gluconeogenesis were upregulated and downregulated, respectively. Most of the upregulated metabolites were small molecules like amino acids and nucleotides, and downregulated metabolites were mainly gluconeogenic intermediates (Table 1). Under our conditions, glucose-6-phosphate and fructose-6phosphate were significantly decreased 5.4-fold and 3.6-fold, respectively. On the other hand, other gluconeogenic intermediates like phosphoenolpyruvate, 3-phosphoglycerate, and dihydroxyacetone phosphate were not changed. These results suggested that moxifloxacin predominantly inhibited the metabolic pathway of gluconeogenesis between phosphoenolpyruvate and fructose-6-phosphate. 3.4. Involvement of fructose 1,6-bisphosphatase in gluconeogenesis suppression by moxifloxacin and gatifloxacin in monkey hepatocytes To identify the key molecule in gluconeogenesis, the effects of additional substrates on glucose production were investigated. In the presence of 10 mM sodium lactate and 1 mM sodium pyruvate with phosphoenolpyruvate or fructose-1,6-bisphosphate, both moxifloxacin and gatifloxacin at 1000 μM suppressed glucose production by 34% to 64% of the control (Table 2). These inhibitory effects were not observed in the presence of phosphoenolpyruvate or fructose-1,6-bisphosphate alone, suggesting that these additional substrates could not enter cytoplasm of the cells under our experimental conditions. In contrast, addition of fructose-6-phosphate increased basal glucose production, but did not show the inhibitory effects of fluoroquinolones either in the presence or absence of sodium lactate and sodium pyruvate. Furthermore, the effect of fluoroquinolones on fructose 1,6-bisphosphatase activity of primary monkey hepatocytes was investigated. Commercially available fructose 1,6-bisphosphatase inhibitor showed 72% inhibition. Under this assay condition, both fluoroquinolones at 1000 μM caused inhibitory effects on the enzyme activity by 86% (moxifloxacin) and 84% (gatifloxacin) compared to the control (Fig. 4). These results indicated that moxifloxacin and gatifloxacin suppressed gluconeogenesis predominantly by inhibiting fructose 1,6-bisphosphatase in primary monkey hepatocytes (Fig. 5). 4. Discussion Gluconeogenesis is a predominant hepatic pathway by which three‑carbon precursors like lactate and pyruvate are enzymatically converted to glucose (Chung et al., 2015; Rui, 2014; Zhang et al., 2019). Most of the enzymes involved in gluconeogenesis also catalyze the reverse reactions in glycolysis, but four unidirectional enzymes like pyruvate carboxylase, phosphoenolpyruvate carboxykinase, fructose 1,6-bisphosphatase, and glucose 6-phosphatase are reportedly involved in the rate-limiting gluconeogenesis steps (Rui, 2014). Drug-induced hypoglycemia was associated with insulin (Gabbay et al., 1996; Dickens et al., 1998), metformin (Kim et al., 2008), or troglitazone (Davies et al., 1999). These drugs have been shown to inhibit the expression of rate-limiting gluconeogenic genes. However, this study revealed that moxifloxacin and gatifloxacin suppressed gluconeogenesis in monkey hepatocytes without any effects on the expression of fructose 1,6-bisphosphatase. Moreover, moxifloxacin and gatifloxacin enzymatically Cytoplasmic extracts from monkey hepatocytes were incubated for 40 min with fluoroquinolones at 1000 μM or fructose 1,6-bisphosphatase inhibitor at 100 μM. Data represent the means ± standard deviations of the enzyme activity in triplicate or quadruplicate. One unit of enzyme activity was defined as that amount which generates 1 μmol of fructose-6-phosphate per min. * P < .05, ** P < .01: Significantly different from the vehicle control group (Student's t-test). Abbreviations: FBPase, fructose 1,6-bisphosphatase.suppressed gluconeogenesis by inhibiting fructose 1,6-bisphosphatase. Fructose 1,6-bisphosphatase catalyzes dephosphorylation of fructose 1,6-bisphosphate to fructose 6-phosphate. Their activities are elevated in insulin-resistant animal models (Andrikopoulos et al., 1993;) and in diabetes patients (Kebede et al., 2008). Fructose 1,6bisphosphatase is reported to be directly regulated by AMP, which binds to an allosteric site as a physiological inhibitor (Gidh-Jain et al., 1994). However, our metabolome analysis suggested that moxifloxacin did not affect cellular AMP levels (data not shown). Therefore, fructose 1,6-bisphosphatase inhibition by moxifloxacin was not caused by the perturbations of metabolite homeostasis like changes to physiological AMP levels. However, further investigation is required to determine whether fluoroquinolones play a role as allosteric ligands for fructose 1,6-bisphosphatase. The suppression of gluconeogenesis induced by fluoroquinolones was reversed by adding fructose-6-phosphate in the glucose production assay. Glycogenolysis is another process that can produce glucose in the liver. In the normal non-diabetic state, the rapid suppression of hepatic glucose production is due to glycogenolysis inhibition, in which glycogen phosphorylase catalyzes degradation of glycogen to glucose-6phosphate (Ramnanan et al., 2010). In this case, ATP can bind to glycogen phosphorylase as an allosteric ligand, resulting in the less active enzyme state. Notably, moxifloxacin and gatifloxacin increased cellular ATP levels in this study, preventing the compensatory increase of glucose production through glycogenolysis. Drozak et al. (2008) suggested that the inhibitory action of gatifloxacin on gluconeogenesis in cortex tubules of the kidney and hepatocytes in rabbits resulted from its impairment of pyruvate transport into mitochondria. This was partially confirmed by our metabolome analysis because most intermediates (α-ketoglutarate, citrate, fumarate, isocitrate, and malate) in the TCA cycle were decreased by moxifloxacin treatment. On the other hand, moxifloxacin had negligible effects on cellular levels of phosphoenolpyruvate which was the first committed gluconeogenic substrate converted from intermediates in the TCA cycle. Taken together, fluoroquinolones might affect glucose synthesis via direct inhibition of fructose 1,6-bisphosphatase rather than interfering with pyruvate uptake into mitochondria in monkey hepatocytes. In this study, the IC50 of moxifloxacin and gatifloxacin on gluconeogenesis in monkey hepatocytes was approximately 100 μM, which is 10-fold higher than the peak plasma concentrations in patients (Turnidge, 1999; Van Bambeke et al., 2005). Simiraly, Ghaly et al. (2009) reported that both moxifloxacin and gatifloxacin at concentrations up to 100 μM did not stimulate insulin secretion from perfused mouse islets in the presence of physiological concentrations of glucose in vitro. Although the contributions to dysglycemia of moxifloxacin and gatifloxacin on gluconeogenesis in the liver remain to be clarified, the inhibitory effects on gluconeogenesis due to fluoroquinolones may be involved in dysglycemia in humans. In conclusion, our in vitro study using monkey hepatocytes provides new insights into the inhibitory mechanism of fluoroquinolones on gluconeogenesis via fructose 1,6-bisphosphatase inhibition, which could help understand the risk of clinically relevant dysglycemia for fluoroquinolones. References Absi, M., Ghareeb, H., Khalil, A., et al., 2013. The effect of levofloxacin and moxifloxacin on cardiovascular functions of rats with streptozotocin-induced diabetes. Diab. Vasc.Dis. Res. 10, 65–71. https://doi.org/10.1177/1479164112445755. Agius, L., 2007. New hepatic targets for glycaemic control in diabetes. Best Pract. Res.Clin. Endocrinol. Metab. 21, 587–605. Andrikopoulos, S., Rosella, G., Gaskin, E., et al., 1993. Impaired regulation of hepatic fructose-1,6-bisphosphatase in the New Zealand obese mouse model of NIDDM.Diabetes. 42, 1731–1736. Aspinall, S.L., Good, C.B., Jiang, R., 2009. Severe dysglycemia with the fluoroquinolones: a class effect? Clin. Infect. Dis. 49, 402–408. https://doi.org/10.1086/600294. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 37, 911–917. Chen, Z., Sheng, L., Shen, H., 2012. Hepatic TRAF2 regulates glucose metabolism through enhancing glucagon responses. Diabetes. 61, 566–573. https://doi.org/10.2337/ db11-0474. Chung, S.T., Chacko, S.K., Sunehag, A.L., et al., 2015. Measurements of gluconeogenesis and glycogenolysis: a methodological review. Diabetes 64, 3996–4010. https://doi. org/10.2337/db15-0640. Davies, G.F., Khandelwal, R.L., Roesler, W.J., et al., 1999. Troglitazone inhibits expression of the phosphoenolpyruvate carboxykinase gene by an insulin-independent mechanism. Biochim. Biophys. Acta 1451, 122–1231. Dickens, M., Svitek, C.A., Culbert, A.A., et al., 1998. Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin. J.Biol. Chem. 273, 20144–20149. Drozak, J., Miecznik, A., Jarzyna, R., et al., 2008. The inhibition of gluconeogenesis by gatifloxacin may contribute to its hypoglycaemic action. Eur. J. Pharmacol. 594, 39–43. https://doi.org/10.1016/j.ejphar.2008.07.050. Frothingham, R., 2005. Glucose homeostasis abnormalities associated with use of gatifloxacin. Clin. Infect. Dis. 41, 1269–1276. Gabbay, R.A., Sutherland, C., Gnudi, L., et al., 1996. Insulin regulation of phosphoenolpyruvate carboxykinase gene expression does not require activation of the Ras/ mitogen-activated protein kinase signaling pathway. J. Biol. Chem. 271, 1890–1897. Gerich, J.E., Meyer, C., Woerle, H.J., et al., 2001. Renal gluconeogenesis: its importance in human glucose homeostasis. Diabetes Care 24, 382–391. Ghaly, H., Kriete, C., Sahin, S., et al., 2009. The insulinotropic effect of fluoroquinolones. Biochem. Pharmacol. 77, 1040–1052. https://doi.org/10.1016/j.bcp.2008.11.019. Gidh-Jain, M., Zhang, Y., van Poelje, P.D., et al., 1994. The allosteric site of human liver fructose-1,6-bisphosphatase. Analysis of six AMP site mutants based on the crystal structure. J. Biol. Chem. 269, 27732–27738. Graumlich, J.F., Habis, S., Avelino, R.R., et al., 2005. Hypoglycemia in inpatients after gatifloxacin or levofloxacin therapy: nested case-control study. Pharmacotherapy. 25, 1296–1302. Ishiwata, Y., Sanada, Y., Yasuhara, M., 2006. Effect of gatifloxacin on serum glucose concentration in normal and diabetic rats. Biol. Pharm. Bull. 29, 527–531. Kebede, M., Favaloro, J., Gunton, J.E., et al., 2008. Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function. Diabetes. 57, 1887–1895. https://doi.org/10.2337/db07-1326. Kim, Y.D., Park, K.G., Lee, Y.S., et al., 2008. Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP. Diabetes. 57, 306–314. Lewis, R.J., Mohr III, J.F., 2008. Dysglycaemias and fluoroquinolones. Drug Saf. 31, 283–292. Maeda, N., Tamagawa, T., Niki, I., et al., 1996. Increase in insulin release from rat pancreatic islets by quinolone antibiotics. Br. J. Pharmacol. 117, 372–376. Mehlhorn, A.J., Brown, D.A., 2007. Safety concerns with fluoroquinolones. Ann.Pharmacother. 41, 1859–1866. Mohr, J.F., McKinnon, P.S., Peymann, P.J., et al., 2005. A retrospective, comparative evaluation of Sodium Pyruvate dysglycemias in hospitalized patients receiving gatifloxacin, levofloxacin, ciprofloxacin, or ceftriaxone. Pharmacotherapy. 25, 1303–1309.
Nagai, M., Nagata, S., Yamagishi, N., et al., 2010. Clinicopathological aspect of dysglycemia in naive and diabetic rats induced by the fluoroquinolone antibacterial gatifloxacin. J. Vet. Med. Sci. 72, 567–573.
Owens Jr., R.C., Ambrose, P.G., 2000. Clinical use of the fluoroquinolones. Med. Clin. North Am. 84, 1447–1469.
Ramnanan, C.J., Edgerton, D.S., Rivera, N., et al., 2010. Molecular characterization of insulin-mediated suppression of hepatic glucose production in vivo. Diabetes. 59, 1302–1311. https://doi.org/10.2337/db09-1625.
Register, T.C., 2009. Primate models in women’s health: inflammation and atherogenesis in female cynomolgus macaques (Macaca fascicularis). Am. J. Primatol. 71, 766–775. https://doi.org/10.1002/ajp.20722.
Roden, M., Bernroider, E., 2003. Hepatic glucose metabolism in humans—its role in health and disease. Best Pract. Res. Clin. Endocrinol. Metab. 17, 365–383.
Rui, L., 2014. Energy metabolism in the liver. Compr. Physiol. 4, 177–1797. https://doi. org/10.1002/cphy.c130024.
Saraya, A., Yokokura, M., Gonoi, T., et al., 2004. Effects of fluoroquinolones on insulin secretion and beta-cell ATP-sensitive K+ channels. Eur. J. Pharmacol. 497, 111–117. Turnidge, J., 1999. Pharmacokinetics and pharmacodynamics of fluoroquinolones. Drugs.58 (Suppl. 2), 29–36.
Uchida, A., Sasaguri, H., Kimura, N., et al., 2012. Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43. Brain. 135, 833–846. https://doi.org/10.1093/brain/awr348.
Van Bambeke, F., Michot, J.M., Van Eldere, J., et al., 2005. Quinolones in 2005: an update. Clin. Microbiol. Infect. 11, 256–280.
Yabe, K., Yamamoto, Y., Suzuki, T., et al., 2019. Functional and morphological characteristics of pancreatic islet lesions induced by quinolone antimicrobial agent Gatifloxacin in rats. Toxicol. Pathol. 47, 35–43. https://doi.org/10.1177/ 0192623318809062.
Yoshimatsu, Y., Ishizaka, T., Chiba, K., et al., 2018. Usefulness of simultaneous and sequential monitoring of glucose level and electrocardiogram in monkeys treated with gatifloxacin under conscious and nonrestricted conditions. Exp. Anim. 67, 281–290. https://doi.org/10.1538/expanim.17-0136.
Zhang, X., Yang, S., Chen, J., et al., 2019. Unraveling the regulation of hepatic gluconeogenesis. Front Endocrinol (Lausanne). 9, 802. https://doi.org/10.3389/fendo.2018.00802.