Administration of Melatonin and Metformin Prevents Deleterious Effects of Circadian Disruption and Obesity in Male Rats

Abstract

Circadian disruption and obesity synergize to predispose to development of type 2 diabetes mellitus (T2DM), signifying that therapeutic targeting of both circadian and metabolic dysfunctions should be considered as a potential treatment approach. To address this hypothesis, we studied rats concomitantly exposed to circadian disruption and diet-induced obesity (CDO), a rat model recently shown to recapitulate phenotypical aspects of obese T2DM (eg, circadian disruption, obesity, insulin resistance, and islet failure). CDO rats were subsequently treated daily (for 12 wk) by timed oral gavage with vehicle, melatonin (a known chronobiotic), metformin, or combination treatment of both therapeutics. Melatonin treatment alone improved circadian activity rhythms, attenuated induction of β-cell failure, and enhanced glucose tolerance. Metformin alone did not modify circadian activity but enhanced insulin sensitivity and glucose tolerance. Importantly, the combination of melatonin and metformin had synergistic actions to modify progression of metabolic dysfunction in CDO rats through improved adiposity, circadian activity, insulin sensitivity, and islet cell failure. This study suggests that management of both circadian and metabolic dysfunctions should be considered as a potential preventative and therapeutic option for treatment of obesity and T2DM.

Figures

Figure 1.

Melatonin and metformin synergistically attenuate body mass gain, caloric intake, and fat accumulation in CDO rats. A, Body mass gain trajectory during the 12-week study period. Mean % body mass gain (B), mean mass of epididymal (C) and retroperitoneal fat depots (D), and mean hepatic mass (E) and mean daily caloric food intake (F) in control (open bar, n = 4), CDO (black bar, n = 9), CDO-MEL (gray bar, n = 10), CDO-MET (yellow bar, n = 9), and CDO-MEL-MET (blue bar, n = 9) rats. Bar graphs represent mean ± SEM; #, P < .05 vs control; *, P < .05 vs CDO; †, P < .05 vs CDO-MEL and ‡, P < .05 vs CDO-MET.

Figure 2.

Melatonin administration improves behavioral circadian rhythms in CDO rats. A, Representative 24-hour locomotor activity (double plotted) actograms in rats monitored for 8 days at baseline under standard LD cycle followed by 6 weeks of recordings under either control, CDO, CDO-MEL, CDO-MET, and CDO-MEL-MET conditions. Shaded areas represent periods of dark. Temporal profiles are presented corresponding to CT as outlined in the methods section. B, Representative χ2 periodograms of activity recordings in control, CDO, CDO-MEL, CDO-MET, and CDO-MEL-MET condition rats. Broken horizontal lines represent statistically significant threshold in determination of dominant circadian period. Note dampening of circadian rhythms in CDO and CDO-MET groups. Mean χ2 periodogram-derived circadian amplitude (C) and average total daily (24 h) activity (D) during corresponding drug treatments (Rx) in control (open bar, n = 4), CDO (black bar, n = 8), CDO-MEL (gray bar, n = 8), CDO-MET (yellow bar, n = 6), and CDO-MEL-MET (blue bar, n = 6) rats. Each bar represents mean ± SEM; #, P < .05 vs control; *, P < .05 vs CDO.

Figure 3.

Effects of melatonin and metformin alone, or in combination, on diurnal glucose homeostasis in CDO rats. Diurnal profiles in plasma glucose (A), plasma insulin (B), and calculated index of insulin resistance HOMA-IR (C) in control (open bar, n = 4), CDO (black bar, n = 9), CDO-MEL (gray bar, n = 10), CDO-MET (yellow bar, n = 9), and CDO-MEL-MET (blue bar, n = 9) rats. Statistical analysis (graphs on the left) was performed by two-way repeated-measures ANOVA for treatment, time, and interaction. Bar graphs (right) display mean ± SEM of AUC for measures of plasma glucose (A), insulin (B), and HOMA-IR (C) across the 24-hour circadian day in respected groups. Plasma samples were obtained at 6-hour intervals. #, P < .05 vs control; *, P < .05 vs CDO; †, P < .05 vs CDO-MEL and ‡, P < .05 vs CDO-MET.

Figure 4.

Effects of melatonin and metformin alone, or in combination, on glucose, insulin, and C-peptide fluxes during an OGTT in CDO rats. Plasma glucose (A), insulin (B), and C-peptide concentrations (C) at baseline (time 0) and after an OGTT performed twice in each rat at CT 4 (left) and CT 16 (right) in control (open circle, n = 4), CDO (black circle, n = 9), CDO-MEL (gray circle, n = 10), CDO-MET (yellow circle, n = 9), and CDO-MEL-MET (blue circle, n = 9) rats. Data are presented as mean ± SEM.

Figure 5.

Effects of melatonin and metformin alone, or in combination, on diurnal glucose tolerance and estimates of β-cell function and insulin sensitivity in CDO rats. A, Mean plasma glucose concentrations during an OGTT performed twice in each rat at CT 4 (left) and CT 16 (right) in control (open circle, n = 4), CDO (black circle, n = 9), CDO-MEL (gray circle, n = 10), CDO-MET (yellow circle, n = 9), and CDO-MEL-MET (blue circle, n = 9) rats. B, Estimate of pancreatic β-cell function expressed as AUC for C-peptide over plasma glucose during the first 30 minutes of an OGTT at CT 4 (left) and CT 16 (right) in control (open circle, n = 4), CDO (black circle, n = 9), CDO-MEL (gray circle, n = 10), CDO-MET (yellow circle, n = 9) and CDO-MEL-MET (blue circle, n = 9) rats. C, Estimate of insulin sensitivity derived from Matsuda index during an OGTT performed at CT 4 (left) and CT 16 (right) in control (open circle, n = 4), CDO (black circle, n = 9), CDO-MEL (gray circle, n = 10), CDO-MET (yellow circle, n = 9), and CDO-MEL-MET (blue circle, n = 9) rats. Data are presented as mean ± SEM; #, P < .05 vs control; *, P < .05 vs CDO; †, P < .05 vs CDO-MEL and ‡, P < .05 vs CDO-MET.

Figure 6.

Effects of melatonin and metformin administration alone, or in combination, on pancreatic islet morphology in CDO rats. A, Representative examples of pancreatic sections imaged at ×5 magnification (top; scale bars, 2000 μm) and individual islets imaged at ×20 magnification (bottom; scale bars, 50 μm) stained for insulin (brown) and hematoxylin (blue) in control, CDO, CDO-MEL, CDO-MET, and CDO-MEL-MET rats. B, Representative examples of islets stained by immunofluorescence for insulin (green) and counterstained (red) with either glucagon, replication marker Ki-67, or DNA damage-induced apoptosis marker TUNEL counterstained with nuclear marker DAPI (blue) imaged at ×20 magnification (scale bars, 50 μm) in control, CDO, CDO-MEL, CDO-MET, and CDO-MEL-MET rats. White arrowheads highlight examples of Ki-67 and TUNEL-positive β-cells.

Figure 7.

Effects of melatonin and metformin administration alone, or in combination, on islet size distribution, islet density and β-cell turnover in CDO rats. A, Scatter plot of islet size distribution obtained from the analysis of whole pancreatic sections in control (open circles, n = 4), CDO (black circles, n = 5), CDO-MEL (gray circles, n = 5), CDO-MET (yellow circles, n = 5), and CDO-MEL-MET (blue circles, n = 5) rats. Mean islet density (B), β-cell fractional area (C), frequency of β-cell proliferation (D), and frequency of DNA damage-induced β-cell apoptosis (E) in control (open bar, n = 4), CDO (black bar, n = 9), CDO-MEL (gray bar, n = 10), CDO-MET (yellow bar, n = 9), and CDO-MEL-MET (blue bar, n = 9) rats. Bar graphs represent mean ± SEM. F, Linear regression analysis between body mass and the frequency of β-cell apoptosis in CDO (black line, r = 0.6: P < .05), CDO-MEL (gray line, r = 0.1: P = .8), CDO-MET (yellow line, r = 0.1: P = .6), and CDO-MEL-MET (blue line, r = 0.2: P = .4) rats. #, P < .05 vs control; *, P < .05 vs CDO; †, P < .05 vs CDO-MEL and ‡, P < .05 vs CDO-MET.