Three human AKR1B genes organized in tandem on chromosome 7q33–35 have been identified (Table 2; Figure 1): AKR1B1 [human aldose reductase (19)], AKR1B10 [also designated as HSI reductase: human small intestine reductase (1, 7)], and AKR1B15 (20). AKR1B1 seems to be ubiquitously expressed, whereas AKR1B10 expression was only reported in small intestine, colon, liver, thymus, and adrenal gland (1, 7). AKR1B15 gene was recently characterized and identified as closely related to the AKR1B1 and AKR1B10 cluster on chromosome 7 (Figure 1). AKR1B15 undergoes alternative splicing, giving rise to two protein isoforms, designated as AK1R1B15.1 and AKR1B15.2, expressed in thyroid gland and testis, respectively, and both in adipose tissue and placenta. AKR1B15.1 transcript encodes a putative protein sharing 68 and 91% sequence identity with AKR1B1 and AKR1B10, respectively (21). Both AKR1B15 transcripts were absent from human adrenal (20).

Four murine Akr1b genes have been described: Akr1b3 (murine aldose reductase), Akr1b7 [previously named MVDP: mouse vas deferens protein (22)], Akr1b8 [previously named FR-1: fibroblast growth factor (FGF)-related protein (23)], and Akr1b16 (21) (Table 2). Murine aldose reductase genes are located on chromosome 6 (locus 6B1), and their tandem arrangement suggests (as for the three human AKR1Bs) that these four genes arise from an ancestral gene duplication event (10, 12) (Figure 1). Several studies had allowed identification of murine Akr1b3, Akr1b8 as the orthologs of the human AKR1B1 and AKR1B10, respectively. However, this phylogenetic analysis between human and mouse AR has some limits and will be commented below. AKR1B1, Akr1b3, and Akr1b16 are rather ubiquitously expressed (11, 21), whereas Akr1b7 and Akr1b8 exhibit a restricted tissue distribution. Indeed, Akr1b7 is detected in vas deferens, adrenal glands, gonads, intestine, white adipose tissue, eye, liver, and kidney (2, 22, 24–26) and Akr1b8 in testis, heart, adrenal glands, intestine, and liver (2, 11, 23).

In studies using murine adrenal cell lines (Y1 adrenocortical cells and MPC862L chromaffin cells), we found that Akr1b3 protein accumulates in both adrenal cortex and medulla. Moreover, in vivo and ex vivo hormonal manipulations demonstrated that unlike the other murine Akr1b7 and Akr1b8 isoforms, Akr1b3 is expressed in the whole gland (27). Finally, cAMP stimulation failed to modulate Akr1b3 expression in Y1 cell line, confirming that Akr1b3 was insensitive to ACTH signaling (28) (Table 3).

Considering their enzymatic properties and expression levels in murine adrenal cortex, Akr1b7 and Akr1b8 are considered as the main isocaproaldehyde reductase and 4-HNE reductase, respectively, while Akr1b3 could rather participate in the elimination of these toxic compounds in basal physiological conditions (27–29). Moreover, Akr1b3 also displays 9-,11-endoperoxide reductase activity that, when coupled to COX-1 (cyclooxygenase type 1), allows prostaglandin F2α (PGF_2α) synthesis in adrenal cortex and medulla (see below).

Despite all these evidences upon Akr1b3 involvement in both lipid aldehyde detoxification and PGF_2α synthesis, in vivo Akr1b3 gene invalidation (Akr1b3^−/− mice) did not highlight any phenotype related to adrenal gland (30, 31). The lack of adrenal dysfunction in Akr1b3^−/− mice may result from the redundancy of enzymatic properties carried by the other murine isoforms expressed in the gland (Akr1b7 and Akr1b8) that could then compensate Akr1b3 loss.

In human adrenal gland, AKR1B1 transcripts have been initially detected using RNA master blot (1) (Table 3). Thereafter, using immunohistochemistry, we confirmed those results and demonstrated that AKR1B1 expression pattern is restricted to the cortex of adrenal gland (27). Treatment of the human adrenocortical tumor cells NC1-H295 with forskolin (adenylyl cyclase inducer) allowed us to suggest that similar to the murine isoform Akr1b7, AKR1B1 expression was sensitive to ACTH (32) (Table 3). The molecular mechanisms and cis-acting elements responsible for ACTH/cAMP responsiveness of AKR1B1 gene have not been investigated to date (Figure 2). Analysis of AKR1B1 expression in stress-related disorders was not explored to date. Moreover, analysis of adrenal samples from Cushing’s disease (ACTH-producing pituitary tumor) revealed unchanged mRNA levels of AKR1B1 gene (32).

Based on its enzymatic properties, AKR1B1 has long been considered as the sole isocaproaldehyde reductase in the human adrenal gland (33) (Figure 3A; Table 4). Interestingly, unlike murine Akr1b7 isoform, NADPH-dependent isocaproaldehyde reductase activity carried by AKR1B1 was inhibited by tolrestat, a potent and specific aldose reductase inhibitor belonging to the carboxylic acids group of AR inhibitors (13, 29, 33). We demonstrated that AKR1B1 was also able to convert PGH₂ into PGF_2α (34) (Figure 3C; Table 4). This 9-,11-endoperoxide reductase activity is also strictly NADPH-dependent and inhibited by tolrestat.

We observed that in the human adrenal gland, AKR1B1 and the cAMP-inducible COX-2 isoform were co-localized in steroidogenic cortical cells (27) (Table 3). Then, we can consider that human adrenal cortex has the potential to produce PGF_2α in response to ACTH surge. The shared properties of human AKR1B1 and mouse Akr1b7, such as hormonal regulation and reductase activity toward common substrates, prompted us to postulate that they can be considered as functional orthologs at least in the adrenal cortex (32). Moreover, increased levels of AKR1B1 transcripts in human were observed in adrenocortical adenomas harboring glucocorticoid autonomous hypersecretion (32). The possibility that AKR1B1 prostaglandin F synthase (PGFS) activity could participate in an intra-adrenal feedback loop between endocrine activities of cortex and medulla in human adrenal gland remains to be explored.

Given the high expression of AKR1B1 in the adrenal cortex, we evaluated alterations in its expression in association with human adrenal disorders. The relative abundance of AKR1B1 mRNA was decreased in adrenocortical carcinomas (ACC) when compared to benign tumors, Cushing’s hyperplasia, or normal adrenals (32). These data were reinforced by de Reyniès et al., who demonstrated that decreased AKR1B1 expression was associated with malignancy using an unsupervised clustering analysis of the human adrenal tumors transcriptome (35). This identified AKR1B1 as a potential negative marker for adrenocortical malignancy.

Akr1b8 mRNA was initially detected in both fetal and adult murine adrenal cortex but remained undetected in the medulla by in situ hybridization (2). In fibroblasts as well as in adrenocortical Y1 cell line, Akr1b8 was previously shown to be controlled by the FGF and phorbol myristate acetate (PMA) (23, 28). On the contrary, dexamethasone-induced ACTH suppression did not influence Akr1b8 mRNA and protein accumulation (27), suggesting that its biological function did not seem to be related to the ACTH-dependent steroidogenic activity present in adrenocortical cells (Table 3). Interestingly, in digestive tract organs, such as liver and small intestine, Akr1b8 gene was recently showed to be a target of NF-E2-related factor2 (Nrf2), which mediates transcriptional response to oxidative stress by binding to antioxidant response element (ARE) sites (36). As expected, Akr1b8 expression was downregulated in digestive tract from Nrf2 knockout mice. However, no adrenal phenotype was described in these mice, and neither oxidative stress nor Nrf2 have been involved in adrenal Akr1b8 expression so far.

Previous studies demonstrated that some AKR1B proteins showed efficient catalytic activity for the reduction of the abundant and highly reactive lipid-derived aldehyde 4-hydroxy-2-nonenal and the phospholipid aldehydes (17). In particular, Akr1b8 displays the most efficient phospholipid aldehyde and HNE-reductase activity in mouse tissues (17, 28, 37) (Figure 3; Table 4). All these enzymatic data suggest that in the adrenal cortex, Akr1b8 isoform could be devoted to detoxify aldehyde lipids abundantly accumulated in this tissue (38). Furthermore, expression of an antisense RNA directed against Akr1b7 suppressed isocaproaldehyde reductase activity in adrenocortical Y1 cells without any alteration of Akr1b8 protein accumulation (29). In view of its enzymatic features and constitutive expression, Akr1b8 is unlikely to be the principal isocaproaldehyde reductase in the adrenocortical gland (28). Akr1b8 gene disruption in mice led to reduced lipid synthesis and diminished proliferation of colonic epithelial cells but had no evident effect on general appearance, body weight, and reproduction. However, in vivo adrenal Akr1b8 physiological role remains to be examined since Akr1b8 gene disruption first report did not notice evident effect on the adrenal physiology (39).

Whether Akr1b8 and human AKR1B10 gene can be considered as ortholog is still a matter of debate since they share high sequence identity, and proteins display several close structural and enzymatic properties (11) (Tables 1 and 4). In contrast to Akr1b8, AKR1B10 gene expression is not controlled by FGF (12). Although AKR1B10 mRNA was initially detected in adrenal glands using a human RNA Master Blot, to date, there is no more information available on its in situ localization and transcriptional control in this organ (1). Moreover, AKR1B10 gene expression pattern only partially overlaps that of Akr1b8, since AKR1B10 transcripts are absent from heart, lung, or testis (7, 11).

Comparative studies demonstrated that AKR1B10 exhibits higher 4-HNE-reductase activity than AKR1B1, while lower than the murine Akr1b8 (11, 16). Ex vivo studies revealed that both human AKR1B1 and -B10 also share the ability to reduce isocaproaldehyde (1, 40). Nevertheless, in a comparative enzymatic study, Hara and colleagues showed that AKR1B1 had a more effective isocaproaldehyde reductase activity than AKR1B10, suggesting that in human steroidogenic organs, the latter was unlikely to play a major role in the detoxification of steroidogenic by-products (41).

The AKR superfamily has been added as a novel group of cytosolic enzymes that could contribute to retinoid–redox conversion. Based on their cofactor specificity (NADPH), AKR work in the reductive direction (42). Retinol (vitamin A) and its derivatives, retinaldehyde and retinoic acid (RA), are essential for the growth and maintenance of many tissues. RA is a key molecule in the development of different vertebrate organs by promoting cell differentiation and apoptosis. The control of retinaldehyde levels is essential in the regulation of RA synthesis and therefore of its signaling role. Once synthesized from β-carotene through the β-carotene 15,15′ monooxygenase 1 (BCO1), retinaldehyde has two alternative fates, its irreversible oxidation to RA (metabolism fate) by the aldehyde dehydrogenases (ADH) or its reduction back to retinol (storage fate) by the retinaldehyde reductase activity of AKR (43) (Figure 3B; Table 4). Comparative in vitro enzymatic studies on murine and human AKRs have fairly evidenced that among AKR1B proteins, AKR1B10 is so far the only retinaldehyde reductase with the highest k_cat value for the retinaldehyde reduction (11, 12, 44). Ex vivo AKR1B10 overexpression in different cell systems demonstrated its contribution in increasing retinol production (45, 46). In rodent, previous data evidenced that normal adrenal gland may function as an important site of retinoic acid synthesis involving class I- and IV-ADH, thus furthering retinaldehyde metabolism rather than its storage through the AKR activity (47). According to its expression, whether the well-established retinaldehyde reductase activity of AKR1B10 is operated in normal human adrenal physiology remains to be explored.

AKR1B10 expression was initially characterized in hepatocellular carcinoma and subsequently found to be altered by tumorigenesis process in several other organs (7, 48–50). Moreover, AKR1B10 expression was associated with smoker’s non-small cell lung carcinomas (48) and was suggested to be involved in drug resistance (51). A putative mechanism by which the activity of AKR1B enzymes could promote tumor growth is the conversion of retinaldehyde to retinol resulting in RA deprivation and blockage of its differentiating effect, promoting cell proliferation and fostering tumorigenesis (43). Furthermore, recent studies have shown that in breast cancer cells, AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), the rate-limiting enzyme of de novo synthesis of long-chain fatty acids, and blocks its ubiquitination and proteasome degradation. Long-chain fatty acids are the building blocks of biomembranes and the precursor of lipid second messengers, playing a critical role in cell growth and proliferation (52). The AKR1B10-mediated regulation on ACCA stability represents a novel regulatory mechanism, in which AKR1B10 promotes cell survival via modulating lipid synthesis, mitochondrial function, oxidative stress, and carbonyl levels (53).

Adrenocortical carcinomas are very aggressive and rare malignant tumors with poor prognosis (54). Microarray analysis was used to seek molecular predictors of malignancy and survival in a large cohort of unilateral adrenocortical tumors (http://www.ebi.ac.uk/arrayexpress, experiment E-TABM-311). Unsupervised clustering analysis allowed robust discrimination of malignant and benign tumors. On the basis of this analysis, AKR1B10 expression was not found to be associated with the ACC group (35).

High levels of Akr1b7 transcripts were initially observed by in situ hybridization in fetal and adult murine adrenal cortex but were undetectable in the medulla (2). We confirmed these results by immunohistochemistry experiments, which allowed us to further restrict major Akr1b7 expression to the zona fasciculata (55). In vivo, ACTH suppression with dexamethasone treatment resulted in a marked decrease of Akr1b7 mRNA levels that were restored when the treated mice were injected with exogenous ACTH. This ACTH/cAMP-induced Akr1b7 transcription was blocked by a PKA inhibitor (H89) in the murine adrenocortical ATC and Y1 cell lines (55, 56).

In the adrenal gland, basal and ACTH-induced expressions of Akr1b7 gene depend on three SF1 response element (SFRE) and on other cis-elements located in the upstream promoter region (Figure 3). Using transgenic mice and transfection experiments, we characterized a cryptic SFRE 102 bp upstream of the transcription start site that supports basal expression of Akr1b7 in the adrenal cortex. Among the two other SFREs identified further upstream, the site at −458 was a bona fide SFRE, essential for both basal and cAMP-stimulated promoter activity. The last SFRE, an Sp1, and C/EBPβ binding sites, respectively, localized at positions −503, −52, and −61 are all involved in cAMP responsiveness (57, 58).

AKR1Bs are capable to handle the large amount of isocaproaldehyde, a toxic by-product coming from the cholesterol side-chain cleavage during the initial step of steroid biosynthesis. Furthermore, isocaproaldehyde accumulation decreased viability of Y1 cells (29). Although Akr1b3, Akr1b7, and Akr1b8 all were able to reduce isocaproaldehyde, the two former seemed to be the more efficient reductases for this substrate (28). However, the silencing of Akr1b7 gene was sufficient to abolish the cAMP-induced isocaproaldehyde reductase activity in Y1 cells. Therefore, Akr1b7 was the main enzyme in charge of isocaproaldehyde detoxification in the adrenal cortex (29). Altogether, these data showed that in the adrenal cortex, ACTH not only controls expression of enzymes synthetizing steroids but also of proteins scavenging toxic by-products derived from steroidogenesis.

Madore et al. established that the bovine 20α-hydroxysteroid dehydrogenase, AKR1B5 was responsible for PGF_2α synthesis in the endometrium (59). Thereafter, we demonstrated by ex vivo studies, that AKR1B1, Akr1b3, and Akr1b7 were also able to reduce PGH₂ into PGF_2α, whereas Akr1b8 and AKR1B10 were devoid of this PGF_2α synthase activity (Table 4). Due to their recent discovery, this 9-,11-endoperoxide reductase activity has not been investigated yet for Akr1b16 and AKR1B15. Moreover, their enzymatic constants suggested that AKR1B1, Akr1b3, and Akr1b7 had a higher 9-,11-endoperoxide reductase activity than the other PGF synthases already described (34). Prostaglandins are paracrine/autocrine signal molecules produced from a common precursor, PGH₂, which is derived from arachidonic acid by COX-1 or COX-2. Unlike COX-1, which is a constitutively expressed enzyme, COX-2 is not expressed in most organs under basal conditions but can be stimulated by inflammation and various mitogenic factors (60). Following these observations, we carefully examined the PGF_2α biosynthetic pathway in the adrenal gland (27).

Prostaglandin F2α was produced by both cortical (steroidogenic cells) and medullary (chromaffin cells) tissue of the adrenal gland. In primary adrenocortical cell culture, PGF_2α release was induced 2.5-fold by ACTH treatment. This secretion was correlated with ACTH responsiveness of both COX-2 and Akr1b7. Using ex vivo gain- and loss-of-function strategies, we demonstrated the pivotal role of Akr1b7 in ACTH-induced PGF_2α release, and it is functionally coupled with COX-2. In the adrenal medulla in which Akr1b7 was not expressed, PGF_2α was produced from the coordinated activities of Akr1b3 and COX-1. Adrenal expression of PGF_2α-specific receptor (FP) was restricted to the chromaffin cells, suggesting that both autocrine and paracrine mechanisms (within the medulla and between steroidogenic and medulla cells, respectively) were relaying PGF_2α action. In agreement with this hypothesis, we demonstrated that PGF_2α repressed both basal and glucocorticoid-induced dopamine release in the chromaffin cell line MPC862L. Comparison of the PGF_2α-responsiveness of isolated cells and whole adrenal tissue cultures showed that PGF_2α-mediated repression of glucocorticoid release is an indirect mechanism relying on a decrease in catecholamine secretion, which in turn decreased cortical steroidogenesis.

These functional data led us to propose an intra-adrenal feedback loop in which adrenal endocrine activities are regulated through the involvement of AKRs [Figure 4 and Ref. (18)]. Surprisingly, however, the absence of Akr1b7 in vivo did not affect basal adrenocortical function as illustrated by normal glucocorticoid plasma levels in Akr1b7^−/− mice (61). Indeed, these mice displayed an obese phenotype that did not rely on adrenal dysfunction but on the lack of Akr1b7-dependent production of PGF_2α within the stromal vascular adipose tissue (3, 61). Adrenal expression of Akr1b3 and b8 is not affected in knockout mice and since they all share redundant enzymatic activities regarding detoxification of lipid aldehydes (see Akr1b3/AKR1B1: Expression Pattern and Relevant Functions and Akr1b8/AKR1B10: Phylogeny, Regulation, and Enzymatic Specificities), the remaining isoforms can compensate the absence of Akr1b7 at least in basal conditions. Importantly, Akr1b7 is the only one out of the three adrenal isoforms to be ACTH-responsive (27) and also the most abundantly expressed (3). Taken together, these hallmarks would predict that physiological importance of Akr1b enzymes in adrenal function should be rather explored under stress conditions during which scavenging capacity of constitutive (and less abundant) isoforms should be exhausted.