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.

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.

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.

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.

Low prolactin levels are associated with visceral adipocyte hypertrophy and insulin resistance in humans.

PURPOSE: Low prolactin (PRL) serum levels are associated with glucose intolerance and type 2 diabetes in adults, and with metabolic syndrome and obesity in children. In obese rodents, PRL treatment promotes insulin sensitivity by maintaining adipose tissue fitness, and lack of PRL signaling exacerbates obesity-derived metabolic alterations. Since adipose tissue dysfunction is a key factor triggering metabolic alterations, we evaluated whether PRL serum levels are associated with adipocyte hypertrophy (a marker of adipose tissue dysfunction), insulin resistance, and metabolic syndrome in lean, overweight, and obese adult men and women. METHODS: Samples of serum and adipose tissue from 40 subjects were obtained to evaluate insulin resistance index (homeostasis model assessment of insulin resistance (HOMA-IR)), signs of metabolic syndrome (glucose levels, high-density lipoproteins, triglycerides, blood pressure, and waist circumference), as well as adipocyte size and gene expression in fat. RESULTS: Lower PRL serum levels are associated with adipocyte hypertrophy, in visceral but not in subcutaneous fat, and with a higher HOMA-IR. Furthermore, low systemic PRL levels together with high waist circumference predict an elevated HOMA-IR whereas low serum PRL values in combination with high blood glucose predicts visceral adipocyte hypertrophy. In agreement, visceral fat from insulin resistant subjects shows reduced expression of prolactin receptor. However, there is no association between PRL levels and obesity or signs of metabolic syndrome. CONCLUSIONS: Our results support that low levels of PRL are markers of visceral fat dysfunction and insulin resistance, and suggest the potential therapeutic value of medications elevating PRL levels to help maintain metabolic homeostasis.

Vasoinhibin Suppresses Nerve Growth Factor-Induced Differentiation and Survival of PC12 Pheochromocytoma Cells.

BACKGROUND: Vasoinhibin, a protein derived from prolactin, regulates various vascular functions including endothelial cell survival. Of note, vasoinhibin is present in the central nervous system, where it triggers neuroendocrine and behavioral responses to stress. Moreover, vasoinhibin compromises nerve growth factor (NGF)-induced neurite outgrowth in primary sensory neurons of the peripheral nervous system. Nonetheless, information on the functions of vasoinhibin in developing neurons remains limited. The present study explored whether vasoinhibin affects the neurotrophic actions of NGF by measuring the cell differentiation and survival of PC12 pheochromocytoma cells. METHODS: The effects of recombinant or lentiviral vector-transduced human vasoinhibin were tested on differentiating PC12 cells. Neurite outgrowth was quantified by measuring their length and density. The MTT assay was employed to assess cell viability, and ELISA was used to quantify DNA fragmentation as an index of apoptosis. Phosphorylated Akt and ERK1/2 were analyzed by Western blotting. RESULTS: The addition of a human recombinant vasoinhibin, and the transduction of a lentiviral vector carrying a human vasoinhibin sequence, significantly reduced NGF-induced neurite outgrowth, cell survival, and phosphorylation of Akt and ERK1/2, and increased DNA fragmentation and caspase 3 activation in PC12 cells. CONCLUSIONS: Vasoinhibin downregulates NGF-induced differentiation and survival of PC12 cells, blocking tropomyosin receptor kinase A-triggered signaling pathways and increasing apoptosis. These results establish that vasoinhibin interaction with NGF and other neurotrophins may be critical in mediating pathways involved in neuronal survival and differentiation.

Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Maternal diet during lactation affects offspring metabolic health throughout life. Prolactin (PRL) is present in high quantities in maternal milk; however, the effects of milk PRL on the offspring remain poorly characterized. In this study, we evaluated whether feeding a high-fat diet (HFD) to rats during lactation alters PRL, both in the mother's serum and in milk, and whether this factor contributes to HFD-induced metabolic dysfunction in the offspring. Maternal HFD resulted in decreased PRL levels in milk (but not in serum), reduced mammary gland (MG) PRL receptor expression, and altered MG structure and function. Offspring from HFD-fed dams had increased body weight and adiposity, and developed fatty liver, hyperinsulinemia, and insulin resistance at weaning. Increasing PRL levels in the HFD-fed mothers by subcutaneous osmotic minipumps releasing PRL normalized MG function and PRL levels in milk. Moreover, PRL treatment in HFD-fed mothers, or directly in their pups via oral PRL administration, increased liver STAT5 phosphorylation, reduced visceral adiposity, ameliorated fatty liver, and improved insulin sensitivity in offspring. Our results show that HFD impairs PRL actions during lactation to negatively affect MG physiology and directly impair offspring metabolism.-De los Ríos, E. A., Ruiz-Herrera, X., Tinoco-Pantoja, V., López-Barrera, F., Martínez de la Escalera, G., Clapp, C., Macotela, Y. Impaired prolactin actions mediate altered offspring metabolism induced by maternal high-fat feeding during lactation.

Prolactin regulates liver growth during postnatal development in mice.

The liver grows during the early postnatal period first at slower and then at faster rates than the body to achieve the adult liver-to-body weight ratio (LBW), a constant reflecting liver health. The hormone prolactin (PRL) stimulates adult liver growth and regeneration, and its levels are high in the circulation of newborn infants, but whether PRL plays a role in neonatal liver growth is unknown. Here, we show that the liver produces PRL and upregulates the PRL receptor in mice during the first 2 wk after birth, when liver growth lags behind body growth. At postnatal week 4, the production of PRL by the liver ceases coinciding with the elevation of circulating PRL and the faster liver growth that catches up with body growth. PRL receptor null mice ( Prlr-/-) show a significant decrease in the LBW at 1, 4, 6, and 10 postnatal weeks and reduced liver expression of proliferation [cyclin D1 ( Ccnd1)] and angiogenesis [platelet/endothelial cell adhesion molecule 1 ( Pecam1)] markers relative to Prlr+/+ mice. However, the LBW increases in Prlr-/- mice at postnatal week 2 concurring with the enhanced liver expression of Igf-1 and the liver upregulation and downregulation of suppressor of cytokine signaling 2 ( Socs2) and Socs3, respectively. These findings indicate that PRL acts locally and systemically to restrict and stimulate postnatal liver growth. PRL inhibits liver and body growth by attenuating growth hormone-induced Igf-1 liver expression via Socs2 and Socs3-related mechanisms.

Prolactin Promotes Adipose Tissue Fitness and Insulin Sensitivity in Obese Males.

Excessive accumulation of body fat triggers insulin resistance and features of the metabolic syndrome. Recently, evidence has accumulated that obesity, type 2 diabetes, and metabolic syndrome are associated with reduced levels of serum prolactin (PRL) in humans and rodents, raising the question of whether low PRL levels contribute to metabolic dysfunction. Here, we have addressed this question by investigating the role of PRL in insulin sensitivity and adipose tissue fitness in obese rodents and humans. In diet-induced obese rats, treatment with PRL delivered via osmotic mini-pumps, improved insulin sensitivity, prevented adipocyte hypertrophy, and reduced inflammatory cytokine expression in visceral fat. PRL also induced increased expression of Pparg and Xbp1s in visceral adipose tissue and elevated circulating adiponectin levels. Conversely, PRL receptor null mice challenged with a high-fat diet developed greater insulin resistance, glucose intolerance, and increased adipocyte hypertrophy compared with wild-type mice. In humans, serum PRL values correlated positively with systemic adiponectin levels and were reduced in insulin-resistant patients. Furthermore, PRL circulating levels and PRL produced by adipose tissue correlated directly with the expression of PPARG, ADIPOQ, and GLUT4 in human visceral and sc adipose tissue. Thus, PRL, acting through its cognate receptors, promotes healthy adipose tissue function and systemic insulin sensitivity. Increasing the levels of PRL in the circulation may have therapeutic potential against obesity-induced metabolic diseases.

Prolactin protects retinal pigment epithelium by inhibiting sirtuin 2-dependent cell death.

The identification of pathways necessary for retinal pigment epithelium (RPE) function is fundamental to uncover therapies for blindness. Prolactin (PRL) receptors are expressed in the retina, but nothing is known about the role of PRL in RPE. Using the adult RPE 19 (ARPE-19) human cell line and mouse RPE, we identified the presence of PRL receptors and demonstrated that PRL is necessary for RPE cell survival via anti-apoptotic and antioxidant actions. PRL promotes the antioxidant capacity of ARPE-19 cells by reducing glutathione. It also blocks the hydrogen peroxide-induced increase in deacetylase sirtuin 2 (SIRT2) expression, which inhibits the TRPM2-mediated intracellular Ca(2+) rise associated with reduced survival under oxidant conditions. RPE from PRL receptor-null (prlr(-/-)) mice showed increased levels of oxidative stress, Sirt2 expression and apoptosis, effects that were exacerbated in animals with advancing age. These observations identify PRL as a regulator of RPE homeostasis.

Prolactin anterior pituitary expression and circulating levels are reduced in obese and diabetic rats: role of TGF-ß and TNF-α.

The levels of the hormone prolactin (PRL) are reduced in the circulation of patients with Type 2 diabetes and in obese children, and lower systemic PRL levels correlate with an increased prevalence of diabetes and a higher risk of metabolic syndrome. The secretion of anterior pituitary (AP) PRL in metabolic diseases may be influenced by the interplay between transforming growth factor ß (TGF-ß) and tumor necrosis factor α (TNF-α), which inhibit and can stimulate AP PRL synthesis, respectively, and are known contributors to insulin resistance and metabolic complications. Here, we show that TGF-ß and TNF-α antagonize the effect of each other on the expression and release of PRL by the GH4C1 lactotrope cell line. The levels of AP mRNA and circulating PRL decrease in high-fat diet-induced obese rats in parallel with increased and reduced AP levels of TGF-ß and TNF-α mRNA, respectively. Likewise, AP expression and circulating levels of PRL are reduced in streptozotocin-induced diabetic rats and are associated with higher AP expression and protein levels of TGF-ß and TNF-α. The opposing effects of the two cytokines on cultured AP cells, together with their altered expression in the AP of obese and diabetic rats suggest they are linked to the reduced PRL production and secretion characteristics of metabolic diseases.