Abstract

   Keratoconus (KC) is a corneal thinning disease with an onset commonly
   immediately post-puberty and stabilization by 40 to 50 years of age.
   The role of hormones in regulating corneal tissue structure in
   homeostatic and pathological conditions is unknown. Our group recently
   linked altered hormone levels to KC. Our current study sought to
   investigate and delineate the effects of exogenous hormones, such as
   androgen, luteotropin, and estrogen, on corneal stroma bioenergetics.
   We utilized our established 3D in vitro model to characterize the
   effects of DHEA, prolactin, 17β-estradiol on insulin-growth factor-1
   and -2 (IGF-1, -2) signaling and metabolic function in primary corneal
   fibroblasts from healthy controls (HCFs) and KC patients (HKCs). Our
   data showed that exogenous DHEA significantly downregulated IGF-1 and
   its receptor in both HCFs and HKCs with HKCs showing consistently lower
   basal pentose phosphate flux. Prolactin caused no significant change in
   IGF-1 levels and an increase in IGF-2 in HKCs correlating with an
   increase in ATP and NADH levels. 17β-estradiol led to a significant
   upregulation in pentose phosphate flux and glycolytic intermediates in
   HCFs. Our results identified hormone-specific responses regulated in
   HKCs compared to HCFs revealing a novel role for hormones on
   bioenergetics in KC.
     __________________________________________________________________

   Sex hormones play a functional role in regulating growth and
   reproduction, systemic metabolism, and cellular differentiation and
   functionality[30]^1,[31]^2,[32]^3. Dehydroandrosterone (DHEA) is a
   steroid produced primarily by the adrenal gland, gonads, and brain that
   is readily converted to androgens or estrogens in a tissue-dependent
   fashion[33]^4,[34]^5 with circulating plasma levels ranging from
   1–6 ng/mL[35]^6. The sulfated form of DHEA, dehydroandrosterone sulfate
   (DHEA-S), is present at much higher concentrations in circulation
   (450–3470 ng/mL) depending on age and sex[36]^7, which upon cellular
   uptake is de-sulfated and converted to steroid metabolites. DHEA-S
   supplementation has been shown to increase plasma levels of insulin
   growth factor-1 (IGF-1)[37]^8,[38]^9 with suggestions that depletions
   in DHEA may be linked to processes associated with
   aging[39]^10,[40]^11,[41]^12. Estrogens, estriol, 17β-estradiol (E2)
   and estrone, are produced by the ovaries in females and in small
   amounts by the prostate in males. Many of these hormones are known to
   regulate IGF-1 signaling directly during development and in
   pathological states, such as cancer[42]^13,[43]^14,[44]^15,[45]^16.
   IGF-1 is a pleiotropic growth factor whose production is stimulated by
   human growth factor serving as a key regulator of anabolic processes
   that promote tissue growth[46]^17,[47]^18. IGFs have been found to be
   essential growth factors during embryonic development mitigating a role
   in ventricular chamber heart development in mice[48]^19. Heterogeneous
   knockout of IGF-1R in mice increased mean lifespan by 26% than
   wild-type controls suggesting that IGF-1 flux may be involved in a
   complicated mechanism in the regulation of aging[49]^20. Localized
   IGF-1 production is suggested to play a fundamental role in regulating
   postnatal tissue growth compared to systemic flux[50]^21. In contrast,
   an overabundance of IGF-1 production due to elevated human growth
   hormone after puberty promotes the development of acromegaly, which
   results in overgrowth of connective tissue, skeletal aberrations, and
   cardiovascular defects[51]^22,[52]^23. Increased corneal thickness has
   also been identified in some acromegaly patients suggesting that
   overproduction of IGF-1 promoted by human growth hormone may promote
   stromal thickening within the eye as well[53]^24,[54]^25. Whether the
   effects of DHEA on corneal extracellular matrix (ECM) deposition are
   mediated via IGF-1 regulation is unknown.

   Corneal thickness varies throughout development to adulthood and from
   person to person[55]^26. A major contributor to corneal thickness has
   been associated with altered hormone levels with varying effects on
   corneal thickness occurring during pregnancy[56]^27 and aging[57]^28.
   In females, corneal thickness has been shown to be greatest at
   ovulation and at the end of the menstrual cycle[58]^29,[59]^30.
   Furthermore, estrogen supplementation has been suggested to promote
   increased corneal thickness in post-menopausal women[60]^31. Hormones
   mediate changes in cell function via binding to their respective
   receptors with both the androgen receptor[61]^32,[62]^33 and estrogen
   receptor[63]^34 being expressed within the human cornea suggesting that
   hormones may influence corneal function directly. A published report
   identified that healthy corneal epithelium and conjunctival cells
   transcribe the genes for enzymes required for intracrine production of
   various hormones[64]^35, including 3β-hydroxysteroid dehydrogenase,
   which converts estrogens and androgens to activated forms via
   hydroxylation at carbon-17 of the steroid ring[65]^36. This study
   suggested that tissues of the anterior segment of the eye may be able
   to produce hormones in situ thereby influencing localized cellular
   signaling in an autocrine or paracrine fashion independent of systemic
   flux.

   The common corneal dystrophy, Keratoconus (KC), is associated with
   significant thinning of the central cornea leading to severe visual
   defects with onset usually post-puberty and stabilization by the fourth
   to fifth decade of life[66]^37,[67]^38. KC has been reported to affect
   a higher predominance of males to females[68]^39,[69]^40. Our previous
   studies have indicated that systemic hormone levels are altered in KC
   patients compared to age- and gender-matched controls suggesting that
   systemic hormone flux may initiate a cascade of downstream signals that
   collectively may be responsible for KC development[70]^41. The KC onset
   at roughly 15 years of age, as well as the gender dependence and
   stabilization of the disease in middle age, support our proposed
   mechanism that a developmental change in a hormone-specific
   relationship may contribute to KC pathogenesis. In our current study,
   we investigated the effects of exogenous hormones in HCFs and HKCs in
   an attempt to delineate the role of hormones in KC using our 3D in
   vitro model. Our data revealed a novel mechanism of action of hormones
   in regulating IGF-1 expression and bioenergetics in corneal fibroblasts
   providing insight into possible causation and progression of corneal
   thinning in KC.

Results

Metabolic Response to DHEA

   KC has been associated with altered cellular metabolism by HKCs with
   increased lactate production and elevated oxidative stress[71]^42. In
   order to determine if exogenous DHEA regulates cellular metabolism, we
   performed a pathway enrichment analysis of metabolites from control and
   DHEA-treated samples. The primary pathways enriched in HCFs following
   DHEA treatment involved lipid metabolism (phospholipid biosynthesis,
   glycerolipid metabolism, glycerol phosphate shuttle), protein
   metabolism (glutamate, tryptophan, methionine, arginine, aspartate, and
   tyrosine metabolism), and glucose metabolism (mitochondrial electron
   transport chain, citric acid cycle, glycolysis, pyruvate metabolism)
   ([72]Fig. 1A). Cholesterol serves as a precursor to
   steroid-biosynthesis, as well as an essential structural component of
   the lipid bilayer. Cholesterol sulfate also functions as a source of
   steroid precursors, including DHEA-S and pregnenolone sulfate, and in
   addition, is a regulator of phosphatidylinositol 3-kinase and protein
   kinase C[73]^43. No change was found in basal cholesterol levels but a
   trend in increasing cholesterol sulfate in both HCFs and HKCs with DHEA
   treatment ([74]Fig. 1B,C). Metabolites important in phospholipid
   biosynthesis and signaling, phosphorylcholine and choline, were
   significantly increased in HCFs (3.4-fold and 7.8–fold, respectively,
   p < 0.05) with DHEA treatment (5 ng/mL) with little change in HKCs
   ([75]Fig. 1D,E). Glycerophosphocholine, ethanolamine, and
   glycerol-3-phosphate, important mediators involved in lipid signaling
   and lipogenesis, were increased in HKCs under all conditions compared
   to HCFs (p < 0.01, [76]Fig. 1F–H). Furthermore, glycerol-3-phosphate,
   which serves as a precursor to many glycerophospholipids, was
   significantly increased with DHEA treatment (5 ng/mL) in HKCs
   highlighting lipid metabolism and phospholipid-mediated signaling as an
   important target of DHEA (1.8-fold, p < 0.05, [77]Fig. 1H). Nucleic
   acid metabolism also showed an increasing trend with DHEA treatment in
   both cell types with elevated uridine and purine flux suggesting DHEA
   may modulate availability of nucleic acids required for gene
   transcription ([78]Fig. 1I–K).

Figure 1. Effects of DHEA on metabolic pathways and IGF-1 expression in
healthy human corneal fibroblasts (HCFs) and human keratoconus cells (HKCs).

   [79]Figure 1
   [80]Open in a new tab

   (A) Chemical structure of DHEA and enrichment pathways identified in
   HCFs by MetaboAnalyst following DHEA treatment. (# denotes pathways
   highlighted by metabolite breakdown). Quantitative determination of
   metabolite levels by targeted LC-MS/MS important in (B,C) steroid
   biosynthesis and metabolism, (D–H) phospholipid biosynthesis and
   signaling, and (I–J) pyridines, and (K) purine metabolism. (L–P)
   NAD+/NADH, ATP, ADP, and AMP flux in HCFs and HKCs detected by
   LC-MS/MS. (Q–T) Representative western blots and quantification of
   protein levels measured by densitometry showing a significant reduction
   in IGF-1 and its active receptor IGF-1R with no change in IGF-2
   expression following DHEA treatment (2.5 ng/mL and 5 ng/mL). Western
   blots were converted to greyscale. Statistical significance determined
   by a two-way ANOVA. Experiments were performed in triplicate. Error
   bars represent standard error of the mean. *p < 0.05, **p < 0.01,
   ***p < 0.001, ***p < 0.0001. Note: controls were treated with vehicle
   (1% DMSO).

   We further determined the levels of nicotinamide adenine dinucleotide
   (NAD+), nicotinamide adenine dinucleotide hydride (NADH), adenosine
   triphosphate (ATP), adenosine diphosphate (ADP), and adenosine
   monophosphate (AMP). We found a significant reduction in NAD+ in HCFs
   compared to HKCs in all conditions (p < 0.05) with a trend of
   increasing levels with the highest concentration of DHEA and no
   significant change in NADH levels ([81]Fig. 1L,M). Furthermore, DHEA
   treatment (5 ng/mL) led to a significant increase in ATP (2.3-fold,
   p < 0.01) and AMP flux (2-fold, p < 0.05) with control HCFs having
   significantly lower levels of ATP compared to control HKCs (2.3-fold,
   p < 0.05) and no difference in ADP production suggesting that DHEA may
   contribute to altered energy production by affecting nucleotide
   availability or possibly glucose metabolism ([82]Fig. 1N–P).

   Since IGFs are one of the major regulators of metabolism[83]^44,[84]^45
   and are known to be affected by hormone flux[85]^17,[86]^46, as well as
   play an important role in corneal epithelial wound healing[87]^47, we
   investigated whether the changes in metabolic regulation were related
   to altered protein expression of localized IGF-1, IGF-2, and the active
   receptor IGF-1R ([88]Fig. 1Q–T). We found a significant reduction in
   IGF-1 production by 4.9-fold with 5 ng/mL DHEA treatment (p < 0.01) in
   HCFs with no significant reduction in HKCs ([89]Fig. 1R). The
   expression of the alternative isoform, IGF-2, was not affected by DHEA
   treatment in either HCFs or HKCs ([90]Fig. 1S). A reduction in the
   primary IGF-1 receptor, IGF-1R, was measured in both HCFs and HKCs by
   2-fold (p < 0.01) with the most significant response at the high dose
   in HKCs suggesting that DHEA may directly regulate localized IGF-1R
   signaling by targeting both ligand and receptor expression ([91]Fig.
   1T). Flux of amino acids, glutamate, asparagine, and alanine, increased
   with DHEA treatment in HCFs with no effect in HKCs ([92]Supplemental
   Fig. 1, p < 0.05). Our results suggest that DHEA downregulates IGF-1
   leading to modulation of net protein synthesis or degradation in HCFs
   with the measured increase in amino acid flux correlating with
   increased catabolism. The lack of change in free amino acid flux in
   HKCs may be related to the sustained IGF-1 levels suggesting that
   ligand expression may be the limiting factor in regulating
   IGF-signaling compared to the active receptor, IGF-1R.

DHEA and the Effects on Glycolysis, TCA, and PPP

   In order to determine the effects of DHEA on the major pathways
   involved in glucose metabolism, we evaluated metabolite flux in
   glycolysis, tricarboxylic acid cycle (TCA), and the pentose phosphate
   pathway (PPP). Surprisingly, no significant change in glycolytic flux
   was measured with exogenous DHEA in either cell type ([93]Fig. 2A–I).
   However, HKCs showed increased flux of glycolytic intermediates,
   including fructose-1,6-bisphosphate (3-fold, p < 0.01),
   phosphoenolpyruvate (5-fold, p < 0.05), and dihydroxyacetone phosphate
   (6.7-fold, p < 0.05), compared to HCFs with DHEA treatment (2.5 ng/mL)
   ([94]Fig. 2C,F,I). Isocitrate was significantly upregulated (2-fold,
   p < 0.01) in HCFs with DHEA treatment (5 ng/mL) with trends of
   increases in other TCA intermediates, including fumarate and malate
   ([95]Fig. 2Q–U). Though HKCs showed increased basal glycolytic flux
   compared to HCFs, they showed no increase in TCA flux correlating with
   an increase in aerobic glycolysis in a Warburg-like phenomena[96]^48
   with KC being previously associated with elevated lactate
   production[97]^42.

Figure 2. Effects of DHEA on glucose metabolism in HCFs and HKCs.

   [98]Figure 2
   [99]Open in a new tab

   (A–I) Glycolytic flux, (J–N) Pentose phosphate flux, and (O–U) TCA
   cycling in HCFs and HKCs following DHEA treatment (2.5 ng/mL and
   5 ng/mL). Experiments were performed in triplicate, error bars
   represent standard error of the mean. Statistical significance was
   determined by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001. (Abbrev.
   F-6-P, fructose-6-phosphate; F-1,6-BP, fructose-1,6-bisphosphate;
   1,3-DPG, 1,3-diphosphoglycerate; 3-PG, 3-phosphoglycerate; PEP,
   phosphoenolpyruvate; Gly-3-P, glyceraldehyde-3-phosphate; DHAP,
   dihydroxyacetone phosphate.) Note: controls were treated with vehicle
   (1% DMSO).

   It has previously been reported that DHEA inhibits the PPP in human
   endometrial stromal cells[100]^49 and tumor cells[101]^50. The PPP
   pathway has also been identified by us as a target of the antioxidant,
   Quercetin, in HKCs with upregulation of glucose-6-phosphate,
   glyceraldehyde-3-phosphate, and erythrose-4-phosphate[102]^51.
   Treatment of HCFs and HKCs with 2.5 ng/mL and 5 ng/mL DHEA did not
   cause a significant reduction in PPP intermediates in our model
   ([103]Fig. 2A,J–N). Interestingly, we measured a significant reduction
   in 6-phospho-D-gluconate and D-sedoheptulose-1,7-phosphate in control
   HKCs compared to control HCFs (2-fold and 3-fold, respectively,
   p < 0.05, [104]Fig. 2K,M). Provided that 6-phospho-D-gluconate is
   converted to sedoheptulose-1,7-phosphate generating NADPH, the reducing
   agent responsible for converting oxidized glutathione to reduced
   glutathione, a reduction in this important substrate may relate to the
   increase in oxidative stress associated with KC.

   Since we found that DHEA treatment influenced IGF-1 expression, we
   further evaluated the effects of DHEA on arginine metabolism, which can
   serve as a source of proline[105]^52 and polyamines[106]^53 affecting
   collagen assembly and cell survival, respectively. The urea cycle is a
   major regulator of arginine metabolism and occurs with the conversion
   of arginine to ornithine, which is then converted to citrulline and
   then to argininosuccinate. Urea is produced as a by-product following
   the conversion of arginine to ornithine. Following DHEA treatment
   (5 ng/mL), we observed a significant increase in carbamoyl production
   (2-fold, p < 0.05) in HCFs, with a slight increase in HKCs
   ([107]Supplemental Fig. 2A). L-argininosuccinate was significantly
   elevated in HKCs with DHEA (2.5 ng/mL and 5 ng/mL, 2-fold and 3-fold,
   respectively, p < 0.01) compared to HCFs ([108]Supplemental Fig. 2C).
   Arginine levels were increased slightly, though not significantly, with
   DHEA treatment suggesting that limiting reagents may restrict arginine
   flux ([109]Supplemental Fig. 2D). Furthermore, both urea and ornithine
   levels were unchanged with DHEA treatment in either cell type
   ([110]Supplemental Fig. 2E,F). The reduction in IGF-1 caused by DHEA
   suggests direct modulation of urea cycling in both HCFs and HKCs, which
   may affect the availability of precursors for proline and
   hydroxyproline biosynthesis involved in collagen assembly.

Metabolic Response of Prolactin

   Prolactin has been shown to increase lactose and lipid production
   within the post-partum mammary gland[111]^54,[112]^55. Corneal
   curvature is known to be modulated during and following pregnancy
   suggesting a potential role for prolactin in regulating post-natal
   corneal structure[113]^56. In order to determine the effects of
   prolactin on bioenergetics in HCFs and HKCs, we measured metabolite
   flux and enrichment pathways following treatment with prolactin. We
   found that prolactin enriched pathways in HCFs associated with
   nicotinamide metabolism, mitochondrial electron transport chain, and
   butyrate metabolism ([114]Fig. 3A). Nicotinamide was significantly
   upregulated 4.3-fold (p < 0.0001) in 50 ng/mL prolactin-treated HCFs
   compared to HKCs ([115]Fig. 3B). Quinolinate is a product of tryptophan
   metabolism in the Kynurenine pathway with reports of elevated levels
   associated with neurodegenerative diseases, including Alzheimer’s and
   Huntington’s diseases, as well as contributing to increased oxidative
   stress[116]^57,[117]^58. We detected a significant reduction in
   quinolinate in control HKCs compared to HCFs with no modulation with
   prolactin treatment (3.3-fold, p < 0.05, [118]Fig. 3C). Furthermore, we
   measured no significant change in free amino acid flux with prolactin
   treatment suggesting that protein synthesis and degradation are not
   affected by prolactin stimulation (25 ng/mL and 50 ng/mL)
   ([119]Supplemental Fig. 3). Geranyl pyrophosphate (geranyl-PP), a
   metabolite important in mediating lipid biogenesis serving as a
   precursor in terpenoid biosynthesis[120]^59, was significantly
   upregulated in control HKCs (2.3-fold, p < 0.05) compared to HCFs with
   a modest reduction in HKC levels with prolactin treatment (25 ng/mL)
   (1.6-fold, p < 0.05, [121]Fig. 3G). Octulose-8-phosphate and
   octulose-1-phosphate (O8P-O1P) are important in L-PPP flux in
   eukaryotic cells[122]^60 and were found to elevated in HCFs (2.5-fold,
   p < 0.05) compared to HKCs with little change following prolactin
   treatment ([123]Fig. 3H).

Figure 3. Effects of prolactin (PRL) (25 ng/mL and 50 ng/mL) on metabolic
pathway and IGF-1 expression.

   [124]Figure 3
   [125]Open in a new tab

   (A) Pathway enrichment predicted by Metaboanalyst and metabolic
   breakdown based on (A,B) Nicotinate and quinolinate metabolism, and
   (D–F) free amino acid flux, (G) geranyl pyrophosphate, (H)
   oculose-8-phosphate-oculose-1-phosphate flux, (I–K) lactate and
   coenzyme A flux, (L-P) NAD+/NADH and ATP, ADP, and AMP flux. (Q-T)
   Representative western blots and quantification of bands by
   densitometry for IGF-1, IGF-2, and IGF-1R. Western blots were converted
   to greyscale. Statistical significance determined by a two-way ANOVA.
   Experiments were performed in triplicate. Error bars represent standard
   error of the mean. *p < 0.05, **p < 0.01, ***p < 0.001.,
   ****p < 0.0001. Note: controls were treated with vehicle (purified
   water).

   We have previously reported elevated lactate production by HKCs in
   vitro[126]^42 and found no significant modulation by prolactin in this
   study with HKCs maintaining significantly higher lactate production
   compared to HCFs showing no improvement in the oxidative tendency of
   HKCs (2-fold, p < 0.001, [127]Fig. 3I). Co-enzyme A and succinyl-CoA
   were upregulated slightly in 25 ng/mL prolactin-treated HCFs compared
   to HKCs (2-fold, p < 0.05) with HKCs showing overall higher levels of
   free coenzyme A with 50 ng/mL prolactin significantly increasing these
   levels in HKCs only (1.5-fold, p < 0.05, [128]Fig. 3J,K). The
   availability of CoA levels would likely affect TCA flux given the
   requirement of this substrate in the biosynthesis of acetyl CoA, while
   the reduced levels in HKCs correlate with higher lactate production and
   glycolytic flux.

   We further determined if prolactin affected production of high-energy
   biomolecules. Interestingly, though HKCs showed lower nicotinamide
   levels, NAD+ levels were upregulated in HKCs control, 25 ng/mL and
   50 ng/mL prolactin-treated samples (2.9-fold, 3.2-fold, and 4.6-fold,
   respectively, p < 0.0001) with an increase in NADH production by
   2.6–fold and 3.5-fold (p < 0.05 and p < 0.001, respectively, [129]Fig.
   3L,M). Correlating with this increase in NADH production by HKCs, ATP
   levels also increased 1.3–fold (p < 0.05) with prolactin treatment with
   no such increase detected in HCFs. In agreement with the DHEA
   experiments ([130]Fig. 1N), basal ATP flux was higher in control HKCs
   compared to HCFs with little change in ADP and AMP levels ([131]Fig.
   3N–P).

   In order to determine if the effects on NADH and ATP production in HKCs
   were attributed to altered IGF-signaling, we measured the protein
   expression of IGF-1, IGF-2, and the receptor IGF-1R ([132]Fig. 3Q).
   Compared to the effects of DHEA, we found little modulation of IGF-1 or
   IGF-1R expression by prolactin but found a significant increase in
   IGF-2 production by HKCs (3.5-fold, p < 0.01) with no effects in HCFs
   ([133]Fig. 3Q–T). Our results agree with previous reports that
   prolactin mediates changes primarily through IGF-2 in breast
   tissue[134]^61 and suggest a potential link to the effects of prolactin
   in regulating post-partum corneal structure via IGF-2 with higher
   responsiveness by HKCs.

Prolactin and the Effects on Glycolysis, TCA, and PPP

   In order to determine if exogenous prolactin resulted in changes in
   glucose metabolism, we investigated glycolytic, TCA, and PPP flux in
   HCFs and HKCs ([135]Fig. 4). 3-Phosphoglycerate (3-PG) flux decreased
   2-fold (p < 0.05) in HCFs with prolactin treatment (25 ng/mL and
   50 ng/mL) ([136]Fig. 4E) with little effect on the other glycolytic
   intermediates ([137]Fig. 4A–I). Both phosphoenolpyruvate (PEP) and
   dihydroxyacetone phosphate (DHAP) were upregulated in all HKC samples
   compared to HCFs again correlating with increased glycolytic activity
   in HKCs with little influence by prolactin ([138]Fig. 4F,I, p < 0.05).
   TCA flux showed higher levels of the intermediates, fumarate,
   isocitrate, and succinate in HCFs with and without prolactin treatment
   with reductions in levels following prolactin treatment ([139]Fig.
   4Q,S,T). While PPP flux was consistently higher in HCFs compared to
   HKCs with elevated erythtrose-4-phosphate ([140]Fig. 4N), prolactin did
   not modulate PPP flux in either cell type ([141]Fig. 4J–N).
   Furthermore, little effect occurred in amino acid flux
   ([142]Supplemental Fig. 3) which agreed with no change in IGF-1 or
   IGF-1R levels induced by prolactin ([143]Fig. 3Q) suggesting that the
   downstream effects of altered IGF-2 levels may not contribute to
   changes in protein synthesis and degradation or significant variations
   in glucose metabolism.

Figure 4. Effects of prolactin (PRL) on glucose metabolism in HCFs and HKCs.

   [144]Figure 4
   [145]Open in a new tab

   (A–I) Glycolytic flux, (J–N) Pentose phosphate flux, and (O–U) TCA
   cycling in HCFs and HKCs following PRL treatment (25 ng/mL and
   50 ng/mL). Experiments were performed in triplicate, error bars
   represent standard error of the mean. Statistical significance was
   determined by ANOVA. *p < 0.05, **p < 0.01, ***p < 0.001,
   ****p < 0.0001. (Abbrev. F-6-P, fructose-6-phosphate; F-1,6-BP,
   fructose-1,6-bisphosphate; 1,3-DPG, 1,3-diphosphoglycerate; 3-PG,
   3-phosphoglycerate; PEP, phosphoenolpyruvate; Gly-3-P,
   glyceraldehyde-3-phosphate; DHAP, dihydroxyacetone phosphate.) Note:
   controls were treated with vehicle (purified water).

Metabolic Response of E2

   Estrogens are known to modulate survival and proliferation of
   cells[146]^62 and promote corneal wound healing in vitro[147]^63. In
   order to determine if E2 influenced cellular metabolism in a similar
   manner as DHEA or prolactin, we investigated the effects of increasing
   concentrations of E2 on metabolic pathways and amino acid flux in HCFs
   and HKCs in our 3D in vitro model. Enriched metabolites associated with
   E2 treatment in HCFs involved pantothenate biosynthesis, histidine
   metabolism, and protein biosynthesis ([148]Fig. 5A). Effects of E2 on
   lipid metabolism differed from DHEA-treated constructs with significant
   reductions in cholesterol sulfate and geranyl-pentaphosphate levels in
   HKCs (2-fold and 2.6-fold, respectively, p < 0.05), but not in HCFs,
   with little change in basal cholesterol levels ([149]Fig. 5B,C).
   Pantothenate is functionally important in coenzyme A
   biosynthesis[150]^64 and may play a role in regulating reactive oxygen
   species flux in dermal fibroblasts[151]^65. Our results show that E2
   reduced pantothenate levels in HCFs by 2.5–fold (p < 0.0001) with HKCs
   showing reduced levels even at basal conditions ([152]Fig. 5E).
   Histidine was differentially regulated as well between HCFs and HKCs
   with E2 treatment (5 ng/mL) increasing it by 1.3-fold (p < 0.001) in
   HCFs and reducing it by 2–fold (p < 0.001) in HKCs ([153]Fig. 5F). This
   trend was also seen with 1-methyl-histidine, a precursor important in
   histidine metabolism, with E2 treatment (2.5 ng/mL) increasing levels
   1.4–fold (p < 0.001) in HCFs and reducing 2–fold (p < 0.001) in HKCs
   ([154]Fig. 5G). Pyridoxine, as a form of Vitamin B6, functions as a
   cofactor for trans-sulfuration reactions with deficiencies associated
   with seizures[155]^66,[156]^67. Pyridoxine and 4-pyridoxic acid were
   both downregulated in HKCs with all treatments potentially identifying
   altered Vitamin B6 metabolism associated with KC (p < 0.01 and
   p < 0.05, respectively, [157]Fig. 5H,I). Interestingly, E2 promoted
   upregulated DHAP levels in HCFs (3.7-fold, p < 0.01) suggesting
   modulations in glycolytic flux with no change in flavone levels (FAD)
   ([158]Fig. 5J,K). In order to determine if amino acid flux was affected
   by E2 treatment ([159]Supplemental Fig. 4), we quantified the
   expression of the following amino acids: phenylalanine, tryptophan,
   asparagine, tyrosine, and threonine ([160]Fig. 5L–P). Significant
   increases were observed in the aromatic amino acids, phenylalanine and
   tyrosine in HCFs (1.4-fold and 1.4–fold, respectively, p < 0.01) with
   an inverse effect in HKCs (down 2.2–fold and 2.7–fold, p < 0.01)
   suggesting an increase in anabolic processes in HKCs, including protein
   biosynthesis, thereby promoting lower availability of amino acids with
   E2 treatment (5 ng/mL). In order to determine if the effects of E2 on
   amino acid metabolism were due to altered IGF signaling, we measured
   protein levels of IGF-1, IGF-2, and IGF-1R ([161]Fig. 5Q). We found
   slight, but insignificant, changes in both IGF-1 and IGF-2 in HCFs with
   little effect in HKCs suggesting that E2 did not modulate energy
   production via the IGF-axis ([162]Supplemental Fig. 5).

Figure 5. Effects of 17β-estradiol (E2) (2.5 ng/mL and 5 ng/mL) on metabolic
pathways and IGF-1 expression in HCFs and HKCs.

   [163]Figure 5
   [164]Open in a new tab

   (A) Chemical structure of E2 and enrichment pathways identified in HCFs
   by MetaboAnalyst. (#denotes pathways highlighted by metabolite
   breakdown). Quantitative determination of metabolite levels by targeted
   LC-MS/MS important in (B–C) steroid biosynthesis and metabolism, (D,E)
   pantothenate and geranyl pyrophosphate flux, (F,G) histidine
   metabolism, (H,I) Vitamin B6 metabolism, and (J,K) glycolytic and
   flavone flux, and (L–P) amino acid flux with increasing E2
   concentrations detected by LC-MS/MS. (Q–T) Representative western blots
   and quantification of protein levels measured by densitometry. Western
   blots were converted to greyscale. Statistical significance determined
   by a two-way ANOVA. Experiments were performed in triplicate. Error
   bars represent standard error of the mean. *p < 0.05, **p < 0.01,
   ***p < 0.001, ****p < 0.0001. Note: controls were treated with vehicle
   (1% DMSO).

E2 and the Effects on Glycolysis, TCA, and PPP

   We sought to further define the effects of E2 on glucose metabolism and
   quantified metabolite flux in glycolysis, TCA, and PPP cycles
   ([165]Fig. 6). As previously shown in [166]Fig. 5J, we measured an
   increase in glycolytic flux with increased dihydroxyacetone phosphate
   in HKCs with E2 treatment (2.5 ng/mL and 5 ng/mL) leading to an
   increase in DHAP in HCFs (4.1-fold and 3.7–fold, respectively,
   p < 0.05, [167]Fig. 6I), as well as elevated fructose-1,6-bisphopshate
   (F-1,6-BP) (2.9-fold and 2.3-fold, respectively, p < 0.05, [168]Fig.
   6C). Phosphoenolpyruvate (PEP) levels in control HKCs in this set of
   experiments were at similar levels as HCFs likely due to vehicle
   differences with E2 treatment (5 ng/mL) significantly reducing PEP in
   HCFs (2.5-fold, p < 0.05, [169]Fig. 6F). Similar to the previous
   experiments showing reduced TCA intermediates in control HKCs compared
   to HCFs, stimulation with 2.5 ng/mL and 5 ng/mL E2 caused a significant
   reduction in isocitrate (1.7-fold and 2.6-fold, respectively,
   p < 0.001) with slight increases in α-ketoglutarate (p < 0.05) in HCFs
   and a 3.5-fold reduction in isocitrate in HKCs (p < 0.05, [170]Fig.
   6Q,R).

Figure 6. Effects of 17β-estradiol (E2) on glucose metabolism in HCFs and
HKCs.

   [171]Figure 6
   [172]Open in a new tab

   (A–I) Glycolytic flux, (J–N) Pentose phosphate flux, and (O–U) TCA
   cycling in HCFs and HKCs following 17β-estradiol (E2) treatment
   (2.5 ng/mL and 5 ng/mL). n = 3, error bars represent standard error of
   the mean. Statistical significance was determined by ANOVA. *p < 0.05,
   **p < 0.01, ***p < 0.001, ****p < 0.0001. (Abbrev. F-6-P,
   fructose-6-phosphate; F-1,6-BP, fructose-1,6-bisphosphate; 1,3-DPG,
   1,3-diphosphoglycerate; 3-PG, 3-phosphoglycerate; PEP,
   phosphoenolpyruvate; Gly-3-P, glyceraldehyde-3-phosphate; DHAP,
   dihydroxyacetone phosphate.) Note: controls were treated with vehicle
   (1% DMSO).

   The most significant effect of E2 on glucose metabolism occurred in the
   PPP flux ([173]Fig. 6J–N). E2 treatment (5ng/mL) led to a significant
   increase in glucono-lactone-6-phosphate (2.7-fold, p < 0.05),
   6-phospho-D-gluconate (1.7-fold, p < 0.05), ribose-phosphate (2.8-fold,
   p < 0.001), and D-erythrose-4-phosphate (1.5-fold, p < 0.05) in HCFs,
   but not HKCs ([174]Fig. 6J–L,N). The observed increase in PPP flux
   induced by E2 stimulation suggests that estrogens may play a direct
   role in regulating the alternative glucose shunt favoring PPP
   conversion over glycolysis.

Discussion

   Hormones are known to influence systemic metabolic rate by altering
   sugar and lipid uptake[175]^68,[176]^69. Various cellular processes,
   including survival and differentiation, proliferation and protein
   degradation, and secretion and assembly of the ECM are dependent on the
   energy-status of the cell[177]^70,[178]^71,[179]^72. It is still
   unclear what role bioenergetics play in regulating corneal keratocyte
   function and how alterations may contribute to cellular responses
   during wounding or pathologically ECM thinning as occurs in KC. In our
   study, we found significant reduction in basal PPP flux in control HKCs
   compared to HCFs. The sex hormones, DHEA and E2, require a lipophilic
   vehicle with utilization of dimethylsulfoxide (DMSO) in our study,
   while prolactin is a water-soluble protein. The differences in PPP
   metabolite flux, involving 6-phospho-D-gluconate,
   sedoheptulose-1,7-phosphate, and erythrose-4-phosphate, in control HCFs
   and HKCs depending on the presence of DMSO suggested that PPP flux is
   sensitive to the presence of a pro-oxidant vehicle though the trend of
   reduced PPP flux persisted in the HKC samples. Since PPP is known as a
   major regulator of antioxidant capacity via the production of NADPH
   which functions to reduce oxidized glutathione to the active
   form[180]^73, our data suggests that reduced pentose phosphate cycling
   in HKCs may be related to the inherent increased oxidative stress
   reported in a number of studies[181]^42,[182]^74,[183]^75,[184]^76. The
   PPP also produces glycolytic intermediates as a by-product, including
   glyceraldehyde-3-phosphate and fructose-6-phosphate, which serve to
   sustain glycolysis. In our studies, we found that HKCs maintained
   elevated glycolytic flux that correlated with increased lactate
   production, as previously reported[185]^42, suggesting that the
   reduction in PPP intermediates does not reduce the overactive
   glycolytic pathway present in HKCs.

   Since KC is a disease with a post-puberty onset when endocrine function
   and sex hormone production is significantly modulated, our study
   focused on the role of exogenous hormones in regulating metabolic
   function within corneal fibroblasts, in self-assembled constructs, to
   determine if KC-derived cells exhibit an altered response. The use of
   fibroblasts compared to the more quiescent keratocyte natively found
   within the corneal stroma was required in long-term culture conditions
   where cells must be grown in the presence of serum. This 3D in vitro
   model has been a useful tool to determine pathological differences
   between HCFs and HKCs[186]^42,[187]^77. Characterization of long-term
   3D in vitro cultures using corneal fibroblasts has been published in
   other studies evaluating the expression of proteoglycans and
   extracellular matrix structure at varying time points[188]^78,[189]^79.

   A number of studies have identified increased oxidative stress,
   mutations in mitochondrial genes, and inflammatory protein expression
   in KC-derived cells in
   vitro[190]^41,[191]^42,[192]^80,[193]^81,[194]^82 and ex
   vivo[195]^41,[196]^83,[197]^84,[198]^85. We posit that oxidative stress
   promoted by altered androgen or estrogen levels during post-pubescence
   may be central to the altered metabolic function observed in HKCs. Our
   proposed mechanism of KC development focuses on early-age hormone
   levels and their role in regulating metabolic function, oxidative
   stress, and ultimately matrix thinning ([199]Fig. 7). Our results from
   this study identified a regulatory role of exogenous hormones on
   localized IGF-1 production and the downstream effects on bioenergetics
   and metabolic function in corneal fibroblasts. We found that exogenous
   DHEA promoted downregulation of IGF-1 and the active IGF-1R receptor
   expression which resulted in an increase in select free amino acids.
   Given the role of choline and phosphorylcholine in regulating protein
   kinase C signaling, the increase in these metabolites in HCFs with DHEA
   treatment suggests that this androgen may modulate calcium signaling
   and a number of downstream pathways related to lipid-mediated signaling
   as well[200]^86.

Figure 7. Summary of metabolic regulation induced by exogenous hormones:
DHEA, prolactin, and E2 in HCFs and HKCs.

   [201]Figure 7
   [202]Open in a new tab

   A number of studies have associated mitochondrial dysfunction with
   KC[203]^80,[204]^93,[205]^94 and altered androgen and estrogen levels
   in KC patients[206]^41. Our previous studies[207]^42,[208]^95 and
   current work support increased lactate production in HKCs compared to
   HCFs correlating with upregulated aerobic glycolysis. We posit that a
   differential response to each hormone is detected in HKCs and HCFs due
   to this aberrant basal metabolism in HKCs.

   While DHEA reduced IGF-1 and IGF-1R expression in both HCFs and HKCs,
   E2 did not significantly modulate expression of these growth factors.
   However, E2 stimulated a strong metabolic response with elevated
   glycolytic and PPP fluxes in HCFs suggesting bioenergetics are
   modulated by estrogen independent of the IGF-1 axis. In contrast, the
   luteotropin prolactin significantly increased IGF-2 expression only in
   HKCs, while having a modest effect on glucose metabolism and ATP
   production. Previous studies have shown that IGF-2 is the primary
   isoform responsible for prolactin-induced morphogenesis within breast
   tissue[209]^61. Our results support the hypothesis that prolactin
   modulates IGF-2 protein expression with the significant response
   occurring in our study in HKCs with no change in IGF-1 or IGF-1R
   expression. The IGF-1R is known to heterodimerize with the insulin
   receptor[210]^87 suggesting that changes in expression of IGF-1R may
   affect responses to insulin as well.

   Though IGF-2 levels were significantly modulated in HKCs and not HCFs,
   glycolytic flux was slightly reduced in HCFs with no change in the
   disparity between the elevated levels in HKCs even at basal conditions.
   These results suggest that though DHEA, prolactin, and E2 influence
   cellular bioenergetics, the inherent defects that drive elevated
   glycolytic flux in HKCs are unable to be overcome with hormone
   stimulation at the physiological levels utilized in this study.
   Furthermore, our study revealed a novel finding that exogenous DHEA may
   contribute to KC development or progression by reducing localized
   production of IGF-1 and autocrine or paracrine signaling contributing
   to altered metabolic function. The increase in carbamoyl phosphate and
   argininosuccinate, key metabolites involved in the urea cycle, suggests
   that arginine and polyamine flux in HKCs may be more sensitive to
   DHEA-induced modulation of collagen precursors, perhaps leading to ECM
   thinning, a trademark of KC. In contrast, HKCs showed less sensitivity
   to E2 treatment compared to HCFs with dramatic increases in glycolysis
   and PPP flux. These results highlight a hormone-specific response in
   healthy versus KC-derived cells which may be a factor related to the
   altered basal metabolism observed in HKCs. Further studies are needed
   to elucidate the effects of hormones in regulating genes associated
   with matrix deposition and mitochondrial function in KC in order to
   determine if the sex chromosomes regulate the cellular responses to
   exogenous hormones in vitro.

Materials and Methods

Isolation of Primary Corneal Fibroblasts

   The ethics committee of the University of Oklahoma Health Sciences
   Center and Aarhus University Hospital approved tissue collection (IRB
   protocols #3450 and #1-10-72-77-14, respectively) with written informed
   consent obtained from patients. All samples were de-identified prior to
   analysis. This study met the tenets of the Declaration of Helsinki.
   Cadaver tissue was provided by NDRI (National Disease Research
   Interchange). KC corneas were obtained from clinical collaborators
   immediately following corneal transplantation. Inclusion/exclusion
   criteria for healthy controls required absence of ophthalmic disease,
   diabetes, or infectious conditions. KC patients required diagnosed by
   an ophthalmologist for inclusion and absence of other corneal diseases.
   KC patients who had previously undergone collagen crosslinking were
   excluded from study. Donor information for HCFs utilized in this study:
   N19 (63 y/o male), N23 (21 y/o male), and N4 (53 y/o, male). Donor
   information for HKCs utilized in this study: DM1 (44 y/o female), WU1
   (62 y/o female), and WU2 (34 y/o male). We isolated both HCFs and HKCs
   as previously described[211]^77,[212]^88. Briefly, the corneal
   epithelium and endothelium were removed using a surgical scalpel. The
   corneal stroma was isolated, washed in sterile PBS, and cut into small
   pieces (2 × 2 × 2 mm) and placed into flasks and allowed to adhere to
   the flask surface. Eagle’s Minimum Essential Media (EMEM) containing
   antibiotic/antimycotic (Anti/Anti, Life Technologies, Grand Island, NY)
   and 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA)
   were added to flask. Explants were grown 2-4 weeks at 37 °C/5%
   CO[2]/95% relative humidity until cells migrated from the tissue
   section into the flask. Cells were then isolated following
   trypsinization and subcultured or frozen using standard cryoprotective
   protocols.

3D In Vitro Model

   The 3D in vitro model has been described extensively[213]^77,[214]^88.
   Briefly, primary corneal fibroblasts were seeded at 10^6 cells/well in
   polycarbonate transwell plates (Corning Costar, Charlotte, NC)
   containing 1.5 mL of EMEM/10% FBS/anti-anti media in both the top and
   bottom wells. Post t = 24 hours following seeding, constructs were
   stimulated with a stable Vitamin C derivative (0.5 mM
   2-O-α-D-glucopyranosyl-L-ascorbic acid, American Custom Chemicals
   Corporation, San Diego, CA) in the media following filter
   sterilization. Media containing hormones were freshly prepared and
   sterilized prior to addition to constructs: (2.5 ng/mL and 5 ng/mL)
   trans-dehydroandrosterone (Sigma Aldrich, St. Louis, MO), (2.5 ng/mL
   and 5 ng/mL) 17β-estradiol (Sigma Aldrich), and (25 ng/mL and 50 ng/mL)
   human prolactin (Sigma Aldrich). DHEA and 17β-estradiol were dissolved
   in sterile DMSO. 1% of DMSO was added to culture media. Lyophilized
   prolactin was dissolved in sterile distilled/deionized water. Media was
   changed every other day for the entire 4 week period, as previously
   described[215]^77.

Metabolite Extraction and Targeted Mass Spectrometry

   Constructs were isolated at the end of week four for metabolite
   isolation. As previously described[216]^42,[217]^89, metabolites were
   isolated using ice-cold (−80 °C) MeOH in water on dry ice, incubated,
   centrifuged, dried, and repeated two times. Dried pellets were stored
   at −80 °C until shipped on dry ice and analyzed. Metabolomics analysis
   was completed at Beth Israel Deaconess Medical Center mass spectrometry
   core facility. HPLC-grade water was used to dissolve pellets and
   analyzed for quantification by LC-MS/MS in a hybrid 5500 QTRAP triple
   quandrupole mass spectrometer (AB/SCIEX). MultiQuant 2.1 was used to
   quantify label-free metabolites. MetaboAnalyst 3.0 software was used to
   determine enrichment pathways, as previously
   described[218]^90,[219]^91. Heat maps were developed using the NCIminer
   server as previously reported by Weinstein et al.[220]^92 to generate a
   one matrix clustered image map with quantile binning.

Protein Isolation and Western Blot

   Constructs were collected, washed 2X with PBS, and total protein was
   isolated using 1X radioimmunoprecipitation assay (RIPA) buffer (50 mM
   Tris pH 8, 150 mM NaCl, 1% Triton X-100, and 0.1% SDS) containing a
   protease inhibitor cocktail (Sigma Aldrich). Cell lysates were
   centrifuged at 4 °C to precipitate insoluble debris, and the
   supernatant was isolated and subjected to a Pierce BCA Protein assay
   (ThermoScientific, Rockford, IL) for protein concentration
   determination. Western blot was performed using a gradient Novex 4–20%
   Tris-glycine gel (Life Technologies, Carlsbad, CA) electrophoresed at
   135–140 V for 1.5 hours, and transferred onto a nitrocellulose membrane
   at 100 V for 1 hour on ice. Blots were blocked in 5% dry milk (Great
   Value, Bentonville, AR) or 5% BSA (Fisher, Fair Lawn, NJ) for 1 hour at
   room temperature with shaking. Anti-human rabbit primary antibodies
   were incubated with membrane overnight at 4 °C with rocking at
   concentrations of 1:500–1:1000: β-actin (Abcam, ab82227, Cambridge,
   MA), IGF-1 (Abcam, ab9572, Cambridge, MA), IGF-1R (Abcam, ab131476,
   Cambridge, MA), IGF-2 (Abcam, ab9574, Cambridge, MA). A fluorescent
   secondary antibody was used at a concentration of 1:1000-1:2000:
   Alexafluor 568 donkey anti-rabbit (Life Technologies, Eugene, OR).
   Detection and quantification of bands was measured using the ChemiDoc
   It2 Imager (UVP, Upland, CA) using densitometry with background
   subtraction and converted to grey scale.

Statistical Analysis

   A two-way ANOVA with multiple comparison analysis was performed in
   GraphPad Prism to determine significance with p < 0.05 considered
   statistically significant. Error bars represent standard error of the
   mean. The n numbers are listed in the appropriate figure legends.

Additional Information

   How to cite this article: McKay, T. B. et al. Differential Effects of
   Hormones on Cellular Metabolism in Keratoconus In Vitro. Sci. Rep. 7,
   42896; doi: 10.1038/srep42896 (2017).

   Publisher's note: Springer Nature remains neutral with regard to
   jurisdictional claims in published maps and institutional affiliations.

Supplementary Material

   Supplementary Information
   [221]srep42896-s1.pdf^ (958.2KB, pdf)

Acknowledgments