An Underlying Mechanism for the Altered Hypoglycemic Effects of Nateglinide in Rats with Acute Peripheral Inflammation

Abstract

The hypoglycemic effects of nateglinide (NTG) were examined in rats with acute peripheral inflammation (API) induced by carrageenan treatment, and the mechanisms accounting for altered hypoglycemic effects were investigated. NTG was administered through the femoral vein in control and API rats, and its plasma concentration profile was characterized. The time courses of the changes in plasma glucose and insulin levels were also examined. Although the plasma concentration profile of NTG in API rats was marginally distinguishable from that in control rats, the hypoglycemic effect of NTG was more persistent in API rats than in control rats. In addition, NTG elevated the plasma level of insulin more intensely in API rats than in control rats. Then, the islets of Langerhans were procured by perfusing the pancreas with collagenase solution in control and API rats, and the pancreatic mRNA expression of preproinsulin (Ins1), as well as that of sulfonylurea receptor ABCC8 (Abcc8), were examined. As a result, the expression of preproinsulin and ABCC8 mRNA increased in API rats. These findings suggest that the hypoglycemic effect of NTG was potentiated in API rats due to increased insulin secretion in the pancreas, which was caused by enhanced preproinsulin synthesis and expression of the sulfonylurea receptor.

INTRODUCTION

Inflammation is a protective reaction that occurs when the body is subjected to objectionable stimulus, and it is commonly characterized by redness, pain, and swelling, which are induced by the chemical mediators, such as histamine and cytokines, released from macrophages and/or the affected tissues. From the viewpoint of pharmacokinetics and therapeutics, because those symptoms reflect the increased permeability of the capillary and the accumulation of bodily fluid in the extracellular space, therapeutic compounds distribute in the bodies of patients under inflammatory conditions to a larger extent than in those of healthy subjects. As a result, as often documented for the dosage optimization of antibiotics in critically ill patients,^1–3) the plasma concentration profile of the compounds is altered in such patients, leading to unintended consequences of the pharmacotherapy.

In addition, the biochemical mechanism behind the symptoms, such as increased gene expression and the increased release of chemical mediators, is recognized as another factor influencing pharmacotherapy.⁴⁾ It is reported that hepatic drug-metabolizing activity decreases under inflammatory conditions, inhibiting the elimination of therapeutic compounds and causing an unexpected increase in plasma concentration.^4–6) The decrease in the drug-metabolizing activity under inflammatory conditions is partly due to suppressed expression of the hepatic drug-metabolizing enzymes, in which the pro-inflammatory factors, such as interleukins and tumor necrosis factors, are known to activate the transcription factor, nuclear factor-kappa B (NF-κB).^7–9) Since NF-κB interferes with the nuclear receptor, pregnane X receptor, which promotes the gene expression of drug-metabolizing enzymes, the activation of NF-κB results in suppression of the enzyme expression.^8–11)

Regarding the pharmacokinetic alteration of the therapeutic compounds mentioned above, the altered pharmacokinetics originates from the altered function and expression of proteins. Thus, it is rational to expect that the pharmacological effects of the compounds also alter under inflammatory conditions, in which the function and/or expression of the proteins involved are affected and changed. The altered pharmacological effects may occur with or without the altered pharmacokinetics of the compounds. Therefore, it is required to assess the extent to which inflammatory conditions affect the pharmacological effects of the compounds to perform suitable pharmacotherapy with adequate optimization and individualization. Such an assessment can be conducted with patients in a clinical situation. Still, it is probably difficult because the altered pharmacological effect is usually observed in a convoluted manner with the altered pharmacokinetics of the therapeutic compound. The pharmacological effects should be monitored with the compound’s pharmacokinetic alteration.

Previously, we conducted an in vivo study on the pharmacokinetics of the hypoglycemic agent nateglinide (NTG) and demonstrated that the plasma concentration profile of NTG changed in rats with carrageenan-induced acute peripheral inflammation (API), where the hepatic protein expression of the drug-metabolizing enzyme that is mainly responsible for NTG elimination was suppressed.¹²⁾ From these findings, we speculated that the hypoglycemic effect of NTG also changed in those API rats. In this study, we examined whether the time courses of the blood glucose and insulin levels change with inflammation, and then we assessed the mechanism responsible for the altered pharmacological effects of NTG in API rats. We set up the experiment condition so that the assessment of the pharmacological effects of NTG would not be hindered by its altered pharmacokinetics. Diabetes, if deteriorated, is often accompanied by inflammatory diseases, such as infectious skin disorders and microvascular complications, leading to retinopathy and nephritis. In some cases, the inflammatory diseases may not be so severe, and the plasma concentration profile of the hypoglycemic compounds may not be perceptibly affected. However, this does not mean that the pharmacological effects of the compounds are unchanged. It is, therefore, necessary to clarify how the pharmacological effects of hypoglycemic agents change under inflammatory conditions to provide pharmacotherapy to diabetic patients.

MATERIALS AND METHODS

Materials

NTG and glimepiride were purchased from Tokyo Chemical Industry (Tokyo, Japan). Glimepiride was used as an internal standard for determining NTG, as described later. λ-Carrageenan, a trypsin inhibitor from soybean (3000–6000 units/mg), and collagenase type V (180–190 units/mg) were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Anti-rat α₁-acid glycoprotein (AGP) polyclonal antibody from rabbits (catalog No. 18130) was purchased from Life Diagnostics Inc. (West Chester, PA, U.S.A.). All other chemicals and materials were analytical grade.

Animals

Male Wistar rats (230–340 g) were obtained from Jackson Laboratory Japan (Yokohama, Japan). They were reared and cared for in an air-conditioned room of the animal facility, where the temperature and relative humidity were maintained at 23–24°C and 40–50%. A 12-h diurnal cycle was provided using the lighting units on the ceiling. The rats had free access to a standard laboratory diet and water. All animal experiments were reviewed and approved by the institutional animal ethics committee (OKU-2021384/OKU-2022264). The experiments were then performed as per the guidelines established for animal experimentation at Okayama University.

The rats were randomly assigned to the control and API groups, and the rats in the API group were subcutaneously injected using the λ-carrageenan solution prepared with saline to their hind paws at a dose of 48 mg/kg, 24 h before the experiments.^12,13) The carrageenan-induced API was retrospectively verified based on an increase in the plasma AGP level observed by western immunoblotting (Fig. 1). The plasma specimen for the AGP measurement was procured from the descending aorta of the rat when it was sacrificed in the experiment for the NTG pharmacokinetics or the islets collection described later. Immunoblotting was carried out as previously reported,^12,13) and the migrated proteins were visualized and semi-quantitatively evaluated based on the densitometric readings. The rats in the control group were used in the experiments without any treatments to avoid uncontrolled inflammation.

Fig. 1. Plasma AGP Levels Evaluated by Western Immunoblotting in Control and API Rats

In panel A, the representative migration patterns of the plasma specimens from control (lanes 1 and 2) and API rats (lanes 3 and 4) are presented. The migration protein with the molecular weight corresponding to AGP is indicated using a closed triangle. In panel B, the plasma AGP levels determined by immunoblotting in control and API rats are shown using a box-and-whisker plot with a median bar. The determination was performed using 10 specimens for control and API rats. All data points are shown in circles, where some points are hidden by others. *p < 0.05: significantly different from the control value. AGP, α1-acid glycoprotein; API, acute peripheral inflammation.

Evaluation of the Plasma NTG Concentration–Time Profile in Rats

The plasma concentration–time profile of NTG was evaluated as previously reported.¹²⁾ After each rat was anesthetized with isoflurane, it was fixed to an animal board on the back, and its left femoral vein was exposed for NTG administration. The NTG solution was prepared with dimethyl sulfoxide at a concentration of 50 mg/mL and gently administered in the exposed femoral vein at a dose of 12.5 mg/kg using a 1-mL tuberculin syringe equipped with a 27-gauge needle. The rat was then quickly unfixed and released in a cage with free water access. The precise dose of administered NTG was determined for normalization by comparing the syringe weight before and after administration. Following NTG administration, a series of 200-μL blood collection was carried out from the tail vein at the scheduled time points of 5, 11, 20, 30, 45, and 60 min. For blood collection, the rat was removed from the cage and fixed in the animal holder (ICN-5; ICM, Tsukuba, Japan) around 1 min before the scheduled time points, and the right side of the tail, near the root, was flicked several times using a finger until that part of the tail got slightly swollen. The swollen part of the tail was then treated with 10% lidocaine solution and warmed using a disposable warming device. At the scheduled time point, the tail was wiped with sterilized cotton, and the blood collection from the tail vein was performed by employing a wing blood collection needle (CL-4597; CLEA, Tokyo, Japan) equipped with a heparinized non-glass hematocrit capillary tube. The collected blood was immediately centrifuged at 20000 × g for 10 min at 4 °C to obtain the plasma specimen, and it was stored at –20 °C until used for NTG determination. The specimens were also used to determine the plasma glucose and insulin levels. After blood collection, the rat was released from the holder and placed back in the cage. These animal experiments were conducted for 6 control and 8 API rats.

The dose-normalized plasma concentration-time profile of NTG was first characterized in a model-independent manner, in which the area under the curve (AUC) regarding the NTG profile was calculated using the trapezoidal rule for the 60-min experimental period. The value of total body clearance (CL) was estimated using the dosed amount of NTG divided by the AUC. In addition, the NTG profile was analyzed using the following equation, and the parameters A, α, B, and β in the equation were estimated by fitting:

  

$$Cp\left(t\right)=A\cdot {\mathrm{e}}^{-\alpha \cdot t}+B\cdot {\mathrm{e}}^{-\beta \cdot t}$$(1)

where Cp(t) indicates the dose-normalized plasma concentration of NTG measured at the time point t.

The concentration of NTG in the plasma specimen was spectrometrically determined by HPLC using an octyl silica column (3 μm, 4.6 × 150 mm, InertSustainSwift C8; GL Science, Tokyo, Japan). The plasma specimen was first mixed with 3 volumes of methanol containing glimepiride as an internal standard. The mixture was then vigorously agitated, and it was centrifuged at 20000 × g for 10 min at 4 °C to collect the supernatant. The supernatant was then subjected to evaporation until dried. After evaporation, the remnant was reconstituted with 120 μL of 40% methanol and then subjected to the solid phase extraction column (MonoSpin C18; GL Science). The extraction process with the column was carried out following the manufacturer’s instructions. NTG was eluted from the column with 120 μL of 60% methanol. The eluent was subjected to HPLC for the determination, in which the mobile phase prepared with 67% methanol and 33% sodium phosphate buffer (10 mM, pH 2.5) was used at a flow rate of 1.0 mL/min. NTG was detected at a wavelength of 210 nm.

Evaluation of the Pharmacological Effects of NTG in Rats

The pharmacological effects of NTG, or the hypoglycemic effect and the insulin secretory effect of NTG, were evaluated by analyzing the changes in the plasma glucose and insulin levels observed following NTG administration. The plasma glucose and insulin levels were determined using the plasma specimens collected for NTG determination. The hypoglycemic effect of NTG was evaluated using the area over the curve (AOC) and the mean effect time (MET) regarding the time course of the plasma glucose level.¹⁴⁾ The values of the AOC and MET were calculated using the following equations and the trapezoidal rule for the 60-min experimental period:

  

$$\mathrm{A}\mathrm{O}\mathrm{C}=\int \left[G_{\mathrm{r}\mathrm{e}\mathrm{f}}-G\left(t\right)\right]dt$$(2)

  

$$\mathrm{M}\mathrm{E}\mathrm{T}={\displaystyle \frac{1}{\mathrm{A}\mathrm{O}\mathrm{C}}\cdot \int t\left[G_{\mathrm{r}\mathrm{e}\mathrm{f}}-G\left(t\right)\right]dt}$$(3)

where G(t) designates the glucose level in the plasma specimen collected at the time point t. G_ref is the value used as a reference, using the plasma glucose level determined 5 min after NTG administration. This time point was the earliest one at which we could collect the plasma specimen following NTG administration. AOC reflects the extent to which the rat was exposed to the NTG-induced hypoglycemic effects, and MET measures the weighed mean regarding how long the hypoglycemic effects last. The NTG-induced insulin secretion was measured as the AUC of the time course for the plasma insulin level, and it was calculated using the following equation and the trapezoidal rule for the experimental period:

  

$$\mathrm{A}\mathrm{U}\mathrm{C}=\int I\left(t\right)dt$$(4)

where I(t) is the insulin level in the plasma specimen collected at the time point t.

The plasma glucose level was evaluated using the commercially available glucose assay kit (LabAssay Glucose, FUJIFILM Wako Pure Chemical Corporation), which determines glucose in a colorimetrical manner based on the mutarotase/glucose oxidase method. The plasma insulin level was also determined in a colorimetrical manner, using the commercial assay kit (LBIS Rat Insulin enzyme-linked immunosorbent assay (ELISA) kit; FUJIFILM Wako Pure Chemical Corporation) following the manufacturer’s instruction.

Evaluation of the Pancreatic mRNA Expression of Preproinsulin and ABCC8 in Rats

The pancreatic expressions of mRNA for preproinsulin (Ins1) and sulfonylurea receptor ABCC8 (Abcc8) were evaluated using the islets of Langerhans procured from control and API rats. The islets were obtained using the collagenase digestion method. The collagenase solution was prepared at the activity of 125 units/mL with Hanks’ balanced salt solution (HBSS) containing the trypsin inhibitor at the activity of 1200 units/mL (HBSS/TI). The collagen solution was kept warm at 37°C while it was used. After each rat was anesthetized, it was fixed on the back, and an abdominal incision was made along the midline. Then, the intestine was gently and carefully pushed aside with surgical cotton, and the duodenum and the bile duct were confirmed and secured. The rat was then sacrificed, and blood was drawn from the descending aorta using a 10-mL syringe. Next, the opening of the bile duct in the duodenum was pinched closed with serrefine, and the bile duct was cannulated with polyethylene tubing (I.D. 0.58 mm, O.D. 0.96 mm; SP-45, Natsume Seisakusho, Tokyo, Japan). The tubing was used after its lumen was washed with the collagen solution to purge the air. For cannulation, the tubing was inserted downward from the liver-adjacent and uppermost part of the bile duct through the area where the pancreas spread. Then, a 10-mL syringe filled with the collagenase solution was attached to the polyethylene tubing, and the solution was carefully introduced into the pancreas until the pancreas became wholly inflated. The polyethylene tubing was then drawn out, and the whole pancreas was carefully excised. The excised pancreas was briefly rinsed with warmed saline, and the tissue debris and vessels were removed using micro scissors and tissue forceps. After that, the pancreas was placed in a 50-mL conical tube containing the collagenase solution, and the tube was then incubated at 37 °C for 60 min, gently shaking the tube every 10–15 min to facilitate digestion, by which a suspension with the tissues and debris was prepared. After incubation, the digestion process was stopped by adding ice-cold HBSS/TI to the tube. The suspension in the tube was transferred by decantation to another conical tube, which was then subjected to centrifugation at 100 × g for 2 min at 4 °C. The precipitation obtained by centrifugation was washed with ice-cold HBSS/TI three times to remove the collagenase. That is, following centrifugation, the supernatant was discarded by aspiration, and the precipitation was then suspended again with ice-cold HBSS/TI for another round of centrifugation and supernatant removal. This process was repeated three times. Next, the obtained precipitate was resuspended, the suspension was poured onto a coarse fiber pad, and the filtrate was further processed using a nylon mesh cell strainer with a pore size of 100 μm. The cell aggregations retained on the strainer were washed and collected with ice-cold HBSS/TI in a petri dish on ice. Finally, the islets of Langerhans were manually picked up and placed into a micro test tube using a mechanical pipette under the stereomicroscope. The collected islets were briefly washed with ice-cold HBSS/TI by centrifugation and supernatant removal, and then the precipitated islets were suspended with ice-cold HBSS containing 20% glycerol and stored at –60°C. The islets were collected from 4 control and 5 API rats.

Then, total RNA was extracted from the pooled specimen of the islets of Langerhans, and the mRNA expression was evaluated. Briefly, the islet specimens collected and stored as described above were thawed and resuspended, and they were centrifuged at 300 × g for 5 min at 4 °C to obtain the precipitate. After that, 2 mg of each islet specimen was weighed and supplied to prepare the pooled specimens of the control and API rats. From the pooled specimen, total RNA was extracted using the commercially available RNA extraction kit (FastGene RNA Basic kit; NIPPON Genetics, Tokyo, Japan), and then the reverse transcription reaction was performed using the commercial kit (ReverTra Ace qPCR RT kit; TOYOBO, Osaka, Japan). The kits were handled according to the manufacturers’ instructions. The reaction product of the reverse transcription was used for the semi-quantitative evaluation of the pancreatic mRNA expression of Ins1 and Abcc8, from which the mRNA expression of glyceraldehyde-3-phosphate dehydrogenase (Gapdh) was also evaluated, and the expressions of Ins1 and Abcc8 were handled relative to that of Gapdh.¹⁵⁾ The expression was evaluated by real-time PCR in quadruplicate using the commercial kit (THUNDERBIRD SYBR qPCR Mix, TOYOBO).¹⁵⁾ The primer pairs used in the evaluation are listed in Table 1.^16–18)

Table 1. Primer Pairs Used for the Evaluation of Pancreatic mRNA Expression in Rats

mRNA	Forward primer	Reverse primer	Location	Amplicon size (bp)	Reference
Ins1	5′-aca gca cct ttg tgg tcc-3′	5′-gga ctc agt tgc agt tc-3′	138‒394	257	Tillmar et al.16)
Abcc8	5′-tga agc aac tgc ctc cat c-3′	5′-gaa gct ttt ccg gct tgt c-3′	4586‒4767	182	Chen et al.17)
Gapdh	5′-gtt acc agg gct gcc ttc tc-3′	5′-ggg ttt ccc gtt gat gag c-3′	121‒288	168	Naruhashi et al.18)

Statistical Analysis

Data are presented as means ± standard error (S.E.) if they were assumed to be normally distributed. The differences between control and API groups were evaluated using Student’s t-test, and p < 0.05 was considered significant. Data that were not assumed to be normally distributed were summarized as the median and quartiles and expressed using a box-and-whisker plot.¹⁹⁾ The differences between the two groups were examined using the Mann–Whitney U-test, and p < 0.05 was considered significant.

RESULTS

Evaluation of the Plasma NTG Concentration–Time Profile in Control and API Rats

As shown in Fig. 2, the plasma concentration of NTG following intravenous administration decreased in a two-exponential manner in control and API rats. The concentration profiles in API rats were barely distinguishable from those in control rats (Fig. 2B), although some pharmacokinetic alterations were indicated in the model-dependent analysis (Table 2). In the model-independent analysis, the differences in AUC and CL values were not significant (Table 2).

Fig. 2. Plasma Concentration Profiles of NTG in Control (Panel A) and API Rats (Panel B)

NTG was intravenously administered in the left femoral vein at a dose of 12.5 mg/kg, followed by blood collection from the tail vein. In both panels, all the plasma profiles given in the experiments are presented using gray symbols and lines, with the mean profile indicated using a black line. In panel B, the mean profile of the control rats shown in panel A is indicated using a dotted line. NTG, nateglinide; API, acute peripheral inflammation.

Table 2. Pharmacokinetic Parameters Characterizing the Plasma Concentration–Time Profiles of NTG in Control and API Rats

Parameters	Control ratsa)	API ratsb)
AUC ( μg·min/mL)	933.1 ± 46.1	992.8 ± 69.2
CL (mL/min/kg)	13.0 ± 0.6	12.5 ± 0.8
A (μg/mL)	58.4 ± 2.0	74.1 ± 0.2c)
B (μg/mL)	11.9 ± 2.4	1.8 ± 0.3c)
α (min–1)	0.098 ± 0.003	0.082 ± 0.005
β (min–1)	0.032 ± 0.002	0.001 ± 0.004

NTG, nateglinide; API, acute peripheral inflammation; AUC, area under the curve; CL, total body clearance.

^a)Data are shown as the mean ± S.E. from 6 experiments. ^b)Data are shown as the mean ± S.E. from 8 experiments. ^c)p < 0.05, significantly different from the corresponding value in the control rats.

Evaluation of the Pharmacological Effects of NTG in Control and API Rats

The hypoglycemic effects of NTG associated with its plasma concentration profile were examined in control and API rats. As shown in Fig. 3, although the observed values widely varied, NTG induced a decrease in the plasma glucose level in control and API rats. However, the plasma glucose level change differed between control and API rats. That is, the decreased glucose level in control rats gradually recovered at a late phase of the experimental period (Fig. 3A). Still, such a recovery was barely observed in API rats (Fig. 3B). These observations were assessed quantitatively by calculating the AOC and MET. No difference in the AOC values was detected between control and API rats (Fig. 4A). Still, the MET value was larger in API rats than in control rats (Fig. 4B), reflecting the observation that the hypoglycemic effect of NTG was more persistent in API rats.

Fig. 3. Changes in the Plasma Glucose Levels Following Intravenous NTG Administration in Control (Panel A) and API Rats (Panel B)

The plasma glucose levels were determined in the specimens obtained by scheduled blood collection during the characterization of the plasma NTG concentration profiles. In both panels, all the plasma glucose levels examined in rats are shown using gray symbols and lines, and the mean profile for those glucose levels is also indicated using a black line. NTG, nateglinide; API, acute peripheral inflammation.

Fig. 4. Comparison of the Hypoglycemic Effects of NTG in Control and API Rats

In panel A, the values of the area over the hypoglycemic time course obtained following the intravenous NTG administration in control and API rats are indicated using a whisker-and-box plot, and all data are presented using circles. In panel B, the values of the mean hypoglycemic effect time of NTG in control and API rats are indicated using a box-and-whisker plot with a median bar. All data are shown using circles. * p < 0.05, significantly different from the control value. AOC, area over the curve regarding the time course of the plasma glucose level; MET, mean effect time regarding the time course of the plasma glucose level; API, acute peripheral inflammation; NTG, nateglinide.

The hypoglycemic effects of NTG were then evaluated by measuring the plasma insulin levels in control and API rats. After NTG administration, the plasma insulin level tended to rise to a higher extent in API rats than in control rats (Figs. 5A, B). Reflecting these observations, the AUC value for the plasma insulin level was larger in API rats than in control rats (Fig. 5C). It appears that NTG induced insulin secretion more potently in API rats.

Fig. 5. Changes in the Plasma Insulin Levels Following Intravenous NTG Administration

The changes in the insulin levels observed in control and API rats are shown in panels A and B, respectively. In panel C, the values of the area under the curve regarding the plasma insulin levels obtained after NTG administration in control and API rats are presented using a box-and-whisker plot with a median bar, and all data are indicated using circles. The median bar is also shown in the box. * p < 0.05, significantly different from the control value. AUC, the area under the curve regarding the time course of the plasma insulin level; API, acute peripheral inflammation. NTG, nateglinide.

Evaluation of the Pancreatic mRNA Expression of Preproinsulin and ABCC8 with NTG Administration in Control and API Rats

With the observation of the insulin level rising to a higher extent in API rats, we then assessed whether the preproinsulin synthesis was altered in API rats by considering the collected islets of Langerhans. Furthermore, the pancreatic expression of the sulfonylurea receptor was evaluated. NTG exerts the hypoglycemic effects through its association with the receptor on pancreatic β cells.²⁰⁾ As shown in Fig. 6A, the mRNA expression of Ins1 markedly increased in API rats compared with that in control rats, suggesting that preproinsulin synthesis was promoted in API rats. Additionally, the mRNA expression of Abcc8 increased in API rats (Fig. 6B). With increased expression of the sulfonylurea receptor, NTG seemed to be more efficiently associated with the pancreatic β cells to exert the hypoglycemic effect in API rats.

Fig. 6. Comparison of the mRNA Expression of Preproinsulin (Ins1, Panel A) and Sulfonylurea Receptor ABCC8 (Abcc8, Panel B) in Control and API Rats

The mRNA expression was examined in the islets of Langerhans procured by pancreatic collagenase perfusion. The mRNA expression was evaluated using the common logarithm of fold change over Gapdh mRNA. The results are presented using a box-and-whisker plot with a media bar, and all data are indicated using circles. * p < 0.05, significantly different from the control value. API, acute peripheral inflammation.

DISCUSSION

In this study, we examined the effects of inflammation on the pharmacological effects of therapeutic compounds in rats, aiming to show that the pharmacological effects of the compounds change, even if the alterations in their pharmacokinetics under inflammatory conditions are small. NTG was employed as the model compound because its hypoglycemic effects are readily measured, and we had some understanding of the pharmacokinetics of NTG in API rats.¹²⁾ Referring to our previous findings,^12,13) API rats were prepared with carrageenan, employing a smaller dose than that used in our previous study, such that the carrageenan-induced inflammation would have little influence on the plasma concentration profile of NTG. As shown in Fig. 2, the plasma concentration profiles in API rats were almost indistinguishable from those in control rats, as expected. However, the values of some of the pharmacokinetic parameters reached significance in API rats (Table 2). On the other hand, the hypoglycemic effects of NTG appeared to be affected in API rats. That is, NTG lowered the plasma glucose level for a longer period in API rats than in control rats (Fig. 3), and thus, the MET was larger in API rats than in control rats (Fig. 4B). These findings indicated that under inflammatory conditions, the pharmacological effects of NTG vary without exhibiting obvious alterations in its plasma concentration profile.

Considering the fact that the plasma glucose level was lowered for a longer period in API rats (Figs. 3 and 4), while the plasma concentration profiles of NTG were nearly the same in control and API rats (Fig. 2), the hypoglycemic effects of NTG were assumed to potentiate with inflammation. Supporting the assumption, it was additionally revealed that the insulin level following NTG administration was noticeably elevated in API rats (Fig. 5). It was demonstrated that insulin secretion was more intensively facilitated in API rats. However, the plasma concentration profile of NTG in API rats was not different from that in control rats. These things suggested that the NTG-induced insulin secretion was likely enhanced in API rats, causing the potentiation of the hypoglycemic effects of NTG.

Since NTG is associated with the sulfonylurea receptor in the islets of Langerhans for insulin secretion,²¹⁾ we then examined the mRNA expression of the sulfonylurea receptor ABCC8, as well as that of preproinsulin in the pancreas, to elucidate the mechanisms underlying the enhanced insulin secretion. For this purpose, we procured the islets of Langerhans from control and API rats. As shown in Fig. 6, the pancreatic expressions of Abcc8 and Ins1 are both increased with inflammation, suggesting that the hypoglycemic effects of NTG were potentiated in API rats, considering that the sulfonylurea receptor is expressed more in API rats than in control rats and preproinsulin synthesis in the pancreas is promoted with inflammation. NTG appeared to exert its hypoglycemic effect more effectively in API rats than in control rats, which probably explains why the hypoglycemic effects of NTG were potentiated while its plasma concentration was barely altered in API rats.

The gene expression of insulin is precisely regulated by various mechanisms and transcription factors.²¹⁾ Among them, the process involving the pancreatic and duodenal homeobox factor-1 and its activation by protein kinase C (PKC) may be mainly related to an increase in the Ins1 mRNA expression observed in this study.^22,23) That is, carrageenan is known to induce inflammation through a process mediated by pattern-recognizing Toll-like receptors, and proinflammatory cytokines are produced.^9,24) The cytokines, some of which may reach the pancreas through the bloodstream, trigger the cellular signaling pathways, in which various protein kinases, including the PKC family, are activated following activation of Janus kinase and phosphoinositide 3-kinase (PI3K).²⁵⁾ The increased expression of Abcc8 in API rats may be partly accounted for because of the fact that the carrageenan-induced inflammation triggers the signaling pathway involving PI3K. Abcc8 expression is regulated by the atypical protein kinase C zeta (PKC ζ),²⁶⁾ and the member proteins of the PKC family are activated through the PI3K-involving signaling process.²⁷⁾

Several issues remain to be clarified in this study. The hypoglycemic effect of NTG, but not its plasma concentration, was affected under the current inflammatory condition, which suggests that the processes regulating the blood glucose level are influenced by the proinflammatory cytokines. In contrast, those involving the NTG pharmacokinetics, such as the protein expression process of the hepatic drug-metabolizing enzymes, are unaffected by the inflammatory condition. We could not elucidate the mechanisms responsible for these responses, but evaluating the inducibility and suppressibility of mRNA expressions may provide insight in future studies. Moreover, it remains unclear whether the inflammation-induced alteration is unique to NTG or applies generally to various therapeutic compounds. We believe that if inflammation influences the mRNA expression and leads to a non-negligible change in the pharmacological process of the compound, inflammation-induced alterations are expected. Most therapeutic compounds fit this case. Besides the patients with progressed diabetes, those suffering from chronic inflammatory diseases, such as rheumatoid arthritis and ulcerative colitis, are considered to receive pharmacotherapy under inflammatory conditions. The pharmacological effects of the compounds in those patients probably vary with their inflammatory conditions. In addition, it is not unusual in daily life to experience intensive inflammation by having an injury, aphthous stomatitis, or other unintentional events, including a herpes virus infection. While the pharmacokinetics of the therapeutic compounds may be unaffected and unchanged in this case, their pharmacological effects are probably different from those expected.

In this study, we demonstrated that the pharmacological effects of the hypoglycemic agent NTG were altered under inflammatory conditions without a noticeable change in its plasma concentration profile. It is necessary to consider that the pharmacological effects of therapeutic compounds may be exerted in an unexpected way in patients under inflammatory conditions.

Acknowledgments

This work was supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (19K07220).

Conflict of Interest

The authors declare no conflict of interest.

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