Altered cellular redox status, sirtuin abundance and clock gene expression in a mouse model of developmentally primed NASH

Abstract

Background: We have previously shown that high fat (HF) feeding during pregnancy primes the development of non-alcoholic steatohepatits (NASH) in the adult offspring. However, the underlying mechanisms are unclear.

Aims: Since the endogenous molecular clock can regulate hepatic lipid metabolism, we investigated whether exposure to a HF diet during development could alter hepatic clock gene expression and contribute to NASH onset in later life.

Methods: Female mice were fed either a control (C, 7%kcal fat) or HF (45%kcal fat) diet. Offspring were fed either a C or HF diet resulting in four offspring groups: C/C, C/HF, HF/C and HF/HF. NAFLD progression, cellular redox status, sirtuin expression (Sirt1, Sirt3), and the expression of core clock genes (Clock, Bmal1, Per2, Cry2) and clock-controlled genes involved in lipid metabolism (Rev-Erbα, Rev-Erbβ, RORα, and Srebp1c) were measured in offspring livers.

Results: Offspring fed a HF diet developed NAFLD. However HF fed offspring of mothers fed a HF diet developed NASH, coupled with significantly reduced NAD(+)/NADH (p<0.05, HF/HF vs C/C), Sirt1 (p<0.001, HF/HF vs C/C), Sirt3 (p<0.01, HF/HF vs C/C), perturbed clock gene expression, and elevated expression of genes involved lipid metabolism, such as Srebp1c (p<0.05, C/HF and HF/HF vs C/C).

Conclusion: Our results suggest that exposure to excess dietary fat during early and post-natal life increases the susceptibility to develop NASH in adulthood, involving altered cellular redox status, reduced sirtuin abundance, and desynchronized clock gene expression.

Keywords: Aging; Circadian; Development; Fatty liver; High fat; Maternal diet.

Fig. 1

15 week old male offspring exposed to high fat diets during development and adulthood have perturbed nycthemeral (day vs night) rhythms of food intake and energy expenditure, and also develop severe fatty liver and imbalanced hepatic cellular redox status. (a.) Total amount of food intake of male offspring from each dietary group, during the 12 h light (daytime, open bars) and 12 h dark (nighttime, closed bars) periods. (b.) Energy expenditure levels in male offspring from each dietary group, during the 12 h light (open bars) and 12 h dark periods (closed bars). (c.) Body weight (recorded at ZT8) of the offspring at 15 weeks of age. (d.) Percentage of steatosis in liver sections determined by point counting (e.) Hematoxylin and eosin stained liver sections from 15 week old male offspring. C/C and HF/C offspring show normal liver architecture. C/HF offspring livers exhibit lipid accumulation. HF/HF livers contain significant lipid accumulation and ballooning degeneration. (f.) Kleiner scores for offspring liver (n = 4 for each offspring group), show that C/C and HF/C are histologically normal, C/HF show a histological phenotype similar to human non-alcoholic fatty liver (NAFL), and HF/HF offspring liver have the appearance of non-alcoholic steatohepatitis (NASH). (g.) Fasting blood glucose over 2 h following bolus intraperitoneal (IP) injection of glucose (inverted arrow). Inset histogram depicts the area under the curve (AUC) levels over the 2-hour monitoring period. (h.) Circulating insulin levels during the 12 h light (daytime, open bars) and 12-hour dark (nighttime, closed bars) periods. For each analysis offspring groups were compared against C/C, where *p < 0.05, **p < 0.01, ***p < 0.001 or ****p < 0.0001 vs C/C.

Fig. 2

As a measure of fibrosis (a.) OPN gene expression was measured in offspring liver. Concentration of pyridine nucleotides (b.) NAD⁺, (c.) NADH in offspring liver (left lobe), and (d.) the NAD⁺/NADH ratio in offspring liver. Circulating levels of (e.) non-esterified fatty acids (NEFAs) and (f.) triglycerides were determined from mouse plasma taken during the light (daytime at ZT8, open bars) or dark (nighttime at ZT20, closed bars) periods (for each analysis offspring groups were compared against C/C, where *p < 0.05, **p < 0.01 or ***p < 0.001 vs C/C.

Fig. 3

Mean relative hepatic mRNA expression of the sirtuins (a.) Sirt1 and (b.) Sirt3; of genes involved in co-ordinating circadian rhythms (c.) Clock, (d.) Bmal1, (e.) Per2, and (f.) Cry2; and downstream lipogenic transcription factors (g.) Rev-Erbα, (h.) Rev-Erbβ, (i.) RORα, and (j.) Srebp1c during the light (daytime at ZT8, open bars) or dark (nighttime at ZT20, closed bars) periods. For each analysis offspring groups were compared against C/C, where *p < 0.05, **p < 0.01 or ***p < 0.001 vs C/C.

Fig. 4

Hypothetical schematic representation of how maternal high fat and post-natal high fat (HF) diet alters sirtuin and clock gene expression leading to severe hepatic steatosis in offspring liver. A HF diet during both early and post-natal life reduces sirtuin levels and alters cellular energy status (reduction in NAD⁺ levels and NAD/NADH ratio). This is associated with desynchronization of circadian clock gene activity and upregulation of downstream lipogenic transcription factors such as Srebp1c to developmentally prime the lipogenic pathway, resulting in further up-regulation of fat accumulation.