Fig 1.
Biphasic effects of bromocriptine on blood glucose levels via the dopamine D2 receptor-independent mechanism.
(A-B) Acute effects of bromocriptine (BC) in dopamine D2 receptor knockout (D2R-/-) and wild-type (D2R+/+) mice. Mice were injected with BC (10 mg/kg, i.p.) or vehicle (10% ethanol). (A) Effects of BC on locomotor activity in D2R+/+ (left) and D2R-/- mice (right) at 4–5 months of age. n = 5 per group. (B) Effects of BC on blood glucose levels under 2-h fasting conditions in D2R+/+ (left) and D2R-/- mice (right) at 8–9 months of age. n = 5 per group. (C-H) Long-term effects of BC on glucose metabolism in D2R+/+ and D2R-/- mice fed a high-fat diet (HFD) for 8 weeks (from 10 weeks of age). HFD-fed D2R+/+ and D2R-/- mice were administered BC (10 mg/kg, i.p.) or vehicle daily at ZT14 for 2 weeks. n = 4–6 per group. (C) Timeline of the experimental procedure. (D) Body weights in D2R+/+ (left) and D2R-/- mice (right). (E-F) Random fed blood glucose levels just after the injection of BC at ZT14 on day 1 (E) and day 10 (F). (G) Glucose tolerance test in D2R+/+ (left) and D2R-/- mice (middle) treated daily with BC or vehicle for 2 weeks. The right panel shows the glucose area under the curves (AUC) calculated on the left and middle panels. (H) Insulin tolerance test in D2R+/+ (left) and D2R-/- mice (right) treated daily with BC or vehicle for 2 weeks. (I-J) Long-term effects of BC on glucose metabolism in dopamine D1 receptor knockout (D1R-/-) mice fed HFD or a normal chow diet (NCD) for 8 weeks (from 3 months of age). Mice were administered BC (10 mg/kg, i.p.) or vehicle daily at ZT14 for 2 weeks. n = 4–5 per group. (I) Body weights after 2 weeks of drug administration. (J) The glucose tolerance test conducted after 2 weeks of drug administration. Values are expressed as the means ± S.D. * p < 0.05 and **p < 0.01 by the Student’s t-test. ##p < 0.01 by a two-way ANOVA with Tukey’s test. †p < 0.05 (vehicle-treated mice fed NCD vs. vehicle-treated mice fed HFD) and ‡p < 0.05 (vehicle-treated mice fed HFD vs. BC-treated mice fed HFD) by a one-way ANOVA with Dunnett’s test.
Fig 2.
Acute effects of bromocriptine on blood glucose levels via an
α2-adrenergic receptor-dependent mechanism. (A-B) Blood glucose levels just after the injection of bromocriptine (BC, 10 mg/kg, i.p.) or vehicle (Veh, 10% ethanol) in 4-month-old C57BL/6J mice under 2-h (A) and 16-h fasting conditions (B). n = 5 per group. (C-I) Effects of a pretreatment with autonomic nervous system (ANS)-blocking agents on the acute glucose-elevating effects of BC in C57BL/6J mice (2–3 months old). (C) Experimental procedures. (D-I) Effects of a pretreatment with hexamethonium (D, HEX), a cocktail of phenoxybenzamine and propranolol (E, POB+PRO), prazosin (F, PZS), yohimbine (G, YHB), propranolol alone (H), atropine (I, ATR), or saline (Sal) on blood glucose levels in mice treated with BC or vehicle under 2-h fasting conditions. n = 4–5 per group. (J) Effects of BC on the expression levels of gluconeogenesis markers (Pepck and G6pase mRNAs) and ER stress marker (Chop mRNA) in the livers of C57BL/6J mice (8 weeks old) fed ad libitum. Liver tissues were isolated 1 h after the injection of BC (10 mg/kg, i.p.) or vehicle at ZT14. n = 6–7 per group. Values are expressed as the means ± S.D. Significant differences in panels A, B, and J were examined by the Student’s t-test: * p < 0.05 and **p < 0.01. Significant differences in panels D-I were assessed by a two-way ANOVA with Tukey’s test: †p < 0.05 and ††p < 0.01 (saline + vehicle treatment vs. saline + BC treatment), #p < 0.05 and ##p < 0.01 (saline + BC treatment vs. blocker + BC treatment), §§p < 0.01 (blocker + vehicle treatment vs. blocker + BC treatment), ap < 0.05 and aap < 0.01 (saline + vehicle treatment vs. blocker + BC treatment), and bp < 0.05 and bbp < 0.01 (saline + BC treatment vs. blocker + vehicle treatment).
Fig 3.
Daily administration of bromocriptine improved glucose metabolism in genetically-obese db/db mice.
(A-D) Diabetic db/db mice (4 months old) fed a normal chow diet were administered bromocriptine (BC, 10 mg/kg, i.p.) or vehicle (10% ethanol) daily at ZT14 for 2 weeks. n = 5–6 per group. (A) The glucose tolerance test was conducted 2 weeks after the daily administration of BC. (B) The insulin tolerance test was conducted 2 weeks after the daily administration of BC. (C) Serum insulin levels after glucose loading. Glucose-stimulated insulin secretion was measured 2 weeks after the daily administration of BC. (D) The levels of Pepck mRNA, a gluconeogenesis marker, in the livers of mice treated daily with BC or vehicle for 4 weeks. Liver tissues were isolated under 16-h fasting conditions. Values are expressed as the means ± S.D. * p < 0.05 and **p < 0.01 by the Student’s t-test.
Fig 4.
Daily administration of bromocriptine improved glucose metabolism in orexin knockout mice under diet-induced obese conditions.
(A-J) Wild-type (Orexin+/+) and orexin knockout (Orexin-/-) mice at 10 weeks of age were fed a high-fat diet for 8 weeks, and bromocriptine (BC, 10 mg/kg, i.p.) or vehicle (10% ethanol) was then administered daily at ZT14 for 2 weeks. n = 4–7 per group. (A-B) Transient increases in blood glucose levels after the BC injection at ZT14 on day 1 and day 2 in Orexin+/+ (A) and Orexin-/- mice (B). (C-D) Random fed blood glucose levels measured at ZT8 after 1 and 2 weeks of the BC treatment in Orexin+/+ (C) and Orexin-/- mice (D). (E-F) The glucose tolerance test conducted 2 weeks after the daily administration of BC in Orexin+/+ (E) and Orexin-/- mice (F). (G-H) The insulin tolerance test conducted 2 weeks after the daily administration of BC in Orexin+/+ (G) and Orexin-/- mice (H). (I-J) Serum insulin levels during the glucose-stimulated insulin secretion test conducted 2 weeks after the daily administration of BC in Orexin+/+ (I) and Orexin-/- mice (J). Values are expressed as the means ± S.D. * p < 0.05 and **p < 0.01 by the Student’s t-test.
Fig 5.
Daily administration of bromocriptine improved glucose tolerance in diet-induced obese mice independently of melatonin and the autonomic nervous system.
C57BL/6J mice (8 weeks old) were fed HFD for 8 weeks. (A-C) Impact of a pretreatment with luzindole on the effects of bromocriptine (BC). Diet-induced obese mice were treated with luzindole (2 mg/kg, i.p.) or vehicle (1% ethanol, i.p.) and, 15 min later, BC (10 mg/kg, i.p.) or vehicle (10% ethanol, i.p.) was administered at ZT14. These drug treatments were repeated for 2 weeks. n = 5–8 per group. (A) Effects of BC or vehicle (10% ethanol) on body weights in mice pretreated daily with vehicle (left, 1% ethanol) or luzindole (right). (B) Transient increases in blood glucose levels by the BC injection in mice pretreated with vehicle (left) or luzindole (right) on day 1. (C) The glucose tolerance test conducted 2 weeks after the administration of BC to mice pretreated with vehicle (left) or luzindole (middle). The right panel shows the glucose area under the curve (AUC) calculated on the left and middle panels. (D-G) Impact of the pretreatment with yohimbine on the effects of BC. Diet-induced obese mice were treated with yohimbine (YHB, 3 mg/kg, s.c.) or vehicle (saline, Sal, s.c.) and, 15 min later, BC (10 mg/kg, i.p.) or vehicle (10% ethanol, i.p.) was administered at ZT14. These drug treatments were repeated for 2 weeks. (D) Effects of BC or vehicle on body weights in mice pretreated daily with saline (left) or yohimbine (right). n = 5–7 per group. (E) Transient increases in blood glucose levels by the BC injection in mice pretreated with saline (left) or yohimbine (right) on day 1. n = 8–10 per group. (F) The glucose tolerance test conducted 3 weeks after the administration of BC to mice pretreated with saline (left) or yohimbine (middle). The right panel shows glucose AUC based on the left and middle panels. n = 8–10 per group. (G) Serum insulin levels during the glucose-stimulated insulin secretion test conducted 4 weeks after the daily administration of BC and/or yohimbine. n = 5–7 per group. (H-J) Impact of the pretreatment with hexamethonium (HEX) on the effects of BC. Diet-induced obese mice were treated with HEX (30 mg/kg, i.p.) or vehicle (saline, i.p.) and, 15 min later, BC (10 mg/kg, i.p.) or vehicle (10% ethanol, i.p.) was administered at ZT14. These drug treatments were repeated for 2 weeks. n = 5–6 per group. (H) Effects of BC or vehicle on body weights in mice pretreated daily with saline (left) or HEX (right). (I) Transient increases in blood glucose levels by the BC injection in mice pretreated with saline (left) or HEX (right) on day 1. (J) The glucose tolerance test conducted 2 weeks after the administration of BC to mice pretreated with saline (left) or HEX (middle). The right panel shows glucose AUC calculated on the left and middle panels. Values are expressed as the means ± S.D. * p < 0.05 and **p < 0.01 by the Student’s t-test. ##p < 0.01 by a two-way ANOVA with Tukey’s test.
Fig 6.
Daily administration of bromocriptine suppressed ER stress in livers of diet-induced obese mice.
C57BL/6J mice (7–8 weeks old) were fed a high-fat diet for 8 weeks and bromocriptine (BC, 10 mg/kg, i.p.) or vehicle (10% ethanol) was then administered daily at ZT14 for 2 weeks. Liver tissues were isolated at ZT16 under pathophysiological ER stress condition (i.e., 2 h of refeeding after 24 h of fasting). n = 6 per group. (A) Experimental protocol. (B) Representative Western blot images. (C-E) Effects of BC on the levels of p-eIF2α/eIF2α, CHOP/α-tubulin, and p-JNK/JNK in the livers of mice treated with or without BC. Values are expressed as the means ± S.D. **p < 0.01 by the Student’s t-test.
Fig 7.
Direct effects of bromocriptine on ER stress levels in HepG2 cells in the presence and absence of thapsigargin.
(A-D) Comparison of bromocriptine (BC)- and thapsigargin (TG)-induced ER stress in HepG2 cells. Cells seeded and incubated for 24 h were treated with vehicle (0.05% DMSO) for 28 h, BC (5 µM) for 28 h, or a vehicle pretreatment (for 24 h) plus TG (100 nM for 4 h). n = 6 per group. (A) Representative Western blot images. (B-D) Effects of BC and TG on the levels of p-eIF2α/eIF2α, CHOP/α-tubulin, and p-JNK/JNK. (E-I) Induction of mild ER stress by BC. HepG2 cells seeded and incubated for 24 h were treated with BC (1–10 µM) or vehicle for 24 h. n = 5 per group. (E) Representative Western blot images. (F-I) Effects of BC on the levels of p-eIF2α/eIF2α, CHOP/α-tubulin, p-IRE1α/IRE1α, and IRE1α/α-tubulin. (J-M) Preconditioning effects of BC to prevent TG-induced severe ER stress. (J) Timeline of experimental procedures. HepG2 cells seeded and incubated for 24 h were pretreated with BC (1–10 µM) or vehicle for 24 h, and then treated with TG (100 nM) or vehicle (0.05% DMSO) for 4 h. n = 5 per group. (K) Representative Western blot images. (L-M) Effects of BC on the levels of p-eIF2α/eIF2α and CHOP/α-tubulin in the presence of TG. Values are expressed as the means ± S.D. * p < 0.05 and **p < 0.01 by a one-way ANOVA with Dunnett’s test.