Prolactin is an Endogenous Antioxidant Factor in Astrocytes That Limits Oxidative Stress-Induced Astrocytic Cell Death via the STAT3/NRF2 Signaling Pathway.

Oxidative stress-induced death of neurons and astrocytes contributes to the pathogenesis of numerous neurodegenerative diseases. While significant progress has been made in identifying neuroprotective molecules against neuronal oxidative damage, little is known about their counterparts for astrocytes. Prolactin (PRL), a hormone known to stimulate astroglial proliferation, viability, and cytokine expression, exhibits antioxidant effects in neurons. However, its role in protecting astrocytes from oxidative stress remains unexplored. Here, we investigated the effect of PRL against hydrogen peroxide (H2O2)-induced oxidative insult in primary cortical astrocyte cultures. Incubation of astrocytes with PRL led to increased enzymatic activity of superoxide dismutase (SOD) and glutathione peroxidase (GPX), resulting in higher total antioxidant capacity. Concomitantly, PRL prevented H2O2-induced cell death, reactive oxygen species accumulation, and protein and lipid oxidation. The protective effect of PRL upon H2O2-induced cell death can be explained by the activation of both signal transducer and activator of transcription 3 (STAT3) and NFE2 like bZIP transcription factor 2 (NRF2) transduction cascades. We demonstrated that PRL induced nuclear translocation and transcriptional upregulation of Nrf2, concurrently with the transcriptional upregulation of the NRF2-dependent genes heme oxygenase 1, Sod1, Sod2, and Gpx1. Pharmacological blockade of STAT3 suppressed PRL-induced transcriptional upregulation of Nrf2, Sod1 and Gpx1 mRNA, and SOD and GPX activities. Furthermore, genetic ablation of the PRL receptor increased astroglial susceptibility to H2O2-induced cell death and superoxide accumulation, while diminishing their intrinsic antioxidant capacity. Overall, these findings unveil PRL as a potent antioxidant hormone that protects astrocytes from oxidative insult, which may contribute to brain neuroprotection.

Dopamine D2 receptor antagonist counteracts hyperglycemia and insulin resistance in diet-induced obese male mice.

Obesity leads to insulin resistance (IR) and type 2 diabetes. In humans, low levels of the hormone prolactin (PRL) correlate with IR, adipose tissue (AT) dysfunction, and increased prevalence of T2D. In obese rats, PRL treatment promotes insulin sensitivity and reduces visceral AT adipocyte hypertrophy. Here, we tested whether elevating PRL levels with the prokinetic and antipsychotic drug sulpiride, an antagonist of dopamine D2 receptors, improves metabolism in high fat diet (HFD)-induced obese male mice. Sulpiride treatment (30 days) reduced hyperglycemia, IR, and the serum and pancreatic levels of triglycerides in obese mice, reduced visceral and subcutaneous AT adipocyte hypertrophy, normalized markers of visceral AT function (PRL receptor, Glut4, insulin receptor and Hif-1α), and increased glycogen stores in skeletal muscle. However, the effects of sulpiride reducing hyperglycemia were also observed in obese prolactin receptor null mice. We conclude that sulpiride reduces obesity-induced hyperglycemia by mechanisms that are independent of prolactin/prolactin receptor activity. These findings support the therapeutic potential of sulpiride against metabolic dysfunction in obesity.

Vasoinhibin's Apoptotic, Inflammatory, and Fibrinolytic Actions Are in a Motif Different From Its Antiangiogenic HGR Motif.

Vasoinhibin, a proteolytic fragment of the hormone prolactin, inhibits blood vessel growth (angiogenesis) and permeability, stimulates the apoptosis and inflammation of endothelial cells, and promotes fibrinolysis. The antiangiogenic and antivasopermeability properties of vasoinhibin were recently traced to the HGR motif located in residues 46 to 48 (H46-G47-R48), allowing the development of potent, orally active, HGR-containing vasoinhibin analogues for therapeutic use against angiogenesis-dependent diseases. However, whether the HGR motif is also responsible for the apoptotic, inflammatory, and fibrinolytic properties of vasoinhibin has not been addressed. Here, we report that HGR-containing analogues are devoid of these properties. Instead, the incubation of human umbilical vein endothelial cells with oligopeptides containing the sequence HNLSSEM, corresponding to residues 30 to 36 of vasoinhibin, induced apoptosis, nuclear translocation of NF-κB, expression of genes encoding leukocyte adhesion molecules (VCAM1 and ICAM1) and proinflammatory cytokines (IL1B, IL6, and TNF), and adhesion of peripheral blood leukocytes. Also, intravenous or intra-articular injection of HNLSSEM-containing oligopeptides induced the expression of Vcam1, Icam1, Il1b, Il6, and Tnf in the lung, liver, kidney, eye, and joints of mice and, like vasoinhibin, these oligopeptides promoted the lysis of plasma fibrin clots by binding to plasminogen activator inhibitor-1 (PAI-1). Moreover, the inhibition of PAI-1, urokinase plasminogen activator receptor, or NF-κB prevented the apoptotic and inflammatory actions. In conclusion, the functional properties of vasoinhibin are segregated into 2 different structural determinants. Because apoptotic, inflammatory, and fibrinolytic actions may be undesirable for antiangiogenic therapy, HGR-containing vasoinhibin analogues stand as selective and safe agents for targeting pathological angiogenesis.

Prolactin Inhibits or Stimulates the Inflammatory Response of Joint Tissues in a Cytokine-dependent Manner.

The close association between rheumatoid arthritis (RA), sex, reproductive state, and stress has long linked prolactin (PRL) to disease progression. PRL has both proinflammatory and anti-inflammatory outcomes in RA, but responsible mechanisms are not understood. Here, we show that PRL modifies in an opposite manner the proinflammatory actions of IL-1ß and TNF-α in mouse synovial fibroblasts in culture. Both IL-1ß and TNF-α upregulated the metabolic activity and the expression of proinflammatory factors (Il1b, Inos, and Il6) via the activation of the nuclear factor-κB (NF-κB) signaling pathway. However, IL-1ß increased and TNF-α decreased the levels of the long PRL receptor isoform in association with dual actions of PRL on synovial fibroblast inflammatory response. PRL reduced the proinflammatory effect and activation of NF-κB by IL-1ß but increased TNF-α-induced inflammation and NF-κB signaling. The double-faceted role of PRL against the 2 cytokines manifested also in vivo. IL-1ß or TNF-α with or without PRL were injected into the knee joints of healthy mice, and joint inflammation was monitored after 24 hours. IL-1ß and TNF-α increased the joint expression of proinflammatory factors and the infiltration of immune cells. PRL prevented the actions of IL-1ß but was either inactive or further increased the proinflammatory effect of TNF-α. We conclude that PRL exerts opposite actions on joint inflammation in males and females that depend on specific proinflammatory cytokines, the level of the PRL receptor, and the activation of NF-κB signaling. Dual actions of PRL may help balance joint inflammation in RA and provide insights for development of new treatments.

The Diversity of Gut Microbiota at Weaning Is Altered in Prolactin Receptor-Null Mice.

Maternal milk supports offspring development by providing microbiota, macronutrients, micronutrients, immune factors, and hormones. The hormone prolactin (PRL) is an important milk component with protective effects against metabolic diseases. Because maternal milk regulates microbiota composition and adequate microbiota protect against the development of metabolic diseases, we aimed to investigate whether PRL/PRL receptor signaling regulates gut microbiota composition in newborn mice at weaning. 16SrRNA sequencing of feces and bioinformatics analysis was performed to evaluate gut microbiota in PRL receptor-null mice (Prlr-KO) at weaning (postnatal day 21). The normalized colon and cecal weights were higher and lower, respectively, in the Prlr-KO mice relative to the wild-type mice (Prlr-WT). Relative abundances (Simpson Evenness Index), phylogenetic diversity, and bacterial concentrations were lower in the Prlr-KO mice. Eleven bacteria species out of 470 differed between the Prlr-KO and Prlr-WT mice, with two genera (Anaerotruncus and Lachnospiraceae) related to metabolic disease development being the most common in the Prlr-KO mice. A higher metabolism of terpenoids and polyketides was predicted in the Prlr-KO mice compared to the Prlr-WT mice, and these metabolites had antimicrobial properties and were present in microbe-associated pathogenicity. We concluded that the absence of the PRL receptor altered gut microbiota, resulting in lower abundance and richness, which could contribute to metabolic disease development.

Differentiated Mouse Adipocytes in Primary Culture: A Model of Insulin Resistance.

Insulin resistance is a reduced effect of insulin on its target cells, usually derived from decreased insulin receptor signaling. Insulin resistance contributes to the development of type 2 diabetes (T2D) and other obesity-derived diseases of high prevalence worldwide. Therefore, understanding the mechanisms underlying insulin resistance is of great relevance. Several models have been used to study insulin resistance both in vivo and in vitro; primary adipocytes represent an attractive option to study the mechanisms of insulin resistance and identify molecules that counteract this condition and the molecular targets of insulin-sensitizing drugs. Here, we have established an insulin resistance model using primary adipocytes in culture treated with tumor necrosis factor-α (TNF-α). Adipocyte precursor cells (APCs), isolated from collagenase-digested mouse subcutaneous adipose tissue by magnetic cell separation technology, are differentiated into primary adipocytes. Insulin resistance is then induced by treatment with TNF-α, a proinflammatory cytokine that reduces the tyrosine phosphorylation/activation of members of the insulin signaling cascade. Decreased phosphorylation of insulin receptor (IR), insulin receptor substrate (IRS-1), and protein kinase B (AKT) are quantified by western blot. This method provides an excellent tool to study the mechanisms mediating insulin resistance in adipose tissue.

Obesity-derived alterations in the lactating mammary gland: Focus on prolactin.

Obesity is a modern pandemic with negative consequences in women's reproductive health. Women with overweight and obesity can develop mammary gland alterations that unable exclusive breastfeeding. Obesity associates with a disturbed lactating mammary gland endocrine environment including a decreased action of the hormone prolactin (PRL), the master regulator of lactation. The PRL receptor and the action of PRL are reduced in the mammary gland of lactating rodents fed an obesogenic diet and are contributing factors to impaired lactation in obesity. Also, treatment with PRL improves milk yield in women with lactation insufficiency. This review focuses on the impact of diet-induced obesity in the lactating mammary gland and how obesity impairs the lactogenic action of PRL. Although obesity alters lactation performance in humans and rodents, the responsible mechanisms have been mainly addressed in rodents.

The beneficial metabolic actions of prolactin.

The role of prolactin (PRL) favoring metabolic homeostasis is supported by multiple preclinical and clinical studies. PRL levels are key to explaining the direction of its actions. In contrast with the negative outcomes associated with very high (>100 µg/L) and very low (<7 µg/L) PRL levels, moderately high PRL levels, both within but also above the classically considered physiological range are beneficial for metabolism and have been defined as HomeoFIT-PRL. In animal models, HomeoFIT-PRL levels counteract insulin resistance, glucose intolerance, adipose tissue hypertrophy and fatty liver; and in humans associate with reduced prevalence of insulin resistance, fatty liver, glucose intolerance, metabolic syndrome, reduced adipocyte hypertrophy, and protection from type 2 diabetes development. The beneficial actions of PRL can be explained by its positive effects on main metabolic organs including the pancreas, liver, adipose tissue, and hypothalamus. Here, we briefly review work supporting PRL as a promoter of metabolic homeostasis in rodents and humans, the PRL levels associated with metabolic protection, and the proposed mechanisms involved. Finally, we discuss the possibility of using drugs elevating PRL for the treatment of metabolic diseases.

Dual Roles of Prolactin and Vasoinhibin in Inflammatory Arthritis.

The term inflammatory arthritis defines a family of diseases, including rheumatoid arthritis (RA), caused by an overactive immune system, and influenced by host aspects including sex, reproductive state, and stress. Prolactin (PRL) is a sexually dimorphic, reproductive, stress-related hormone long-linked to RA under the general assumption that it aggravates the disease. However, this conclusion remains controversial since PRL has both negative and positive outcomes in RA that may depend on the hormone circulating levels, synthesis by joint tissues, and complex interactions at the inflammatory milieu. The inflamed joint is rich in matrix metalloproteases that cleave PRL to vasoinhibin, a PRL fragment with proinflammatory effects and the ability to inhibit the hyperpermeability and growth of blood vessels. This review addresses this field with the idea that explanatory mechanisms lie within the PRL/vasoinhibin axis, an integrative framework influencing not only the levels of systemic and local PRL, but also the proteolytic conversion of PRL to vasoinhibin, as vasoinhibin itself has dual actions on joint inflammation. In this review, we discuss recent findings from mouse models suggesting the upregulation of endogenous vasoinhibin by the pro-inflammatory environment and showing dichotomous actions and signaling mechanisms of PRL and vasoinhibin on joint inflammation that are cell-specific and context-dependent. We hypothesize that these opposing actions work together to balance the inflammatory response and provide new insights for understanding the pathophysiology of RA and the development of new treatments.

Vasoinhibin is Generated and Promotes Inflammation in Mild Antigen-induced Arthritis.

Inflammatory arthritis defines a family of diseases influenced by reproductive hormones. Vasoinhibin, a fragment of the hormone prolactin (PRL), has antiangiogenic and proinflammatory properties. We recently showed that vasoinhibin reduces joint inflammation and bone loss in severe antigen-induced arthritis (AIA) by an indirect mechanism involving the inhibition of pannus vascularization. This unexpected finding led us to hypothesize that a severe level of inflammation in AIA obscured the direct proinflammatory action of vasoinhibin while allowing the indirect anti-inflammatory effect via its antiangiogenic properties. In agreement with this hypothesis, here we show that the intra-articular injection of an adeno-associated virus type-2 vector encoding vasoinhibin reduced joint inflammation in a severe AIA condition, but elevated joint inflammation in a mild AIA model. The proinflammatory effect, unmasked in mild AIA, resulted in joint swelling, enhanced leukocyte infiltration, and upregulation of expression of genes encoding proinflammatory mediators (Il1b, Il6, Inos, Mmp3), adhesion molecule (Icam1), and chemokines (Cxcl1, Cxcl2, Cxcl3, Ccl2). Furthermore, vasoinhibin induced the expression of proinflammatory mediators and chemokines in cultured synovial fibroblasts through nuclear factor-κB. Finally, matrix metalloproteases and cathepsin D, upregulated in the arthritic joint, cleaved PRL to vasoinhibin, and vasoinhibin levels increased in the circulation of mice subjected to AIA. We suggest that vasoinhibin is generated during inflammatory arthritis and acts on synovial fibroblasts and endothelial cells to initially promote and later inhibit inflammation, respectively. These opposite effects may work together to help keep joint inflammation under balance.

The spike protein of SARS-CoV-2 induces endothelial inflammation through integrin α5ß1 and NF-κB signaling.

Vascular endothelial cells (ECs) form a critical interface between blood and tissues that maintains whole-body homeostasis. In COVID-19, disruption of the EC barrier results in edema, vascular inflammation, and coagulation, hallmarks of this severe disease. However, the mechanisms by which ECs are dysregulated in COVID-19 are unclear. Here, we show that the spike protein of SARS-CoV-2 alone activates the EC inflammatory phenotype in a manner dependent on integrin ⍺5ß1 signaling. Incubation of human umbilical vein ECs with whole spike protein, its receptor-binding domain, or the integrin-binding tripeptide RGD induced the nuclear translocation of NF-κB and subsequent expression of leukocyte adhesion molecules (VCAM1 and ICAM1), coagulation factors (TF and FVIII), proinflammatory cytokines (TNFα, IL-1ß, and IL-6), and ACE2, as well as the adhesion of peripheral blood leukocytes and hyperpermeability of the EC monolayer. In addition, inhibitors of integrin ⍺5ß1 activation prevented these effects. Furthermore, these vascular effects occur in vivo, as revealed by the intravenous administration of spike, which increased expression of ICAM1, VCAM1, CD45, TNFα, IL-1ß, and IL-6 in the lung, liver, kidney, and eye, and the intravitreal injection of spike, which disrupted the barrier function of retinal capillaries. We suggest that the spike protein, through its RGD motif in the receptor-binding domain, binds to integrin ⍺5ß1 in ECs to activate the NF-κB target gene expression programs responsible for vascular leakage and leukocyte adhesion. These findings uncover a new direct action of SARS-CoV-2 on EC dysfunction and introduce integrin ⍺5ß1 as a promising target for treating vascular inflammation in COVID-19.

Sulpiride-induced hyperprolactinaemia increases retinal vasoinhibin and protects against diabetic retinopathy in rats.

Excessive vasopermeability and angiogenesis compromise vision in diabetic macular oedema (DME) and diabetic retinopathy (DR). Vasoinhibin is a fragment of the hormone prolactin (PRL) that inhibits diabetes-induced retinal hypervasopermeability and ischaemia-induced retinal angiogenesis in rodents. Hyperprolactinaemia generated by the dopamine D2 receptor antagonist, levosulpiride, is associated with higher levels of vasoinhibin in the vitreous of patients with DR, implying a beneficial outcome due to vasoinhibin-mediated inhibition of retinal vascular alterations. Here, we tested whether hyperprolactinaemia induced by racemic sulpiride increases intraocular vasoinhibin levels and inhibits retinal hypervasopermeability in diabetic rats. Diabetes was generated with streptozotocin and, 4 weeks later, rats were treated for 2 weeks with sulpiride or osmotic minipumps delivering PRL. ELISA, Western blot, and Evans blue assay were used to evaluate serum PRL, retinal vasoinhibin, and retinal vasopermeability, respectively. Hyperprolactinaemia in response to sulpiride or exogenous PRL was associated with increased levels of vasoinhibin in the retina and reduced retinal hypervasopermeability. Furthermore, sulpiride decreased retinal haemorrhages in response to the intravitreal administration of vascular endothelial growth factor (VEGF). Neither sulpiride nor exogenous PRL modified blood glucose levels or bodyweight. We conclude that sulpiride-induced hyperprolactinaemia inhibits the diabetes- and VEGF-mediated increase in retinal vasopermeability by promoting the intraocular conversion of endogenous PRL to vasoinhibin. These findings support the therapeutic potential of sulpiride and its levorotatory enantiomer, levosulpiride, against DME and DR.

The HGR motif is the antiangiogenic determinant of vasoinhibin: implications for a therapeutic orally active oligopeptide.

The hormone prolactin acquires antiangiogenic and antivasopermeability properties after undergoing proteolytic cleavage to vasoinhibin, an endogenous prolactin fragment of 123 or more amino acids that inhibits the action of multiple proangiogenic factors. Preclinical and clinical evidence supports the therapeutic potential of vasoinhibin against angiogenesis-related diseases including diabetic retinopathy, peripartum cardiomyopathy, rheumatoid arthritis, and cancer. However, the use of vasoinhibin in the clinic has been limited by difficulties in its production. Here, we removed this barrier to using vasoinhibin as a therapeutic agent by showing that a short linear motif of just three residues (His46-Gly47-Arg48) (HGR) is the functional determinant of vasoinhibin. The HGR motif is conserved throughout evolution, its mutation led to vasoinhibin loss of function, and oligopeptides containing this sequence inhibited angiogenesis and vasopermeability with the same potency as whole vasoinhibin. Furthermore, the oral administration of an optimized cyclic retro-inverse vasoinhibin heptapeptide containing HGR inhibited melanoma tumor growth and vascularization in mice and exhibited equal or higher antiangiogenic potency than other antiangiogenic molecules currently used as anti-cancer drugs in the clinic. Finally, by unveiling the mechanism that obscures the HGR motif in prolactin, we anticipate the development of vasoinhibin-specific antibodies to solve the on-going challenge of measuring endogenous vasoinhibin levels for diagnostic and interventional purposes, the design of vasoinhibin antagonists for managing insufficient angiogenesis, and the identification of putative therapeutic proteins containing HGR.

Plasmin generates vasoinhibin-like peptides by cleaving prolactin and placental lactogen.

Vasoinhibin is an antiangiogenic, profibrinolytic peptide generated by the proteolytic cleavage of the pituitary hormone prolactin by cathepsin D, matrix metalloproteinases, and bone morphogenetic protein-1. Vasoinhibin can also be generated when placental lactogen or growth hormone are enzymatically cleaved. Here, it is investigated whether plasmin cleaves human prolactin and placental lactogen to generate vasoinhibin-like peptides. Co-incubation of prolactin and placental lactogen with plasmin was performed and analyzed by gel electrophoresis and Western blotting. Mass spectrometric analyses were carried out for sequence validation and precise cleavage site identification. The cleavage sites responsible for the generation of the vasoinhibin-like peptides were located at K170-E171 in prolactin and R160-T161 in placental lactogen. Various genetic variants of the human prolactin and placental lactogen genes are projected to affect proteolytic generation of the vasoinhibin-like peptides. The endogenous counterparts of the vasoinhibin-like peptides generated by plasmin may represent vasoinhibin-isoforms with inhibitory effects on vasculature and coagulation.

Thrombin Cleaves Prolactin Into a Potent 5.6-kDa Vasoinhibin: Implication for Tissue Repair.

Vasoinhibin is an endogenous prolactin (PRL) fragment with profibrinolytic, antivasopermeability, and antiangiogenic effects. The fact that blood clotting, vascular permeability, and angiogenesis are functionally linked during the wound-healing process led us to investigate whether thrombin, a major protease in tissue repair, generates vasoinhibin. Here, we have incubated human PRL with thrombin and analyzed the resulting proteolytic products by Western blot, mass spectrometry, high-performance liquid chromatography purification, recombinant production, and bioactivity. We unveil a main thrombin cleavage site at R48-G49 that rapidly (< 10 minutes) generates a 5.6-kDa fragment (residues 1-48) with full vasoinhibin activity, that is, it inhibited the proliferation, invasion, and permeability of cultured endothelial cells and promoted the lysis of a fibrin clot in plasma with a similar potency to that of a conventional 14-kDa vasoinhibin (residues 1-123). The R48-G49 cleavage site is highly conserved throughout evolution and precedes the intramolecular disulfide bond (C58-C174), thereby allowing the 5.6-kDa vasoinhibin to be released without a reduction step. Furthermore, the 5.6-kDa vasoinhibin is produced by endogenous thrombin during the clotting process. These findings uncover the smallest vasoinhibin known, add thrombin to the list of PRL-cleaving proteases generating vasoinhibin, and introduce vasoinhibin as a thrombin-activated mechanism for the regulation of hemostasis, vasopermeability, and angiogenesis in response to tissue injury.

Development of Vasoinhibin-Specific Monoclonal Antibodies.

Vasoinhibin is a protein hormone with antiangiogenic, antivasodilatatory, and antivasopermeability effects generated by the proteolytic cleavage of prolactin. The discovery of its role in diabetic retinopathy and peripartum cardiomyopathy led to the evaluation of new pharmacological treatments in clinical interventional trials. However, the quantitative evaluation of vasoinhibin in biological samples from patients has not been possible due to the lack of vasoinhibin-specific antibodies. Recently, loop 1 of vasoinhibin was identified to have a different three-dimensional structure compared to PRL, and thus to contain vasoinhibin-specific epitopes. Here, we report the development of two sets of vasoinhibin-specific monoclonal antibodies against two neighboring regions of the vasoinhibin loop 1. An experimental sandwich ELISA with two monoclonal anti-vasoinhibin antibodies was developed, which had no cross-reactivity to recombinant human full-length prolactin. The ELISA had a quantitation limit of 100 ng/ml, and intra-assay- and inter-assay coefficients of variation of 12.5% and 14%, respectively. The evaluation of 15 human serum samples demonstrated concentrations of below limit of detection (n=3), below limit of quantitation (n=1) and between 0.23 µg/ml (230 ng/ml) to 605 µg/ml (n=12) in the quantifiable range. Despite the high specificity of the monoclonal-monoclonal antibody sandwiches which discriminate vasoinhibin from PRL, there might be cross-reactivities by serum proteins other than vasoinhibin. A fully established vasoinhibin ELISA may support diagnostic and therapeutic measures in vascular diseases.

Global Deletion of the Prolactin Receptor Aggravates Streptozotocin-Induced Diabetes in Mice.

Prolactin (PRL) levels are reduced in the circulation of rats with diabetes or obesity, and lower circulating levels of PRL correlate with increased prevalence of diabetes and a higher risk of metabolic alterations in the clinic. Furthermore, PRL stimulates ß-cell proliferation, survival, and insulin production and pregnant mice lacking PRL receptors in ß-cells develop gestational diabetes. To investigate the protective effect of endogenous PRL against diabetes outside pregnancy, we compared the number of cases and severity of streptozotocin (STZ)-induced hyperglycemia between C57BL/6 mice null for the PRL receptor gene (Prlr-/- ) and wild-type mice (Prlr+/+ ). STZ-treated diabetic Prlr-/- mice showed a higher number of cases and later recovery from hyperglycemia, exacerbated glucose levels, and glucose intolerance compared to the Prlr+/+ mice counterparts. Consistent with the worsening of hyperglycemia, pancreatic islet density, ß-cell number, proliferation, and survival, as well as circulating insulin levels were reduced, whereas α-cell number and pancreatic inflammation were increased in the absence of PRL signaling. Deletion of the PRL receptor did not alter the metabolic parameters in vehicle-treated animals. We conclude that PRL protects whole body glucose homeostasis by reducing ß-cell loss and pancreatic inflammation in STZ-induced diabetes. Medications elevating PRL circulating levels may prove to be beneficial in diabetes.

Levosulpiride Increases the Levels of Prolactin and Antiangiogenic Vasoinhibin in the Vitreous of Patients with Proliferative Diabetic Retinopathy.

Purpose: High circulating levels of the hormone prolactin (PRL) protect against experimental diabetic retinopathy (DR) due to the retinal accumulation of vasoinhibin, a PRL fragment that inhibits blood vessel permeability and growth. A phase 2 clinical trial is investigating a new therapy for DR based on elevating serum PRL levels with levosulpiride, a prokinetic dopamine D2 receptor blocker. Here, we tested whether levosulpiride-induced hyperprolactinemia elevates PRL and vasoinhibin in the vitreous of volunteer patients with proliferative DR (PDR) undergoing elective pars plana vitrectomy. Methods: Patients were randomized to receive placebo (lactose pill, orally TID; n = 19) or levosulpiride (25 mg orally TID; n = 18) for the 7 days before vitrectomy. Vitreous samples from untreated non-diabetic (n = 10) and PDR (n = 17) patients were also studied. Results: Levosulpiride elevated the systemic (101 ± 13 [SEM] vs. 9.2 ± 1.3 ng/mL, P < 0.0001) and vitreous (3.2 ± 0.4 vs. 1.5 ± 0.2 ng/mL, P < 0.0001) levels of PRL, and both levels were directly correlated (r = 0.58, P < 0.0002). The vitreous from non-diabetic patients or from PDR patients treated with levosulpiride, but not from placebo-treated PDR patients, inhibited the basic fibroblast growth factor (bFGF)- and vascular endothelial growth factor (VEGF)-induced proliferation of endothelial cells in culture. Vasoinhibin-neutralizing antibodies reduced the vitreous antiangiogenic effect. Matrix metalloproteases (MMPs) in the vitreous cleaved PRL to vasoinhibin, and their activity was higher in non-diabetic than in PDR patients. Conclusions: Levosulpiride increases the levels of PRL in the vitreous of PDR patients and promotes its MMP-mediated conversion to vasoinhibin, which can inhibit angiogenesis in DR. Translational Relevance: These findings support the potential therapeutic benefit of levosulpiride against vision loss in diabetes.

Prolactin stimulates the vascularisation of the retina in newborn mice under hyperoxia conditions.

The hormone prolactin (PRL) is emerging as an important regulator of ocular blood vessels. PRL is pro-angiogenic and acquires anti-angiogenic properties after undergoing proteolytic cleavage to the PRL fragment, vasoinhibin. The vascularisation of the rodent retina develops after birth when it rapidly expands until completion at the end of the first postnatal week. Exposure of newborn mice to high oxygen levels lowers the rate of blood vessel growth. In the present study, we investigated whether PRL treatment modifies the vascularisation of the retina in newborn mice exposed to high oxygen or to normoxia and whether the retinal conversion of PRL to vasoinhibin may be altered in the neonate. Newborn mice and their nursing mothers were subjected to 75% oxygen or to normoxia from postnatal day (P) 6 to P8 (group 1) or from P2 to P5 (group 2). PRL (2 µg g-1 , i.p., twice a day) or vehicle was injected from P5 to P8 in group 1 and from P1 to P5 in group 2. PRL treatment reduced the retinal inhibition of blood vessel growth and the increase in vascular regression induced by hyperoxia as revealed by immunofluorescence staining of blood vessels and the expression of angiogenesis and apoptosis markers. The pro-angiogenic effect may involve a reduced conversion of PRL to vasoinhibin. Incubation of PRL with retinal extracts showed reduced activity of the PRL-cleaving protease, cathepsin D, in the neonate vs the adult retina that was further reduced under hyperoxia. PRL and the PRL receptor mRNA were expressed at higher levels in the retina at P8 than in the adult, whereas endogenous PRL was undetectable in the circulation at P8. We conclude that PRL has a pro-angiogenic effect in the neonate retina as a result of its reduced conversion to vasoinhibin and that PRL produced by the retina may help promote physiological vascularisation after birth.