Impacts métaboliques et thérapeutiques de la vitamine A, sous forme d’acide rétinoïque, dans l’obésité, la résistance à l’insuline et le diabète de type 2 chez la souris ob/ob = Metabolic and Therapeutic Impacts of Vitamin A as Retinoic Acid on Obesity, Insulin Resistance, and Type 2 Diabetes in ob/ob Mice

Abstract  Introduction: Obesity and type 2 diabetes (T2DM) are associated with the impaired metabolism of insulin, energy and vitamin A (ROL-retinol; RE-retinyl esters; RA-retinoic acid) and with cardiovascular diseases. Hepatic retinol binding protein (RBP4) is released into the serum at an equimolar ratio to ROL, and is also secreted as an adipokine. Serum RBP4 is elevated in obesity, insulin resistance (IR) and T2DM. However, limited information is available on the rate of vitamin A metabolism in diseases such as diabetes or as to whether vitamin A (ROL) transport is enhanced by the elevation of RBP4 in the serum. Reducing the serum RBP4 level decreases IR; however, it is challenging to avoid depletion in circulating ROL following an artificial reduction in RBP4. Our laboratory has demonstrated that RA treatment reduces serum ROL in normal rats, with no vitamin A depletion effects (given that RA is the most bioactive metabolite of ROL). Others have suggested that RA may have variable impacts on body weight, IR and the heart in some obese and diabetic rodents. A standardized RA treatment protocol for IR and T2DM does not yet exist, as the mechanisms, optimal doses, duration and routes of administration are still under investigation. Furthermore, the serum and tissue ROL and RBP4 concentrations were not investigated under these conditions. It is of high interest to investigate these ROL and RBP4 concentrations in a certified, yet relatively unstudied for retinoid, obese and diabetic mouse model and to determine whether and how treatment with a small dose of RA influences their metabolic rates associated with IR, T2DM and eventual post-treatment improvements in these animals.   Hypothesis: Serum concentrations of ROL and RBP4 are elevated in the ob/ob mouse, a certified severely obese and diabetic animal model. The high serum levels of ROL and RBP4 are accompanied by high tissue concentrations of ROL and reduced tissue levels of RBP4. Short-term treatment with a low dose of RA will reduce serum ROL and RBP4, will normalize tissue ROL and RBP4 levels, and will decrease body fat and weight. All of these effects will contribute to lowering IR, glucose and insulin intolerance and blood glucose. These improvements in retinoid metabolism and the diabetic state will be associated with tissue-specific expression profiles for genes involved in vitamin A metabolism, and with genomic, histological and physiological indices of improved energy metabolism. A direct cardioprotective effect of RA may also be observed.    Aims: To evaluate the impact of our RA treatment in ob/ob mice on: A) serum and tissue levels of vitamin A and RBP4, blood glucose, IR, glucose and insulin intolerance, body weight and fat cell size; B) the expression (mRNA) of genes involved in vitamin A metabolism in subcutaneous fat (SF), visceral fat (VF), muscle (SM) and liver (LIV); the expression of the PGC1α and UCP1 genes, which are involved in the energy metabolism of beige/brown adipose tissue (BAT); the expression of  natriuretic peptide (NP) cardioprotective system genes and of those associated with the apoptosis and fibrosis of cardiomyocytes; and C) the morphology of SF and VF adipose tissue and of the cardiac muscle (HT).     Methods: Sixteen 9-week-old  B6.V-Lep/J ob/ob  mice were certified by Jackson Labs USA through the use of metabolic cages and motor protocols, to be hypometabolic, lethargic, subfertile, hyperphagic, obese, IR, glucose intolerant and severely hyperglycemic (≥ 20 mmol / L). These mice were divided into two equal groups. For 16 days, half of the mice (n=8) were treated daily by stomach intubation with 100 μg at-RA/day (2 μg/g body weight/day) in 100 μl of corn oil (vehicle), and the other half (n=8) were treated with only the vehicle, thus serving as diabetic controls. Normal and healthy-weight mice (n=5-9) were used as healthy controls. All mice received the international No.2018 standard rodent diet from Harlan Teklad-USA, which has an energy content of 13.7 kJ/g, and access to water ad libitum. Body weight and food consumption were monitored daily using a precision scale. Glycaemia was measured after 4 hours of fasting on days 9 and 16. Intraperitoneal glucose tolerance tests (IPGTTs) and insulin tolerance tests (IPITTs) were performed on days 15 and 16. The protein concentrations of RBP4 in the blood and in the SF and VF, SM and LIV, as well as protein in the cardiac NP system, were measured by Western blot (WB). Circulating and tissue vitamin A levels were quantified by HPLC. The RNA was extracted from tissue by the TRIZOL method and gene expression (mRNA) was evaluated by semi-quantitative RT-qPCR. Tissue morphologies were evaluated by staining and histology. Motor reflexes were evaluated daily with valid SHIRPA standard protocol stimuli and a minimum of two daily direct observations.    Results: Our RA treatment was associated with: 1) a decrease in body weight under similar food intake conditions and decreased motor-reflexes compared with untreated mice; 2) a decrease in serum levels of ROL and RBP4, blood glucose, and IR, as well as of glucose and insulin intolerance; 3) tissue-specific expression profiles for genes involved in: a) vitamin A metabolism, b) energy metabolism (increased UCP1 and PGC1α in SF and VF), c) the cardio-NP system (normalized anti-apoptotic, anti-fibrotic and NP); 4) a normalization of the RBP4, ROL and RE levels in tissues; 5) a reduction in adipocyte size; 6) reduced fibrosis and apoptosis of cardiomyocytes;7) increased poly unsaturated fatty acids (PUFAs) in the adipose tissue of  RA treated mice, documented by cryoseparation of liquid and solid phases at -20ºC; and 8) histology potentially suggesting adipocyte angiogenesis and autophagy.   Conclusion: Our RA treatment: 1) decreases serum ROL and RBP4 levels, glycaemia and IR; 2) reduces the expression (mRNA) of the RBP4 adipokine; 3) modulates the expression of genes involved in vitamin A metabolism, improving its homeostasis; 4) normalizes tissue RBP4 and vitamin A levels; 5) increases PGC1α and UCP1 gene expression, a signature of brown/beige fat; 6) improves energy metabolism, reducing adipocyte size, fat mass and body weight; 7) induces  cardioprotective, anti-apoptotic and anti-fibrotic effects; 8) is associated with increased PUFAs presence in the adipose tissue of RA-treated mice, suggesting that RA may stimulate BAT genesis due to typical dominance of PUFAs in beige/brown fat; 9) is associated with histologic structures indicating angiogenesis and autophagy in fat tissues, suggesting (pending further investigation) a contribution to improved metabolism in these tissues; 10) clearly demonstrates that in obesity and diabetes, cardio-metabolic improvements are possible out of the signaling pathways strictly controlled by leptin and sexual hormones, known to be beneficial for the energetic metabolism but decreased in men and women with obesity, insulin-resistance, diabetes, and with age (ex: strongly after menopause). An RA treatment conjugated effect is possible when these hormones are partially functional!

Keyphrases: Retinol Binding Protein RBP4, Retinoic acid, Vitamin A, Retinol Binding Protein RBP4, Insulin resistance, Diabetes, Brown / Beige fat, Energy metabolism, Natriuretic peptides ANP / BNP, Fibrosis prevention, Apoptosis, Autophagy