Therapeutic Chemical Screen Identifies Phosphatase Inhibitors to Reconstitute PKB Phosphorylation and Cardiac Contractility in ILK-Deficient Zebrafish

Patients with inherited dilated cardiomyopathy (DCM) often suffer from severe heart failure based on impaired cardiac contractility leading to increased morbidity and mortality. Integrin-linked kinase (ILK) as a part of the cardiac mechanical stretch sensor was found to be an essential genetic regulator of cardiac contractility. Integrin-linked kinase localizes to z-disks and costameres in vertebrate hearts and regulates the activity of the signaling molecule protein kinase B (PKB/Akt) by controlling its phosphorylation. Despite identification of several potential drug targets in the ILK signaling pathway, pharmacological treatment strategies to restore contractile function in ILK-dependent cardiomyopathies have not been established yet. In recent years, the zebrafish has emerged as a valuable experimental system to model human cardiomyopathies as well as a powerful tool for the straightforward high-throughput in vivo small compound screening of therapeutically active substances. Using the ILK deficient zebrafish heart failure mutant main squeeze (msq), which shows reduced PKB phosphorylation and thereby impaired cardiac contractile force, we identified here, in an automated small compound screen, the protein phosphatase inhibitors calyculin A and okadaic acid significantly restoring myocardial contractile function by reconstituting PKB phosphorylation in msq ILK-deficient zebrafish embryos.

Abstract 13133: Silencing of Angiopoietin-like Protein 3 ( ANGPTL3 ) in IHH- Hepatocytes Results in Increased Insulin Sensitivity and Reduced Triglyceride-rich VLDL Secretion

AIMS: Homozygosity of loss-of-function mutations in ANGPTL3 -gene results in familial combined hypolipidemia (FHBL2,OMIM #605019) characterized by reduction of all major plasma lipoprotein classes VLDL, LDL, HDL and low circulating free fatty acids, glucose and insulin levels. Thus complete lack of ANGPTL3 in humans not only affects lipid metabolism but also whole-body insulin and glucose balance. Our aim was to investigate the function of ANGPTL3, a hepatic secretory protein, in promoting hypolipidemia and hepatic insulin sensitivity. METHODS: We used wild type and ANGPTL3-silenced human immortalized hepatocytes (IHH) to investigate the effect of ANGPTL3-silencing on hepatic VLDL secretion and glucose uptake. RESULTS: We demonstrate that insulin downregulates hepatic secretion of ANGPTL3 and triglyceride-enriched VLDL1-type particles in a dose dependent manner. Similar effect on VLDL secretion was demonstrated with a treatment of PPAR[[Unable to Display Character: ƴ]] agonist rosiglitazone. We further show that ANGPTL3-silenced cells display a more pronounced shift from the secretion of TG-enriched VLDL1-type particles to a secretion of lipid poor VLDL2-type particles during insulin stimulation. Silencing of ANGPTL3 improved hepatic glucose uptake by 20-50 % depending on the glucose and insulin concentration, resulted in a trend towards increased AKT/PKB phosphorylation upon insulin stimulation and downregulated fasting induced transcription factor PGC1α and its downstream targets. CONCLUSION: Our results indicate a similar function of both insulin and rosiglitazone regarding regulation of ANGPTL3 and VLDL secretion in hepatocytes, and suggest that insulin and PPAR[[Unable to Display Character: ƴ]] might mediate some of their functions via ANGPTL3. Our results give more insight into the liver specific role of ANGPTL3 and links silencing of ANGPTL3 with Insulin sensitivity. Since humans with elevated levels of ANGPTL3 display hyperlipidemia and insulin resistance it might be beneficial to target ANGPTL3 silencing in the liver, the major site of ANGPTL3 expression, to balance lipid and glucose homeostasis and lower risk for cardiovascular diseases.

Development aggravates the severity of skeletal muscle catabolism induced by endotoxemia in neonatal pigs

Accretion rates of muscle protein are elevated in normal neonates, but this anabolic drive decreases with maturation. As this change occurs, it is not known whether development also influences muscle protein catabolism induced by sepsis. We hypothesize that protein degradation in skeletal muscle induced by endotoxemia becomes more severe as the neonate develops. Fasted 7- and 26-day-old pigs were infused for 8 h with LPS (0 and 10 μg·kg−1·h−1), while plasma amino acids (AA), 3-methylhistidine (3-MH), and α-actin concentrations and muscle protein degradation signal activation were determined ( n = 5–7/group/age). Plasma full-length α-actin was greater in 7- than 26-day-old pigs, suggesting a higher baseline protein turnover in neonatal pigs. LPS increased plasma total AA, 3-MH, and full-length and cleaved α-actin in 26- than in 7-day-old pigs. In muscle of both age groups, LPS increased AMPK and NF-κB phosphorylation, the abundances of activated caspase 3 and E-3 ligases MuRF1 and atrogin1, as well as the abundance of cleaved α-actin, suggesting activation of muscle proteolysis by endotoxin in muscle. LPS decreased Forkhead box 01 (Fox01) and Fox04 phosphorylation and increased procaspase 3 abundance in muscle of 26-day-old pigs despite the lack of effect of LPS on PKB phosphorylation. The results suggest that skeletal muscle in healthy neonatal pigs maintains high baseline degradation signal activation that cannot be enhanced by endotoxin, but as maturation advances, the effect of LPS on muscle protein catabolism manifests its severity.

IκB kinase β (IKKβ) does not mediate feedback inhibition of the insulin signalling cascade

In the present study, we have examined whether IKKβ [IκB (inhibitor of nuclear factor κB) kinase β] plays a role in feedback inhibition of the insulin signalling cascade. Insulin induces the phosphorylation of IKKβ, in vitro and in vivo, and this effect is dependent on intact signalling via PI3K (phosphoinositide 3-kinase), but not PKB (protein kinase B). To test the hypothesis that insulin activates IKKβ as a means of negative feedback, we employed a variety of experimental approaches. First, pharmacological inhibition of IKKβ via BMS-345541 did not potentiate insulin-induced IRS1 (insulin receptor substrate 1) tyrosine phosphorylation, PKB phosphorylation or 2-deoxyglucose uptake in differentiated 3T3-L1 adipocytes. BMS-345541 did not prevent insulin-induced IRS1 serine phosphorylation on known IKKβ target sites. Secondly, adenovirus-mediated overexpression of wild-type IKKβ in differentiated 3T3-L1 adipocytes did not suppress insulin-stimulated 2-deoxyglucose uptake, IRS1 tyrosine phosphorylation, IRS1 association with the p85 regulatory subunit of PI3K or PKB phosphorylation. Thirdly, insulin signalling was not potentiated in mouse embryonic fibroblasts lacking IKKβ. Finally, insulin treatment of 3T3-L1 adipocytes did not promote the recruitment of IKKβ to IRS1, supporting our findings that IKKβ, although activated by insulin, does not promote direct serine phosphorylation of IRS1 and does not contribute to the feedback inhibition of the insulin signalling cascade.

Insulin stimulation of Akt/PKB phosphorylation in the placenta of preeclampsia patients

CONTEXT AND OBJECTIVE: Preeclampsia is a multi-systemic disease and one of the most frequent severe health problems during pregnancy. Binding of insulin triggers phosphorylation and activates cytoplasmic substrates such as phosphatidylinositol 3 kinase (PI3K). Phosphorylation of membrane phosphoinositide 2 (PIP2) to phosphoinositide 3 (PIP3) by PI3K starts Akt/PKB activation. Defects in phosphorylation of the insulin receptor and its substrates have an important role in insulin resistance. Studies have shown that insulin resistance is associated with preeclampsia and its pathophysiology. The aim here was to investigate insulin stimulation of the Akt/PKB pathway in the placenta, in normal and preeclampsia parturients. DESIGN AND SETTING: Cross-sectional study in a tertiary public university hospital. METHODS: Placentas were collected from 12 normal and 12 preeclampsia patients. These were stimulated and analyzed using Western blot to quantify the Akt/PKB phosphorylation. RESULTS: The insulin stimulation was confirmed through comparing the stimulated group (1.14 ± 0.10) with the non-stimulated group (0.91 ± 0.08; P < 0.001). The phosphorylation of Akt/PKB did not differ between the placenta of the normal patients (1.26 ± 0.16) and those of the preeclampsia patients (1.01 ± 0.11; P = 0.237). CONCLUSIONS: In vitro insulin stimulation of the human placenta has been well established. There was no difference in Akt/PKB phosphorylation, after stimulation with insulin, between placentas of normal and preeclampsia patients. Nevertheless, it cannot be ruled out that the Akt/PKB signaling pathway may have a role in the pathophysiology of preeclampsia, since the substrates of Akt/PKB still need to be investigated.