Circadian disruption by short light exposure and a high energy diet impairs glucose tolerance and increases cardiac fibrosis in Psammomys obesus

Abstract

Type 2 diabetes mellitus (T2DM) increases cardiac inflammation which promotes the development of cardiac fibrosis. We sought to determine the impact of circadian disruption on the induction of hyperglycaemia, inflammation and cardiac fibrosis.

Methods: Psammomys obesus (P. obesus) were exposed to neutral (12 h light:12 h dark) or short (5 h light:19 h dark) photoperiods and fed a low energy (LE) or high energy (HE) diet for 8 or 20 weeks. To determine daily rhythmicity, P. obesus were euthanised at 2, 8, 14, and 20 h after 'lights on'.

Results: P. obesus exposed to a short photoperiod for 8 and 20 weeks had impaired glucose tolerance following oral glucose tolerance testing, compared to a neutral photoperiod exposure. This occurred with both LE and HE diets but was more pronounced with the HE diet. Short photoperiod exposure also increased myocardial perivascular fibrosis after 20 weeks on LE (51%, P < 0.05) and HE (44%, P < 0.05) diets, when compared to groups with neutral photoperiod exposure. Short photoperiod exposure caused elevations in mRNA levels of hypertrophy gene Nppa (atrial natriuretic peptide) and hypertrophy transcription factors Gata4 and Mef2c in myocardial tissue after 8 weeks.

Conclusion: Exposure to a short photoperiod causes impaired glucose tolerance in P. obesus that is exacerbated with HE diet and is accompanied by an induction in myocardial perivascular fibrosis.

Figure 1

High energy diet and Short photoperiod cause abnormal glucose tolerance. Oral glucose tolerance tests (OGTT) were performed in P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 (a) or 20 weeks (b). P. obesus were fasted for 4 h. A dose of 2 g glucose/kg body weight was administered via gavage. Blood was collected at time 0 and 120 min later. Mean ± SEM, n = 6–15/group. *P < 0.05 compared to time point 0.

Figure 2

20 weeks of high energy diet and short photoperiod increases myocardial perivascular fibrosis. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 or 20 weeks. (a) Representative images of Masson’s trichrome-stained hearts depicting collagen deposition surrounding the vessels after 8-weeks of treatment (n = 11–15/group). Perivascular fibrosis was analysed by determining area of blue staining around selected vessels and normalised to the vessel area. (b) Average Col1a1 gene expression normalised using the ^ΔΔCt method to Cyclophilin and to the 12:12LE group. (c) Daily Col1a1 rhythms at hourly time points post-‘lights on’. (d) Representative images of perivascular fibrosis in myocardial sections after 20 weeks of treatment with analyses (n = 6–12/group). (e) Average Col1a1 gene expression. Mean ± SEM. *P < 0.05, **P < 0.01.

Figure 3

8 Weeks of High energy diet and shorter photoperiod increase hypertrophy gene expression. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 or 20 weeks. Cardiac hypertrophy genes were measured by qPCR including: (a) Gata4, (b) Merf2c, (c) Nppa and (d) the Myh7:Myh6 ratio. (e) Representative images of H&E-stained hearts after 8 weeks of treatment. Myocyte size (area) was analysed by determining the area of eosin staining divided by the number of nuclei/image. Mean ± SEM, n = 11–15/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

20 weeks of high energy diet and shorter photoperiod reduce cardiomyocyte size. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 or 20 weeks. Cardiac hypertrophy genes were measured by qPCR including: (a) Merf2c and (b) the Myh7:Myh6 ratio. (c) Representative images of H&E-stained hearts after 20 weeks of treatment. Myocyte size (area) was analysed by determining the area of eosin staining divided by the number of nuclei/image. Mean ± SEM, n = 6–12/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Expression of Per2 and inflammatory markers after 8 weeks of high energy diet and/or shorter photoperiod. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 weeks. Expression of (a,b) Per2, (c,d) Rela, (e,f) Ccl2 and (g,h) Tgfb1 genes in the myocardial tissues. Average gene expression was determined across the 4 groups and normalised using the ^ΔΔCt method to Cyclophilin and to the 12:12LE group (left panels). Daily rhythms were determined by measuring gene expression at hourly time points post-lights on. Mean ± SEM, n = 11–15/group. ^†P < 0.05 within 12:12LE group; ^#P < 0.05, ^##P < 0.01 within 12:12HE group; ^{^}P < 0.05, ^{^^}P < 0.01, within 5:19LE group.

Figure 6

Expression of apoptosis markers and Serca2 after 8 weeks of high or low energy diet and/or shorter photoperiod. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 8 weeks. Expression of (a,b) Bax, (c,d) Bcl2, (e,f) Bax/Bcl2 and (g,h) Serca2 in the 8-week cohort. Average gene expression was determined and normalised using the ^ΔΔCt method to Cyclophilin and to the 12:12LE group (n = 11–15/group, left panels). Daily rhythms were determined by measuring gene expression at hourly time points post-lights on (n = 2–6/group, right panels). Mean ± SEM ^†P < 0.05 within 12:12LE group.

Figure 7

Effect of 20 Weeks of low or high energy diet and/or shorter photoperiod on gene expression. P. obesus were exposed to low energy (LE) or high energy (HE) diet and either a neutral photoperiod (12 light:12 dark) or short photoperiod (5 light:19 dark) for 20 weeks. Average gene expression of (a) Per2, (b) Rela, (c) Ccl2, (d) Tgfb1, (e) Bax, (f) Bcl2, (g) Bax/Bcl2 and (h) Serca2 was determined across the 4 groups and normalised using the ^ΔΔCt method to Cyclophilin and to the 12:12LE group of the 20-week cohort. Mean ± SEM, n = 6–12/group. *P < 0.05.