Paliperidone-Induced Acute Hyperglycemia Is Caused by Adrenaline Secretion via the Activation of Hypothalamic AMP-Activated Protein Kinase

Abstract

Although paliperidone-related hyperglycemia has been extensively examined, the underlying mechanisms have not yet been elucidated. We investigated the effects of a single intravenous injection of paliperidone (0.2, 0.4, or 0.6 mg/kg) on serum concentrations of glucose and other endogenous metabolites in rats. We also examined the effects of a single intravenous injection of paliperidone (0.4 mg/kg) on AMP-activated protein kinase (AMPK) activity in the hypothalamus and liver. To clarify the relationship between AMPK activity and adrenaline secretion, the effects of berberine, which inhibits hypothalamic AMPK, on paliperidone-induced hyperglycemia were assessed. Significant increases were observed in serum glucose, adrenaline, and insulin concentrations following intravenous injections of paliperidone at doses of 0.4 and 0.6 mg/kg. A propranolol pretreatment attenuated paliperidone-induced increases in serum concentrations of glucose, but not adrenaline. Significant increases were also noted in phosphorylated AMPK concentrations in the hypothalamus following the administration of paliperidone at a dose of 0.4 mg/kg. A berberine pretreatment attenuated paliperidone-induced increases in blood concentrations of glucose, adrenaline, and insulin and phosphorylated AMPK concentrations in the hypothalamus. Collectively, the present results demonstrated that an acute treatment with paliperidone induced hyperglycemia, which was associated with the effects of hypothalamic AMPK activation on the secretion of adrenaline.

INTRODUCTION

The greater broad-spectrum efficacy and lower prevalence of extrapyramidal symptoms associated with atypical antipsychotics, such as olanzapine, quetiapine, and paliperidone, than with typical antipsychotics has resulted in their wide use in clinical therapy.¹⁾ However, hyperglycemia induced by these drugs in therapeutic applications has been an issue of great concern in recent years.^2–4) Emergency safety information for olanzapine and quetiapine was issued in Japan in 2002 due to reported cases of diabetic ketoacidosis and diabetic coma in clinical treatment. Paliperidone, a newer atypical antipsychotic, has been shown to exert promising therapeutic effects with a lower prevalence of extrapyramidal symptoms and motor dysfunction^5,6); however, hyperglycemia is a potential adverse event.⁷⁾ A previous study demonstrated that paliperidone significantly increased glucose concentrations over those with a placebo in both short-term and long-term trials.⁸⁾

We previously examined the mechanisms underlying the development of hyperglycemia in rats treated with atypical antipsychotics. A single intravenous injection of olanzapine increased serum glucose concentrations over those of adrenaline, whereas clozapine increased serum glucose concentrations over those of both adrenaline and glucagon.^9,10) Based on these findings, the mechanisms underlying drug-induced hyperglycemia by atypical antipsychotics appear to differ. However, the molecular processes underlying the relationships among atypical antipsychotics, endogenous metabolites, and hyperglycemia remain unclear. AMP-activated protein kinase (AMPK) is crucially involved in glucose metabolism, with its activation in the liver inhibiting glucose metabolism and that in the hypothalamus promoting the production of glucose.¹¹⁾ Ikegami et al. previously reported that an intracerebroventricular injection of olanzapine activated hypothalamic AMPK, which, in turn, promoted the hepatic production of glucose via sympathetic efferent nerves.¹²⁾ These effects of activated hypothalamic AMPK were attributed to the antagonism of histamine H₁, dopamine D₂, and α₁-adrenergic receptors¹³⁾; therefore, the suppression of the hypothalamic AMPK pathway may reduce the risk of hyperglycemia in mice treated with atypical antipsychotics.¹⁴⁾ However, the effects of the systemic administration of olanzapine on hepatic AMPK have not yet been clarified. The mechanisms underlying hyperglycemia induced by atypical antipsychotics appear to be more complex than expected. Moreover, limited information is currently available on the mechanisms underlying paliperidone-induced hyperglycemia.

Therefore, the present study examined the effects of a single intravenous dose of paliperidone on the serum concentrations of several endogenous metabolites as well as hepatic and hypothalamic AMPK activities in rats. The effects of the combination of paliperidone and berberine, which inhibits hypothalamic AMPK,¹⁵⁾ were also assessed. To the best of our knowledge, this is the first study to systemically investigate the mechanisms underlying paliperidone-induced hyperglycemia based on the relationships between hypothalamic and hepatic AMPK and endogenous metabolites in vivo, which may provide an important theoretical basis for effective clinical treatments with atypical antipsychotics.

MATERIALS AND METHODS

Animals

The present study was conducted using male Wistar rats (Japan SLC, Hamamatsu, Japan). Rats were fasted overnight in a controlled environment prior to the cannulation experiment. During the cannulation experiment, fasted rats were anesthetized with isoflurane and indwelling cannulas were inserted into the left carotid artery and jugular vein for blood collection and intravenous injections, respectively. The Institutional Animal Care and Use Committee of Tokyo Medical and Dental University approved all animal experiments (Approval Nos. A2020-060A, A2021-149A, and A2021-252A).

Materials

Paliperidone, berberine, and clozapine (the internal standard (IS) in the paliperidone assay) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ten milligrams of paliperidone dissolved in 333 µL of 0.1 M hydrochloric acid was adjusted to doses of 0.2, 0.4, and 0.6 mg/mL using 10 mM sodium acetate buffer (pH 4.0) prior to its intravenous administration. Berberine (40 mg/mL) was dissolved into a liquid suspension using saline for its oral administration. (±)-Propranolol hydrochloride and (1R,2R)-(+)-1,2-diphenylethylenediamine were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan), and (±)-propranolol hydrochloride (0.8 mg/mL) was dissolved in saline. (R)-(−)-Epinephrine and isoproterenol hydrochloride (the IS in the (R)-(−)-epinephrine assay) were obtained from FUJIFILM Wako Pure Chemical Corporation and potassium ferricyanide (III) from Sigma-Aldrich (St. Louis, MO, U.S.A.). All chemicals used were of analytical grade.

Effects of a Single Intravenous Injection of Paliperidone on Blood Concentrations of Glucose and Other Endogenous Metabolites

One hour after recovery from anesthesia, rats were administered a single intravenous dose of paliperidone at 0.2, 0.4, or 0.6 mg/kg for 10 s. Control rats were administered with an equivalent volume of 10 mM sodium acetate buffer (pH 4.0). Blood collection (450 µL) was performed 0, 15, 30, 60, 120, and 180 min after the administration of paliperidone for the measurement of serum glucose, adrenaline, insulin, glucagon, and paliperidone concentrations. Blood samples were subjected to centrifugation at 2000 × g for 10 min, and the resulting serum was placed into test tubes and stored at −80 °C for later analyses.

Effects of Propranolol on Paliperidone-Induced Increases in Serum Concentrations of Glucose and Other Endogenous Metabolites

Rats were divided into the following groups: propranolol/paliperidone, propranolol/10 mM sodium acetate buffer (pH 4.0), and saline/paliperidone. At least 30 min after recovery from anesthesia, each rat was intravenously administered propranolol (2 mg/kg) or saline for 30 s. Blank blood samples were collected 30 min after the propranolol or saline injection. Paliperidone (0.4 mg/kg) or 10 mM sodium acetate buffer (pH 4.0) was then administered for 10 s, respectively. Blood collection (450 µL) was performed after 15, 30, 60, 120, and 180 min for the measurement of serum glucose, adrenaline, and paliperidone concentrations. All blood samples were subjected to centrifugation at 2000 × g for 10 min, and the resulting serum was placed into test tubes and then stored at −80 °C for later analyses.

Effects of a Single Intravenous Injection of Paliperidone on Hypothalamic and Hepatic AMPK Activities

At least 1 h after recovery from anesthesia, rats received a single intravenous dose of paliperidone at 0.4 mg/kg or 10 mM sodium acetate buffer (pH 4.0) for 10 s. Rats were sacrificed 15 min after the paliperidone injection. The hypothalamus and liver tissues were harvested, rapidly washed with ice-cold Tris-buffered saline (TBS), and then snap-frozen in liquid nitrogen. The flash-frozen hypothalamus and liver tissues were placed into test tubes and stored at −80 °C for later analyses.

Effects of Berberine on Paliperidone-Induced Increases on Hypothalamic and Hepatic AMPK Activities

Rats were divided into the following groups: berberine/paliperidone, berberine/10 mM sodium acetate buffer (pH 4.0), and saline/paliperidone. Each rat was orally administered berberine (400 mg/kg) or saline once a day for 28 d. The same method was used to examine the effects of a single intravenous injection of paliperidone on AMPK activity.

Effects of Berberine on Paliperidone-Induced Increases in Serum Concentrations of Glucose and Other Endogenous Metabolites

Rats were divided into the following groups: berberine/paliperidone, berberine/10 mM sodium acetate buffer (pH 4.0), and saline/paliperidone. Each rat was orally administered berberine (400 mg/kg) or saline orally once a day for 28 d. The same method was used to examine the effects of propranolol on paliperidone-induced increases in the serum concentrations of glucose and other endogenous metabolites.

Analytical Methods

Serum Concentrations of Glucose, Paliperidone, and Other Endogenous Metabolites

A previously reported method was employed to measure serum glucose, adrenaline, insulin, and glucagon concentrations.^9,16) Serum paliperidone concentrations were assessed using the liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay with a slight modification.¹⁷⁾ The LC-MS/MS system consisted of ExionLC AC and Triple Quad 5500 (AB Sciex, Foster City, CA, U.S.A.) and was controlled using Analyst 1.7.2 software. MS/MS conditions are summarized in Table 1. The mobile phase comprised 10 mM ammonium acetate consisting of 0.1% formic acid–acetonitrile (50 : 50, v/v) at a total flow rate of 0.3 mL/min. Stock solutions of paliperidone (1 mg/mL) and clozapine (IS, 1 mg/mL) were prepared in methanol. The calibration curve for paliperidone was prepared by methanol/water (50/50, v/v) and the linear portion was between the concentration range of 1–200 ng/mL. Thirty microliters of serum was mixed with 10 µL of the IS working solution (0.5 µg/mL of clozapine). Extraction of the analyte was performed using 100 µL of acetonitrile–methanol (70 : 30, v/v). This mixture was then vortexed and centrifuged at 15000 × g at room temperature for 5 min to remove the protein pellet. One hundred and twenty microliters of the resulting supernatant organic layer was transferred to a 1-mL polypropylene tube and completely evaporated at 40 °C. Samples were resuspended in 200 µL of the mobile phase mixture and 2 µL was injected into the LC-MS/MS apparatus for analyses. Paliperidone and clozapine (IS) retention times were 0.76 and 0.89 min, respectively. The column was a DOS C₁₈ (100 × 2.1 mm, 2.6 µm, Kinetex) protected by a C₁₈ security guard (4 × 3.0 mm, ID 5 µm) and maintained at 40 °C.

Table 1. Ion Source and Analyte-Dependent MS Parameters

Ion source parameters
Ionization mode	Electrospray ionization
Polarity mode	Positive
Ionspray voltage	5500 V
Capillary temperature	650 °C
Curtain gas	30 psi
Collision gas	8 psi
Ion source gas 1	70 psi
Ion source gas 2	80 psi
Analyte-dependent MS parameters
	Paliperidone	Clozapine (IS)
Precursor ion	427.093 m/z	327.026 m/z
Product ion	207.000 m/z	270.000 m/z
Declustering potential	151 V	156 V
Entrance potential	10 V	10 V
Collision energy	37 V	31 V
Collision cell exit potential	28 V	8 V

Western Blotting

AMPKα and phosphorylated AMPKα concentrations in the hypothalamus and liver tissues were measured by Western blotting. The hypothalamus and liver tissues were homogenized in ice-cold radio immunoprecipitation assay (RIPA) buffer (Nacalai Tesque, Inc. Kyoto, Japan) containing 50 mM Tris–HCl buffer (pH 7.6), 150 mM NaCl, 1% Nonidet P-40 substitute, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulphate. A 1% protease inhibitor cocktail (Nacalai Tesque, Inc.) and 1% phosphatase inhibitor cocktail (Nacalai Tesque, Inc.) were then added. The homogenate was centrifuged at 15000 × g at 4 °C for 20 min, and the resulting supernatant was retained as the total tissue lysate. The Protein Assay BCA Kit (Nacalai Tesque, Inc.) was employed to measure protein concentrations. The dilution of samples to the same protein concentration (3 µg/µL) was performed using RIPA buffer. Loading samples were also diluted to the same protein concentration (15.75 µg/7 µL) using 4× Laemmli sample buffer (containing 10% 2-mercaptoethanol). The separation of proteins was performed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (7.5% gel; Bio-Rad, Hercules, CA, U.S.A.) for 34 min. The Trans-Blot Turbo system (Bio-Rad) was used for 7 min to transfer proteins to polyvinylidene difluoride membranes, which were then rinsed in TBS/T for 2 min and soaked in EveryBlot blocking buffer (Bio-Rad) at room temperature for 5 min with gentle agitation. Membranes were again rinsed in TBS/T for 2 min and immunoblotted with rabbit antibodies for AMPKα (1 : 2500; Cell Signaling Technology, Danvers, MA, U.S.A.) and AMPKα phosphorylated at threonine 172 (Thr-172) (1 : 2500; Cell Signaling Technology) at 4 °C for 16 h with gentle agitation. Membranes were then rinsed three times in TBS/T, washed three times in TBS/T at intervals of 10 min, and incubated in Anti-Rabbit immunoglobulin G (IgG) (1 : 10000; Cell Signaling Technology) at room temperature for 60 min with gentle agitation. After rinsing three times in TBS/T and washing three times in TBS/T at intervals of 10 min, membranes were immersed in Clarity Western ECL Substrate solution (Bio-Rad) for 5 min. The ChemiDoc MP Imaging System (Bio-Rad) was used to detect the antigen-antibody peroxidase complex. Membranes were washed again three times in water, added to WB Stripping Solution Strong (Nacalai Tesque, Inc.) for 15 min with gentle agitation for stripping, and then washed in TBS/T for 5 min and soaked in EveryBlot blocking buffer (Bio-Rad) at room temperature for 5 min with gentle agitation. Membranes were again rinsed in TBS/T for 2 min and incubated with an antibody against β-actin (1 : 10000; Cell Signaling Technology) at room temperature for 1 h with gentle agitation. Membranes were rinsed three times in TBS/T and washed three times in TBS/T at intervals of 10 min. Membranes were then incubated with Anti-mouse IgG (1 : 10000; Cell Signaling Technology) at room temperature for 60 min with gentle agitation. After being rinsed three times in TBS/T and washed three times in TBS/T at intervals of 10 min, membranes were immersed in the Clarity Western ECL Substrate solution (Bio-Rad) for 5 min, and image development was conducted. Image Lab (Bio-Rad) was used to analyze and quantify band intensities. Values for AMPKα and phosphorylated AMPKα in the hypothalamus and liver tissues were normalized by the respective values for β-actin.

Statistical Analysis

Data are shown as means and standard deviations. Statistical analyses were conducted using the Student’s t-test or Tukey–Kramer multiple comparison test. p-Values <0.05 were considered to indicate a significant difference.

RESULTS

Effects of the Single Intravenous Injection of Paliperidone on Blood Concentrations of Glucose and Other Endogenous Metabolites

Dose-dependent increases were observed in serum paliperidone concentrations following the intravenous injection of paliperidone (Fig. 1). Furthermore, significant increases were noted in serum glucose, adrenaline, and insulin concentrations following the injection of paliperidone at doses of 0.4 and 0.6 mg/kg, with similar increases being detected at both doses (Figs. 2A–C). Serum glucagon concentrations were not significantly affected by the intravenous injection of paliperidone at either dose (Fig. 2D).

Fig. 1. Serum Concentration–Time Profiles of Paliperidone after Intravenous Injection of Paliperidone at a Dose of 0.2 mg/kg (■, n = 6), 0.4 mg/kg (●, n = 7), or 0.6 mg/kg (▲, n = 6) in Rats

Fig. 2. Serum Concentration–Time Profiles of (A) Glucose, (B) Adrenaline, (C) Insulin, and (D) Glucagon after Intravenous Injection of Acetate Buffer (○, as Control, n = 4), or Paliperidone at a Dose of 0.2 mg/kg (■, n = 5), 0.4 mg/kg (●, n = 6), or 0.6 mg/kg (▲, n = 5) in Rats

Each point represents the mean ± standard deviation (S.D.). * p < 0.05 vs. control group, ^† p < 0.05 vs. paliperidone at 0.2 mg/kg (the Tukey–Kramer test).

Effects of Propranolol on Paliperidone-Induced Increases in Serum Concentration of Glucose and Other Endogenous Metabolites

Paliperidone-induced increases in blood glucose concentrations were completely inhibited by the propranolol pretreatment (2 mg/kg) (Fig. 3A). Serum adrenaline concentrations increased in the saline/paliperidone and propranolol/paliperidone groups (Fig. 3B). No significant differences were observed in serum paliperidone concentrations in the propranolol/paliperidone group or saline/paliperidone group, except for at 15 min (Fig. 3C).

Fig. 3. Effects of Propranolol (2 mg/kg) on Serum Concentrations of (A) Glucose, (B) Adrenaline, and (C) Paliperidone after Intravenous Injection of Paliperidone (0.4 mg/kg) in Rats, for Rats Treated with Saline/Acetate Buffer (○, as Control, n = 5), Rats Treated with Saline/Paliperidone (●, n = 5), and Rats Treated with Propranolol/Paliperidone (▲, n = 5)

Each point represents the mean ± S.D. * p < 0.05 vs. control group, ^† p < 0.05 vs. propranolol/paliperidone (the Tukey–Kramer test).

Effects of the Single Intravenous Injection of Paliperidone on Hypothalamic and Hepatic AMPK Activities

To confirm the paliperidone-mediated activation of hypothalamic and hepatic AMPK, we examined the effects of paliperidone on the phosphorylation of AMPKα at Thr-172, namely, the activated form of AMPKα. A significant increase was observed in the concentration of AMPKα phosphorylated at Thr-172 in the hypothalamus following the intravenous injection of paliperidone (Fig. 4A), whereas the level of phosphorylated AMPKα in the liver remained unchanged (Fig. 4B).

Fig. 4. Effects of Paliperidone (0.4 mg/kg) on the Amount of AMPKα Phosphorylated at Threonine 172 [pAMPKα (Thr172)] and Total AMPKα in the Rat (A) Hypothalamus and (B) Liver

Samples were collected 15 min after paliperidone (0.4 mg/kg, i.v.) administration. Upper panel: Representative immunoblots for pAMPKα (Thr172) and β-actin in the hypothalamus and liver of rats treated with acetate buffer (as control, n = 7) or paliperidone (n = 6). Middle panel: Representative immunoblots for total AMPKα and β-actin in the hypothalamus and liver of rats treated with acetate buffer (as control, n = 7) or paliperidone (n = 6). Lower panel: Semiquantitative analysis of the intensity of each band. The immunoblots of pAMPKα (Thr172) and total AMPKα were normalized by β-actin. Each column represents the mean ± S.D. * p < 0.05 vs. control group (Student’s t-test).

Effects of Berberine on Paliperidone-Induced Increases in Hypothalamic and Hepatic AMPK Activities

The concentration of AMPKα phosphorylated at Thr-172 in the hypothalamus significantly increased in the saline/paliperidone group, but only slightly increased in the berberine/paliperidone group (Fig. 5A). The concentration of AMPK phosphorylated at Thr-172 in the liver remained unchanged in the berberine/paliperidone and saline/paliperidone groups (Fig. 5B).

Fig. 5. Effects of Berberine (400 mg/kg, Oral, 28 d) on the Amount of AMPKα Phosphorylated at Threonine 172 [pAMPKα (Thr172)] and Total AMPKα in the Rat (A) Hypothalamus and (B) Liver after Paliperidone Administration

Samples were collected 15 min after paliperidone (0.4 mg/kg, i.v.) administration. Upper panel: Representative immunoblots for pAMPKα (Thr172) and β-actin in the hypothalamus and liver of rats treated with berberine/acetate buffer (as control, n = 8), berberine/paliperidone (n = 8), and saline/paliperidone (n = 7). Middle panel: Representative immunoblots for total AMPKα and β-actin in the hypothalamus and liver of rats treated with berberine/acetate buffer (as control, n = 8), berberine/paliperidone (n = 8), and saline/paliperidone (n = 7). Lower panel: Semiquantitative analysis of the intensity of each band. The immunoblots of pAMPKα (Thr172) and total AMPKα were normalized by β-actin. Each column represents the mean ± S.D. * p < 0.05 vs. control group (the Tukey–Kramer test).

Effects of Berberine on Paliperidone-Induced Increases in Serum Concentrations of Glucose and Other Endogenous Metabolites

The berberine pretreatment (400 mg/kg) attenuated paliperidone-induced increases in the blood concentrations of glucose, adrenaline, and insulin (Figs. 6A–C). No changes were observed in serum paliperidone concentrations following the berberine treatment (Fig. 6D).

Fig. 6. Effects of Berberine (400 mg/kg, Oral, 28 d) on Serum Concentrations of (A) Glucose, (B) Adrenaline, (C) Insulin, and (D) Paliperidone after the Intravenous Injection of Paliperidone (0.4 mg/kg) in Rats, for Rats Treated with Berberine/Acetate Buffer (○, as Control, n = 6), Rats Treated with Berberine/paliperidone (▲, n = 8), and Rats Treated with Saline/paliperidone (●, n = 8)

Each point represents the mean ± S.D. * p < 0.05 vs. control group, ^† p < 0.05 vs. berberine/paliperidone (the Tukey–Kramer test).

DISCUSSION

The present study examined the effects of an acute paliperidone treatment on the concentrations of glucose and several endogenous metabolites in rats. Increases were observed in serum glucose concentrations following the administration of paliperidone (Fig. 2A). Furthermore, serum adrenaline and insulin concentrations were increased by the administration of paliperidone, whereas those of glucagon were not (Figs. 2B–D). Paliperidone-induced increases in blood glucose, but not adrenaline, concentrations were completely suppressed by the pretreatment with propranolol (a β-adrenergic antagonist) (Figs. 3A, B). Therefore, adrenaline, but not glucagon, appears to play a role in the development of paliperidone-induced hyperglycemia following the administration of a single dose of paliperidone. These results are consistent with previous findings on olanzapine, but not clozapine, indicating distinct mechanisms for atypical antipsychotic-induced acute hyperglycemia.^9,10) Atypical antipsychotics exert antagonistic effects on a number of receptors, including dopamine, 5-hydroxytryptamine, and muscarinic receptors, and affinity for several receptors varies among these drugs.¹⁸⁾ This may be one of the reasons why atypical antipsychotic drugs induce hyperglycemia through different mechanisms.

Despite a decrease in blood adrenaline levels 60 min after the administration of paliperidone, serum levels of glucose and insulin remained significantly elevated. Boyda et al. reported insulin resistance during paliperidone-induced hyperglycemia.¹⁹⁾ Furthermore, insulin resistance has been implicated in the regulation of AMPK.²⁰⁾ These findings suggest that the single administration of paliperidone induced insulin resistance.

Previous studies examined the therapeutic range of paliperidone.⁵⁾ Liu et al. proposed a therapeutic concentration range of 20–60 ng/mL.²¹⁾ A comparison of D₂ receptor occupancy and serum drug concentrations supported a target of 40–80 ng/mL for paliperidone.²²⁾ In the present study, significant increases were observed in serum glucose concentrations following the administration of paliperidone at doses of 0.4 and 0.6 mg/kg (Fig. 2A), and serum paliperidone concentrations ranged between 92 and 184 ng/mL in a dose-dependent manner 15 min after the administration of paliperidone (Fig. 1). Since serum paliperidone concentrations in the present study were consistent with previously reported therapeutic ranges in clinical practice, blood glucose concentrations need to be carefully monitored in patients receiving paliperidone.

AMPK is a phylogenetic serine/threonine kinase that functions as a master metabolic regulator, and its modulation has attracted the interest of researchers on type 2 diabetes mellitus and metabolic syndrome. AMPK dysregulation was recently closely implicated in the development of insulin receptors and type 2 diabetes mellitus.²³⁾ AMPK may also play important roles in the hypothalamus and liver tissues, with previous findings showing that atypical antipsychotics significantly altered glucose metabolism.²⁴⁾ The inhibition of AMPK in the liver or its activation of AMPK in the hypothalamus may promote glucose production.¹¹⁾ Recent studies found that atypical antipsychotics, such as olanzapine- and clozapine-induced hyperglycemia, were associated with activated hypothalamic AMPK; however, it currently remains unclear whether hyperglycemia induced by these drugs is associated with hepatic AMPK activity.^12–14,25) Therefore, we investigated whether hypothalamic and hepatic AMPK activities contributed to the development of paliperidone-induced hyperglycemia. The concentration of AMPK phosphorylated at Thr-172 significantly increased in the hypothalamus only following the administration of paliperidone (Fig. 4), which demonstrated that paliperidone-induced hyperglycemia is mediated by the activation of hypothalamic AMPK rather than hepatic AMPK in normal rats.

AMPK, which is abundantly expressed in the arcuate and ventromedial hypothalamus, is critical for energy balance and metabolism and is regulated by various neurotransmitters and neuropeptides via receptors targeted by atypical antipsychotics.²⁴⁾ In addition, concentrations of D₂ and D₃ receptors are high in the hypothalamus arcuate nucleus, and their blockade has been suggested to contribute to the deregulation of glucose and lipid metabolism.²⁴⁾ Atypical antipsychotics have been shown to increase hypothalamic AMPK activity, mostly by their function as antagonists of D₂, D₃, α₁, and H₁ receptors.¹⁸⁾ A previous study reported that an intracerebroventricular injection of D₂ antagonists may cause hyperglycemia in mice, which was associated with the activation of hypothalamic AMPK.¹³⁾ The administration of highly selective serotonin (5-HT)_2A antagonists to animals was shown to increase insulin secretion.²⁶⁾ A pharmacodynamic-pharmacoepidemiological investigation also identified a relationship between 5-HT_2C antagonism and antipsychotic-induced hyperglycemia, which was aggravated by simultaneous H₁ antagonism.²⁷⁾ Furthermore, AMPK activated hepatic gluconeogenesis and glycogenolysis by stimulating the sympathetic nervous system, which increased the secretion of hormones, such as glucagon, corticosterone, and adrenaline.²⁸⁾ Paliperidone exhibited strong activity by antagonizing central D_2–4, 5-HT_2A, and α₁ receptors.⁶⁾ Interestingly, a single dose of paliperidone increased adrenaline concentrations and hypothalamic AMPK activity in the present study (Figs. 2B, 4). Therefore, paliperidone-induced hyperglycemia may be associated with the antagonistic effects of D₂, D₃, 5-HT_2A, and α₁ receptors, which activate hypothalamic AMPK and promote the secretion of adrenaline.

Berberine has been consistently shown to function as a powerful inducer of the phosphorylation of AMPK at Thr-172 in adipocytes, myotubes, and the liver.^29–32) However, Zhang et al. recently demonstrated that a four-week treatment with berberine decreased AMPK phosphorylation levels in the hypothalamus of obese C57BLKS/J-Leprdb/Leprdb mice.¹⁵⁾ To confirm the relationship between AMPK activity and paliperidone-induced increases in adrenaline/glucose concentrations, we investigated the effects of paliperidone combined with 28 d of oral berberine on paliperidone-induced increases in serum concentrations of glucose and other endogenous metabolites as well as hypothalamic and hepatic AMPK activities. In comparisons with the saline/paliperidone group, the berberine pretreatment more strongly attenuated paliperidone-induced increases in blood concentrations of glucose, adrenaline, and insulin as well as the concentration of hypothalamic AMPK phosphorylated at Thr-172 (Figs. 5, 6). These results suggest that the paliperidone-induced release of adrenaline is associated with the activation of hypothalamic AMPK. The present results are consistent with the findings reported by McCrimmon et al. showing that the down-regulation of AMPK in the ventromedial hypothalamus suppressed the response of adrenaline to acute hypoglycemia.³³⁾ On the other hand, 15 min after the administration of paliperidone, the berberine pretreatment inhibited AMPK activity in the hypothalamus and decreased adrenaline levels, whereas blood glucose levels remained unchanged. These results suggest the involvement of mechanisms other than the inhibition of hypothalamic AMPK activity in paliperidone-induced hyperglycemia, such as the glucose transporter type 4 pathway in muscle cells.²⁴⁾

Berberine is present in several traditional Chinese medications, such as Huang-Lian-Jie-Du-Decoction and Sanhuang Xiexin Tang, and has attracted significant interest because of its potential to cure metabolic illnesses.^34,35) Animal and clinical research demonstrated its beneficial therapeutic effects on type 2 diabetes mellitus.^36,37) Berberine also exerts hypoglycemic effects with the same activation mechanism and molecular target as metformin, and a similar impact on hypoglycemia was observed in diabetic rats.^38,39) In clinical investigations, berberine is an oral hypoglycemic agent that exerts a similar hypoglycemic effect as metformin in adult patients diagnosed with type 2 diabetes mellitus.⁴⁰⁾ Berberine also has extremely low toxicity at the typical dosages and was found to be clinically beneficial without severe adverse effects.⁴¹⁾ In the present study, berberine suppressed paliperidone-induced hyperglycemia, indicating the utility of berberine or traditional herbal medicines containing berberine as prophylactic agents for atypical antipsychotic-induced hyperglycemia in humans.

A potential limitation of the present study is that we used a single dose of paliperidone to show that berberine reduced blood glucose concentrations. The mechanisms underlying hyperglycemia induced by a long-term treatment with antipsychotics may differ from those of a single-dose treatment. To obtain a more detailed understanding of the mechanisms responsible for antipsychotic-induced hyperglycemia, further research on the effects of berberine in long-term antipsychotic therapy is needed.

CONCLUSION

Acute paliperidone-induced hyperglycemia is caused by an increase in the secretion of adrenaline via the activation of hypothalamic AMPK.

Acknowledgments

The authors would like to thank T. Akahoshi for technical assistance with experiments. This work was supported by JSPS KAKENHI Grant Nos. JP19K07189 and JP22K06720.

Conflict of Interest

The authors declare no conflict of interest.

REFERENCES

• 1) Orsolini L, Tomasetti C, Valchera A, Vecchiotti R, Matarazzo I, Vellante F, Iasevoli F, Buonaguro EF, Fornaro M, Fiengo AL, Martinotti G, Mazza M, Perna G, Carano A, De Bartolomeis A, Di Giannantonio M, De Berardis D. An update of safety of clinically used atypical antipsychotics. Expert Opin. Drug Saf., 15, 1329–1347 (2016).
• 2) Aichhorn W, Whitworth AB, Weiss EM, Marksteiner J. Second-generation antipsychotics: is there evidence for sex differences in pharmacokinetic and adverse effect profiles? Drug Saf., 29, 587–598 (2006).
• 3) Werner FM, Coveñas R. Safety of antipsychotic drugs: focus on therapeutic and adverse effects. Expert Opin. Drug Saf., 13, 1031–1042 (2014).
• 4) Grajales D, Ferreira V, Valverde ÁM. Second-generation antipsychotics and dysregulation of glucose metabolism: beyond weight gain. Cells, 8, 1336 (2019).
• 5) Urban AE, Cubała WJ. Therapeutic drug monitoring of atypical antipsychotics. Psychiatr. Pol., 51, 1059–1077 (2017).
• 6) Wesołowska A, Jastrzębska-Więsek M, Cios A, Partyka A. The preclinical discovery and development of paliperidone for the treatment of schizophrenia. Expert Opin. Drug Discov, 15, 279–292 (2020).
• 7) Emsley R, Berwaerts J, Eerdekens M, Kramer M, Lane R, Lim P, Hough D, Palumbo J. Efficacy and safety of oral paliperidone extended-release tablets in the treatment of acute schizophrenia: pooled data from three 52-week open-label studies. Int. Clin. Psychopharmacol., 23, 343–356 (2008).
• 8) De Hert M, Yu W, Detraux J, Sweers K, van Winkel R, Correll CU. Body weight and metabolic adverse effects of asenapine, iloperidone, lurasidone and paliperidone in the treatment of schizophrenia and bipolar disorder: a systematic review and exploratory meta-analysis. CNS Drugs, 26, 733–759 (2012).
• 9) Nagata M, Nakajima M, Ishiwata Y, Takahashi Y, Takahashi H, Negishi K, Yasuhara M. Mechanism underlying induction of hyperglycemia in rats by single administration of olanzapine. Biol. Pharm. Bull., 39, 754–761 (2016).
• 10) Nagata M, Kimura Y, Ishiwata Y, Takahashi H, Yasuhara M. Clozapine-induced acute hyperglycemia is accompanied with elevated serum concentrations of adrenaline and glucagon in rats. Biol. Pharm. Bull., 41, 1286–1290 (2018).
• 11) Xue B, Kahn BB. AMPK integrates nutrient and hormonal signals to regulate food intake and energy balance through effects in the hypothalamus and peripheral tissues. J. Physiol., 574, 73–83 (2006).
• 12) Ikegami M, Ikeda H, Ohashi T, Ohsawa M, Ishikawa Y, Kai M, Kamei A, Kamei J. Olanzapine increases hepatic glucose production through the activation of hypothalamic adenosine 5′-monophosphate-activated protein kinase. Diabetes Obes. Metab., 15, 1128–1135 (2013).
• 13) Ikegami M, Ikeda H, Ohashi T, Kai M, Osada M, Kamei A, Kamei J. Olanzapine-induced hyperglycemia: possible involvement of histaminergic, dopaminergic and adrenergic functions in the central nervous system. Neuroendocrinology, 98, 224–232 (2013).
• 14) Ikegami M, Ikeda H, Ishikawa Y, Ohsawa M, Ohashi T, Kai M, Kamei A, Kamei J. Olanzapine induces glucose intolerance through the activation of AMPK in the mouse hypothalamus. Eur. J. Pharmacol., 718, 376–382 (2013).
• 15) Zhang Z, Zhang H, Li B, Meng X, Wang J, Zhang Y, Yao S, Ma Q, Jin L, Yang J, Wang W, Ning G. Berberine activates thermogenesis in white and brown adipose tissue. Nat. Commun., 5, 5493 (2014).
• 16) Ishiwata Y, Sanada Y, Yasuhara M. Effects of gatifloxacin on serum glucose concentration in normal and diabetic rats. Biol. Pharm. Bull., 29, 527–531 (2006).
• 17) Vanwong N, Prommas S, Puangpetch A, Hongkaew Y, Nuntamool N, Nakorn CN, Ngamsamut N, Limsila P, Sukasem C. Development and validation of liquid chromatography/tandem mass spectrometry analysis for therapeutic drug monitoring of risperidone and 9-hydroxyrisperidone in pediatric patients with autism spectrum disorders. J. Clin. Lab. Anal., 30, 1236–1246 (2016).
• 18) Siafis S, Tzachanis D, Samara M, Papazisis G. Antipsychotic drugs: from receptor-binding profiles to metabolic side effects. Curr. Neuropharmacol., 16, 1210–1223 (2018).
• 19) Boyda HN, Procyshyn RM, Tse L, Yuen JWY, Honer WG, Barr AM. A comparison of the metabolic side-effects of the second-generation antipsychotic drugs risperidone and paliperidone in animal models. PLOS ONE, 16, e0246211 (2021).
• 20) Ruderman NB, Carling D, Prentki M, Cacicedo JM. AMPK, insulin resistance, and the metabolic syndrome. J. Clin. Invest., 123, 2764–2772 (2013).
• 21) Liu HY, Hwang TJ, Tsai IL, Kuo CH. Use of high-conductivity sample solution with sweeping-micellar electrokinetic capillary chromatography for trace-level quantification of paliperidone in human plasma. Electrophoresis, 36, 534–542 (2015).
• 22) Muly EC, Votaw JR, Ritchie J, Howell LL. Relationship between dose, drug levels, and D2 receptor occupancy for the atypical antipsychotics risperidone and paliperidone. J. Pharmacol. Exp. Ther., 341, 81–89 (2012).
• 23) Coughlan KA, Valentine RJ, Ruderman NB, Saha AK. AMPK activation: a therapeutic target for type 2 diabetes? Diabetes Metab. Syndr. Obes., 7, 241–253 (2014).
• 24) Carli M, Kolachalam S, Longoni B, Pintaudi A, Baldini M, Aringhieri S, Fasciani I, Annibale P, Maggio R, Scarselli M. Atypical antipsychotics and metabolic syndrome: from molecular mechanisms to clinical differences. Pharmaceuticals (Basel), 14, 238 (2021).
• 25) Kim SF, Huang AS, Snowman AM, Teuscher C, Snyder SH. From the Cover: Antipsychotic drug-induced weight gain mediated by histamine H1 receptor-linked activation of hypothalamic AMP-kinase. Proc. Natl. Acad. Sci. U.S.A., 104, 3456–3459 (2007).
• 26) Guenette MD, Giacca A, Hahn M, Teo C, Lam L, Chintoh A, Arenovich T, Remington G. Atypical antipsychotics and effects of adrenergic and serotonergic receptor binding on insulin secretion in-vivo: an animal model. Schizophr. Res., 146, 162–169 (2013).
• 27) Montastruc F, Palmaro A, Bagheri H, Schmitt L, Montastruc JL, Lapeyre-Mestre M. Role of serotonin 5-HT2C and histamine H1 receptors in antipsychotic-induced diabetes: a pharmacoepidemiological-pharmacodynamic study in VigiBase. Eur. Neuropsychopharmacol., 25, 1556–1565 (2015).
• 28) Steinberg GR, Kemp BE. AMPK in health and disease. Physiol. Rev., 89, 1025–1078 (2009).
• 29) Lee YS, Kim WS, Kim KH, Yoon MJ, Cho HJ, Shen Y, Ye JM, Lee CH, Oh WK, Kim CT, Hohnen-Behrens C, Gosby A, Kraegen EW, James DE, Kim JB. Berberine, a natural plant product, activates AMP-activated protein kinase with beneficial metabolic effects in diabetic and insulin-resistant states. Diabetes, 55, 2256–2264 (2006).
• 30) Cheng Z, Pang T, Gu M, Gao AH, Xie CM, Li JY, Nan FJ, Li J. Berberine-stimulated glucose uptake in L6 myotubes involves both AMPK and p38 MAPK. Biochim. Biophys. Acta, Gen. Subj., 1760, 1682–1689 (2006).
• 31) Kim SH, Shin EJ, Kim ED, Bayaraa T, Frost SC, Hyun CK. Berberine activates GLUT1-mediated glucose uptake in 3T3-L1 adipocytes. Biol. Pharm. Bull., 30, 2120–2125 (2007).
• 32) Yin J, Gao Z, Liu D, Liu Z, Ye J. Berberine improves glucose metabolism through induction of glycolysis. Am. J. Physiol. Endocrinol. Metab., 294, E148–E156 (2008).
• 33) McCrimmon RJ, Shaw M, Fan X, Cheng H, Ding Y, Vella MC, Zhou L, McNay EC, Sherwin RS. Key role for AMP-activated protein kinase in the ventromedial hypothalamus in regulating counterregulatory hormone responses to acute hypoglycemia. Diabetes, 57, 444–450 (2008).
• 34) Chen M, Liao Z, Lu B, Wang M, Lin L, Zhang S, Li Y, Liu D, Liao Q, Xie Z. Huang-Lian-Jie-Du-decoction ameliorates hyperglycemia and insulin resistant in association with gut microbiota modulation. Front. Microbiol., 9, 2380 (2018).
• 35) Wei X, Tao J, Shen Y, Xiao S, Jiang S, Shang E, Zhu Z, Qian D, Duan J. Sanhuang Xiexin Tang ameliorates type 2 diabetic rats via modulation of the metabolic profiles and NF-κB/PI-3K/Akt signaling pathways. Front Pharmacol, 9, 955 (2018).
• 36) Liang Y, Xu X, Yin M, Zhang Y, Huang L, Chen R, Ni J. Effects of berberine on blood glucose in patients with type 2 diabetes mellitus: a systematic literature review and a meta-analysis. Endocr. J., 66, 51–63 (2019).
• 37) Xu X, Yi H, Wu J, Kuang T, Zhang J, Li Q, Du H, Xu T, Jiang G, Fan G. Therapeutic effect of berberine on metabolic diseases: both pharmacological data and clinical evidence. Biomed. Pharmacother., 133, 110984 (2021).
• 38) Tang LQ, Wei W, Chen LM, Liu S. Effects of berberine on diabetes induced by alloxan and a high-fat/high-cholesterol diet in rats. J. Ethnopharmacol., 108, 109–115 (2006).
• 39) Kim J, Yang G, Kim Y, Kim J, Ha J. AMPK activators: mechanisms of action and physiological activities. Exp. Mol. Med., 48, e224 (2016).
• 40) Yin J, Xing H, Ye J. Efficacy of berberine in patients with type 2 diabetes mellitus. Metabolism, 57, 712–717 (2008).
• 41) Imenshahidi M, Hosseinzadeh H. Berberine and barberry (Berberis vulgaris): A clinical review. Phytother. Res., 33, 504–523 (2019).