Biguanide metformin acts on tau phosphorylation via mTOR/protein phosphatase 2A (PP2A) signaling

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.     

Resolution of adenylate cyclase sensitive and insensitive to Ca2+ and calcium-dependent regulatory protein (CDR) by CDR-sepharose affinity chromatography.

Partially purified adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from bovine brain cortex was fractionated into two separate forms by calcium-dependent regulatory protein (CDR)-Sepharose affinity chromatography. The major form of the enzyme, comprising approximately 80% of the applied activity, did not bind to the affinity column in the presence of Ca2+ and was insensitive to the CDR. Approximately 20% of adenylate cyclase activity was absorbed to CDR-Sepharose in the presence of Ca2+. This activity was stimulated by Ca2+ and CDR. This study directly demonstrates that brain cortex contains Ca2+-CDR-sensitive and -insensitive forms of adenylate cyclase and indicates that CDR-Sepharose may be a useful tool for purification of adenylate cyclase. The Ca2+ -stimulated adenylate cyclase was purified at least 55-fold with a 13% yield.     

Developmental differences in the expression of the cholera toxin sensitive subunit (Gs alpha) of adenylate cyclase in the rat small intestine.

BACKGROUND: The stimulatory guanosine triphosphate (GTP) binding protein alpha subunit (Gs alpha) of adenylate cyclase is the target protein for cholera toxin. AIMS/METHODS: The expression of this signal transducer was analysed in the small intestine of developing rats by RNA transfer (northern blot) analysis by immunoblotting, and by ADP-ribosylation of membrane proteins. RESULTS: Intestinal Gs alpha mRNA (about 1.9 kb) was increased in the neonate compared with the adult rat. Two isoforms of Gs alpha proteins, a 45,000 and a 52,000 form, were expressed in the small intestinal epithelial cell and both were ADP-ribosylated by cholera toxin. A significant increase in the larger isoform (52,000) and in its ribosylation was noted in the 2 week old suckling compared with post-weaned older animals. The protein content or ribosylation of the smaller form (45,000) did not significantly change with age. CONCLUSION: These data show that a developmental decline of intestinal Gs alpha expression seems to be, in part, regulated at the mRNA level. An increased Gs alpha expression in the immature intestine may help to explain a previously reported, dose dependent increased adenylate cyclase response and an increase in fluid secretion to cholera toxin in neonates compared with adults.     

Characterization of a metabotropic glutamate receptor: direct negative coupling to adenylyl cyclase and involvement of a pertussis toxin-sensitive G protein.

We have characterized a G-protein-coupled glutamate receptor in primary cultures of striatal neurons. Glutamate, quisqualate, or trans-1-aminocyclopentane-1,3-dicarboxylate inhibited by 30-40% either forskolin-stimulated cAMP production in intact cells or forskolin plus vasoactive intestinal peptide-activated adenylyl cyclase assayed in neuronal membrane preparations. These inhibitory effects were suppressed after treatment of striatal neurons with Bordetella pertussis toxin, suggesting the involvement of a heterotrimeric guanine nucleotide-binding protein (G protein) of the G(i)/G(o) subtype. The pharmacological profile of this glutamate receptor negatively coupled to adenylyl cyclase was different from that of the metabotropic Qp glutamate receptor coupled to phospholipase C in striatal neurons and from that of the recently cloned "mGluR2" glutamate receptor, which is negatively coupled to adenylyl cyclase when expressed in non-neuronal cells.     

Cholecystokinin (CCK) stimulates aldosterone secretion from human adrenocortical cells via CCK2 receptors coupled to the adenylate cyclase/protein kinase A signaling cascade

Cholecystokinin (CCK) IS a regulatory peptide that acts via two receptor subtypes, CCK1-R and CCK2-R. RT-PCR demonstrated the expression of both CCK1-R and CCK2-R in the zona glomerulosa (ZG), but not zona fasciculata-reticularis cells of the human adrenal cortex. CCK and the CCK2-R agonist pentagastrin enhanced basal aldosterone secretion from ZG cells without affecting cortisol production from zona fasciculata-reticularis cells. The aldosterone response to CCK and pentagastrin was suppressed by a CCK2-R antagonist, but not by a CCK1-R antagonist. Pentagastrin evoked a sizeable cAMP, but not inositol triphosphate, response from ZG cells, whereas CCK plus CCK2-R antagonist was ineffective. The cAMP response to pentagastrin was abrogated by CCK2-R antagonist or the adenylate cyclase inhibitor SQ-22536, and the aldosterone response was abolished by both SQ-22536 and the protein kinase A inhibitor H-89. Both CCK and pentagastrin increased steroidogenic acute regulatory protein mRNA expression in ZG cells; the effect was abrogated by CCK2-R antagonist. We conclude that CCK exerts secretagogue action on human ZG cells, acting through CCK2-Rs coupled to the adenylate cyclase/protein kinase A signaling cascade, which, in turn, stimulates the expression of steroidogenic acute regulatory protein, the rate-limiting step of steroidogenesis.     

Adrenal glucocorticoid permissive regulation of muscle glycogenolysis: action on protein phosphatase(s) and its inhibitor(s).

Adrenal glucocorticoids exert a permissive action on the glycogen phosphorylase cascade. Epinephrine activation of muscle phosphorylase and phosphorylase b kinase is depressed in adrenalectomized rats. Phosphorylase phosphatase activity is increased by steroid lack, and normal epinephrine inhibition of the enzyme does not occur. Phosphorylase b kinase phosphatase activity is also increased; epinephrine, however, does not inhibit activity in muscle from normal or adrenalectomized rats. Protein phosphatase inhibitor activity is depressed in boiled dialyzed preparations made from adrenalectomized rat muscle. Cortisol resplacement therapy restores protein phosphatase inhibitor activity, decreases increased protein phosphatase activity, and restores normal epinephrine-induced activation of phosphorylase b kinase and phosphorylase and epinephrine inhibition of phosphorylase phosphatase.     

Facilitation of noradrenaline release by activation of adenosine A(2A) receptors triggers both phospholipase C and adenylate cyclase pathways in rat tail artery

OBJECTIVE: The present work is aimed at elucidating the signalling pathway(s) triggered by activation of A(2A) receptors involved in the facilitation of noradrenaline release in rat tail artery as an attempt to clarify their role in the cardiovascular system. METHODS: Electrically evoked (5 Hz, 100 pulses, 1 ms) tritium overflow was evaluated in preparations of rat tail artery, pre-incubated with [(3)H]-noradrenaline (0.1 microM), in the absence or in the presence of adenosine receptor agonists and antagonists and/or activators and inhibitors of phospholipase C (PLC)-protein kinase C (PKC) and of adenylate cyclase (AC)-cyclic adenosine-3',5'-monophosphate (cAMP)-protein kinase A (PKA) pathways. RESULTS: Activation of A(2A) receptors by 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680; 100 nM) enhanced tritium overflow, an effect prevented by the A(2A) receptor antagonist 5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo-[4,3]-1,2,4-triazolo[1,5]pyrimidine (SCH 58261; 20 nM), by the protein kinase A (PKA) inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide (H-89; 1 microM), or the PKC inhibitor 2-(8-[(dimethylamino)methyl-6,7,8,9-tetrahydropyrido[1,2]indol-3-yl]-3-(1-methylindol-3-yl)maleimide (Ro 32-0432; 1 microM). The PKC activator phorbol 12-myristate 13-acetate (PMA; 1 microM) and the PKA activator 8-bromo-cAMP (0.5 mM) also enhanced tritium overflow. The effect caused by PMA was blunted both by Ro 32-0432 and by H-89 whereas that caused by 8-bromo-cAMP was only prevented by H-89. CONCLUSIONS: In rat tail artery, the A(2A) receptor-mediated facilitation of noradrenaline release requires activation of both PKC and PKA, and PKA activation seems to occur downstream of PKC activation.     

Depletion of the catalytic subunit of protein phosphatase-2A (PP2Ac) markedly attenuates glucose-stimulated insulin secretion in pancreatic beta-cells

Among various phosphatases, the protein phosphatase 2A (PP2A) is relatively well studied in the islet. Previously, we have demonstrated that the catalytic subunit of PP2A (PP2Ac) undergoes okadaic acid (OKA)-sensitive, reversible carboxylmethylation (CML), which appears to be requisite for glucose-stimulated insulin secretion (GSIS). Using the siRNA approach, we examined, herein, the contributory roles of PP2Ac in GSIS from insulin-secreting pancreatic β-(INS-1 832/13) cells. Immunologically, PP2Ac was detectable in all the subcellular fractions studied in rank order of: cytosol > microsomes > secretory granules = nucleus > mitochondria. Transfection of PP2Ac-specific, but not scrambled-siRNA, markedly attenuated PP2A activity and GSIS in these cells. Together, our findings provide a direct evidence for a positive modulatory role for PP2Ac in signaling steps leading to GSIS.     

Structural requirements for the activation of rat anterior pituitary adenylate cyclase by growth hormone-releasing factor (GRF): discovery of (N-Ac-Tyr1, D-Arg2)-GRF(1-29)-NH2 as a GRF antagonist on membranes

The efficacy and potency of 14 GH-releasing factor (GRF) analogs, substituted in position 1 to 7, on adenylate cyclase activation in crude homogenates from rat anterior pituitary were related to those of human pancreatic GRF(1-29)-amide and vasoactive intestinal peptide. Among several D-amino acid substitutions, that in position 2 was the only one to yield a super-agonist [with a Kact (concentration required for half-maximal adenylate cyclase activation) 2 times lower than that of GRF(1-29)-NH2]. By contrast, D-isomer substitution in position 1 and 3 was without effect and D-isomer substitution in position 4, 6, or 7 decreased the affinity of the analog. The N-acetylated analog of GRF was as potent and active as the parent peptide, and the identity of the amino acid in position 2 of (N-Ac-Tyr1)-GRF(1-29)-NH2 proved to be determining for enzyme activation, with D-Phe2 and D-Trp2 derivatives acting as partial agonists and the (N-Ac-Tyr1,D-Arg2) analog being an efficient competitive antagonist of GRF(1-29)-NH2. With use of this antagonist, it was possible to demonstrate that GRF and vasoactive intestinal peptide receptors represent distinct entities in the rat anterior pituitary.     

蛋白酪氨酸磷酸酶2自身抗体微量平板放射结合检测法的建立与临床应用

目的建立蛋白酪氨酸磷酸酶2自身抗体（IA-2A）的微量平板放射结合检测法（RBA）,并初步评价其临床应用价值。方法经纯化的^35S-IA-2抗原与血清在96孔V形底平板中缓慢振荡孵育24h,而后转入已包被蛋白A的Millipore平板中,洗涤后采用多功能液体闪烁发光仪计数。检测162例1型糖尿病（T1DM）患者、210例新诊2型糖尿病（T2DM）患者和224名健康者血清中IA-2A浓度,初步评价其临床应用效果。结果（1）该法检测IA-2A批内CV4．1％～10．0％,批间CV5．7％～12．8％。（2）国际第4次糖尿病自身抗体检测标准化评估回报结果显示,其灵敏度为72％,特异度为98％。与放射配体法（RLA）结果判定一致率为96．5％,检测值呈显著正相关（r=0．962,P〈0．01）。（3）IA-2A在T1DM患者中阳性率为22．8％,明显高于健康者的0．89％（P〈0．01）;在T2DM患者中阳性率为2．4％,与健康者相比差异无统计学意义。（4）该法检测指血IA-2A与RLA法检测静脉血结果判定一致率为100％。结论微量平板RBA法灵敏度、特异度、重复性好,能适用于末梢血IA-2A检测,具有较好的临床应用价值。     

Cancerous inhibitor of PP2A (CIP2A) at diagnosis of chronic myeloid leukemia is a critical determinant of disease progression

Prospective identification of patients whose chronic myeloid leukemia (CML) will progress to blast crisis is currently not possible. PP2A is a phosphatase and tumor suppressor that regulates cell proliferation, differentiation, and survival. Cancerous inhibitor of PP2A (CIP2A) is a recently described inhibitor of PP2A in breast and gastric cancer. The aim of this study was to investigate whether CIP2A played a role in CML and whether PP2A or its inhibitor proteins CIP2A or SET could predict clinical outcome. At the time of diagnosis of CML, patients who will later progress to blast crisis have significantly higher levels of CIP2A protein (P < .0001) than patients who do not progress, suggesting that PP2A is functionally inactive. We show that the potential mechanism for disease progression is via altered phosphorylation of the oncogene c-Myc. Knockdown of CIP2A results in increased PP2A activity, decreased c-Myc levels, and a decrease in BCR-ABL1 tyrosine kinase activity. We demonstrate that CIP2A levels at diagnosis can consistently predict patients who will progress to blast crisis. The data show that CIP2A is biologically and clinically important in CML and may be a novel therapeutic target.     

Endothelin-1 (ET-1)-potentiated insulin secretion: involvement of protein kinase C and the ET(A) receptor subtype

Endothelin-1 (ET-1), a potent vasoconstrictor peptide of endothelial origin, is capable of influencing hormone secretion from endocrine tissues, eg, pancreatic islet cells. We have shown a direct stimulatory effect of ET-1 on insulin secretion from isolated mouse islets of Langerhans. However, it is unknown as to whether the peptide acts through specific receptors on the islet cells and which mechanisms are involved in this insulinotropic action. We have therefore used the specific ET(A) receptor antagonist BQ123, the ET(B) receptor agonist BQ3020, and classic alpha- and beta-adrenergic and cholinergic antagonists. ET-1 (100 nmol/L) stimulated insulin secretion from islets incubated at 8.3, 11.1, 16.7, and 25 mmol/L glucose (P < .05). At 3.3 mmol/L glucose, no alteration in insulin secretion was found. The cholinergic receptor antagonist atropine (5 micromol/L) or the adrenergic receptor antagonists propranolol (5 micromol/L) or phentolamine (5 micromol/L) did not affect ET-1 (100 nmol/L)-stimulated insulin secretion. BQ123 (10 pmol/L to 10 nmol/L) and BQ3020 (1 nmol/L to 1 micromol/L) had no effect on glucose (16.7 mmol/L)-stimulated insulin secretion, but BQ123 counteracted the stimulatory effect of ET-1 (100 nmol/L) at concentrations of 1 nmol/L to 10 micromol/L (P < .01). We also studied the relative role of protein kinase C (PKC) and a Wortmannin-sensitive pathway for ET-1-induced insulin secretion using 12-O-tetradecanoyl phorbol-13-acetate (TPA), Calphostin C, and Wortmannin, respectively. At 5.6 mmol/L glucose, ET-1 (100 nmol/L) had no effect per se, whereas in the presence of 1 micromol/L TPA, which acutely stimulates PKC, the peptide did potentiate insulin secretion (P < .05). Furthermore, the insulinotropic effect of ET-1 at 16.7 mmol/L glucose was counteracted by the PKC inhibitor Calphostin C (P < .05) and by downregulation of PKC by 24 hours of exposure of islets to TPA (0.5 micromol/L, P < .05). Wortmannin (1 micromol/L) did not alter ET-1-potentiated insulin secretion. In conclusion, our results suggest that ET-1 acts through specific ET-1 receptors, most likely the ETA subtype. Furthermore, PKC plays an essential role in the insulinotropic action of ET-1 in mouse islets.     

Pituitary adenylate cyclase polypeptide (PACAP) stimulates cyclic AMP formation in pituitary fibroblasts and 3T3 tumor fibroblasts: lack of enhancement by protein kinase C activation.

A number of neuropeptides were shown to produce potent mitogenic effects on Swiss 3T3 fibroblasts by activating the phospholipase C pathway. Here we provide evidence for the activation by PACAP of the adenylate cyclase pathway in 3T3, as well as in non-tumoral pituitary fibroblasts, similarly to what was seen in pituitary endocrine cells. In these cells, PACAP triggered elevation of both intracellular and extracellular contents of cAMP and the effect was time- and dose-dependent, with half-maximal stimulations being induced with about 0.1 nM. Following activation of protein kinase C (PKC) by the phorbol ester phorbol 12-myristate 13-acetate (PMA), PACAP-induced cAMP production was amplified in pituitary endocrine cells, but was either unchanged or dampened in 3T3 and pituitary fibroblasts, respectively. Pretreatment of cells with pertussis toxin (PT) failed to change the effect of PMA on PACAP-stimulated adenylate cyclase activity, irrespective of the cell type being used. However, PT dramatically reduced the potentiation by PMA of cAMP production enhanced by forskolin in 3T3 cells. These results provide new evidence pointing to the presence in fibroblasts of receptors for PACAP, coupled to cAMP production, which may play a role in the modulation of the mitogenic signal. They also indicate that, compared with pituitary endocrine cells, PKC activation in fibroblasts differentially affected PACAP-induced cAMP formation and that these effects were unaltered upon inhibition by PT of Gi-like proteins.     

Mice lacking phosphatase PP2A subunit PR61/B'delta (Ppp2r5d) develop spatially restricted tauopathy by deregulation of CDK5 and GSK3beta

Functional diversity of protein phosphatase 2A (PP2A) enzymes mainly results from their association with distinct regulatory subunits. To analyze the functions of one such holoenzyme in vivo, we generated mice lacking PR61/B'δ (B56δ), a subunit highly expressed in neural tissues. In PR61/B'δ-null mice the microtubule-associated protein tau becomes progressively phosphorylated at pathological epitopes in restricted brain areas, with marked immunoreactivity for the misfolded MC1-conformation but without neurofibrillary tangle formation. Behavioral tests indicated impaired sensorimotor but normal cognitive functions. These phenotypical characteristics were further underscored in PR61/B'δ-null mice mildly overexpressing human tau. PR61/B'δ-containing PP2A (PP2A(T61δ)) poorly dephosphorylates tau in vitro, arguing against a direct dephosphorylation defect. Rather, the activity of glycogen synthase kinase-3β, a major tau kinase, was found increased, with decreased phosphorylation of Ser-9, a putative cyclin-dependent kinase 5 (CDK5) target. Accordingly, CDK5 activity is decreased, and its cellular activator p35, strikingly absent in the affected brain areas. As opposed to tau, p35 is an excellent PP2A(T61δ) substrate. Our data imply a nonredundant function for PR61/B'δ in phospho-tau homeostasis via an unexpected spatially restricted mechanism preventing p35 hyperphosphorylation and its subsequent degradation.