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Energy Metabolism Regulates Retinoic Acid Synthesis and Homeostasis in Physiological Contexts

  • Author(s): Obrochta, Kristin Marie
  • Advisor(s): Napoli, Joseph L
  • et al.
Abstract

Mounting evidence supports a regulated and reciprocal relationship between retinoid homeostasis and energy metabolism, with a physiologically relevant consequence of disrupted energy balance. This research was motivated by an observation that all-trans-retinoic acid (atRA), and biosynthetic precursors, were responsive to acute shifts in energy status, in wild type animals with normal body weight and glucose tolerance, i.e. not consequent to metabolic syndrome. My dissertation was designed to characterize associations of retinoid changes under different metabolic conditions by targeting synthesis of retinoic acid, identifying underlying mechanism(s), and describing consequent function(s) in four contexts.

A model that compared fasted versus re-fed ad-libitum animals was used to evaluate the impact of an acute shift in whole body metabolism on retinoid status. In liver, atRA was elevated in fasted mice, and reduced by 50% in re-fed counterparts. Consistent with synthesis driving the atRA concentration in liver, expression of retinol dehydrogenase (Rdh) genes, Rdh10 and Rdh1, were elevated by fasting, and reduced 50% in re-fed as well as glucose and insulin treated mice, which preceded the reduction in atRA. Subsequent characterization of Rdh10 and Rdh16 (human orthologue of mouse Rdh1) regulation in a human hepatoma (HepG2) cell line identified transcriptional repression by insulin that required PI3K, Akt (PKB) and suppression of the transcription factor FoxO1, which was required for optimal expression of Rdhs. Conversely, expressions of Rdhs were elevated and stabilized in serum free medium conditions. This study illustrated that liver atRA, and specifically its synthesis, is regulated by acute changes in feeding status. This controlled fluctuation in atRA concentration enables transcriptional regulation by atRA in a physiological context, and reinforces an established link between atRA and liver glucose metabolism via phosphoenolpyruvate carboxy kinase.

In pancreas tissue, the 9-cis-retinoic acid (9cRA) concentration was reduced in animals that were fed ad-libitum, versus a fasted state, and also decreased rapidly and transiently in response to a glucose bolus versus a fasted state. 9cRA functions in pancreas to attenuate insulin secretion and synthesis, with the physiological impact of preventing hypoglycemia. In a fed, or glucose treated state, reduced 9cRA sensitizes pancreas for optimum insulin secretion. To understand metabolic regulation of 9cRA, synthesis was characterized with focus on Rdh5 expression in a beta cell model (832/13 rat insulinoma cells). Rdh5 expression and activity were reduced by glucose and cAMP, and overexpression of Rdh5 was sufficient to increase 9c-retinal and 9cRA synthesis. This work identified Rdh5 as a physiologically relevant regulator of 9cRA synthesis in beta cells.

A third project aimed to characterize a regulatory function of atRA in central nervous system (CNS) mediated energy balance. The hypothalamus brain region is an integrative center for peripheral signals, and regulates a range of functions including reproduction, autonomic nervous system, immune response, sleep, thermoregulation, fluid homeostasis, food intake and energy expenditure. Research established active atRA metabolism by regionally distinct concentrations of atRA, expression of genes for synthesis and response to atRA, and in situ synthesis of atRA in hypothalamus tissue explants. However, the atRA concentration did not change during dietary interventions of acute fasting and feeding, high fat diet feeding, or to cold exposure. Nuclear receptor PPARδ localized to nuclei and axon structures of neurons in hypothalamus and other brain regions, as demonstrated by immuno-histochemistry and confocal microscopy. This work set a foundation for study of atRA activity, and non-genomic function of PPARδ in hypothalamus, but was unsuccessful in establishing a regulatory function of atRA on CNS mediated energy balance.

The impact of dietary vitamin A on retinoid levels was evaluated in multiple tissues of five mouse strains, over three generations. The model transitioned mice from chow diet, containing copious vitamin A, to a semi-purified, vitamin A sufficient (VAS) diet. Three generations of VAS feeding decreased atRA in most tissues of most strains, in some cases more than 50%, and maintained an order of liver = testis > kidney > white adipose tissue = serum. Neither serum retinol nor atRA reflected tissue atRA concentrations. Strain and tissue-specific differences in retinol and atRA that reflect different amounts of dietary vitamin A could have profound effects on retinoid action, and are predictive of strain dependent differences in enzymes regulating atRA synthesis and catabolism. Consequently, the lab has continued to provide semi-purified VAS diet in mouse studies that evaluate retinoid homeostasis.

In total, this collection of work provides original and novel insights into metabolic regulation of atRA biosynthesis. The controlled fluctuation in atRA concentrations, as demonstrated in multiple contexts of physiologically relevant shifts in energy status, enables dynamic regulation of downstream transcriptional targets of atRA.

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