Dosing Time-Dependent Difference in the Suppressive Effect of Empagliflozin on the Development of Mechanical Pain Hypersensitivity in Diabetic Mice.

A problem for patients with diabetes is the rise of complications, such as peripheral neuropathy, nephropathy, and retinopathy. Among them, peripheral neuropathy, characterized by numbness and/or hypersensitivity to pain in the extremities, is likely to develop in the early stages of diabetes. Empagliflozin (EMPA), a sodium-glucose cotransporter-2 inhibitor, exerts hypoglycemic effects by preventing glucose reabsorption in proximal tubular cells. EMPA can improve cardiovascular and renal outcomes in diabetic patients, but its suppressive effect on the development of diabetic neuropathy remains unclear. In this study, we demonstrated that optimizing the dosing schedule of EMPA suppressed the development of pain hypersensitivity in streptozotocin (STZ)-induced diabetic model mice maintained under standardized light/dark cycle conditions. A single intraperitoneal administration of STZ to mice induced hyperglycemia accompanied by pain hypersensitivity. Although EMPA did not exert anti-hypersensitivity effect on STZ-induced diabetic mice after the establishment of neuropathic pain, the development of pain hypersensitivity in the diabetic mice was significantly suppressed by daily oral administration of EMPA at the beginning of the dark phase. On the other hand, the suppressive effect was not observed when EMPA was administered at the beginning of the light phase. The hypoglycemic effect of EMPA and its stimulatory effect on urinary glucose excretion were also enhanced by the administration of the drug at the beginning of the dark phase. Nocturnal mice consumed their food mainly during the dark phase. Our results support the notion that morning administration of EMPA may be effective in suppressing the development of peripheral neuropathy in diabetic patients. SIGNIFICANCE STATEMENT: Empagliflozin, a sodium-glucose cotransporter-2 inhibitor suppressed the development of neuropathic pain hypersensitivity in streptozotocin-induced diabetic model mice in a dosing time-dependent manner.

Prostaglandin F2α Affects the Cycle of Clock Gene Expression and Mouse Behavior.

Prostaglandins are bioactive compounds, and the activation of their receptors affects the expression of clock genes. However, the prostaglandin F receptor (Ptgfr) has no known relationship with biological rhythms. Here, we first measured the locomotor period lengths of Ptgfr-KO (B6.129-Ptgfrtm1Sna) mice and found that they were longer under constant dark conditions (DD) than those of wild-type (C57BL/6J) mice. We then investigated the clock gene patterns within the suprachiasmatic nucleus in Ptgfr-KO mice under DD and observed a decrease in the expression of the clock gene cryptochrome 1 (Cry1), which is related to the circadian cycle. Moreover, the expression of Cry1, Cry2, and Period2 (Per2) mRNA were significantly altered in the mouse liver in Ptgfr-KO mice under DD. In the wild-type mouse, the plasma prostaglandin F2α (PGF2α) levels showed a circadian rhythm under a 12 h cycle of light-dark conditions. In addition, in vitro experiments showed that the addition of PTGFR agonists altered the amplitude of Per2::luc activity, and this alteration differed with the timing of the agonist addition. These results lead us to hypothesize that the plasma rhythm of PGF2α is important for driving clock genes, thus suggesting the involvement of PGF2α- and Ptgfr-targeting drugs in the biological clock cycle.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

Senescence-induced alteration of circadian phagocytic activity of retinal pigment epithelium cell line ARPE-19.

Renewal of retinal photoreceptor outer segments is conducted through daily shedding of distal photoreceptor outer segment tips and subsequent their phagocytosis by the adjacent retinal pigment epithelium (RPE) monolayer. Dysregulation of the diurnal clearance of photoreceptor outer segment tips has been implicated in age-related retinal degeneration, but it remains to be clarified how the circadian phagocytic activity of RPE cells is modulated by senescence. In this study, we used the human RPE cell line ARPE-19 to investigate whether hydrogen peroxide (H2O2)-induced senescence in ARPE-19 cells alters the circadian rhythm of their phagocytic activity. After synchronization of the cellular circadian clock by dexamethasone treatment, the phagocytic activity of normal ARPE-19 cells exhibited significant 24-h oscillation, but this oscillation was modulated by senescence. The phagocytic activity of senescent ARPE-19 cells increased constantly throughout the 24-h period, which still exhibited blunted circadian oscillation, accompanied by an alteration in the rhythmic expression of circadian clock genes and clock-controlled phagocytosis-related genes. The expression levels of REV-ERBα, a molecular component of the circadian clock, were constitutively increased in senescent ARPE-19 cells. Furthermore, pharmacological activation of REV-ERBα by its agonist SR9009 enhanced the phagocytic activity of normal ARPE-19 cells and increased the expression of clock-controlled phagocytosis-related genes. Our present findings extend to understand the role of circadian clock in the alteration of phagocytic activity in RPE during aging. Constitutive enhancement of phagocytic activity of senescent RPE may contribute to age-related retinal degeneration.

Estradiol regulation of P-glycoprotein expression in mouse kidney and human tubular epithelial cells, implication for renal clearance of drugs.

P-glycoprotein (P-gp/ABCB1) is an ATP-binding cassette drug efflux transporter expressed in a variety of tissues that affects the pharmacokinetic disposition of many drugs. Although several studies have reported gender-dependent differences in the expression of P-gp, the role of sex hormones in regulating the expression of P-gp and its transport activity has not been well understood. In this study, we demonstrated that 17ß-estradiol has the ability to induce the expression of P-pg in mouse kidneys and cultured human renal proximal tubular epithelial cells. After intravenous injection of a typical P-gp substrate, digoxin, renal clearance in female mice was approximately 2-fold higher than that in male mice. The expression of murine P-gp and its mRNA (Abcb1a and Abcb1b) were also higher in female mice than in male mice. The expression of P-gp in cultured renal tissues prepared from female and male mice was significantly increased by 17ß-estradiol, but not testosterone. Similar 17ß-estradiol-induced expression of P-gp was also detected in cultured human tubular epithelial cells, accompanied by the enhancement of its transport activity of digoxin. The present findings suggest the contribution of estradiol to female-predominant expression of P-gp in renal cells, which is associated with sex-related disparities in the renal elimination of digoxin.

Epigenetic repression of de novo cysteine synthetases induces intra-cellular accumulation of cysteine in hepatocarcinoma by up-regulating the cystine uptake transporter xCT.

BACKGROUND: The metabolic reprogramming of amino acids is critical for cancer cell growth and survival. Notably, intracellular accumulation of cysteine is often observed in various cancers, suggesting its potential role in alleviating the oxidative stress associated with rapid proliferation. The liver is the primary organ for cysteine biosynthesis, but much remains unknown about the metabolic alterations of cysteine and their mechanisms in hepatocellular carcinoma cells. METHODS: RNA-seq data from patients with hepatocarcinoma were analyzed using the TNMplot database. The underlying mechanism of the oncogenic alteration of cysteine metabolism was studied in mice implanted with BNL 1ME A.7 R.1 hepatocarcinoma. RESULTS: Database analysis of patients with hepatocellular carcinoma revealed that the expression of enzymes involved in de novo cysteine synthesis was down-regulated accompanying with increased expression of the cystine uptake transporter xCT. Similar alterations in gene expression have also been observed in a syngeneic mouse model of hepatocarcinoma. The enhanced expression of DNA methyltransferase in murine hepatocarcinoma cells caused methylation of the upstream regions of cysteine synthesis genes, thereby repressing their expression. Conversely, suppression of de novo cysteine synthesis in healthy liver cells induced xCT expression by up-regulating the oxidative-stress response factor NRF2, indicating that reduced de novo cysteine synthesis repulsively increases cystine uptake via enhanced xCT expression, leading to intracellular cysteine accumulation. Furthermore, the pharmacological inhibition of xCT activity decreased intracellular cysteine levels and suppressed hepatocarcinoma tumor growth in mice. CONCLUSIONS: Our findings indicate an underlying mechanism of the oncogenic alteration of cysteine metabolism in hepatocarcinoma and highlight the efficacy of alteration of cysteine metabolism as a viable therapeutic target in cancer.

Suppression of neuropathic pain in the circadian clock-deficient Per2m/m mice involves up-regulation of endocannabinoid system.

Neuropathic pain often results from injuries and diseases that affect the somatosensory system. Disruption of the circadian clock has been implicated in the exacerbation of the neuropathic pain state. However, in this study, we report that mice deficient in a core clock component Period2 (Per2m/m mice) fail to develop tactile pain hypersensitivity even following peripheral nerve injury. Similar to male wild-type mice, partial sciatic nerve ligation (PSL)-Per2m/m male mice showed activation of glial cells in the dorsal horn of the spinal cord and increased expression of pain-related genes. Interestingly, α1D-adrenergic receptor (α1D-AR) expression was up-regulated in the spinal cord of Per2m/m mice, leading to increased production of 2-arachidonoylglycerol (2-AG), an endocannabinoid receptor ligand. This increase in 2-AG suppressed the PSL-induced tactile pain hypersensitivity. Furthermore, intraspinal dorsal horn injection of adeno-associated viral vectors expressing α1D-AR also attenuated pain hypersensitivity in PSL-wild-type male mice by increasing 2-AG production. Our findings reveal an uncovered role of the circadian clock in neuropathic pain disorders and suggest a link between α1D-AR signaling and the endocannabinoid system.

Basis for diurnal exacerbation of neuropathic pain hypersensitivity and its application for drug development.

In addition to diurnal rhythms in physiology and behavior, a variety of pathological conditions also exhibit marked day-night changes in symptom intensity, exemplified by allergic rhinitis, arthritis, asthma, myocardial infarction, congestive heart failure, stroke and chronic pain disorders. Currently, novel therapeutic approaches are facilitated by the development of chemical compounds targeted to key proteins that cause diurnal exacerbation of pathological events. Neuropathic pain is a chronic condition that occurs by tumor-induced nerve compression, cancer cell infiltration into the nerve, diabetes and herpes virus infection. One troublesome hallmark symptom of neuropathic pain is hypersensitivity to normally innocuous stimuli known as 'mechanical allodynia' that is often refractory to common analgesic therapies. Millions of patients worldwide presently endure neuropathic pain. We summarize the recent insights gained into the mechanism of diurnal exacerbation of neuropathic pain hypersensitivity and introduce the strategy of circadian clock-based drug development.

Sulfasalazine alleviates neuropathic pain hypersensitivity in mice through inhibition of SGK-1 in the spinal cord.

Diurnal variations in pain hypersensitivity are common in chronic pain disorders. Temporal exacerbation of neuropathic pain hypersensitivity is dependent on diurnal variations in glucocorticoid secretion from the adrenal glands. We previously demonstrated that spinal expression of serum- and glucocorticoid-inducible kinase-1 (SGK-1) is associated with glucocorticoid- induced exacerbation of pain hypersensitivity, but there are no available strategies to inhibit SGK-1 in the spinal cord. By screening a clinically approved drug library (more than 1,200 drugs), we found that sulfasalazine (SSZ) has inhibitory effects on SGK-1. SSZ is a prodrug composed of 5-aminosalicylic acid and sulfapyridine linked by NN bond, which is therapeutically effective for inflammatory bowel diseases. However, the NN bond in SSZ was necessary for its inhibitory action against SGK-1. Although intrathecal injection of SSZ to nerve-injured mice significantly alleviated mechanical pain hypersensitivity, no significant anti- neuropathic pain effects of SSZ were detected after oral administration due to its low bioavailability and limited spinal distribution, which were associated with efflux by the xenobiotic transporter breast cancer resistance protein (BCRP). Concomitant oral administration of SSZ with febuxostat (FBX), which is an approved drug to inhibit BCRP, improved the distribution of SSZ to the spinal cord. The concomitant oral administration with FBX also increased the anti-neuropathic pain effects of SSZ. Our study revealed a previously unrecognized pharmacological effect of SSZ to alleviate SGK-1-induced painful peripheral neuropathy, and concomitant oral administration of SSZ with FBX may also be a preventative option for diurnal exacerbation of neuropathic pain hypersensitivity.

Diurnal expression of MRP4 in bone marrow cells underlies the dosing-time dependent changes in the oxaliplatin-induced myelotoxicity.

The expression and function of some xenobiotic transporters varies according to the time of day, causing the dosing time-dependent changes in drug disposition and toxicity. Multidrug resistance-associated protein-4 (MRP4), an ATP-binding cassette (ABC) efflux transporter encoded by the Abcc4 gene, is highly expressed in bone marrow cells (BMCs) and protects them against xenobiotics, including chemotherapeutic drugs. In this study, we demonstrated that MRP4 was responsible for the extrusion of oxaliplatin (L-OHP), a platinum (Pt)-based chemotherapeutic drug, from BMCs of mice, and that the efflux transporter expression exhibited significant diurnal variation. Therefore, we investigated the relevance of the diurnal expression of MRP4 in BMCs for L-OHP-induced myelotoxicity in mice maintained under standardized light/dark cycle conditions. After intravenous injection of L-OHP, the Pt content in BMCs varied according to the injection time. Lower Pt accumulation in BMCs was detected in mice after injection of L-OHP at the mid-dark phase, during which the expression levels of MRP4 increased. Consistent with these observations, the myelotoxic effects of L-OHP were attenuated when mice were injected with L-OHP during the dark phase. This dosing schedule also alleviated the L-OHP-induced reduction of the peripheral white blood cell count. The present results suggest that the myelotoxicity of L-OHP is attenuated by optimizing the dosing schedule. Diurnal expression of MRP4 in BMCs is associated with the dosing time-dependent changes in L-OHP-induced myelotoxicity.

Contribution of the clock gene DEC2 to VEGF mRNA upregulation by modulation of HIF1α protein levels in hypoxic MIO-M1 cells, a human cell line of retinal glial (Müller) cells.

PURPOSE: Clock genes are components of the molecular clock. Their malfunction is thought to increase the risk of numerous diseases, including cancer. Vascular endothelial growth factor (VEGF) has a pivotal role in angiogenesis, and its expression levels are controlled by clock genes in tumor cells. Ophthalmic diseases such as age-related macular degeneration, proliferative diabetic retinopathy, and neovascular glaucoma are also associated with abnormal angiogenesis followed by upregulation of VEGF in the eye. In the present study, we aimed to uncover the relationship between clock genes and VEGF in the eye. STUDY DESIGN: Laboratory investigation METHODS: Oxygen-induced retinopathy (OIR) mice were prepared to mimic hypoxic conditions in the eye. Deferoxamine (DFO) was used to mimic hypoxic conditions in human Müller cell line MIO-M1 cells. Expression levels of mRNA and protein were quantified by quantitative reverse transcription polymerase chain reaction and Western blot analysis, respectively. RESULTS: In the retinas of OIR mice, the expression levels of Vegf and the clock gene Dec2 increased transiently, and their temporal profiles were correlated. Knockdown of DEC2 resulted in a significant (26.7%) reduction of VEGF expression in MIO-M1 cells under hypoxia-mimicking conditions induced by DFO (P < .05). Levels of HIF1α protein were also reduced significantly, by 60.2%, in MIO-M1 cells treated with siRNA against the DEC2 gene (P < .05). Moreover, HIF1α levels showed a significant (2.5-fold) increase in MIO-M1 cells overexpressing DEC2 (P < .05). CONCLUSION: DEC2 could upregulate retinal VEGF gene expression through modulation of HIF1α levels under hypoxic conditions.