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Niels L Mulder, Rick Havinga, Joost Kluiver, Albert K Groen and Janine K Kruit

MicroRNAs have emerged as essential regulators of beta cell function and beta cell proliferation. One of these microRNAs, miR-132, is highly induced in several obesity models and increased expression of miR-132 in vitro modulates glucose-stimulated insulin secretion. The aim of this study was to investigate the therapeutic benefits of miR-132 overexpression on beta cell function in vivo. To overexpress miR-132 specifically in beta cells, we employed adeno-associated virus (AAV8)-mediated gene transfer using the rat insulin promoter in a double-stranded, self-complementary AAV vector to overexpress miR-132. Treatment of mice with dsAAV8-RIP-mir132 increased miR-132 expression in beta cells without impacting expression of miR-212 or miR-375. Surprisingly, overexpression of miR-132 did not impact glucose homeostasis in chow-fed animals. Overexpression of miR-132 did improve insulin secretion and hence glucose homeostasis in high-fat diet-fed mice. Furthermore, miR-132 overexpression increased beta cell proliferation in mice fed a high-fat diet. In conclusion, our data show that AAV8-mediated gene transfer of miR-132 to beta cells improves beta cell function in mice in response to a high-fat diet. This suggests that increased miR-132 expression is beneficial for beta cell function during hyperglycemia and obesity.

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Renata C Uliani, Alan J Conley, C Jo Corbin, Aimê M Friso, Luciana F S Maciel and Marco A Alvarenga

Anti-Müllerian hormone (AMH) is used as a marker of follicle population numbers and potential fertility in several species including horses but limited data exist across the lifespan. No one has decreased ovarian reserve experimentally to investigate whether a corresponding, quantitative decrease in AMH results. Concentrations of AMH across the lifespan were compiled from 1101 equine females sampled from birth to >33 years of age. Young and old mares (averaging 6 and 19 years) were hemi-ovariectomized and circulating AMH was assessed before and daily thereafter for 15 days. The remaining ovary was removed later and blood was drawn again before and after this second surgery for AMH determination. Polynomial regression analysis and analysis of mares grouped by 5-year intervals of age demonstrated AMH concentrations to be higher in mares aged 5–10 and 10–15 years than 0–5 years of age and lower in mares after 20 years of age. There was high variability in AMH concentrations among neonatal fillies, some of which had concentrations typical of males. Hemi-ovariectomy was followed by a decrease of AMH, almost exactly halving concentrations in intact mares. Concentrations of AMH had returned to intact levels in old mares before complete ovariectomy, as if exhibiting ovarian compensatory hypertrophy, but recovery of AMH was not evident in young mares. AMH may reflect ovarian senescence in mares after 20 years of age but is too variable to do so in the first two decades of life. The ovarian endocrine response to hemi-ovariectomy in mares appears to change with age.

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Ebony T Gilbreath, Lakshmikripa Jaganathan, Madhan Subramanian, Priya Balasubramanian, Katrina D Linning, Sheba M J MohanKumar and Puliyur S MohanKumar

Women are chronically exposed to estrogens through oral contraceptives, hormone replacement therapy or environmental estrogens. We hypothesized that chronic exposure to low levels of estradiol-17β (E2) can induce inflammatory and degenerative changes in the tuberoinfundibular dopaminergic (TIDA) system leading to reduced dopamine synthesis and hyperprolactinemia. Young (Y; 3–4 months) and middle-aged (MA; 10–12 months) Sprague–Dawley rats that were intact or ovariectomized (OVX) were either sham-implanted or implanted with a slow-release E2 pellet (20 ng E2/day for 90 days). To get mechanistic insight, adult 3- to 4-month-old WT, inducible nitric oxide synthase (iNOS) and IL-1 receptor (IL-1R) knockout (KO) mice were subjected to a similar treatment. Hypothalamic areas corresponding to the TIDA system were analyzed. E2 treatment increased IL-1β protein and nitrate levels in the arcuate nucleus of intact animals (Y and MA). Nitration of tyrosine hydroxylase in the median eminence increased with E2 treatment in both intact and OVX animals. There was no additional effect of age. This was accompanied by a reduction in dopamine levels and an increase in prolactin in intact animals. E2 treatment increased nitrate and reduced dopamine levels in the hypothalamus and increased serum prolactin in WT mice. In contrast, the effect of E2 on nitrate levels was blocked in IL-1R-KO mice and the effect on dopamine and prolactin were blocked in iNOS KO animals. Taken together, these results show that chronic exposure to low levels of E2 decreases TIDA activity through a cytokine-nitric oxide-mediated pathway leading to hyperprolactinemia and that aging could promote these degenerative changes.

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Maria Namwanje, Longhua Liu, Michelle Chan, Nikki Aaron, Michael J Kraakman and Li Qiang

Fat remodeling has been extensively explored through protein deacetylation, but not yet acetylation, as a viable therapeutic approach in the management of obesity and related metabolic disorders. Here, we investigated the functions of key acetyltransferases CBP/p300 in adipose remodeling and their physiological effects by generating adipose-specific deletion of CBP (Cbp-AKO), p300 (p300-AKO) and double-knockout (Cbp/p300-AKO) models. We demonstrated that Cbp-AKO exhibited marked brown remodeling of inguinal WAT (iWAT) but not epididymal WAT (eWAT) after cold exposure and that this pattern was exaggerated in diet-induced obesity (DIO). Despite this striking browning phenotype, loss of Cbp was insufficient to impact body weight or glucose tolerance. In contrast, ablation of p300 in adipose tissues had minimal effects on fat remodeling and adiposity. Surprisingly, double-knockout mice (Cbp/p300-AKO) developed severe lipodystrophy along with marked hepatic steatosis, hyperglycemia and hyperlipidemia. Furthermore, we demonstrated that pharmacological inhibition of Cbp and p300 activity suppressed adipogenesis. Collectively, these data suggest that (i) CBP, but not p300, has distinct functions in regulating fat remodeling and that this occurs in a depot-selective manner; (ii) brown remodeling occurs independently of the improvements in glucose metabolism and obesity and (iii) the combined roles of CBP and p300 are indispensable for normal adipose development.

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Eun Soo Lee, Mi-Hye Kwon, Hong Min Kim, Nami Kim, You Mi Kim, Hyeon Soo Kim, Eun Young Lee and Choon Hee Chung

Dibenzoylmethane (DBM) is a beta-diketone analog of curcumin. Numerous studies have shown the beneficial effects of curcumin on diabetes, obesity and diabetic complications including diabetic nephropathy. Recently, we investigated the beneficial metabolic effects of DBM on high-fat diet-induced obesity. However, the effects and mechanisms of action of DBM in the kidney are currently unknown. To investigate the renoprotective effects of DBM in type 2 diabetes, we administered DBM (100 mg/kg) orally for 12 weeks to high-fat diet-induced diabetic model mice. We used mouse renal mesangial (MES13) and macrophage (RAW 264.7) cells to examine the mechanism of action of DBM (20 μM). After DBM treatment, the albumin-to-creatinine ratio was significantly decreased compared to that of the high-fat-diet group. Moreover, damaged renal ultra-structures and functions including increased glomerular volume, glomerular basement membrane thickness and inflammatory signals were ameliorated after DBM treatment. Stimulation of MES13 and RAW264.7 cells by palmitate or high-dose glucose with lipopolysaccharides increased inflammatory signals and macrophage migration. However, these changes were reversed by DBM treatment. In addition, DBM inhibited NADPH oxidase 2 and 4 expression and oxidative DNA damage. Collectively, these data suggested that DBM prevented diabetes-induced renal injury through its anti-inflammatory and antioxidant effects.

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Liliana del V Sosa, Juan P Petiti, Florencia Picech, Sabrina Chumpen, Juan P Nicola, Pablo Perez, Ana De Paul, Javier Valdez-Taubas, Silvina Gutierrez and Alicia I Torres

The molecular mechanisms underlying the ERα nuclear/cytoplasmic pool that modulates pituitary cell proliferation have been widely described, but it is still not clear how ERα is targeted to the plasma membrane. The aim of this study was to analyse ERα palmitoylation and the plasma membrane ERα (mERα) pool, and their participation in E2-triggered membrane-initiated signalling in normal and pituitary tumour cell growth. Cell cultures were prepared from anterior pituitaries of female Wistar rats and tumour GH3 cells, and treated with 10 nM of oestradiol (E2). The basal expression of ERα was higher in tumour GH3 than in normal pituitary cells. Full-length palmitoylated ERα was observed in normal and pituitary tumour cells, demonstrating that E2 stimulation increased both, ERα in plasma membrane and ERα and caveolin-1 interaction after short-term treatment. In addition, the Dhhc7 and Dhhc21 palmitoylases were negatively regulated after sustained stimulation of E2 for 3 h. Although the uptake of BrdU into the nucleus in normal pituitary cells was not modified by E2, a significant increase in the GH3 tumoural cell, as well as ERK1/2 activation, with this effect being mimicked by PPT, a selective antagonist of ERα. These proliferative effects were blocked by ICI 182780 and the global inhibitor of palmitoylation. These findings indicate that ERα palmitoylation modulated the mERα pool and consequently the ERK1/2 pathway, thereby contributing to pituitary tumour cell proliferation. These results suggest that the plasma membrane ERα pool might be related to the proliferative behaviour of prolactinoma and may be a marker of pituitary tumour growth.

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Ana P Pinto, Alisson L da Rocha, Eike B Kohama, Rafael C Gaspar, Fernando M Simabuco, Fabiani G Frantz, Leandro P de Moura, José R Pauli, Dennys E Cintra, Eduardo R Ropelle, Ellen C de Freitas and Adelino S R da Silva

The endoplasmic reticulum (ER) stress and inflammation relationship occurs at different levels and is essential for the adequate homeostatic function of cellular systems, becoming harmful when chronically engaged. Intense physical exercise enhances serum levels of interleukin 6 (IL-6). In response to a chronic exhaustive physical exercise protocol, our research group verified an increase of the IL-6 concentration and ER stress proteins in extensor digitorium longus (EDL) and soleus. Based on these results, we hypothesized that IL-6-knockout mice would demonstrate a lower modulation in the ER stress proteins compared to the wild-type mice. To clarify the relationship between exercise-induced IL-6 increased and ER stress, we studied the effects of an acute exhaustive physical exercise protocol on the levels of ER stress proteins in the skeletal muscles of IL-6-knockout (KO) mice. The WT group displayed a higher exhaustion time compared to the IL-6 KO group. After 1 h of the acute exercise protocol, the serum levels of IL-6 and IL-10 were enhanced in the WT group. Independent of the experimental group, the CHOP and cleaved caspase 12/total caspase 12 ratio in EDL as well as ATF6 and CHOP in soleus were sensitive to the acute exercise protocol. Compared to the WT group, the oscillation patterns over time of BiP in EDL and soleus as well as of peIF2-alpha/eIF2-alpha ratio in soleus were attenuated for the IL-6 KO group. In conclusion, IL-6 seems to be related with the ER stress homeostasis, once knockout mice presented attenuation of BiP in EDL and soleus as well as of pEiF2-alpha/EiF2-alpha ratio in soleus after the acute exhaustive physical exercise protocol.

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Marie M Devillers, Florence Petit, Victoria Cluzet, Charlotte M François, Frank Giton, Ghislaine Garrel, Joëlle Cohen-Tannoudji and Céline J Guigon

Anti-Müllerian hormone (AMH) regulates ovarian function in cyclic females, notably by preventing premature follicle-stimulating hormone (FSH)-mediated follicular growth and steroidogenesis. Its expression in growing follicles is controlled by FSH and by estradiol (E2). In infantile females, there is a transient increase in the activity of the gonadotrope axis, as reflected by elevated levels of both gonadotropins and E2. We previously demonstrated in mice that elevated FSH concentrations are necessary to induce E2 production by preantral/early antral follicles through the stimulation of aromatase expression without supporting their growth. However, whether this action of FSH could involve AMH is unknown. Here, we show that Amh mRNA and protein abundance and serum AMH levels are elevated in infantile mouse females, compared with those in adults. By experimentally manipulating FSH and E2 levels in infantile mice, we demonstrate that high FSH concentrations lower Amh expression specifically in preantral/early antral follicles, whereas E2 has no effect. Importantly, treatment of infantile ovaries in organotypic cultures with AMH decreases FSH-mediated expression of Cyp19a1 aromatase, but it does not alter the expression of cyclin D2-mediating granulosa cell proliferation. Overall, our data indicate that the infantile elevation in FSH levels suppresses Amh expression in preantral/early antral follicles, thereby favoring Cyp19a1 aromatase expression and E2 production. Together with recent discoveries that AMH can act on both the hypothalamus and the pituitary to increase gonadotropin levels, this work suggests that AMH is a critical regulator of the gonadotrope axis during the infantile period, thereby contributing to adult reproductive function programming.

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Sara Pepe, Márta Korbonits and Donato Iacovazzo

While 95% of pituitary adenomas arise sporadically without a known inheritable predisposing mutation, in about 5% of the cases they can arise in a familial setting, either isolated (familial isolated pituitary adenoma or FIPA) or as part of a syndrome. FIPA is caused, in 15–30% of all kindreds, by inactivating mutations in the AIP gene, encoding a co-chaperone with a vast array of interacting partners and causing most commonly growth hormone excess. While the mechanisms linking AIP with pituitary tumorigenesis have not been fully understood, they are likely to involve several pathways, including the cAMP-dependent protein kinase A pathway via defective G inhibitory protein signalling or altered interaction with phosphodiesterases. The cAMP pathway is also affected by other conditions predisposing to pituitary tumours, including X-linked acrogigantism caused by duplications of the GPR101 gene, encoding an orphan G stimulatory protein-coupled receptor. Activating mosaic mutations in the GNAS gene, coding for the Gα stimulatory protein, cause McCune–Albright syndrome, while inactivating mutations in the regulatory type 1α subunit of protein kinase A represent the most frequent genetic cause of Carney complex, a syndromic condition with multi-organ manifestations also involving the pituitary gland. In this review, we discuss the genetic and molecular aspects of isolated and syndromic familial pituitary adenomas due to germline or mosaic mutations, including those secondary to AIP and GPR101 mutations, multiple endocrine neoplasia type 1 and 4, Carney complex, McCune–Albright syndrome, DICER1 syndrome and mutations in the SDHx genes underlying the association of familial paragangliomas and phaeochromocytomas with pituitary adenomas.

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Yuefei Huang, Pei Yee Ting, Tham M Yao, Tsuyoshi Homma, Danielle Brooks, Isis Katayama Rangel, Gail K Adler, Jose R Romero, Jonathan S Williams, Luminita H Pojoga and Gordon H Williams

Human risk allele carriers of lysine-specific demethylase 1 (LSD1) and LSD1-deficient mice have salt-sensitive hypertension for unclear reasons. We hypothesized that LSD1 deficiency causes dysregulation of aldosterone’s response to salt intake resulting in increased cardiovascular risk factors (blood pressure and microalbumin). Furthermore, we determined the effect of biological sex on these potential abnormalities. To test our hypotheses, LSD1 male and female heterozygote-knockout (LSD1+/−) and WT mice were assigned to two age groups: 18 weeks and 36 weeks. Plasma aldosterone levels and aldosterone production from zona glomerulosa cells studied ex vivo were greater in both male and female LSD1+/− mice consuming a liberal salt diet as compared to WT mice consuming the same diet. However, salt-sensitive blood pressure elevation and increased microalbuminuria were only observed in male LSD1+/− mice. These data suggest that LSD1 interacts with aldosterone’s secretory response to salt intake. Lack of LSD1 causes inappropriate aldosterone production on a liberal salt diet; males appear to be more sensitive to this aldosterone increase as males, but not females, develop salt sensitivity of blood pressure and increased microalbuminuria. The mechanism responsible for the cardiovascular protective effect in females is uncertain but may be related to estrogen modulating the effect of mineralocorticoid receptor activation.