Loss of oligophrenin1 leads to uncontrolled Rho activation and increased thrombus formation in mice.

BACKGROUND: Platelet cytoskeletal reorganization is essential for platelet adhesion and thrombus formation in hemostasis and thrombosis. The Rho GTPases RhoA, Rac1 and Cdc42 are the main players in cytoskeletal dynamics of platelets and induce filopodia and lamellipodia formation and actin polymerization to strongly increase the platelet surface upon activation. Moreover, they are important for platelet secretion, integrin activation and arterial thrombus formation. OBJECTIVES: Rho GTPases are regulated by GTPase-activating proteins (GAPs) that stimulate their GTPase activity to terminate Rho signaling. The regulation of Rho GTPase activity in platelets is not well defined. Recently, we identified oligophrenin1 (OPHN1), a RhoGAP in platelets that exhibits strong GTPase-stimulating activity towards RhoA, Cdc42 and Rac1. RESULTS: In the present study we show for the first time, that deficiency of OPHN1 led to abnormal Rho activation and increased platelet cytoskeletal reorganization, including cell adhesion and lamellipodia formation on fibrinogen. Furthermore, platelets from ophn1(-/-) mice showed enhanced susceptibility to platelet activation with alterations in actin distribution and early release of granules. Platelet activation was enhanced following GPVI and PAR4 stimulation. This translated into elevated platelet thrombus formation and promoted arterial thrombosis under low shear conditions with altered hemostasis, as detected by tail bleeding time. CONCLUSIONS: The results of the present study identified OPHN1 as an important regulator of platelet cytoskeletal reorganization and demonstrate that abnormal regulation of Rho proteins leads to increased platelet adhesion and thrombus formation under low shear conditions in vitro and in vivo, suggesting a prothrombotic phenotype of mice critical for acute thrombotic occlusions.

Specific lentiviral shRNA-mediated knockdown of cyclin D1 in mantle cell lymphoma has minimal effects on cell survival and reveals a regulatory circuit with cyclin D2.

Cyclin D1 overexpression is the hallmark of mantle cell lymphoma (MCL). However, the importance of cyclin D1 in the maintenance and progression of the disease remains to be defined. The aim of this study was to elucidate the role of cyclin D1 overexpression using an efficient cyclin D1-shRNA and a lentiviral system in well-characterized MCL cell lines. Surprisingly, the knockdown of cyclin D1 led to a moderate retardation in growth, without induction of apoptosis. The cyclin D1-shRNA-transduced MCL cells showed a 15% shift from S phase to G(1) phase of the cell cycle, a weak induction of p27(Kip1), decreased Rb (Ser807/811) phosphorylation, and a consistent upregulation of cyclin D2 mRNA and protein expression. However, double knockdown of cyclins D1 and D2 did not intensify the effects observed with cyclin D1 knockdown alone. These data suggest that the moderate effects of cyclin D1 downregulation on survival and proliferation are likely the result of compensatory cyclin-independent mechanisms governing proliferation or alternatively, secondary genetic events that make cyclin D1 dispensable. These findings have important implications for MCL therapy, as strategies targeting only cyclin D1 function might be hampered by compensatory regulatory mechanisms, resulting in a low probability of treatment response.

Insulin resistance-inducing cytokines differentially regulate SOCS mRNA expression via growth factor- and Jak/Stat-signaling pathways in 3T3-L1 adipocytes.

Various cytokines, including tumor necrosis factor (TNF) alpha, growth hormone (GH) and interleukin (IL)-6, induce insulin resistance. Recently, it was demonstrated that induction of suppressor of cytokine signaling (SOCS)-3 by TNFalpha and GH is an important mechanism by which these cytokines impair insulin sensitivity. The current study investigated in 3T3-L1 adipocytes whether TNFalpha and GH also upregulate SOCS-1 and SOCS-6, which have both been shown to inhibit insulin signaling potently, and whether IL-6 might alter synthesis of SOCS-1, -3 and -6. Interestingly, 10 ng/ml TNFalpha, 500 ng/ml GH and 30 ng/ml IL-6 induced SOCS-1 mRNA time-dependently with maximal stimulation detectable after 8 h of TNFalpha and 1 h of GH and IL-6 addition respectively. Furthermore, TNFalpha and GH caused sustained upregulation of SOCS-1 for up to 24 h, whereas stimulation by IL-6 was only transient, with SOCS-1 mRNA returning to basal levels 2 h after effector addition. Induction of SOCS-1 was dose-dependent, and significant stimulation was detectable at concentrations as low as 3 ng/ml TNFalpha, 50 ng/ml GH and 10 ng/ml IL-6. Furthermore, stimulation experiments and studies using pharmacologic inhibitors suggested that the positive effect of TNFalpha, GH and IL-6 on SOCS-1 mRNA is, at least in part, mediated by Janus kinase (Jak) 2. Finally, SOCS-3 expression was dose- and time-dependently induced by IL-6, at least in part via Jak2, but none of the cytokines affected SOCS-6 expression. Taken together, our results show a differential regulation of SOCS mRNA by insulin resistance-inducing hormones, and suggest that SOCS-1, as well as SOCS-3, may be an important intracellular mediator of insulin resistance in fat cells and a potential pharmacologic target for the treatment of impaired insulin sensitivity.

GH is a positive regulator of tumor necrosis factor alpha-induced adipose related protein in 3T3-L1 adipocytes.

Tumor necrosis factor (TNF) alpha-induced adipose-related protein (TIARP) has recently been cloned as a TNFalpha-stimulated protein expressed in adipocytes. Its expression is differentiation-dependent and potentially involved in mediating TNFalpha-induced insulin resistance. To further characterize regulation of TIARP gene expression, 3T3-L1 adipocytes were treated with key hormones modulating insulin sensitivity and influencing adipocyte metabolism, and TIARP gene expression was determined by quantitative real-time RT-PCR. Interestingly, TIARP mRNA expression was stimulated almost 9-fold after 500 ng/ml GH were added for 16 h whereas addition of 10 microM isoproterenol, 100 nM insulin and 100 nM dexamethasone for 16 h significantly decreased TIARP gene expression to between 35 and 50% of control levels. In contrast, angiotensin 2 (10 microM) and triiodothyronine (1 microM) did not have any effect. The stimulatory effect of GH was time- and dose-dependent with stimulation occurring as early as 1 h after effector addition and at concentrations as low as 5 ng/ml GH. Moreover, pharmacological inhibition of Janus kinase 2 and p42/44 mitogen-activated protein kinase reversed the stimulatory effect of GH, suggesting that both signaling molecules are involved in activation of TIARP gene expression by GH. Furthermore, an increase of TIARP mRNA could be completely reversed to control levels by withdrawal of GH for 24 h. Taken together, these results show that TIARP is not only responsive to TNFalpha but also to important other hormones influencing glucose homeostasis and adipocyte metabolism. Thus, this factor may play an integrative role in the pathogenesis of insulin resistance and its link to obesity.

Suppression of aquaporin adipose gene expression by isoproterenol, TNFalpha, and dexamethasone.

Aquaporin adipose (AQPap) is a putative glycerol channel in adipocytes. It has recently been shown to be upregulated in insulin resistance stimulated by thiazolidinediones and inhibited by insulin. To further clarify regulation of AQPap gene expression, 3T3-L1 adipocytes were chronically treated with various hormones known to influence insulin sensitivity and adipocyte metabolism, and AQPap mRNA was measured by quantitative real-time reverse transcription-polymerase chain reaction. Interestingly, treatment of 3T3-Ll adipocytes with 10 micro M isoproterenol, 10 ng/ml TNFalpha, and 100 nM dexamethasone for 16 h inhibited AQPap gene expression by 62 %, 60 %, and 39 %, respectively; angiotensin 2, growth hormone, and triiodothyronine did not have any effect. The inhibitory effects were dose-dependent with significant suppression detectable at concentrations as low as 1 nM isoproterenol, 1 ng/ml TNFalpha, and 10 nM dexamethasone. Furthermore, inhibition of AQPap gene expression could be almost completely reversed by pretreating 3T3-L1 adipocytes with the beta-adrenoceptor antagonist propranolol. Moreover, stimulation of Gs-proteins with cholera toxin and adenylyl cyclase with forskolin and dibutyryl-cAMP dramatically downregulated AQPap mRNA. Taken together, our results suggest that AQPap is an adipocyte-expressed glycerol channel selectively regulated and profoundly downregulated by hormones implicated in the pathogenesis of insulin resistance and dyslipidemia.

[Diagnostic possibilities and limits of 4-electrode rheoencephalography in premature infants with risk of brain damage]. / Diagnostische Möglichkeiten und Grenzen der 4-Elektroden-Rheoenzephalografie bei Frühgeborenen mit drohender Hirnschädigung.

Diagnostic value of cerebral electrical impedance for recognizing the risk of cerebral damage was analysed in 63 premature infants with postnatal respiratory disturbance during the first 4 weeks of life. Relationship between clinical risk factors like gestational age, Apgar scores, blood gas values on the one hand and impedance parameter of pulse volume, flow velocity and periphereal vascular resistance on the other were studied by means of correlation as well as variance analysis. Decrease of gestational age, Apgar score and increasing severity of respiratory disturbance cause statistically significant lowering of impedance parameters indicating reduction of volume, flow velocity and periphereal resistance in cerebral circulation by extreme immaturity, serious asphyxia as well as idiopathic respiratory distress syndrome. On contrast no statistically significant difference could be found between infants with and without intraventricular haemorrhages. However, significant differences were seen between group of patients only. Because of intercorrelation of risk factors and distinct variance no critical level of impedance parameters could be defined for the individual patient.

Effect of beta-casomorphins and analogs on insulin release in dogs.

Opioid-active substances have been isolated from bovine beta-casein peptone (beta-casomorphin). Since the ingestion of beta-casomorphin-containing foodstuff elicits an increase in postprandial insulin release, the present study was designed to determine the effect of iv infused beta-casomorphins on insulin release. The infusion of beta-casomorphin-7, -5, -4, and -3 did not alter basal insulin secretion. During prestimulation of insulin release with amino acids and glucose the infusion of beta-casomorphin-7, -5, -4, and -3 at a dose of 1 nmol/ kg . h augmented insulin release, whereas at a concentration of 100 nmol/kg . h no further stimulatory effect was observed except for the infusion of beta-casomorphin-4. In comparison, the infusion of morphine elicited a potentiation of insulin release at the lower dose, whereas no effect was observed at the higher infusion rate. Leucine-enkephalin had no effect at the lower dose but elicited an inhibitory effect at the higher rate. The infusion of opiate-active and inactive analogs of beta-casomorphin-4 resulted in a loss of its stimulatory effect, indicating that the full tetrapeptide structure of the molecule is important for its stimulatory activity on beta-cell secretion. All stimulatory and inhibitory effects of the opioid-active substances were blocked by the specific opiate-receptor antagonist naloxone. Additionally, the present data support the concept that beta-cell secretion is stimulated by mu-receptor activation and inhibited by delta-receptor activation. The K-receptor antagonist ethyl-ketocyclazocine did not alter insulin secretion. The fact that iv administered beta-casomorphins stimulate insulin release raises the possibility that ingested food-derived opioid-active substances modulate pancreatic endocrine function also after their absorption, provided the doses employed in the present study reflect physiological concentrations of circulating beta-casomorphins.

Effect of acetylcholine on the release of pancreatic and gastric somatostatin-like immunoreactivity in normal, chemically sympathectomized and indomethacin-treated dogs.

The present study was designed to determine the effects of intravenously infused acetylcholine on the release of pancreatic and gastric somatostatin-like immunoreactivity (SLI), in anesthetized normal, chemically sympathectomized and Indomethacin-treated prostaglandin deficient dogs. In normal dogs acetylcholine infusion (500 micrograms/min) elicited a significant rise in pancreatic vein, inferior vena cava, fundic and antral vein SLI levels. In the sympathectomized animals the rise in pancreatic and antral vein SLI was not different from the controls, while the rise in fundic vein SLI was abolished, and inferior vena cava plasma SLI levels were reduced. During Indomethacin-induced prostaglandin deficiency, basal SLI levels were reduced significantly, and the acetylcholine-induced stimulation was completely abolished from both stomach and pancreas. It is concluded that in anesthetized dogs the intravenous infusion of acetylcholine stimulates pancreatic and gastric SLI release, and this stimulatory effect depends on the presence of prostaglandins and is modulated by adrenergic mechanisms.

Rate dependent stimulation of insulin and glucagon but not gastrin and somatostatin release during the intestinal phase of a meal.

Previous studies have indicated a possible influence of gastric emptying on postprandial pancreatic endocrine function and the present study was designed to determine if the rate at which nutrients enter the small intestine may play a role in the postprandial regulation of insulin, glucagon, somatostatin and gastrin release in conscious dogs. In response to an intraduodenal instillation of a liver extract--sucrose test meal postprandial insulin and glucagon levels increased significantly with increasing infusion rates of the test meal, whereas somatostatin and gastrin levels did not change. The rise of the endocrine factors preceded any increase of peripheral vein plasma glucose levels. The present data demonstrate that during the intestinal phase of a meal the rate of nutrient entry into the duodenum favours insulin and glucagon but not somatostatin and gastrin release. This mechanism could be of importance in the regulation of nutrient homeostasis during the ingestion of certain carbohydrate containing meals.

Effect of histamine H2-receptor stimulation on postprandial pancreatic and gastric endocrine function in dogs.

The effect of histamine H2-receptor stimulation via the infusion of impromidine was assessed with regard to postprandial plasma insulin, pancreatic polypeptide (PP), somatostatin, and gastrin levels. The effect of impromidine was assessed in the postprandial state during a liver extract/sucrose test meal which had a buffer capacity to maintain the intragastric pH at a constant level for the time impromidine was infused. Postprandial plasma insulin and gastrin levels were not changed by impromidine (10 micrograms/kg X h-1). Plasma somatostatin levels rose significantly, whereas the postprandial increase of plasma PP levels was attenuated. The effects on somatostatin and PP were antagonized by the infusion of cimetidine, a specific histamine H2-receptor blocker. In conclusion the present data demonstrate that in the postprandial state activation of H2-receptors stimulates somatostatin and inhibits PP release while insulin and gastrin release are not affected.

Effect of physiological increments of blood glucose on plasma somatostatin and pancreatic polypeptide levels in dogs.

The present study was designed to determine the effects of physiological increments of plasma glucose levels upon basal and stimulated plasma somatostatin and pancreatic polypeptide levels. In seven conscious dogs the elevation of plasma glucose levels by 30-40 mg/dl did not change basal somatostatin and pancreatic polypeptide levels. During stimulation of these two hormones by acetylcholine and the octapeptide of cholecystokinin intravenous infusion of glucose elicited a significant decrease of somatostatin levels by 30 pg/ml and of pancreatic polypeptide levels by 300 pg/ml. The present data demonstrate that a physiological elevation of plasma glucose levels inhibits stimulated but not basal somatostatin and pancreatic polypeptide levels which may be of importance for nutrient entry and metabolism.

Effect of solid and liquid carbohydrates upon postprandial pancreatic endocrine function.

The present study was designed to determine the effect of sucrose-containing liquids upon postprandial pancreatic endocrine function in comparison to an identical quantity of sucrose contained in the solid part of the test meal. In 10 normal subjects, the ingestion of a solid-liquid meal with sucrose in the liquid part elicited a significantly greater increase in the plasma insulin and glucose levels during the first 20 min than did the ingestion of the same meal in homogenized form. Plasma glucagon levels fell below baseline during the early phase of the meal in response to the solid-liquid meal, whereas values increased immediately upon ingestion of the homogenized meal. To determine the effect of repeated ingestion of sucrose-containing liquids, 6 subjects ingested a meal containing sucrose in solid form together with water (solid sucrose); on another occasion, the same subjects ingested the sucrose in liquid form. In response to the liquid-sucrose meal, mean postprandial plasma insulin levels were significantly higher than those observed in response to the solid-sucrose meal (110 +/- 11.3 vs. 80 +/- 8.5 microunits/ml; P less than 0.01) as were plasma glucagon levels (284 +/- 12.2 vs. 198 +/- 8.2 pg/ml; P less than 0.001). Mean postprandial plasma glucose levels and the insulin to glucagon ratio were not different. The present data demonstrate that the ingestion of sucrose in the liquid part of a meal results in a significant elevation of plasma insulin concentrations compared to the ingestion of sucrose in the solid part of the meal.

Evidence for an effect of exorphins on plasma insulin and glucagon levels in dogs.

Recently, peptides with opioid-like activity have been demonstrated in peptic digests of dietary protein. The present study was designed to determine the effect of digested and undigested gluten on postprandial insulin and glucagon levels in conscious dogs. The intragastric instillation of digested gluten (25 g) elicited a more rapid and a significantly greater rise in postprandial peripheral vein insulin and glucagon levels compared with the effect of 25 g undigested gluten. The incremental insulin level was 104 +/- 20 microU/ml after digested gluten and only 58 +/- 7 microU/ml (P less than 0.01) after undigested gluten; the respective values for glucagon are 426 +/- 25 pg/ml versus 302 +/- 20 pg/ml (P less than 0.01). The intragastric administration of naloxone (4 mg), a specific opiate receptor antagonist, reduced the insulin response and augmented the glucagon response to the digested gluten test meal, whereas the response of both hormones to the undigested gluten meal was not affected by naloxone. Intravenously infused naloxone during the digested gluten meal did not influence insulin or glucagon levels. The present data suggest that in dogs the peptic digest of gluten contains an opioid-like material that stimulates postprandial insulin and glucagon release.

Comparison of the biologic activity of biosynthetic human insulin and natural pork insulin in juvenile-onset diabetic subjects assessed by the glucose controlled insulin infusion system.

To assess the biologic activity of biosynthetic human insulin (BHI) synthesized by Escherichia coli, six insulin-dependent juvenile-onset diabetic subjects were studied with BHI and natural pork insulin, by means of the glucose controlled insulin infusion system (GCIIS). First, after an overnight normalization of blood glucose levels, the 24-h insulin requirement was determined while the patients were consuming a diet of 30 kcal/kg. Then, the amount of glucose necessary to maintain normal blood glucose levels during a 5-h intravenous infusion of BHI and pork insulin, respectively, was assessed. Both studies demonstrate that in the insulin-dependent diabetic subject, BHI is at least as effective as natural pork insulin and may, therefore, be useful for the treatment of insulin-dependent diabetes mellitus.