The effects of magnesium sulfate on placental vascular endothelial growth factor expression in preeclampsia.

OBJECTIVE: To evaluate the effect of magnesium sulfate (MgSO(4)) on placental expression levels of vascular endothelial growth factor (VEGF). MATERIALS AND METHODS: Cotyledons of term normotensive and preeclamptic placentas were dually perfused for 6 h, with MgSO(4) (6-7 mg%) in the maternal reservoir [normotensive (n = 3); preeclamptic (n = 4)] and with the control medium (without MgSO(4)) [normotensive (n = 3); preeclamptic (n = 6)]. After perfusion, placental tissue samples were collected from four different placental compartments (amnion, chorion, placental villous and decidua). The collected placental tissues were homogenized and examined for VEGF by ELISA. Statistical significance was determined using a two-way analysis of variance. RESULTS: After perfusion with control medium, significantly lower levels of VEGF were detected in the chorion and placental villous compartments of preeclamptic placentas (70 ± 24 pg/g protein and 29 ± 11 pg/g protein; respectively), as compared with normotensive placentas (172 ± 80 pg/g protein and 51 ± 17 pg/g protein; respectively; p < 0.05). Exposure of preeclamptic placentas to MgSO(4) resulted in decreased VEGF levels by the amnion (57 ± 26 pg/g protein), as compared with the control group (153 ± 62 pg/g protein) (p < 0.05). On the other hand, MgSO(4) significantly increased VEGF levels by the placental villous and the decidua (58 ± 15 pg/g protein, 70 ± 29 pg/g protein; respectively), as compared with the control group (29 ± 11 pg/g protein, 33 ± 14 pg/g protein; respectively) (p < 0.01, p < 0.05; respectively). Exposure to MgSO(4) did not affect VEGF levels in normotensive placentas. CONCLUSION: Reduced levels of VEGF are expressed by some placental compartments in preeclampsia compared with normotensive pregnancy. Perfusion with MgSO(4) affects VEGF expression differently by preeclamptic and normotensive placentas. Increased production of placental VEGF in preeclampsia may play a role in the therapeutic action of MgSO(4).

Efficacy and safety of immunization with phosphorylated tau against neurofibrillary tangles in mice.

As an abnormally folded and aggregated protein, tau composed of neurofibrillary tangles (NFTs) in Alzheimer's disease and other tauopathies seems to be a candidate for immunotherapy. Yet, the encephalitogenicity of full-length tau protein, recently reported by us in immunized mice, demands to carefully and selectively target pathological tau and address both efficacy (anti-NFT effect) and safety (free of encephalitis). We immunized NFT mice with NFT-related phosphorylated (phos) tau peptides, using an immunization protocol aimed to predispose a proinflammatory milieu in CNS as a set up to detect biohazard, an approach we used when the neurotoxicity of full-length tau was detected [use of complete Freund adjuvant (CFA) with pertussis toxin (PT)]. A decrease of about 40% in NFT burden in CNS was demonstrated and was accompanied with an increase in microglial burden. Anti-phos-tau antibodies were detected in serum and blood vessels in the CNS, while no encephalitogenicity (free of clinical neurological deficits, of adverse effects on brain inflammatory cells and of axonal damage) was recorded. The level of the lysosomal proteases, cathepsins D and L, was affected in the immunized mice suggesting the possible involvement of the lysosomal system in the decrease of NFTs. The robust anti-NFT effect and the lack of encephalitogenicity in NFT mice immunized with phos-tau peptides, even though CFA with PT was included in vaccine, point to their anti-NFT therapeutic potential.

PKCepsilon mediates glucose-regulated insulin production in pancreatic beta-cells.

Endocrine cells produce large amounts of one or more peptides. The post-translational control of selective production of a single protein is often unknown. We used 3 unrelated approaches to diminish PKCepsilon in rat islets to evaluate its role in preferential glucose-mediated insulin production. Transfection with siRNA (siR-PKCepsilon) or expression of inactive PKCepsilon (PKCepsilon-KD) resulted in a significant reduction in insulin response to glucose (16.7 mmol/l). Glucose stimulation resulted in concentration of PKCepsilon in the perinuclear region, an area known to be rich in ER-Golgi systems, associated with insulin-containing structures. ss'COP1 (RACK2) is the anchoring protein for PKCepsilon. Glucose-stimulated proinsulin production was diminished by 50% in islets expressing PKCepsilon-KD, and 60% in islets expressing RACK2 binding protein (epsilonV1-2); total protein biosynthesis was not affected. In islets expressing epsilonV1-2, a chase period following glucose stimulus resulted in a reduced proinsulin conversion to mature insulin. We propose that PKCepsilon plays a specific role in mediating the glucose-signal into insulin production: binding to ss'COP1 localizes the activated enzyme to the RER where it modulates the shuttling of proinsulin to the TGN. Subsequently the enzyme may be involved in anterograde trafficking of the prohormone or in its processing within the TGN.

Dynamics of glucose-induced localization of PKC isoenzymes in pancreatic beta-cells: diabetes-related changes in the GK rat.

Glucose metabolism affects most major signal pathways in pancreatic beta-cells. Multiple protein kinases, including protein kinase C (PKC) isoenzymes, are involved in these effects; however, their role is poorly defined. Moreover, the dynamics of kinase isoenzyme activation in reference to the biphasic insulin secretion is unknown. In perfused pancreas of Wistar rats, PKCalpha staining was strongly associated with insulin staining, jointly accumulating in the vicinity of the plasma membrane during early first-phase insulin response. The signal declined before the onset of second phase and reappeared during second-phase insulin release as foci, only weekly associated with insulin staining; this signal persisted for at least 15 min after glucose stimulation. In the GK rat, glucose had minimal effect on beta-cell PKCalpha. In control beta-cells, PKCdelta stained as granulated foci with partial association with insulin staining; however, no glucose-dependent translocation was observed. In the GK rat, only minimal staining for PKCdelta was observed, increasing exclusively during early first-phase secretion. In Wistar beta-cells, PKCepsilon concentrated near the nucleus, strongly associated with insulin staining, with dynamics resembling that of biphasic insulin response, but persisting for 15 min after cessation of stimulation. In GK rats, PKCepsilon staining lacked glucose-dependent changes or association with insulin. PKCzeta exhibited bimodal dynamics in control beta-cells: during early first phase, accumulation near the cell membrane was observed, dispersing thereafter. This was followed by a gradual accumulation near the nucleus; 15 min after glucose stimulus, clear PKCzeta staining was observed within the nucleus. In the GK rat, a similar response was only occasionally observed. In control beta-cells, glucose stimulation led to a transient recruitment of PKCtheta, associated with first-phase insulin release, not seen in GK beta-cell. Data from this and related studies support a role for PKCalpha in glucose-induced insulin granule recruitment for exocytosis; a role for PKCepsilon in activation of insulin granules for exocytosis and/or in the glucose-generated time-dependent potentiation signal for insulin release; and a dual function for PKCzeta in initiating insulin release and in a regulatory role in the transcriptional machinery. Furthermore, diminished levels and/or activation of PKCalpha, PKCepsilon, PKCtheta, and PKCzeta could be part of the defective signals downstream to glucose metabolism responsible for the deranged insulin secretion in the GK rat.

New insights into fatty acid modulation of pancreatic beta-cell function.

Insulin resistance states as found in type 2 diabetes and obesity are frequently associated with hyperlipidemia. Both stimulatory and detrimental effects of free fatty acids (FFA) on pancreatic beta cells have long been recognized. Acute exposure of the pancreatic beta cell to both high glucose concentrations and saturated FFA results in a substantial increase of insulin release, whereas a chronic exposure results in desensitization and suppression of secretion. Reduction of plasma FFA levels in fasted rats or humans severely impairs glucose-induced insulin release but palmitate can augment insulin release in the presence of nonstimulatory concentrations of glucose. These results imply that changes in physiological plasma levels of FFA are important for regulation of beta-cell function. Although it is widely accepted that fatty acid (FA) metabolism (notably FA synthesis and/or formation of LC-acyl-CoA) is necessary for stimulation of insulin secretion, the key regulatory molecular mechanisms controlling the interplay between glucose and fatty acid metabolism and thus insulin secretion are not well understood but are now described in detail in this review. Indeed the correct control of switching between FA synthesis or oxidation may have critical implications for beta-cell function and integrity both in vivo and in vitro. LC-acyl-CoA (formed from either endogenously synthesized or exogenous FA) controls several aspects of beta-cell function including activation of certain types of PKC, modulation of ion channels, protein acylation, ceramide- and/or NO-mediated apoptosis, and binding to and activating nuclear transcriptional factors. The present review also describes the possible effects of FAs on insulin signaling. We have previously reported that acute exposure of islets to palmitate up-regulates some key components of the intracellular insulin signaling pathway in pancreatic islets. Another aspect considered in this review is the potential source of fatty acids for pancreatic islets in addition to supply in the blood. Lipids can be transferred from leukocytes (macrophages) to pancreatic islets in coculture. This latter process may provide an additional source of FAs that may play a significant role in the regulation of insulin secretion.

p57Kip2 (cdkn1c): sequence, splice variants and unique temporal and spatial expression pattern in the rat pancreas.

The cyclin-dependent kinase (CDK) inhibitor p57Kip2 (CDKN1C) is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities in vitro. The p57Kip2 gene is imprinted and the maternal allele is expressed in terminally differentiated cells, including human beta-cells. Somatic loss of p57Kip2 expression is associated with increased beta-cell proliferation in the focal form of Hyperinsulinism of Infancy. We cloned and sequenced the rat ortholog of p57Kip2, and demonstrate that it is highly homologous to the mouse gene. However, the human and rodent genes are quite divergent. Despite having highly homologous C- and N-terminal domains, the mid-portion of the human gene is entirely different from that of its rodent counterparts. Expression of p57Kip2 was evaluated during fetal and postnatal development, and a highly cell-specific, temporal and spatial expression profile was found. In contrast to other tissues, the expression pattern in rat pancreas was entirely opposite from that previously reported in man, with high levels of expression in rodent exocrine cells, but no expression in beta-cells during any stage of development. These findings demonstrate that p57Kip2 expression is highly regulated. In the pancreas, the functional significance of this gene appears to be quite different in humans when compared with rodents, suggesting that a better understanding of the function of this protein may provide new insights into the mechanisms involved in the control of human beta-cell mass.

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets.

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.

Palmitate acutely raises glycogen synthesis in rat soleus muscle by a mechanism that requires its metabolization (Randle cycle).

The acute effect of palmitate on glucose metabolism in rat skeletal muscle was examined. Soleus muscles from Wistar male rats were incubated in Krebs-Ringer bicarbonate buffer, for 1 h, in the absence or presence of 10 mU/ml insulin and 0, 50 or 100 microM palmitate. Palmitate increased the insulin-stimulated [(14)C]glycogen synthesis, decreased lactate production, and did not alter D-[U-(14)C]glucose decarboxylation and 2-deoxy-D-[2,6-(3)H]glucose uptake. This fatty acid decreased the conversion of pyruvate to lactate and [1-(14)C]pyruvate decarboxylation and increased (14)CO(2) produced from [2-(14)C]pyruvate. Palmitate reduced insulin-stimulated phosphorylation of insulin receptor substrate-1/2, Akt, and p44/42 mitogen-activated protein kinases. Bromopalmitate, a non-metabolizable analogue of palmitate, reduced [(14)C]glycogen synthesis. A strong correlation was found between [U-(14)C]palmitate decarboxylation and [(14)C]glycogen synthesis (r=0.99). Also, palmitate increased intracellular content of glucose 6-phosphate in the presence of insulin. These results led us to postulate that palmitate acutely potentiates insulin-stimulated glycogen synthesis by a mechanism that requires its metabolization (Randle cycle). The inhibitory effect of palmitate on insulin-stimulated protein phosphorylation might play an important role for the development of insulin resistance in conditions of chronic exposure to high levels of fatty acids.

Melatonin inhibits insulin secretion and decreases PKA levels without interfering with glucose metabolism in rat pancreatic islets.

The effect of melatonin (0.1 microM) on freshly isolated islets from adult rats was investigated. Melatonin caused a marked decrease of insulin secretion by islets in response to glucose. The mechanism involved was then examined. Melatonin did not interfere with glucose metabolism as indicated by the measurement of glucose oxidation. However, the content of the protein kinase A (PKA) catalytic alpha-subunit was significantly decreased in islets exposed to melatonin for 1 hr in the presence of 8.3 mM glucose, whereas that of the protein kinase C (PKC) alpha-subunit remained unchanged. Melatonin also inhibited forskolin-induced insulin secretion, a well known activator of adenylate cyclase (AC) activity. This may explain the low content of insulin found in islets incubated in the presence of melatonin for 3 hr. In fact, 3',5' -cyclic adenosine monophosphate (cAMP), a product of AC activity, stimulates insulin synthesis. These findings led us to postulate that a down-regulation of the PKA signaling pathway may be the mechanism involved in the melatonin inhibition of the process of glucose-induced insulin secretion.

Daily rhythm of glucose-induced insulin secretion by isolated islets from intact and pinealectomized rat.

It is well known that pinealectomy induces in rats a diminished glucose tolerance, insulin resistance, a reduction in GLUT4 content in adipose and muscular tissues, a decrease in hepatic and muscular glycogenesis, impairment of glucagon action and an increase in blood pyruvate concentration. In addition, it has been shown that melatonin suppresses insulin secretion in several experimental conditions. The objective of the present study was to investigate the daily rhythm of glucose-induced insulin secretion and glucose oxidation by isolated pancreatic islets and to investigate the effect of chronic absence of melatonin (30 days of pinealectomy) on this rhythmic process. The data obtained confirmed the presence of a strong 24-hr rhythm of insulin secretion by isolated pancreatic islets. In addition, it was demonstrated that the glucose-metabolizing ability of the B-cell follows a daily rhythm phase locked to insulin secretion rhythm. Most interesting, however, was the demonstration that the daily rhythmic processes of insulin secretion and B-cell -[U-14C]-glucose oxidation by isolated pancreatic islets is completely modified by the chronic absence of the pineal gland. Thus, pinealectomy induced in all groups an increase in 24-hr mean glucose-stimulated insulin secretion and [U-14C]-glucose oxidation, in addition to some alterations in the rhythmic amplitude and a remarkable phase-advancing of the daily curves for 8.3 mm glucose (a condition similar to that observed in fed animals and where the B-cells are supposedly more active). These observations strongly suggest that the presence of the pineal gland may be necessary for the proper synchronization of these metabolic rhythms with other circadian rhythms like activity-rest and feeding.

Secreçäo da insulina: efeito autócrito da insulina e modulaçäo por ácidos graxos / Insulin secretion: insulin autocrinous effect and modulation by fatty acids

A insulina exerce um papel central na regulação da homeostase da glicose e atua de maneira coordenada em eventos celulares que regulam os efeitos metabólicos e de crescimento. A sub-unidade 13 do receptor de insulina possui atividade tirosina quinase intrínseca. A autofosforilação do receptor, induzida pela insulina, resulta na fosforilação de substratos protéicos intracelulares, como o substrato-l do receptor de insulina (IRS-1). O IRS-1 fosforilado associa-se a domínios SH2 e SH3 da enzima PI 3-quinase, transmitindo, desta maneira, o sinal insulínico. A insulina parece exercer feedback positivo na sua secreção, pela interação com seu receptor em células B pancreáticas. Alterações nos mecanismos moleculares da via de sinalização insulínica sugerem uma associação entre resistência à insulina e diminuição da secreção deste hormônio, semelhante ao observado em diabetes mellitus tipo 2. Uma das anormalidades associadas à resistência à insulina é a hiperlipidemia. O aumento do pool de ácidos graxos livres circulantes pode modular a atividade de enzimas e de proteínas que participam na exocitose da insulina. Essa revisão descreve também os possíveis mecanismos de modulação da secreção de insulina pelos ácidos graxos em ilhotas pancreáticas.

Bioensaio de hormônio de crescimento humano por incremento de peso de ratos hipofisectomizados: análise computadorizada de esquemas fatoriais 2x2 / Bioassay of human growth hormone by the increase of body weight of hypophysectomized rats: computerized analysis of factorial 2x2 schedules

O bioensaio de hormônio de crescimento humano por incremento de peso corpóreo de ratos hipofisectomizados tem sido estudado com enfoque especial na economia de tempo e material, além de melhorar ou ao menos manter a qualidade dos parâmetros estatísticos do ensaio fatorial 2x2 clássico, que usa doses de 20 e 100microng/rato/dia. A determinaçäo da atividade biológica e dos parâmetros estatísticos relativos foi obtida através de um programa de computaçäo BASSY, em linguagem FORTRAN IV, descrito para a análise de ensaio fatorial 2x2. Com uma sensibilidade calculada da ordem de 2migrong/rato/dia, o ensaio pode ainda ser realizado em condiçöes de maior economia usando-se apenas um total de 0,6mg de hGH, com índice de precisäo inferior a 0,3. O presente estudo também mostra a excelente estabilidade das preparaçöes padräo (WHO) e Ipen ampolizadas e a precisäo interensaio relativamente alta deste bioensaio in vivo: CV = 8,7 e 18,9% respectivamente, usando-se o mesmo (n = 3) e diferentes (n = 6) esquemas de ensaio em um período superior a um ano