Discrimination between neuronal and glial cell tumours by pyruvate kinase electrophoresis

Summary Pyruvate kinase is a tetrameric enzyme, of which different isozymes are known (M, K and L). In normal human brain of adults mainly the M₄ type is found with little or none of type K₄ or the hybrid K₃M. In gliomas of adults as well as in children a shift in the synthesis of isozymes of pyruvate kinase occurs from the M-towards the K-type. In these tumours the hybrid K₂M₂ is not expressed. In gliomas of children only a spur of K₂M₂ is found. There exists a peculiar difference in isozyme distribution of pyruvate kinase between gliomas on the one hand and neuro-, retino- and medulloblastomas on the other. In the latter group of tumours often the whole isozyme set is observed and in all cases the hybrids K₂M₂ and KM₃ are found. These findings provide enzymological criteria for the discrimination of neuronal tumours from glial-cell tumours.     

The pyruvate kinase isoenzyme shift in human gliomas: a potential marker in the treatment of gliomas

In primary human brain tumours a shift occurs in the synthesis of isoenzymes of pyruvate kinase from the M towards the K-type. In astrocytomas, oligodendrogliomas and glioblastomas, which were localised in the cerebral hemispheres of adult patients over 20 years of age, the shift correlated well with histological grading and growth rate as observed in postoperative survival. Gliomas of adults, localised in midline structures, as well as childrens gliomas were characterised too by a strong shift from M towards the K type. However, in these tumours, less correlation with histological grading and growth rate was found. The isoenzyme shift can be rapidly demonstrated with an alanine inhibition test. The application of this assay may have a diagnostic value during operation for gliomas in grading of malignancy in adults as well as demarcation of the resection of gliomas in all age groups. The test can be performed within 10-15 min and can thus fit easily into a surgical procedure. A case report is presented for illustration.     

Oedema formation in experimental photo-irradiation therapy of brain tumours using 5-ALA

Summary Background. Five-aminolevulinic acid (5-ALA) induces the specific accumulation of photosensitising porphyrins in malignant gliomas and has been explored for photo-irradiation therapy of these tumours. However, information is unavailable on whether and to what extent this treatment modality may induce the formation of brain oedema, and how potential oedema might be treated.Methods. Rats were implanted with C6 gliomas. Eight days later magnetic resonance images (MRI) were obtained. On day 9 rats received 100mg 5-ALA/kg b.w. and were craniotomized for photo-irradiation of tumours 6 hours later (100J/cm², 635nm argon-dye laser). Part of the animals was treated with daily dexamethasone injections (0.3mg/kg), beginning 6 hours before phototherapy. 72 hours later, brains were removed and dissected according to tumour dimensions on pre-therapy MRI into tumour, brain around tumour (BAT), residual cortex and basal ganglia, for measurements of water contents. Measurements were also performed in untreated animals with tumours, with or without steroid treatment and in control animals. An additional group of animals lacking tumours, with or without steroid treatment, underwent 5-ALA-phototherapy to determine effects on normal brain.Results. C6 gliomas induced brain oedema, which responded to steroid treatment. 5-ALA-phototherapy resulted in additional oedema, which responded partly to steroids. 5-ALA-phototherapy of normal brain increased water content moderately in irradiated cortex. This oedema was also partly counteracted by steroids.Conclusions. Photo-irradiation therapy with 5-ALA induces oedema which is partly counteracted by steroid therapy. The possibility of steroid resistant oedema formation should be considered when planning human trials with this treatment modality.     

Effects of vitamin D and retinoic acid on human glioblastoma cell lines

Summary The biological significance of vitamin D receptors expressed by glioblastoma and other glial tumours is still unclear. In an effort to clarify this issue we studied the effects of increasing concentrations of 25-dihydroxyvitamin D₃ and its metabolite 1 , 25-dihydroxyvitamin D₃ on two human glioblastoma cell lines. Both substances were capable of inducing a significant (> 50%) reduction in growth of the two glioblastoma cell lines at dosages over 5 M. When the HU 70 cell line was treated by increasing dilutions of 25-dihydroxyvitamin D₃ combined with 1 M all trans-retinoic acid, significant inhibition was apparent even after addition of 25-dihydroxyvitamin D³ in the nanomolar range. Reduction of growth index was mainly due to induced cell death.Our results providein vitro evidence that vitamin D metabolites alone or in combination with retinoids may be potentially useful agents in the differentiation therapy of human malignant gliomas.     

Cytochemical changes in lactate dehydrogenase isoenzymes in human brain tumours

Summary The lactate dehydrogenase (LDH) isoenzyme patterns in benign and malignant brain tumours were determined by means of electrophoresis of the cell extracts and selective cytochemical stain of the smears.The LDH isoenzyme distribution of the cell extracts showed a pronounced cathodal shift in the malignant gliomas and metastatic carcinomas. Normal brain tissues and histologically benign gliomas, however, showed an anodal pattern with a dominance of the H-type LDH. Schwannomas and meningiomas had a midzone isoenzyme pattern with a dominant LDH₃ fraction. Pituitary adenomas usually showed the LDH pattern similar to that of the normal cerebrum.The LDH M fraction could be cytochemically verified using an inhibitory effect by 2.6 M urea in staining. Astrocytomas grades 3–4 and metastatic carcinomas were characterized by loss or marked reduction of stainability by urea treatment, while astrocytomas grades 1–2 and oligodendrogliomas were resistant to urea inhibition.     

Increase in ω3 (Peripheral-Type Benzodiazepine) binding site densities in different types of human brain tumours

Summary The density of ₃ (peripheral type benzodiazepine) binding sites, a marker of reactive and tumoural cells, has been measured in different types of human brain tumours; ₃ sites were quantified autoradiographically in sections from biopsy or autopsy specimens labelled with the specific radioligand³H-PK 11195. Compared to normal brain parenchyma, up to 12-fold increase in ₃ site densities were found in appparently viable areas of high grade astrocytoma and glioblastoma specimens, whereas more limited increases (2 to 3-fold) in this marker were observed in areas of necrosis. Low grade gliomas (astrocytomas) and meningiomas exhibited only moderate increases (2 to 3-fold) in this autoradiographic marker. Metastases of lung or kidney origin were characterized by greatly elevated (up to 20-fold) ₃ site densities as compared to normal brain parenchyma. In every case, there was a good spatial correspondence between the histopathological limits of the tumour and the anatomical location of the increase in ₃ site densities. These results suggest that ₃ site densities in human brain tumours reflect their proliferative activity and point to a possible future usefulness of positron or gamma-ray emitting ₃ site ligands for the clinical investigation and detection of human brain proliferative diseases.     

The effects of malignant glioma on the EEG and seizure thresholds: An experimental study

Summary Generalised or partial seizures are a common problem with many supratentorial gliomas. Their underlying pathophysiological mechanisms are poorly understood. To investigate this problem clinical and EEG seizure thresholds were investigated in experimental rodent gliomas using the epileptogenic drug pentylenetetrazole (PTZ). Mixed C6/A15A5 malignant gliomas were grown in adult Wistar rats after unilateral stereotactic implantation of a 50 50 cell mix into the caudoputaminal region. Eleven to 14 days later EEG (raw and spectrally analysed) was recorded bilaterally from the frontal and parietal regions under mixed -chloralose and urethane anaesthesia. Baseline EEG (15 minutes), EEG during and after (30 minutes) PTZ infusion (100 l/min) and the time to appearance of seizure manifestations after starting PTZ were recorded. Fourteen animals were studied (5 normal, 5 with tumours, 4 sham implants) and mean BP, PaCO₂, PaO₂ and temperature were similar in the three groups. Baseline raw EEG showed predominate slow wave activity with lower amplitude and less spontaneous activity overlying tumours. Following PTZ infusion a sequence of vibrissal twitching (following a mean of 14.5 mg/kg PTZ in control and sham animals); jaw/nasal twitches (17.5 mg/kg); fore and hind limb jerking (46 mg/kg); myoclonic jerking (47 mg/kg); and status (77.5 mg/kg) was observed. The seizure thresholds for all PTZ induced seizure phenomena were, except for status epilepticus, highest in the tumour bearing animals. The time to 70% seizure activity on the EEG was also significantly longer in the tumour bearing animals. Spectral analysis of the EEG, although showing increased alpha and theta activity after PTZ infusion, did not discriminate between the three experimental groups either before or after PTZ activation. These studies have confirmed that experimental gliomas alter baseline EEG and both the EEG and behavioural response to PTZ. The reasons for the raised seizure threshold in the glioma bearing animals and the relevance of this experimental paradigm to human tumour associated epilepsy are discussed.     

Potential of O6-methylguanine or O6-benzylguanine in the enhancement of chloroethylnitrosourea cytotoxicity on brain tumours

Summary The purine analogues O⁶-methylguanine and O⁶-benzylguanine are well-known as a chemical modulator of the DNA repair enzyme O⁶-methylguanine-DNA methyltransferase. Inactivation of the enzyme by O⁶-methylguanine or O⁶-benzylguanine is expected to enhance sensitivity of tumours to chloroethylnitrosoureas.We studied the effect of O⁶-methylguanine or O⁶-benzylguanine pretreatment on cytotoxity of 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (ACNU) in brain tumour cells and transplanted brain tumours. Two-hour exposure of O⁶-methylguanine at higher concentrations (500 M, 1,000 M) increased ACNU cytotoxicity by only 2 times in ACNU-resistant C6-1 brain tumour cells. O⁶-Benzylguanine at concentrations between 10 and 100 M markedly enhanced the cytotoxic effecct. The ACNU sensitivity of the tumour cels pretreated with O⁶-benzylguanine was 5–40 times that of the cells without O⁶-benzylguanine. Neither O⁶-methylguanine nor O⁶-benzylguamne appreciably enhanced ACNU cytotoxicity of 9 L cells, which were origininally sensitive to ACNU. Intracarotid ACNU with O⁶-methylguanine or O⁶-benzylguanine decreased proliferating activity of transplanted C6-1 brain tumours significantly during 48 hours. O⁶-Benzylguanine pretreatment resulted in a greater degree of suppression for a long time. The C6-1 tumours treated only with intracarotid ACNU showed a transient inhibition and a rapid regrowth during 24 hours after the treatment.These results indicate that O⁶-methylguanine or O⁶-benzylguanine increases ACNU cytotoxicity and may be feasible for effective combination therapy with chloroethylnitrosourea in the chemotherapy of malignant brain tumours.     

Resorption of peritumoural oedema in cerebral gliomas during dexamethasone treatment evaluated by NMR relaxation time imaging

Summary Peritumoural brain oedema is a prominent feature of malignant brain tumours. Glucocorticoids diminish the neurological symptoms and signs caused by the oedema and reduce the abnormally high cerebral water content. The exact mechanisms of action of the glucocorticoids are unknown.The present study investigates the influence of dexamethasone on NMR relaxation time T₁ in peritumoural oedema in 13 patients with gliomas. It is shown that NMR T₁ images can be used as a potent monitor of brain oedema, and that dexamethasone significantly reduces mean T₁ after 1, 3, and 7 days of treatment by 2%, 6%, and 13% respectively.Using an image histogram analysis technique the term superoedema was defined as the 50% of the total oedema area with the highest t₁, corresponding to the highest water content. It is shown, that with this technique the treatment effect of steroids on superoedema was a reduction of 13%, 33%, and 57% after 1, 3, and 7 days of treatment respectively. The mean change after 24 hours of treatment was statistically significant (p < 0.01).The method can be used in all situations where the anti-oedematous effect of a given treatment is to be monitored.     

Linkage between O6-methylguanine-DNA methyltransferase (O6-MT) activity and cellular resistance to antitumour nitrosoureas in cultured rat brain tumour cell strains

Summary We have examined O⁶-methylguanine-DNA methyltransferase (O⁶-MT) activity of rat brain tumour cell strains with reference to cellular resistance to antitumour nitrosoureas, 1-(4-amino-2-methyl-5-pyrimidinyl) methyl-3-(2-chloroethyl)-3-nitrosourea hydrochloride (nimustine, ACNU) and methyl-6-[3-(2-chloroethyl)-3-nitrosoureido]-6-deoxy--D-glucopyranoside (ramustine, MCNU). The values of O⁶-MT activity were 52 and 160 fmol/mg protein extract in 9 L and C 6 rat brain tumour cells, respectively; while HeLa S 3 cells, as a methyl excision repair positive (Mer⁺) cell strain, revealed a rather high value of 488 fmol/mg. 9 L cells indicative of a low O⁶-MT activity showed 13 M for ACNU and 18 M for MCNU at a 10% survival dose (SD₁₀), determined by a clonogenic cell assay as an index of cellular resistance. In contrast to this, C 6 cells revealed a SD₁₀ value of 67 M and 36 M for ACNU and MCNU, respectively, indicating higher resistance than 9 L cells. HeLa S 3 cells showed the highest SD₁₀ value as follows: 84 M for ACNU and 73 M for MCNU. The relationship between the O⁶-MT activity and the cellular resistance was almost linear, with relatively resistant cell lines exhibiting the higher levels of the O⁶-MT activity. This correlation between the O⁶-MT activity and the cellular resistance to nitrosoureas as ACNU and MCNU was not observed among other antitumour drugs, which included bleomycin (BUM), neocarzinostatin (NCS),cis-diamminedichloroplatinum (II) (CDDP), and etoposide (VP-16) in clinical use for brain tumour chemotherapy. This indicates that O⁶-MT activity can be an indicator of cellular resistance to antitumour nitrosoureas in the chemotherapy of brain tumours.     

Congenital hyperinsulinism and glucose hypersensitivity in homozygous and heterozygous carriers of Kir6.2 (KCNJ11) mutation V290M mutation: K(ATP) channel inactivation mechanism and clinical management

OBJECTIVE

The ATP-sensitive K⁺ channel (K_ATP) controls insulin secretion from the islet. Gain- or loss-of-function mutations in channel subunits underlie human neonatal diabetes and congenital hyperinsulinism (HI), respectively. In this study, we sought to identify the mechanistic basis of K_ATP-induced HI in two probands and to characterize the clinical course.

RESEARCH DESIGN AND METHODS

We analyzed HI in two probands and characterized the course of clinical treatment in each, as well as properties of mutant K_ATP channels expressed in COSm6 cells using Rb efflux and patch-clamp methods.

RESULTS

We identified mutation V290M in the pore-forming Kir6.2 subunit in each proband. In vitro expression in COSm6 cells supports the mutation resulting in an inactivating phenotype, which leads to significantly reduced activity in intact cells when expressed homomerically, and to a lesser extent when expressed heteromerically with wild-type subunits. In one heterozygous proband, a fluoro-DOPA scan revealed a causal focal lesion, indicating uniparental disomy with loss of heterozygosity. In a second family, the proband, homozygous for the mutation, was diagnosed with severe diazoxide–unresponsive hypersinsulinism at 2 weeks of age. The patient continues to be treated successfully with octreotide and amlodipine. The parents and a male sibling are heterozygous carriers without overt clinical HI. Interestingly, both the mother and the sibling exhibit evidence of abnormally enhanced glucose tolerance.

CONCLUSIONS

V290M results in inactivating K_ATP channels that underlie HI. Homozygous individuals may be managed medically, without pancreatectomy. Heterozygous carriers also show evidence of enhanced glucose sensitivity, consistent with incomplete loss of K_ATP channel activity.The ATP-sensitive K⁺ channel (K_ATP) regulates insulin secretion from the pancreatic β-cell by coupling changes in metabolism to changes in electrical activity. K_ATP overactivity suppresses insulin release and causes neonatal diabetes (1,2), whereas K_ATP underactivity causes hypersecretion and congenital hyperinsulinemia (HI) (3–5).HI mutations can cause aberrant channel synthesis or trafficking or altered channel gating (5,6). Mature K_ATP channels are hetero-octomers of four pore-forming Kir6.2 subunits (KCNJ11) and four sulfonylurea receptor subunits (ABCC8) (7–9). We report a novel Kir6.2 mutation (V290M), identified in two unrelated HI probands. V290M reduces channel activity by causing an inactivating phenotype, which explains the HI outcome. Importantly, the V290M mutation is present in the homozygous state in one of the HI-affected probands and is heterozygous in the unaffected parents and one sibling. Oral glucose tolerance tests (OGTTs) on the heterozygous mother and sibling suggest hyper-responsivity in both individuals.     

Glucose Deprivation Regulates KATP Channel Trafficking via AMP-Activated Protein Kinase in Pancreatic ��-Cells

OBJECTIVE

AMP-activated protein kinase (AMPK) and the ATP-sensitive K⁺ (K_ATP) channel are metabolic sensors that become activated during metabolic stress. AMPK is an important regulator of metabolism, whereas the K_ATP channel is a regulator of cellular excitability. Cross talk between these systems is poorly understood.

RESEARCH DESIGN AND METHODS

Rat pancreatic β-cells or INS-1 cells were pretreated for 2 h at various concentrations of glucose. Maximum K_ATP conductance (G_max) was monitored by whole-cell measurements after intracellular ATP washout using ATP-free internal solutions. K_ATP channel activity (NPo) was monitored by inside-out patch recordings in the presence of diazoxide. Distributions of K_ATP channel proteins (Kir6.2 and SUR1) were examined using immunofluorescence imaging and surface biotinylation studies. Insulin secretion from rat pancreatic islets was measured using an enzyme immunoassay.

RESULTS

G_max and NPo in cells pretreated with glucose-free or 3 mmol/l glucose solutions were significantly higher than in cells pretreated in 11.1 mmol/l glucose solutions. Immunofluorescence imaging and biotinylation studies revealed that glucose deprivation induced an increase in the surface level of Kir6.2 without affecting the total cellular amount. Increases in G_max and the surface level of Kir6.2 were inhibited by compound C, an AMPK inhibitor, and siAMPK transfection. The effects of glucose deprivation on K_ATP channels were mimicked by an AMPK activator. Glucose deprivation reduced insulin secretion, but this response was attenuated by compound C.

CONCLUSIONS

K_ATP channel trafficking is regulated by energy status via AMPK, and this mechanism may play a key role in inhibiting insulin secretion under low energy status.ATP-sensitive K⁺ (K_ATP) channels are metabolic sensors that couple cellular energy status to electrical activity and play key roles in energy-dependent insulin secretion in pancreatic β-cells (1). The molecular mechanisms underlying the regulation of K_ATP channel activity have been investigated extensively. Adenine nucleotides are well known to induce K_ATP channel closure by binding to the pore-forming subunit Kir6.2 (2), yet activate channel opening by interacting with the regulatory subunit SUR in a Mg²⁺-dependent manner (3,4). Therefore, energy-dependent regulations of K_ATP currents are believed to be because of the direct effects of these nucleotides on K_ATP channel gating. However, the total conductance of an ion channel is determined not only by open probabilities but also by the available channel numbers. Our work addresses the latter, focusing on whether K_ATP channel numbers at the surface membrane can be regulated by cellular energy status.The importance of the trafficking mechanism for K_ATP channels was first recognized in studies on mutant channels involved in insulin secretion disorders. For some mutations causing congenital hyperinsulinism the forward trafficking is impaired (5,6), whereas mutations that affect the signaling motif responsible for endocytic trafficking cause neonatal diabetes (7). The trafficking of normal K_ATP channels has been reported to be regulated in several recent studies. High-glucose conditions have led to the recruitment of K_ATP channels to the β-cell plasma membrane in a Ca²⁺ and PKA-dependent manner, resulting in an increase in K_ATP currents (8), whereas a protein kinase C activator facilitated endocytic trafficking of K_ATP, resulting in decreased K_ATP currents (9). These studies suggest that regulation of the surface density of K_ATP channels is a dynamic process involving various steps of trafficking and that each step is subject to regulation by various cellular signaling mechanisms. However, the involvement of energy-dependent signaling mechanisms in the regulation of K_ATP channel trafficking has not been fully studied.AMP-activated protein kinase (AMPK) is an evolutionarily conserved metabolic sensor that is activated under conditions of energy deficiency and plays key roles as a regulator of energy metabolism (10). Recent studies have found that AMPK also plays important roles in coupling membrane transport to cellular metabolism (11). AMPK has been shown to upregulate glucose transporters and fatty acid translocase (12) but downregulate ion-transport proteins such as cystic fibrosis transmembrane conductance regulator (CFTR) Cl^– channels (13) and epithelial Na⁺ channels (14). Although the mechanisms involved in these effects are not fully understood, AMPK-dependent downregulation of CFTR has been shown to be associated with decreased CFTR surface expression in colonic epithelium (15), whereas AMPK increases GLUT4 translocation to the sarcolemma in skeletal and cardiac muscle (16,17). These results may suggest that AMPK regulates the mechanisms involved in the trafficking of surface proteins.Pancreatic β-cells are a key player in the regulation of whole-body energy balance. They are specialized to synthesize and secrete insulin, a key anabolic hormone of the body. Insulin secretion is controlled tightly by blood glucose concentration, and the ability of the K_ATP channel to couple its activity to cellular energy status is generally believed to be responsible for glucose-dependent insulin secretion. AMPK activity is also controlled by glucose concentration in insulin-secreting cells (18), but little is known about the roles of AMPK in pancreatic β-cells. In the present study, we investigated whether AMPK activation contributes to the activation of K_ATP channels in pancreatic β-cells and INS-1 cells. We found that the activation of AMPK by glucose deprivation induces an increase in the surface levels of K_ATP channels, and this increase contributes to the increased K_ATP conductance.     

Mechanisms of Action of the Gasotransmitter Hydrogen Sulfide in Modulating Contractile Activity of Longitudinal Muscle of Rat Ileum

Aim

This study aims to determine mechanisms of action of the gasotransmitter hydrogen sulfide (H₂S) on contractile activity in longitudinal muscle of rat ileum.

Methods

Ileal longitudinal muscle strips were prepared to measure isometric contractions. Effects of sodium hydrosulfide (NaHS), a donor of H₂S, were evaluated on spontaneous contractile activity and after enhanced contractile activity with bethanechol. l-cysteine was evaluated as a potential endogenous donor of H₂S. We evaluated involvement of extrinsic nerves, enteric nervous system, visceral afferent nerves, nitric oxide, and K _ATP ⁺ channel and K _Ca ⁺ channel activity on the action of H₂S using non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, l-N^G-nitro arginine (l-NNA), glibenclamide, and apamin, respectively, as well as electrical field stimulation.

Result

NaHS dose-dependently and reversibly inhibited spontaneous and bethanechol-stimulated contractile activity (p?<?0.05). l-cysteine had no inhibitory effect. Non-adrenergic/non-cholinergic conditions, tetrodotoxin, capsaicin, l-NNA, glibenclamide, or apamin had no major effect on total contractile activity by NaHS, although both tetrodotoxin and apamin decreased the frequency of bethanechol-enhanced contractile activity (p?<?0.05). We could not demonstrate H₂S release by electrical field stimulation but did show that inhibition of cystathionine ?? synthase, an endogenous source of H₂S, augmented the inhibitory effect of low-frequency electrical field stimulation.

Conclusion

H₂S inhibits contractile activity of ileal longitudinal muscle dose-dependently but not through pathways mediated by the extrinsic or enteric nervous system, visceral afferent nerves, nitric oxide, K _ATP ⁺ channels, or K _Ca ⁺ channels.     

Deoxythymidine-kinase in cerebrospinal fluid: A new potential “marker” for brain tumours

Summary A recently developed method for deoxythymidine kinase (dTK) determination was applied to cerebrospinal fluid (CSF) and serum samples, derived from healthy individuals and from patients with non-neoplastic or neoplastic disorders of the brain. No dTK activity could be detected in the CSF of healthy individuals or in patients with hydrocephalus or cranio-cerebral trauma. dTK levels ranging from detectable to high were found in the CSF of patients with malignant primary brain tumours or secondary brain tumours. The highest values were found in the patients with the most rapidly growing tumours, while in some cases of low grade primary brain tumours dTK could not be detected in the CSF.CSF samples taken before and after treatment were available both in some patients with CNS involvement of malignant lymphomas and in some with primary brain tumours. The dTK activity rapidly declined after chemotherapy intrathecally and irradiation, as well as after surgery.Enhanced CSF dTK was not only a feature of malignant CNS processes, but was also found in patients with cerebral haemorrhage penetrating into the CSF. The origin of dTK in CSF and the practical use of quantifying it is discussed.     

Altered MAPK Signaling in Progressive Deterioration of Endothelial Function in Diabetic Mice

We aimed to investigate specific roles of mitogen-activated protein kinases (MAPK) in the deterioration of endothelial function during the progression of diabetes and the potential therapeutic effects of MAPK inhibitors and agonists in the amelioration of endothelial function. Protein expression and phosphorylation of p38, c-Jun NH₂-terminal kinase (JNK), and extracellular signal–regulated kinase (Erk) were assessed in mesenteric arteries of 3- (3M) and 9-month-old (9M) male diabetic and control mice. The expression of p38, JNK, and Erk was comparable in all groups of mice, but the phosphorylation of p38 and JNK was increased in 3M and further increased in 9M diabetic mice, whereas the phosphorylation of Erk was substantially reduced in 9M diabetic mice. NADPH oxidase–dependent superoxide production was significantly increased in vessels of two ages of diabetic mice. Inhibition of either p38 with SB203580 or JNK with SP600125 reduced superoxide production and improved shear stress–induced dilation (SSID) in 3M, but not in 9M, diabetic mice. Treating the vessels of 9M diabetic mice with resveratrol increased Erk phosphorylation and shear stress–induced endothelial nitric oxide synthase (eNOS) phosphorylation and activity, but resveratrol alone did not improve SSID. Administration of resveratrol and SB203580 or resveratrol and SP600125 together significantly improved SSID in vessels of 9M diabetic mice. The improved response was prevented by U0126, an Erk inhibitor. Thus, p38/JNK-dependent increase in oxidative stress diminished nitric oxide–mediated dilation in vessels of 3M diabetic mice. Oxidative stress and impaired Erk-dependent activation of eNOS exacerbates endothelial dysfunction in the advanced stage of diabetes.Diabetes is associated with various cardiovascular complications. In particular, the increased oxidative stress, which inactivates NO and hence impairs endothelium-dependent vasodilator responses and induces the dysfunctionality of endothelial progenitor cells (1–3), contributes significantly to the cardiovascular dysfunction in diabetes. We also demonstrated that inhibition of superoxide production improved endothelium-dependent shear stress–induced dilation (SSID) in arteries of young diabetic mice. In aged diabetic mice, however, impaired endothelial nitric oxide (NO) synthase (eNOS) activation prevented the antioxidative effect on ameliorating endothelial function (4). Thus, oxidative stress and impaired eNOS activation are two separate but mechanistically connected events, especially during the cardiovascular complications in late stages of diabetes.Among the family of mitogen-activated protein kinase (MAPK), p38 kinase (p38) and c-Jun NH₂-terminal kinase (JNK) are activated in response to hyperglycemia, oxidative stress, and proinflammtory cytokines. Increased activation of p38 and JNK has become a fundamental mechanism responsible for cardiovascular dysfunction in diabetes (5,6). Indeed, inhibition of p38/JNK improved nitric oxide–mediated vasodilatation and reduced inflammation in hypercholesterolemic patients (7) and prevented tumor necrosis factor-α (TNF-α)– and hypercholesterolemia-induced endothelial dysfunction (8,9). On the other hand, extracellular signal–regulated kinase (Erk), another member of the MAPK family, is mainly involved in regulating mitogen-induced cellular growth. Understanding of the specific role of Erk in endothelial dysfunction of diabetes remains incomplete, although some studies have suggested that the activation of Erk is increased in cultured endothelial cells isolated from subcutaneous tissues of type 2 diabetic subjects (10). However, in normal vascular endothelium, fluid shear stress quickly activates Erk-related signaling pathways (11,12), implying that Erk activation involves shear stress–induced regulation of endothelial function. Moreover, insulin and proinsulin C-peptide–induced eNOS activation are linked to the activation of Erk (13,14); and the cardiovascular protective effects of estrogen and estrogen receptor agonists are mediated through Erk-dependent mechanisms (15). Thus, the physiological activation of Erk is important for maintaining cardiovascular homeostasis. Despite the fact that the importance of MAPK in the regulation of vascular function has been described, changes in function of MAPK during the progression of diabetes have not yet been studied in resistance arteries. In particular, based on our previous findings that in addition to an increased oxidative stress, inactivation of eNOS plays a significant role in the endothelial dysfunction of 9-month-old (9M) diabetic mice (4), the question arises as to whether the specific modulation of MAPK activity can ameliorate endothelial function in advanced diabetes. Thus, in the current study, we aimed to assess the causative relationship between the MAPK activity and the endothelial dysfunction in blood vessels of diabetic mice. We hypothesized that an altered vascular MAPK is responsible for the exacerbation of endothelial dysfunction during the progression of diabetes, and therefore, normalizing MAPK activity improves endothelial function. To accomplish this goal, we used 3-month-old (3M) and 9M Lepr^db−/− mice as models for the early and advanced stages of type 2 diabetes. As observed, Lepr^db−/− mice develop obesity, hyperglycemia, and hyperinsulinemia after their first month and do not survive longer than 10 months. The heterozygous (Lepr^db+/−) littermates are lean and have normal plasma insulin and glucose and a normal life span. Therefore, age-matched male Lepr^db+/− mice were used as normal control mice.     

Double inhibition of cAMP and mTOR signalling may potentiate the reduction of cell growth in ADPKD cells

Background

ADPKD is a renal pathology caused by mutations of PKD1 and PKD2 genes, which encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. PC1 plays an important role regulating several signal transducers, including cAMP and mTOR, which are involved in abnormal cell proliferation of ADPKD cells leading to the development and expansion of kidney cysts that are a typical hallmark of this disease. Therefore, the inhibition of both pathways could potentiate the reduction of cell proliferation enhancing benefits for ADPKD patients.

Methods

The inhibition of cAMP- and mTOR-related signalling was performed by Cl-IB-MECA, an agonist of A3 receptors, and rapamycin, respectively. Protein kinase activity was evaluated by immunoblot and cell growth was analyzed by direct cell counting.

Results

The activation of A₃AR by the specific agonist Cl-IB-MECA causes a marked reduction of CREB, mTOR, and ERK phosphorylation in kidney tissues of Pkd1 ^flox/?: Ksp-Cre polycystic mice and reduces cell growth in ADPKD cell lines, but not affects the kidney weight. The combined sequential treatment with rapamycin and Cl-IB-MECA in ADPKD cells potentiates the reduction of cell proliferation compared with the individual compound by the inhibition of CREB, mTOR, and ERK kinase activity. Conversely, the simultaneous application of these drugs counteracts their effect on cell growth, because the inhibition of ERK kinase activity is lost.

Conclusion

The double treatment with rapamycin and Cl-IB-MECA may have synergistic effects on the inhibition of cell proliferation in ADPKD cells suggesting that combined therapies could improve renal function in ADPKD patients.

     

Vitamin K2 promotes 1α,25(OH)2 vitamin D3-induced mineralization in human periosteal osteoblasts

The effect of vitamin K on mineralization by human periosteal osteoblasts was investigated in the absence and presence of 1,25 dihydroxyvitamin D₃ (1,25(OH)₂D₃). Vitamin K₁ and K₂, but not vitamin K₃, at 2.5 M enhanced in vitro mineralization when cells were cultured with vitamin K for 20 days after reaching confluence in vitro. Vitamin K₂ (2-methyl-3-all-trans-tetraphenyl-1,4-naphthoquinone: menatetrenone) was the most potent of these vitamin K analogs; it slightly inhibited alkaline phosphatase (ALP) activity. Human osteoblasts were mineralized and showed the enhanced ALP activity on treatment with 10^-9 M of 1,25(OH)₂D₃ for 20 or 25 days after confluence. Vitamin K₂ promoted the 1,25(OH)₂D₃-induced mineralization, but slightly inhibited the 1,25(OH)₂D₃-induced ALP activity. Moreover, vitamin K₂ enhanced the 1,25(OH)₂D₃-induced osteocalcin accumulation in the cells and the extracellular matrix (cell layer), but inhibited the osteocalcin content in the medium produced by the 1,25(OH)₂D₃ treatment. However, vitamin K₂ alone did not induce osteocalcin production in the human osteoblasts. On Northern blot analysis, osteocalcin mRNA expression on 1,25(OH)₂D₃-treated cells was enhanced by vitamin K₂ treatment, but vitamin K₂ alone did not induce osteocalcin mRNA expression. Warfarin blocked both the 1,25(OH)₂D₃-induced osteocalcin production and the accumulation in the cell layer, and also blocked the 1,25(OH)₂D₃ plus vitamin K₂-induced osteocalcin production and the accumulation in the cell layer. The 1,25(OH)₂D₃-induced mineralization promoted by vitamin K₂ was probably due to the enhanced accumulation of osteocalcin induced by vitamin K₂ in the cell layer. However, we concluded that the mineralization induced by vitamin K₂ alone was due to the accumulation of osteocalcin in bovine serum on the cell layer, since osteocalcin extracted from the cell layer was not identified by specific antiserum against human osteocalcin, which does not cross-react with bovine osteocalcin. These results suggest that the mechanism underlying the mineralization induced by vitamin K₂ in the presence of 1,25(OH)₂D₃ was different from that of vitamin K₂ alone, and that osteocalcin plays an important role in mineralization by osteoblasts in vitro.     

Experimental and clinical standards,and evolution of lasers in neurosurgery

Summary From initial experiments of ruby, argon and CO₂ lasers on the nervous system so far, dramatic progress was made in delivery systems technology as well as in knowledge of laser-tissue interaction effects and hazards through various animal experiments and clinical experience. Most surgical effects of laser light on neural tissue and the central nervous system (CNS) are thermal lesions. Haemostasis, cutting and vaporization depend on laser emission parameters — wavelength, fluence and mode — and on the exposed tissues optical and thermal properties — water and haemoglobin content, thermal conductivity and specific heat. CO₂ and Nd-YAG lasers have today a large place in the neurosurgical armamentarium, while new laser sources such as high power diode lasers will have one in the near future. Current applications of these lasers derive from their respective characteristics, and include CNS tumour and vascular malformation surgery, and stereotactic neurosurgery. Intracranial, spinal cord and intra-orbital meningiomas are the best lesions for laser use for haemostasis, dissection and tissue vaporization. Resection of acoustic neuromas, pituitary tumours, spinal cord neuromas, intracerebral gliomas and metastases may also benefit from lasers as accurate, haemostatic, non-contact instruments which reduce surgical trauma to the brain and eloquent structures such as brain stem and cranial nerves. Coagulative lasers (1.06 m and 1.32 m Nd-YAG, argon, or diode laser) will find an application for arteriovenous malformations and cavernomas. Any fiberoptic-guided laser will find a use during stereotactic neurosurgical procedures, including image-guided resection of tumours and vascular malformations and endoscopie tumour resection and cysts or entry into a ventricle. Besides these routine applications of lasers, laser interstitial thermotherapy (LITT) and photodynamic therapy (PDT) of brain tumours are still in the experimental stage.The choice of a laser in a neurosurgical operating room implies an evaluation of the laser use (applications, frequency), of the available budget and costs-including purchase, maintenance and staff training-, and material that will be necessary: unit, peripherals, safety devices and measures, training programme.Future applications of lasers in neurosurgery will come from technological advances and refined experimental applications. The availability of new wavelength, tunable, small sized and smart laser units, will enlarge the thermal and non-thermal interactions between laser energy and neural tissue leading to new surgical applications. Tissue photo-ablation, photohynamic therapy using second generation of photosensitizers, updated thermotherapy protocols, are current trends for further use of lasers in neurosurgery.     

Hypothalamic protein kinase C regulates glucose production

OBJECTIVE—A selective rise in hypothalamic lipid metabolism and the subsequent activation of SUR1/Kir6.2 ATP-sensitive K⁺ (K_ATP) channels inhibit hepatic glucose production. The mechanisms that link the ability of hypothalamic lipid metabolism to the activation of K_ATP channels remain unknown.RESEARCH DESIGN AND METHODS—To examine whether hypothalamic protein kinase C (PKC) mediates the ability of central nervous system lipids to activate K_ATP channels and regulate glucose production in normal rodents, we first activated hypothalamic PKC in the absence or presence of K_ATP channel inhibition. We then inhibited hypothalamic PKC in the presence of lipids. Tracer-dilution methodology in combination with the pancreatic clamp technique was used to assess the effect of hypothalamic administrations on glucose metabolism in vivo.RESULTS—We first reported that direct activation of hypothalamic PKC via direct hypothalamic delivery of PKC activator 1-oleoyl-2-acetyl-sn-glycerol (OAG) suppressed glucose production. Coadministration of hypothalamic PKC-δ inhibitor rottlerin with OAG prevented the ability of OAG to activate PKC-δ and lower glucose production. Furthermore, hypothalamic dominant-negative Kir6.2 expression or the delivery of the K_ATP channel blocker glibenclamide abolished the glucose production-lowering effects of OAG. Finally, inhibition of hypothalamic PKC eliminated the ability of lipids to lower glucose production.CONCLUSIONS—These studies indicate that hypothalamic PKC activation is sufficient and necessary for lowering glucose production.The hypothalamus senses nutrients and hormones to regulate energy and glucose homeostasis (1–9), but the associated central nervous system (CNS) sensing mechanisms remain unclear. A selective increase in long-chain fatty acyl-coenzyme A (LCFA-CoA) level in the hypothalamus leads to the activation of SUR1/Kir6.2-containing ATP-sensitive K⁺ (K_ATP) channels and lowers glucose production (10). In contrast, an elevation of LCFA-CoA level in the liver actually increases glucose production during hyperinsulinemia (1). These observations led us to hypothesize that lipid-sensing mechanisms share similar biochemical (i.e., LCFA-CoA accumulation) but have opposing physiological mechanisms (i.e., glucose production regulation) in operation (1).In the peripheral tissues such as the liver and muscle, an elevation of lipids (especially the long-chain fatty acids [LCFAs]) activates the novel isoforms of protein kinase C (PKC) (i.e., -δ, -ɛ, and -θ) to induce insulin resistance during hyperinsulinemic-euglycemic clamps (11–16). Although novel isoforms of PKC (especially -δ and -ɛ) are expressed in the brain (17), it is currently unknown whether LCFAs activate hypothalamic, novel isoforms of PKC to regulate glucose production. It has been reported that activation of PKC leads to phosphorylation of the conserved threonine residue (T180) in the pore-forming subunit Kir6.2 of the K_ATP channels in the pancreatic β-cells (18). These channels are expressed in both β-cells and neurons (18,19), and direct activation of the hypothalamic K_ATP channels has been shown to lower glucose production (19). Both the PKC-induced K_ATP channel activation (18) and hypothalamic K_ATP channels’ regulation of glucose production (19) are blocked by pretreatment with the K_ATP channel blocker glibenclamide (18,19). It is possible that the mechanism of activation of K_ATP channels in the β-cells by PKC is also found in the hypothalamus.Based on these independent yet parallel findings, we tested the hypothesis that activation of hypothalamic PKC is sufficient and necessary for CNS lipid-sensing mechanisms to lower glucose production and regulate glucose homeostasis (Fig. 1A).Open in a separate windowFIG. 1.Hypothalamic PKC activation lowers glucose production. A: Working hypothesis: lipids activate hypothalamic PKC to phosphorylate and activate the hypothalamic Kir6.2/SUR1-containing K_ATP channels to lower glucose production. Direct MBH administration of PKC activator OAG increased glucose infusion rate (B) and lowered glucose production (C) during the clamps. MBH OAG coinfused with general PKC inhibitor BIM (n = 5), specific PKC-δ inhibitor Rot (n = 6), or K_ATP channel blocker glibenclamide (n = 5) or in MBH DN Kir6.2 AAA-injected rats (n = 5) failed to increase glucose infusion rate (B) and lower glucose production (C). D: Glucose uptake was comparable in all groups. MBH vehicle (VEH) (n = 6) consisted of MBH saline (n = 3) and MBH 5% DMSO (n = 3). MBH OAG (n = 7) consisted of MBH OAG in normal rats (n = 4) and in MBH GFP-injected rats (n = 3). *P < 0.001 (ANOVA) and P < 0.01 vs. other individual groups.     

Renal Cortical Pyruvate Depletion during AKI

Pyruvate is a key intermediary in energy metabolism and can exert antioxidant and anti-inflammatory effects. However, the fate of pyruvate during AKI remains unknown. Here, we assessed renal cortical pyruvate and its major determinants (glycolysis, gluconeogenesis, pyruvate dehydrogenase [PDH], and H₂O₂ levels) in mice subjected to unilateral ischemia (15–60 minutes; 0–18 hours of vascular reflow) or glycerol-induced ARF. The fate of postischemic lactate, which can be converted back to pyruvate by lactate dehydrogenase, was also addressed. Ischemia and glycerol each induced persistent pyruvate depletion. During ischemia, decreasing pyruvate levels correlated with increasing lactate levels. During early reperfusion, pyruvate levels remained depressed, but lactate levels fell below control levels, likely as a result of rapid renal lactate efflux. During late reperfusion and glycerol-induced AKI, pyruvate depletion corresponded with increased gluconeogenesis (pyruvate consumption). This finding was underscored by observations that pyruvate injection increased renal cortical glucose content in AKI but not normal kidneys. AKI decreased PDH levels, potentially limiting pyruvate to acetyl CoA conversion. Notably, pyruvate therapy mitigated the severity of AKI. This renoprotection corresponded with increases in cytoprotective heme oxygenase 1 and IL-10 mRNAs, selective reductions in proinflammatory mRNAs (e.g., MCP-1 and TNF-α), and improved tissue ATP levels. Paradoxically, pyruvate increased cortical H₂O₂ levels. We conclude that AKI induces a profound and persistent depletion of renal cortical pyruvate, which may induce additional injury.Increasing evidence indicates the diverse role that pyruvate can play as a potential modulator of tissue injury. Pyruvate is classically viewed as a key determinant of cellular energy metabolism. Under aerobic conditions, pyruvate, generated from glucose by glycolysis, undergoes decarboxylation by the enzyme complex pyruvate dehydrogenase (PDH), forming acetyl CoA. Alternatively, pyruvate can be carboxylated by pyruvate carboxylase, yielding oxaloacetate, with subsequent acetyl CoA/oxaloacetate metabolism through the Krebs cycle and increased aerobic ATP production results. Conversely, under anaerobic conditions, pyruvate is metabolized by lactate dehydrogenase (LDH) to lactate, and in the process, NAD is generated, which is required for additional glycolytic ATP production.In addition to its central role in supporting both aerobic and anaerobic energy production, pyruvate may exert a variety of cytoprotective effects. For example, studies by Salahudeen et al.¹ and Nath et al.² showed that pyruvate is a potent H₂O₂ scavenger, which was assessed in an in vitro system. Furthermore, Salahudeen et al.¹ and Nath et al.² reported that pyruvate administration decreased renal injury after intrarenal H₂O₂ injection and during the course of the glycerol model of rhabdomyolysis-induced ARF.In addition to its antioxidant properties, pyruvate may also exert anti-inflammatory and cytoprotective effects. For example, Miyaji et al.³ and Ulloa et al.⁴ reported that ethyl pyruvate conferred protection against the cecal ligation and puncture model of sepsis-induced AKI. Ulloa et al.⁴ also noted that ethyl pyruvate decreased lethality in sepsis-induced inflammatory states.⁴ Ethyl pyruvate has been shown to protect against experimental ischemic-reperfusion injury and shock.^5–7 Finally, pyruvate administration has been noted to decrease myocardial inflammation after cardiopulmonary bypass⁸ as well as stroke.⁹ The importance of these latter studies is underscored by the fact that Na, rather than ethyl-pyruvate, was used, given that the ethyl pyruvate formulation may exert compound-specific effects.³Despite the critical role of pyruvate in cellular energy production as well as its potential to impact experimental tissue injury, the impact of ischemic or nephrotoxic AKI on tissue pyruvate levels and its determinants has remained undefined. Hence, the goals of this study were threefold: first, document the influence of evolving ischemic and toxic AKI on pyruvate levels within renal cortex; second, ascertain the metabolic pathways that ultimately alter pyruvate expression; third, further elucidate the potential effects of pyruvate on the course and selected potential mediators of experimental models of ARF.