Response to the photoperiod in the white and brown adipose tissues of Fischer 344 rats fed a standard or cafeteria diet

 Researchers are identifying new factors that contribute to the obesity epidemic

• changes in the photoperiod are one promising risk factor
• Here are some of the findings:
• In one experiment, Fischer 344 rats were treated for 14 weeks with either a long day (18 h light:6 h dark; LD) or a short day (6h light:18h dark; SD) and fed a standard diet (STD).
• Biometric measures, postprandial plasmatic parameters, gene expression in the retroperitoneal white adipose tissue (RWAT) and brown adiposa tissue (BAT) were analyzed.
• Both experiments showed in the SD a decrease in the BAT expression of lipid uptake and β-oxidation genes, while only the STD additionally showed a reduction in Ucp1 expression.

Obesity is a major health problem, defined by the World Health Organization as an "abnormal or excessive fat accumulation that presents a risk to health," caused by an imbalance between caloric intake and calorie expenditure

• Its primary consequence is the predisposition to insulin resistance, cardiovascular diseases, musculoskeletal disorders and a major risk factor for some cancers, among others.

Animals and treatments

• The 12 animals used in the first experiment were 2-month-old male Fischer 344/IcoCrl rats (Charles River Laboratories, Barcelona, Spain) fed with a standard chow diet (STD) with a caloric distribution (3.2 kcal/g) of 19.3% protein, 8.4% fat and 72.8% carbohydrates.
• Animals were housed two animals per cage at 22°C and 55% humidity and with free access to food and water. The animals were randomly distributed into two groups (n=6) depending on the photoperiod to which they were exposed, LD (18 h light: 6 h dark) and SD (6h light: 18 h dark), for 14 weeks.
• For the second experiment, 20 Fischer 344/​IcoCral rats, 2 months old, were fed a CAF ad libitum, consisting of bacon, biscuits with pâté and cheese, muffin, carrots and sweetened milk (22% sucrose w/v).
• This high-fat, high-sugar diet model is based on appetizing ingredients that induce voluntary hyperphagia, increasing fat and sugar ingestion.

RNA extraction and quantification by real-time qRT-PCR

• Total RNA from RWAT and BAT was extracted using Trizol reagent following the manufacturer's instructions.
• RNA yield was quantified in a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA).
• Overall, 0.5 μg of total RNA was reverse transcribed using a high-capacity cDNA reverse transcription kit (Applied Biosystems, Madrid, Spain).

Western blot

• Ucp1 protein content in BAT was determined by Western blot.
• Protein content was quantified using the BCA protein assay kit (Pierce, Rockford, IL, USA).
• Gel transference into a PVDF membrane was done using the Trans-Blot Transfer System (Bio-Rad).

Histology

• Frozen RWAT samples were thawed and fixed in 4% formaldehyde.
• Sections were observed and acquired at ×10 magnification using AxioVision Zeiss Imaging software (Carl Zeiss Iberia, S.L., Madrid, Spain).
• The area of adipocytes was measured using the Adiposoft open source software (CIMA, University of Navarra, Spain.)
• adipocyte area was calculated from the average value of the area of cells in all measured fields for each sample.

Statistical Analysis

• Twelve samples per experiment and 27 variables were used: biometric and metabolic parameters, gene expression in RWAT and BAT, and adipocyte area and number.
• A varimax-rotated principal component analysis (PCA) was performed with XLSTAT (Addinsoft, Paris, France) to assess the relationships between our variables.
• The Kaiser-Meyer-Olkin index of our variables was >0.5. After data scaling, the analysis was based on the correlation matrix, and a principal component was considered to be significant if it contributed to >5% of the variance.

Biometric and metabolic parameters

• There were no significant differences in final body weight and food intake between the studied photoperiods.
• The lean mass of rats in the SD group was increased compared to the LD group, although the increase was not statistically significant (P=.067).
• In rats fed the CAF diet, a significant increase in BAT (%) and a tendency for BAT (gr) were observed in SD group compared to LD group.
• No significant differences were found in plasmatic parameters.

Gene expression in RWAT

• In animals fed the STD, the photoperiod altered the expression of lipogenesis genes.
• Fasn expression levels were significantly lower in the SD animals than in the LD animals, and a similar effect was observed with Acacα expression levels; however, the differences were not significant (P=.066).
• The expression levels of Mgl, Atgl, and Hsl, which are involved in lipolysis, and C/ebpα and Pparγ, both of which are related to adipogenesis, were significantly reduced in SD animals compared to LD animals (Fig. 1A).

Fasn protein levels in the RWAT

• In all, protein levels were significantly increased in the SD animals compared to the LD animals (Fig. 2).

Histology of RWAT

• Rats fed with STD and treated with the SD photoperiod showed a significant increase in the frequency of adipocytes smaller than 5000 μm2 (Fig. 3A).
• Adipocyte area, though not significant (P=.06), was also smaller in the SD group.
• No differences between the groups were observed in the total adipocytes in the RWAT

Gene expression in the BAT

• In rats fed the STD, the expression levels of the genes involved in β-oxidation were significantly decreased for CPT1b and Had and significantly increased for Pparα in the SD animals compared to the LD animals.
• No differences were observed in the expression of genes involved with thermogenesis, except for Ucp1.

UCP1 protein levels on BAT

• Due to significant changes on Ucp1 gene expression levels between the groups of animals fed the STD, we decided to quantify the protein levels in these animals.

Principal components analysis

• In rats fed the STD diet, PC1 explained 44.73% of the variance and PC2 explained 17.56% (Supplemental Fig. 2A).
• PC1 clearly discriminated between the LD and SD groups and was characterized by TAG, adipocyte number and Pparα gene expression on the left side and fat (gr, %), adipocyte area and the expression of all genes analyzed, except for PParα and Prdm16 in the BAT, on the right side.
• PC2 did not discriminate between the photoperiods; however, accumulated caloric intake, weight, lean mass, and fat were positively associated.
• Fat was related to both PCs but stronger to PC2.

Discussion Obesity is primarily caused by an imbalance between caloric intake and expenditure, meaning that excess calories will be accumulated as lipids

• Seasonal changes in the photoperiod help mammals adapt to the approaching season and augment the timing of reproduction to the changing environment
• There is a large body of evidence regarding the presence of seasonal or circannual effects in rodents
• Fischer 344 rats grown during a short day (SD) show slower gonadal growth, which suggests that cycles of light:dark affect their development
• Though all research to date agrees on the effect of the Photoperiod on body weight, the effect on energy intake seems to remain unsettled and needs further research
• The tendency observed in fat changes might be due to other mechanisms
• Body temperature, satiety hormones and sleep to be the causes

Reference

10.1016/j.jnutbio.2019.04.005