Description: Dysregulation of the IGF axis is implicated in the development of benign prostatic hyperplasia (BPH) and prostate cancer (PCa), 2 of the most common diseases affecting elderly males. PCa is the second leading cause of male-related cancer death in Western societies. Although distinct pathologies, BPH and PCa are both characterized by extensive stromal remodeling, in particular fibroblast-to-myofibroblast differentiation, thought to be induced by elevated local production of TGFβ1. We previously showed that TGFβ1-mediated fibroblast-to-myofibroblast differentiation of primary human prostatic stromal cells resulted in the dsyregulation of several components of the IGF axis, including the induction of IGF binding protein 3 (IGFBP3). Using isoform-specific lentiviral-mediated knockdown, we demonstrate herein that IGFBP3 is essential for TGFβ1-mediated differentiation. Although recombinant human IGFBP3 alone was not sufficient to induce differentiation, IGFBP3 synergistically potentiated TGFβ1-mediated stromal remodeling predominantly via an IGF-independent mechanism. Consistent with these in vitro findings, IGFBP3 immunohistochemistry revealed elevated levels of IGFBP3 in the hyperplastic fibromuscular stroma of BPH specimens and in the tumor-adjacent stroma of high-grade PCa. Collectively these data indicate that the dysregulation of the stromal IGF axis, in particular elevated IGFBP3, plays a crucial role in fibroblast-to-myofibroblast differentiation in the diseased prostatic stroma and indicate the therapeutic potential of inhibiting stromal remodeling and the resulting dysregulation of the stromal IGF axis as a novel strategy for the treatment of advanced PCa and BPH.

Description: In obesity, reduced cardiac glucose uptake and mitochondrial abnormalities are putative causes of cardiac dysfunction. However, high-fat diet (HFD) does not consistently induce cardiac insulin resistance and mitochondrial damage, and recent studies suggest HFD may be cardioprotective. To determine cardiac responses to HFD, we investigated cardiac function, glucose uptake, and mitochondrial respiration in young (3-month-old) and middle-aged (MA) (12-month-old) male Ldlr –/– mice fed chow or 3 months HFD to induce obesity, systemic insulin resistance, and hyperinsulinemia. In MA Ldlr –/– mice, HFD induced accelerated atherosclerosis and nonalcoholic steatohepatitis, common complications of human obesity. Surprisingly, HFD-fed mice demonstrated increased cardiac glucose uptake, which was most prominent in MA mice, in the absence of cardiac contractile dysfunction or hypertrophy. Moreover, hearts of HFD-fed mice had enhanced mitochondrial oxidation of palmitoyl carnitine, glutamate, and succinate and greater basal insulin signaling compared with those of chow-fed mice, suggesting cardiac insulin sensitivity was maintained, despite systemic insulin resistance. Streptozotocin-induced ablation of insulin production markedly reduced cardiac glucose uptake and mitochondrial dysfunction in HFD-fed, but not in chow-fed, mice. Insulin injection reversed these effects, suggesting that insulin may protect cardiac mitochondria during HFD. These results have implications for cardiac metabolism and preservation of mitochondrial function in obesity.

Description: Menopause is characterized by the rapid age-related decline of circulating 17β-estradiol (E 2 ) levels in women, which can sometimes result in cognitive disorders such as impaired memory and increased anxiety. Hormone therapy (HT) is a widely used treatment for the adverse effects associated with menopause; however, evidence suggests that HT administered to postmenopausal women age 65 years and over can lead to increased risks for cognitive disorders. We hypothesized that these age-related changes in E 2 action are due to posttranscriptional gene regulation by microRNAs (miRNAs). miRNAs are a class of small noncoding RNAs that regulate gene expression by binding to the 3'-untranslated region of target mRNAs and subsequently target these transcripts for degradation. In the present study, 3- and 18-month-old female rats were oophorectomized (OVX) and treated 1 week after surgery with 2.5 μg E 2 once per day for 3 days. Total RNA was isolated from the ventral and dorsal hippocampus, central amygdala, and paraventricular nucleus. Our results showed that E 2 differentially altered miRNA levels in an age- and brain region-dependent manner. Multiple miRNA target prediction algorithms revealed putative target genes that are important for memory and stress regulation, such as BDNF , glucocorticoid receptor, and SIRT-1 . Indeed, quantitative RT-PCR analyses of some of the predicted targets, such as SIRT1 , showed that the mRNA expression levels were the inverse of the targeting miRNA, thereby confirming the prediction algorithms. Taken together, these data show that E 2 regulates miRNA expression in an age- and E 2 -dependent manner, which we hypothesize results in differential gene expression and consequently altered neuronal function.

Description: Cocaine- and amphetamine-regulated transcript (CART) is a hypothalamic neuropeptide implicated in both metabolic and reproductive regulation, raising the possibility that CART plays a role in reproductive inhibition during negative metabolic conditions. The current study characterized CART's regulatory influence on GnRH and kisspeptin (Kiss1) cells and determined the sensitivity of different CART populations to negative energy balance. CART fibers made close appositions to 60% of GnRH cells, with the majority of the fibers (〉80%) originating from the arcuate nucleus (ARH) CART/pro-opiomelanocortin population. Electrophysiological recordings in GnRH-green fluorescent protein rats demonstrated that CART postsynaptically depolarizes GnRH cells. CART fibers from the ARH were also observed in close contact with Kiss1 cells in the ARH and anteroventral periventricular nucleus (AVPV). Recordings in Kiss1-GFP mice demonstrated CART also postsynaptically depolarizes ARH Kiss1 cells, suggesting CART may act directly and indirectly, via Kiss1 populations, to stimulate GnRH neurons. CART protein and mRNA levels were analyzed in 2 models of negative energy balance: caloric restriction (CR) and lactation. Both CART mRNA levels and the number of CART-immunoreactive cells were suppressed in the ARH during CR but not during lactation. AVPV CART mRNA was suppressed during CR, but not during lactation when there was a dramatic increase in CART-immunoreactive cells. These data suggest differing regulatory signals of CART between the models. In conclusion, both morphological and electrophysiological methods identify CART as a novel and potent stimulator of Kiss1 and GnRH neurons and suppression of CART expression during negative metabolic conditions could contribute to inhibition of the reproductive axis.

Description: Kisspeptin neurons located in the arcuate nucleus (ARN) coexpress dynorphin and neurokinin B (NKB) and may interact to influence gonadotropin secretion. Using a kisspeptin-green fluorescent protein mouse model, the present study examined whether the neuropeptides kisspeptin, dynorphin, and NKB modulate the electrical activity of ARN kisspeptin neurons in the adult male mouse. Cell-attached recordings showed that kisspeptin itself had no effect on kisspeptin neuron firing. Dynorphin and the -opioid receptor agonist U50-488 evoked a potent suppression of all ARN kisspeptin neuron firing that was blocked completely by the -opioid receptor antagonist nor -Binaltorphimine. Both NKB and Senktide, a neurokinin 3 receptor agonist, exerted a potent stimulatory action on ~95% of ARN kisspeptin neurons. Although the selective neurokinin 3 receptor antagonists SB222200 and SR142801 blocked the effects of Senktide on kisspeptin neurons, they surprisingly had no effect on NKB activation of firing. Studies with selective neurokinin 1 receptor (SDZ-NKT343) and neurokinin 2 receptor (GR94800) antagonists revealed that the activation of kisspeptin neurons by NKB was only blocked completely by a cocktail of antagonists against all 3 tachykinin receptors. Whole-cell recordings revealed that individual kisspeptin neurons were activated directly by all 3 tachykinins substance, P, neurokinin A, and NKB. These experiments show that dynorphin and NKB have opposing actions on the electrical activity of kisspeptin neurons supporting the existence of an interconnected network of kisspeptin neurons in the ARN. However, the effects of NKB result from an unexpected activation of multiple tachykinin receptors.

Description: The development of insulin resistance is tightly linked to fatty liver disease and is considered a major health concern worldwide, although their mechanistic relationship remains controversial. Activin has emerging roles in nutrient homeostasis, but its metabolic effects on hepatocytes remain unknown. In this study, we investigated the effects of increased endogenous activin bioactivity on hepatic nutrient homeostasis by creating mice with inactivating mutations that deplete the circulating activin antagonists follistatin-like-3 ( FSTL3 ) or the follistatin 315 isoform ( FST315 ; FST288 -only mice). We investigated liver histology and lipid content, hepatic insulin sensitivity, and metabolic gene expression including the HepG2 cell and primary hepatocyte response to activin treatment. Both FSTL3 -knockout and FST288 -only mice had extensive hepatic steatosis and elevated hepatic triglyceride content. Unexpectedly, insulin signaling, as assessed by phospho-Akt (a.k.a. protein kinase B), was enhanced in both mouse models. Pretreatment of HepG2 cells with activin A increased their response to subsequent insulin challenge. Gene expression analysis suggests that increased lipid uptake, enhanced de novo lipid synthesis, decreased lipolysis, and/or enhanced glucose uptake contribute to increased hepatic triglyceride content in these models. However, activin treatment recapitulated only some of these gene changes, suggesting that increased activin bioactivity may be only partially responsible for this phenotype. Nevertheless, our results indicate that activin enhances hepatocyte insulin response, which ultimately leads to hepatic steatosis despite the increased insulin sensitivity. Thus, regulation of activin bioactivity is critical for maintaining normal liver lipid homeostasis and response to insulin, whereas activin agonists may be useful for increasing liver insulin sensitivity.

Description: Antiestrogens such as tamoxifen (TAM) provided a successful treatment for estrogen receptor (ER)-positive breast cancer for the past four decades. However, most breast tumors are eventually resistant to TAM therapy. The molecular mechanisms underlying TAM resistance have not been well established. Recently, we reported that breast cancer patients with tumors expressing high concentrations of ER-α36, a variant of ER-α, benefited less from TAM therapy than those with low concentrations of ER-α36, suggesting that increased ER-α36 concentration is one of the underlying mechanisms of TAM resistance. Here, we investigated the function and underlying mechanism of ER-α36 in TAM resistance. We found that TAM increased ER-α36 concentrations, and TAM-resistant MCF7 cells expressed high concentrations of ER-α36. In addition, MCF7 cells with forced expression of recombinant ER-α36 and H3396 cells expressing high concentrations of endogenous ER-α36 were resistant to TAM. ER-α36 down-regulation in TAM-resistant cells with the short hairpinRNA method restored TAM sensitivity. We also found that TAM acted as a potent agonist by activating phosphorylation of the AKT kinase in ER-α36-expressing cells. Finally, we found that cells with high concentration of ER-α36 protein were hypersensitive to estrogen, activating ERK phosphorylation at picomolar range. Our results thus demonstrated that elevated ER-α36 concentration is one of the mechanisms by which ER-positive breast cancer cells escape TAM therapy and provided a rational to develop novel therapeutic approaches for TAM-resistant patients by targeting ER-α36.

Description: The most effective treatment for obesity is bariatric surgery. However, there is increasing concern that bariatric surgery can cause nutrient deficiencies that translate into metabolic bone disease. Whether this is true for all surgery types is not yet clear. We therefore investigated the effects of 2 commonly applied bariatric surgeries (Roux-en-Y gastric bypass [RYGB] and vertical sleeve gastrectomy) on energy and bone metabolism in rats 60 days after surgery. Both surgeries resulted in similar reductions of body weight, body fat, and food intake. Glucose tolerance was improved to a similar extent after both surgeries and was accompanied by increased postprandial secretion of glucose-dependent insulinotropic peptide. Using microcomputed tomography, we found that, relative to sham-operated rats, bone volume was significantly reduced after RYGB but not vertical sleeve gastrectomy. RYGB rats also had markedly reduced lipid absorption from the intestine and significantly lower serum 25-hydroxyvitamin D and calcium levels. Importantly, dietary supplementation with calcium and vitamin D could not fully rescue the reduced bone volume after RYGB surgery. Both surgeries resulted in a significant increase in stomach pH, which may have worsened the malabsorption in RYGB rats. Our findings suggest that bone loss in RYGB rats is not exclusively driven by calcium and vitamin D malabsorption but also by additional factors that may not be rescuable by dietary supplementation. These data point toward important similarities and differences between bariatric procedures that should be considered in clinical settings as guidance for which procedure will be best for specific patient populations.

Description: Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4'-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

Description: Chemerin is an adipokine involved in obesity, inflammation, and innate immune system that is highly expressed in the liver. In the present study, we find that chemerin mRNA expression is decreased in the livers of rodents with nonalcoholic fatty liver disease as well as in HepG2 cells after lipid overloading. Moreover, we report that chemerin expression and secretion are induced in HepG2 cells and primary hepatocytes from wild-type mice, but not farnesoid X receptor (FXR)–/– mice, in response to the synthetic FXR ligand GW4064. Hepatic chemerin expression is decreased in FXR–/– mice but up-regulated by GW4064 administration in wild-type mice. Dual-luciferase reporter assay and chromatin immunoprecipitation analyses further identified a functional FXR response element located in the –258-bp /+121-bp region of the chemerin gene. These data demonstrate that chemerin, a novel target gene of FXR, is related to nonalcoholic steatohepatitis.