An intimate interaction between vitamin A (retinol) and energy homeostasis is demonstrated by retinoid regulation of metabolic enzymes and transcription factors, alterations in retinoid levels and proteins during different metabolic states, as well as retinoid control over the development of many tissues critical to energy status. Despite these associations, little progress has been made in understanding the molecular mechanisms governing these interactions and the impact on systemic physiology. Retinol is the precursor for two biologically active metabolites, all-trans-RA (atRA) and 9-cis -RA (9cRA). Currently, 9cRA is the only high affinity ligand known to activate the retinoid X receptor (RXR), the obligate dimer for all type II nuclear receptors, many of which are involved in regulating energy metabolism. The spectrum of pathways regulated by RXR predicts 9cRA as the retinoid with the broadest impact on energy regulation. Despite this potential, the widespread presence of atRA in many tissues, and the undetectable levels of 9cRA in vivo have evoked skepticism in the physiological relevance of 9cRA activation.
Investigation into pancreatic retinoid metabolism and whether retinoids govern insulin secretion was prompted by islet expression of several members of the retinoid pathway, in conjunction with the hyperinsulinemic effects in patients taking the drug Accutane®; the active ingredient being large doses of the biologically inactive RA isomer, 13-cis-RA. Using a highly sensitive LC/MS/MS assay with complete separation of geometric isomers, we reveal the first in vivo identification of 9cRA as an endogenous retinoid in mouse pancreas, at levels higher than atRA. Comparing opposing mouse models of β-cell mass, we demonstrate pancreatic 9cRA levels reflect β-cell number and determine the β-cell line 832/13 can synthesize 9cRA enzymatically. Decreased 9cRA levels accompany states of insulin demand, including fasting and following a glucose bolus. The absence of this decreases results in impaired insulin secretion and hyperglycemia, as demonstrated by aberrant retinoid synthesis in the cellular retinol binding protein I (CRBPI) null mice. Isolated islets and 832/13 cells treated with 9cRA have a dual response, both increasing and decreasing insulin secretion depending upon fluctuation of 9cRA levels. Variation in pancreatic 9cRA disrupts the balance of factors required for proper islet function and retinoid synthesis, ultimately dictating systemic insulin and glucose levels.
New aspects are identified in both the biological synthesis of 9cRA and retinoid metabolism. The ability to detect 9cRA only in the pancreas is a function of decreased metabolic clearance of pancreatic 9cRA in 832/13 cells and isolated microsomal fractions. CRBPII expression is induced in islets of two mouse models of dysregulated insulin secretion and 9cRA levels, the CRBPI nulls and ob/ob mice. This is the first identification of strong CRBPII expression outside of the intestine in the adult mouse and we introduce an oxidative capacity of CRBPII similar to CRBPI previously unacknowledged. These studies confirm the biological significance of 9cRA activation, expand the current understanding of mechanisms regulating retinoid metabolism and identify new pancreatic targets of therapeutic potential.
Changes in pancreatic 9cRA levels during states of insulin demand, such as starvation, indicated retinoid levels fluctuate with energy status. Whether this phenomenon occurs in other tissues, and the metabolic conditions and factors stimulating changes to retinoid metabolism remain unclear. Peroxisome proliferator-activated receptor α (PPARα) is widely acknowledged for its role in governing fatty acid oxidation and the starvation response. The large ligand-binding pocket of PPARα allows low affinity binding and activation by several dietary lipids. This ligand promiscuity has suggested PPARα acts as a lipid sensor, altering gene transcription designed to clear and catabolize activating lipids under different metabolic conditions. Identification of a retinal reductase with peroxisomal localization regulated by clofibrate supported PPARα regulation of retinoid metabolism, leading us to hypothesize PPARα activation is serving as an interface between the sensing of energy status and the subsequent alterations in retinoid homeostasis.
Using different mouse models and pharmacological induction, we determined states of PPARα activation and/or increased fatty acid oxidation initiate changes in systemic retinoid flux. Mice displaying constitutively elevated levels of PPARα and increased fatty acid oxidation (PPARα agonist activation and ob/ob mice) display more than 2-fold elevations in serum retinol, increased serum and adipose levels of RBP4 and almost total ablation of liver retinyl esters (RE). Depletion of liver RE can be invoked in fasted mice and restored upon feeding of a vitamin A deficient diet, indicating a transient redistribution of retinoids throughout the body. This mobilization is facilitated by a PPARα regulated peroxisomal RE hydrolyzing capacity crucial for both storage and movement of liver RE depots. (Abstract shortened by UMI.)