Integrated or Independent Actions of Metformin in Target Tissues Underlying Its Current Use and New Possible Applications in the Endocrine and Metabolic Disorder Area.

Metformin is considered the first-choice drug for type 2 diabetes treatment. Actually, pleiotropic effects of metformin have been recognized, and there is evidence that this drug may have a favorable impact on health beyond its glucose-lowering activity. In summary, despite its long history, metformin is still an attractive research opportunity in the field of endocrine and metabolic diseases, age-related diseases, and cancer. To this end, its mode of action in distinct cell types is still in dispute. The aim of this work was to review the current knowledge and recent findings on the molecular mechanisms underlying the pharmacological effects of metformin in the field of metabolic and endocrine pathologies, including some endocrine tumors. Metformin is believed to act through multiple pathways that can be interconnected or work independently. Moreover, metformin effects on target tissues may be either direct or indirect, which means secondary to the actions on other tissues and consequent alterations at systemic level. Finally, as to the direct actions of metformin at cellular level, the intracellular milieu cooperates to cause differential responses to the drug between distinct cell types, despite the primary molecular targets may be the same within cells. Cellular bioenergetics can be regarded as the primary target of metformin action. Metformin can perturb the cytosolic and mitochondrial NAD/NADH ratio and the ATP/AMP ratio within cells, thus affecting enzymatic activities and metabolic and signaling pathways which depend on redox- and energy balance. In this context, the possible link between pyruvate metabolism and metformin actions is extensively discussed.

Autophagy in normal pituitary and pituitary tumor cells and its potential role in the actions of somatostatin receptor ligands in acromegaly.

Autophagy is an evolutionary conserved process for the self-degradation and recycling of cellular components in the cytoplasm. It is involved in both physiological and pathological conditions. In detail, the term "autophagy" refers to intracellular degradative pathways that lead to packaging and deliver of cellular components to lysosomes or to plant and yeast vacuoles. Autophagy is triggered by a variety of stimuli like nutrient deprivation, hypoxia, mitochondrial dysfunction, endoplasmic reticulum stress, and is regulated by immune- and hormonal factors. The role of autophagy in tumor cells is complex. Indeed, autophagy may act as a tumor suppressor as well as a tumor survival factor, in a context-dependent manner. The research into autophagy in normal pituitary and pituitary tumors has not gained great consideration, yet. Nevertheless, some recent articles joint to previous case studies, suggest that this process plays a role in the modulation and fluctuation of normal pituitary cell functions and in the response of pituitary tumor cells to drug therapy, including the response to somatostatin receptor ligand (SRLs), the first-line medical therapy of acromegaly. Although it is not possible to draw any conclusion, the aim of this review was to highlight some considerations and perspectives in this research field. Reports on the effects of octreotide on autophagy induction and autophagic flux in extra-pituitary target tissues, have also been discussed.

Role of Bioactive Peptide Sequences in the Potential Impact of Dairy Protein Intake on Metabolic Health.

For years, there has been an increasing move towards elucidating the complexities of how food can interplay with the signalling networks underlying energy homeostasis and glycaemic control. Dairy foods can be regarded as the greatest source of proteins and peptides with various health benefits and are a well-recognized source of bioactive compounds. A number of dairy protein-derived peptide sequences with the ability to modulate functions related to the control of food intake, body weight gain and glucose homeostasis have been isolated and characterized. Their being active in vivo may be questionable mainly due to expected low bioavailability after ingestion, and hence their real contribution to the metabolic impact of dairy protein intake needs to be discussed. Some reports suggest that the differential effects of dairy proteins-in particular whey proteins-on mechanisms underlying energy balance and glucose-homeostasis may be attributed to their unique amino acid composition and hence the release of free amino acid mixtures enriched in essential amino acids (i.e., branched-chain-amino acids) upon digestion. Actually, the research reports reviewed in this article suggest that, among a number of dairy protein-derived peptides isolated and characterized as bioactive compounds in vitro, some peptides can be active in vivo post-oral administration through a local action in the gut, or, alternatively, a systemic action on specific molecular targets after entering the systemic circulation. Moreover, these studies highlight the importance of the enteroendocrine system in the cross talk between food proteins and the neuroendocrine network regulating energy balance.

Differential effects of metformin on reductive activity and energy production in pituitary tumor cells compared to myogenic precursors.

PURPOSE: Given the multiple targets of metformin within cells, the mechanism by which it may exert a growth-inhibitory action on pituitary tumor cells in vitro remains to be explored. Previous research stressed metformin-induced changes in the activity of signaling pathways regulating cell growth and cell death. In this work, we investigated the effects of metformin on cell viability markers related to cell metabolic activity in rat pituitary tumor cells versus rat myogenic precursors as a model of normal proliferating somatic cells. METHODS: We designed our experiments in order to use the MTT reduction as a marker of cellular reductive activity and the total cellular ATP levels as a marker of energy supply during short incubations with different metabolic substrates (sodium pyruvate, D-glucose, L-glutamine, sodium citrate). Then, we extended the analysis to extracellular glucose consumption, extracellular medium acidification and pyruvate dehydrogenase (PDH) complex activity. RESULTS: Metformin was found to be effective in both cell types at the same concentrations, although the outcome of the treatment was quite the opposite. Unexpectedly, metformin increased the viability of subconfluent rat myoblasts. Rat pituitary tumor cells and myoblasts differed in the utilization of distinct metabolic substrates and the PDH complex activity. Metformin actions on reductive activity and ATP production were substrate-dependent. CONCLUSIONS: Overall, this work points out that metformin actions at the cellular level depend on metabolic features and metabolic requirements of cells. The pyruvate metabolic branch point is most likely to play a main role in the variability of cell response to metformin.

Effects of octreotide on autophagy markers and cell viability markers related to metabolic activity in rat pituitary tumor cells.

PURPOSE: The aim of this work was to investigate possible direct effects of the somatostatin analog octreotide on autophagy markers and markers of cellular metabolic activity using in vitro cultured rat pituitary tumor cells (GH3 cell line). METHODS: We measured two markers of the autophagic flux in cell lysates by Western blot and MTT reductive activity, total cellular ATP levels, pyruvate dehydrogenase (PDH) complex activity in cells lysates as markers of cell viability related to metabolic activity. RESULTS: Octreotide (100 nM) treatment induced autophagy activation (increased LC3-I protein lipidation) and enhanced the autophagic flux (SQSTM1/p62 protein downregulation) in GH3 cells in different incubation media, in detail in Hank's balanced salt solution (HBSS) as well as in maintenance medium with serum. We did not observe any decrease of redox activity and energy production related to the induction of autophagy by octreotide. On the other hand, short-term treatments with octreotide in HBSS tended to enhance MTT reduction activity and to increase PDH complex enzymatic activity and ATP levels measured in GH3 cell lysates. CONCLUSIONS: We provided evidence that octreotide can affect autophagy in pituitary tumor cells. The observed effects of octreotide were not related to a decrease of cellular metabolic activity. Finally, the induction of autophagy was either short-lived or overshadowed by other factors in the long term and this limit does not help clarifying their real impact on the pharmacological activity of somatostatin analogs.

How treatments with endocrine and metabolic drugs influence pituitary cell function.

A variety of endocrine and metabolic signals regulate pituitary cell function acting through the hypothalamus-pituitary neuroendocrine axes or directly at the pituitary level. The underlying intracellular transduction mechanisms in pituitary cells are still debated. AMP-activated protein kinase (AMPK) functions as a cellular sensor of low energy stores in all mammalian cells and promotes adaptive changes in response to calorie restriction. It is also regarded as a target for therapy of proliferative disorders. Various hormones and drugs can promote tissue-specific activation or inhibition of AMPK by enhancing or inhibiting AMPK phosphorylation, respectively. This review explores the preclinical studies published in the last decade that investigate the role of AMP-activated protein kinase in the intracellular transduction pathways downstream of endocrine and metabolic signals or drugs affecting pituitary cell function, and its role as a target for drug therapy of pituitary proliferative disorders. The effects of the hypoglycemic agent metformin, which is an indirect AMPK activator, are discussed. The multiple effects of metformin on cell metabolism and cell signalling and ultimately on cell function may be either dependent or independent of AMPK. The in vitro effects of metformin may also help highlighting differences in metabolic requirements between pituitary adenomatous cells and normal cells.

International Union of Basic and Clinical Pharmacology. CV. Somatostatin Receptors: Structure, Function, Ligands, and New Nomenclature.

Somatostatin, also known as somatotropin-release inhibitory factor, is a cyclopeptide that exerts potent inhibitory actions on hormone secretion and neuronal excitability. Its physiologic functions are mediated by five G protein-coupled receptors (GPCRs) called somatostatin receptor (SST)1-5. These five receptors share common structural features and signaling mechanisms but differ in their cellular and subcellular localization and mode of regulation. SST2 and SST5 receptors have evolved as primary targets for pharmacological treatment of pituitary adenomas and neuroendocrine tumors. In addition, SST2 is a prototypical GPCR for the development of peptide-based radiopharmaceuticals for diagnostic and therapeutic interventions. This review article summarizes findings published in the last 25 years on the physiology, pharmacology, and clinical applications related to SSTs. We also discuss potential future developments and propose a new nomenclature.

Effects of metformin on cell growth and AMPK activity in pituitary adenoma cell cultures, focusing on the interaction with adenylyl cyclase activating signals.

For a few years we have been investigating AMP-activated protein kinase (AMPK) as a target for drug therapy of GH-secreting pituitary adenomas. Aim of this study was to investigate the direct effects of metformin, which causes AMPK activation in different cell types, on rat pituitary adenoma cell growth and on related cell signalling pathways. Our results suggest that metformin can exert a growth-inhibitory activity in rat pituitary tumor cells mediated by AMPK activation, although multiple mechanisms are most likely involved. Membrane proteins, including growth factor receptors, are valuable targets of AMPK. The inhibition of the mTOR-p70S6 kinase signalling pathway plays a role in the suppressive effect of metformin on pituitary tumor cell growth. Metformin did not affect the MTT reduction activity in energetic stress conditions. Finally, metformin was still able to induce AMPK activation and to inhibit cell growth in cells treated with forskolin and in transfected cells overexpressing GHRH-receptor and treated with GHRH. Hence, adenylyl cyclase over-activation does not account for the lack of response of some human pituitary tumors to AMPK-activating compounds in vitro.

MURC/cavin-4 Is Co-Expressed with Caveolin-3 in Rhabdomyosarcoma Tumors and Its Silencing Prevents Myogenic Differentiation in the Human Embryonal RD Cell Line.

The purpose of this study was to investigate whether MURC/cavin-4, a plasma membrane and Z-line associated protein exhibiting an overlapping distribution with Caveolin-3 (Cav-3) in heart and muscle tissues, may be expressed and play a role in rhabdomyosarcoma (RMS), an aggressive myogenic tumor affecting childhood. We found MURC/cavin-4 to be expressed, often concurrently with Cav-3, in mouse and human RMS, as demonstrated through in silico analysis of gene datasets and immunohistochemical analysis of tumor samples. In vitro expression studies carried out using human cell lines and primary mouse tumor cultures showed that expression levels of both MURC/cavin-4 and Cav-3, while being low or undetectable during cell proliferation, became robustly increased during myogenic differentiation, as detected via semi-quantitative RT-PCR and immunoblotting analysis. Furthermore, confocal microscopy analysis performed on human RD and RH30 cell lines confirmed that MURC/cavin-4 mostly marks differentiated cell elements, colocalizing at the cell surface with Cav-3 and labeling myosin heavy chain (MHC) expressing cells. Finally, MURC/cavin-4 silencing prevented the differentiation in the RD cell line, leading to morphological cell impairment characterized by depletion of myogenin, Cav-3 and MHC protein levels. Overall, our data suggest that MURC/cavin-4, especially in combination with Cav-3, may play a consistent role in the differentiation process of RMS.

Interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK) and the estrogen receptor activities in regulating rat pituitary tumor cell (GH3) growth in vitro.

Estrogen receptor α has a role in regulating rat somatolactotroph tumor cell growth (GH3 cells). AMP-activated protein kinase (AMPK) is a metabolic checkpoint which is able to negatively regulate intracellular signaling downstream of growth factors receptors in conditions increasing cellular AMP levels. We have recently reported on the role of AMPK activation in affecting viability and proliferation of GH3 cells. In the present study, we investigated the interplay between ER- and AMPK-pathways. Results can be regarded as relevant to the development of novel multi-targeted pharmacological therapies against pituitary tumors. We confirmed that estradiol (E2) and the ER antagonist fulvestrant exert stimulatory and inhibitory effects, respectively on GH3 cell growth in a competitive manner. The upstream kinase LKB1 is known to phosphorylate and activate AMPK. Here we showed that neither E2 nor fulvestrant caused a downregulation of LKB1 expression and phospho-AMPK levels in GH3 cells. Actually, fulvestrant strongly reduced the phosphorylation of ACC, which is a direct target of AMPK and a known index of AMPK activity. 2-deoxyglucose, a compound reducing glucose utilization, caused an increase in AMPK activity vs baseline and was able to hinder the stimulatory effect of E2 on cell viability, confirming that the exposure of GH3 cells to estrogens does not prevent them from being responsive to the inhibitory activity of compounds activating AMPK. Finally, the AMPK activator AICAR (AMP analog) did not cause further decrease in cell viability in the course of co-treatments with fulvestrant versus fulvestrant alone, in agreement with impaired phospho-AMPK activity in the presence of the anti-estrogen.

Effects of AMPK activation and combined treatment with AMPK activators and somatostatin on hormone secretion and cell growth in cultured GH-secreting pituitary tumor cells.

We investigated the effects of the AMPK activator AICAR as compared to somatostatin-14 on cell viability and GH secretion in human GH-secreting pituitary adenomas in vitro and in rat GH3 cells. Overnight treatment with AICAR increased phospho-(threonine-172) AMPK levels (activated AMPK) in cultured human adenomas. As to the effects on cell viability, four adenomas out of 15 were responsive to AICAR (0.4mM) and five adenomas were responsive to SS-14 (100 nM). One adenoma was responsive to both somatostatin and AICAR. The effects of cotreatment with SS-14 and AICAR were investigated in eight adenomas. In two adenomas, the effects of AICAR+SS-14 did not exceed the effect of AICAR. In two adenomas which were not responsive to either AICAR or SS-14, the cotreatment was able to reduce cell viability versus control. Two adenomas were not responsive to any treatment. As to the effects on GH secretion, nine adenomas out of 15 were responsive to AICAR. Twelve adenomas were responsive to SS-14. Eight adenomas were responsive to both AICAR and SS-14. Cotreatment exceeded the effect of single treatments in 4 out of 10 adenomas. In GH3 cells, AICAR reduced the activity of p70S6 kinase, which plays an important role in cell growth. SS-14 did not affect significantly AMPK phosphorylation and p70S6K activity but it was able to enhance the inhibitory effect of AICAR on phospho-S6 levels. Moreover, AICAR and SS-14 reduced ERK phosphorylation with a different time course. The combined treatment reduced phospho-ERK levels at any time point.

Points of integration between the intracellular energy sensor AMP-activated protein kinase (AMPK) activity and the somatotroph axis function.

AMP-activated protein kinase (AMPK), an enzyme functioning as a cellular sensor of low energy, stores and promotes adaptive changes in growth, differentiation, and metabolism. While AMPK is primarily thought of as a regulator of systemic metabolism, it has been clearly established that it also has a role in the regulation of cell growth and may be a therapeutic target for proliferative disorders. Growth hormone (GH) secretion from the anterior pituitary and GH-induced synthesis and release of insulin-like-growth-factor-1 (IGF-1) from the liver determine linear growth before puberty. Actually, GH and IGF-1 are potent growth factors affecting cell growth and differentiation in different tissues, and still have anabolic functions and serve as essential regulators of fuel metabolism in adulthood, as well. A variety of peripheral hormonal and metabolic signals regulate GH secretion either by acting directly on the anterior pituitary and/or modulating GH-releasing hormone or somatostatin release from the hypothalamus. Actually, intracellular transduction of endocrine and metabolic signals regulating somatotroph function is still debated. Based on the previously summarized contents, the aim of the present work has been to review currently available data suggesting a role of AMPK in the interplay between GH axis activity and metabolic functions.

Pharmacological characterization of the ghrelin receptor mediating its inhibitory action on inflammatory pain in rats.

Recent research suggests a role for ghrelin in the modulation of inflammatory disorders. However, the type of ghrelin receptor (GHS-R) involved in both the anti-inflammatory and anti-hyperalgesic actions of ghrelin remains to be characterized. In this study, we examined whether the inhibitory effect of ghrelin in the development of hyperalgesia and edema induced by intraplantar carrageenan administration depends on an interaction with GHS-R1a. Both central (1 nmol/rat, i.c.v.) and peripheral (40 nmol/kg, i.p.) administration of the selective GHS-R1a agonist EP1572 had no effect on carrageenan-induced hyperalgesia measured by Randall-Selitto test and paw edema. Furthermore, pre-treatment with the selective GHS-R1a antagonist, D-lys(3)-GHRP-6 (3 nmol/rat, i.c.v.) failed to prevent the anti-hyperalgesic and anti-inflammatory effects exerted by central ghrelin administration (1 nmol/rat), thus indicating that the type 1a GHS-R is not involved in these peptide activities. Accordingly, both central (1 and 2 nmol/rat, i.c.v.) and peripheral (40 and 80 nmol/kg, i.p.) administration of desacyl-ghrelin (DAG), which did not bind GHS-R1a, induced a significant reduction of the hyperalgesic and edematous activities of carrageenan. In conclusion, we have shown for the first time that DAG shares with ghrelin an inhibitory role in the development of hyperalgesia, as well as the paw edema induced by carrageenan and that a ghrelin receptor different from type 1a is involved in the anti-inflammatory activities of the peptide.

Characterization of the mechanisms involved in the gastric antisecretory effect of TLQP-21, a vgf-derived peptide, in rats.

TLQP-21, a vgf-derived peptide modulates gastric emptying and prevents ethanol-induced gastric lesions in rats. However, it remains to be studied whether or not TLQP-21 affects gastric acid secretion. In this study, we evaluated the effects of central (0.8-8 nmol/rat) or peripheral (48-240 nmol/kg, intraperitoneally) TLQP-21 administration on gastric acid secretion in pylorus-ligated rats. The mechanisms involved in such activity were also examined. Central TLQP-21 injection significantly reduced gastric acid volume and dose-dependently inhibited total acid output (ED(50) = 2.71 nmol), while peripheral TLQP-21 administration had no effect. The TLQP-21 antisecretory activity was prevented by cysteamine (300 mg/kg, subcutaneously), a depletor of somatostatin, by indomethacin (0.25 mg/rat, intracerebroventricularly), a non-selective cyclooxygenase inhibitor, and by functional ablation of sensory nerves by capsaicin. We conclude that TLQP-21 could be considered a new member of the large group of regulatory peptides affecting gastric acid secretion. The central inhibitory effect of TLQP-21 on gastric acid secretion is mediated by endogenous somatostatin and prostaglandins and requires the integrity of sensory nerve fibres.

Whey proteins as source of dipeptidyl dipeptidase IV (dipeptidyl peptidase-4) inhibitors.

Preclinical and clinical studies suggest that whey proteins can reduce postprandial glucose levels and stimulate insulin release in healthy subjects and in subjects with type 2 diabetes by reducing dipeptidyl peptidase-4 (DPP-4) activity in the proximal bowel and hence increasing intact incretin levels. Our aim was to identify DPP-4 inhibitors among short peptides occurring in hydrolysates of ß-lactoglobulin, the major whey protein found in the milk of ruminants. We proved that the bioactive peptide Ile-Pro-Ala can be regarded as a moderate DPP-4 inhibitor.

AMP-activated protein kinase regulates normal rat somatotroph cell function and growth of rat pituitary adenomatous cells.

AMP-activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP and elevate AMP levels such as glucose deprivation and hypoxia. The AMPK system is primarily thought of as a regulator of metabolism and cell proliferation. Little is known about the regulation and the effects of AMPK in somatotroph cells. We present results from "in vitro" studies showing that AMPK activity has a role in regulating somatotroph function in normal rat pituitary and is a promising target for the development of new pharmacological treatments affecting cell proliferation and viability of pituitary adenomatous cells. In parallel, we show "in vivo" data obtained in the rat suggesting that AMPK is an intracellular transducer that may play a role in mediating the effects of the pharmacological treatment with dexamethasone on somatotrophs. In rat pituitary cell cultures, the AMP analog AICAR induced a rapid and clear-cut activation of AMPK. AICAR decreased GH release and total cellular GH content. An appropriate level of AMPK activation was essential for GH3 adenomatous cells. Remarkably, over-activation by AICAR induced apoptosis of GH3 whereas the AMPK inhibitor compound C was more effective at reducing cell proliferation. The role of endocrine or paracrine factors in regulating AMPK phosphorylation and activity in GH3 cells has been also studied. As to "in vivo" studies, western blot analysis revealed a significant decrease of phosphorylated AMPK alpha-subunit in pituitary homogenates of DEX-treated rats versus controls, suggesting reduced AMPK activity. In conclusion, our studies showed that AMPK has a role in regulating somatotroph function in normal rat pituitary and proliferation of pituitary adenomatous cells.

Direct effects of casein phosphopeptides on growth and differentiation of in vitro cultured osteoblastic cells (MC3T3-E1).

Casein phosphopeptides (CPPs) obtained by enzymatic hydrolysis in vitro of caseins, have been shown to enhance calcium solubility and to increase the calcification of embryonic rat bones in their diaphyseal area. Little is known about the direct effects of CPPs on cultured osteoblastic cells. Calcium in the microenvironment surrounding bone cells is not only important for the mineralization of the extracellular matrix, but it is believed to provide preosteblasts with a signal that modulates their proliferation and differentiation. The aim of the present study was to investigate the direct effects of four selected casein phosphopeptides on osteoblastic cell (MC3T3-E1 cells) viability and differentiation. The selected peptides have been obtained by chemical synthesis and differed in the number of phosphorylated sites and in the amino acid spacing out two phosphorylated sites, in order to further characterize the relationship between structure and function. The results obtained in this work demonstrated that CPPs may directly affect osteoblast-like cell growth, calcium uptake and ultimately calcium deposition in the extracellular matrix. The effects exerted by distinct CPPs on osteogenesis in vitro can be either stimulatory or inhibitory. Differential short amino acid sequences in their molecules, like the -SpEE- and the -SpTSpEE-motifs, are likely the molecular determinants for their biological activities on osteoblastic cells. Moreover, two genetic variants of CPPs showing one amino acid change in their sequence may profoundly differ in their biological activities. Finally, our data may also suggest important clues about the role of intrinsic phosphorylated peptides derived from endogenous phosphorylated proteins in bone metabolism, apart from extrinsic CPPs.

Chronic treatment with polychlorinated biphenyls (PCB) during pregnancy and lactation in the rat: Part 1: Effects on somatic growth, growth hormone-axis activity and bone mass in the offspring.

Polychlorinated biphenyls (PCBs) are pollutants detected in animal tissues and breast milk. The experiments described in the present paper were aimed at evaluating whether the four PCB congeners most abundant in animal tissues (PCB-138, -153, -180 and -126), administered since fetal life till weaning, can induce long-term alterations of GH-axis activity and bone mass in the adult rat. We measured PCB accumulation in rat brain and liver, somatic growth, pituitary GH expression and plasma hormone concentrations at different ages. Finally, we studied hypothalamic somatostatin expression and bone structure in adulthood, following long-term PCB exposure. Dams were treated during pregnancy from GD15 to GD19 and during breast-feeding. A constant reduction of the growth rate in both male and female offspring from weaning to adulthood was observed in exposed animals. Long-lasting alterations on hypothalamic-pituitary GH axis were indeed observed in PCB-exposed rats in adulthood: increased somatostatin expression in hypothalamic periventricular nucleus (both males and females) and lateral arcuate nucleus (males, only) and decreased GH mRNA levels in the pituitary of male rats. Plasma IGF-1 levels were higher in PCB-exposed male and female animals as compared with controls at weaning and tended to be higher at PN60. Plasma testosterone and thyroid hormone concentrations were not significantly affected by exposure to PCBs. In adulthood, PCBs caused a significant reduction of bone mineral content and cortical bone thickness of tibiae in male rat joint to increased width of the epiphyseal cartilage disk. In conclusion, the developmental exposure to the four selected PCB compounds used in the present study induced far-reaching effects in the adult offspring, the male rats appearing more sensitive than females.