Muscle-specific Pten deletion protects against insulin resistance and diabetes

Pten (phosphatase with tensin homology), a dual-specificity phosphatase, is a negative regulator of the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. Pten regulates a vast array of biological functions including growth, metabolism, and longevity. Although the PI3K/Akt pathway is a key determinant of the insulin-dependent increase in glucose uptake into muscle and adipose cells, the contribution of this pathway in muscle to whole-body glucose homeostasis is unclear. Here we show that muscle-specific deletion of Pten protected mice from insulin resistance and diabetes caused by high-fat feeding. Deletion of muscle Pten resulted in enhanced insulin-stimulated 2-deoxyglucose uptake and Akt phosphorylation in soleus but, surprisingly, not in extensor digitorum longus muscle compared to littermate controls upon high-fat feeding, and these mice were spared from developing hyperinsulinemia and islet hyperplasia. Muscle Pten may be a potential target for treatment or prevention of insulin resistance and diabetes.     

Polymerase chain reaction-restriction fragment-length polymorphism method to distinguish Liriomyza huidobrensis from L. Langei (Diptera: Agromyzidae) applied to three recent leafminer invasions.

A molecular method is presented for differentiating the morphologically cryptic leafminers Liriomyza langei Frick and L. huidobrensis (Blanchard). This method requires polymerase chain reaction (PCR) amplification of a 1031-bp region of mitochondrial cytochrome oxidase DNA followed by restriction fragment analysis using the restriction enzymes SpeI and EcoRV. Spel cuts the mitochondrial fragment of L. langei into two fragments, but does not cut the L. huidobrensis fragment. EcoRV cuts the L. huidobrensis fragment into two fragments, but does not cut the L. langei fragment. This PCR-restriction fragment-length polymorphism (RFLP) method is faster and less costly than DNA sequencing,which is currently the only other way to differentiate these two species. We apply the method to samples from recently introduced leafminer populations in Sri Lanka, Canada, and South Africa and find that the invasive leafminer in all three locations is L. huidobrensis.     

Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK

Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.     

Biological activities and potential health benefits of bioactive peptides derived from marine organisms

Marine organisms have been recognized as rich sources of bioactive compounds with valuable nutraceutical and pharmaceutical potentials. Recently, marine bioactive peptides have gained much attention because of their numerous health beneficial effects. Notably, these peptides exhibit various biological activities such as antioxidant, anti-hypertensive, anti-human immunodeficiency virus, anti-proliferative, anticoagulant, calcium-binding, anti-obesity and anti-diabetic activities. This review mainly presents biological activities of peptides from marine organisms and emphasizing their potential applications in foods as well as pharmaceutical areas.     

Protective effect of chitin oligosaccharides against lipopolysaccharide-induced inflammatory response in BV-2 microglia

Chitin oligosaccharides (NA-COS) of two different molecular weight ranges (below 1 and 1-3kDa) were examined for their capabilities against lipopolysaccharide-induced inflammatory responses in BV-2 murine microglia. It was found that NA-COS reduced the level of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production by suppressing the expression of NO synthase (iNOS) and cyclooxygenase (COX)-2 without significant cytotoxicity. Furthermore, the inhibitory effects of NA-COS on generation of interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α were determined. Notably, NA-COS exerted anti-inflammatory activities via blocking degradation of inhibitor of kappaB-alpha (IκB-α), translocation of nuclear factor (NF)-κB, and phosphorylation of mitogen-activated protein kinases (MAPKs) in a dose-dependent manner. These findings provide mechanistic insights into the anti-inflammatory and neuroprotective actions of NA-COS in BV-2 microglia.     

Effects of high-fat diet feeding on Znt8-null mice: differences between β-cell and global knockout of Znt8

Genomewide association studies have linked a polymorphism in the zinc transporter 8 (Znt8) gene to higher risk of developing type 2 diabetes. Znt8 is highly expressed in pancreatic β-cells where it is involved in the regulation of zinc transport into granules. However, Znt8 is also expressed in other tissues including α-cells, where its function is as yet unknown. Previous work demonstrated that mice lacking Znt8 globally were more susceptible to diet-induced obesity (Lemaire et al., Proc Natl Acad Sci USA 106: 14872-14877, 2009; Nicolson et al., Diabetes 58: 2070-2083, 2009). Therefore, the main goal of this study was to examine the physiological impact of β-cell-specific Znt8 deficiency in mice during high-fat high-calorie (HFHC) diet feeding. For these studies, we used β-cell-specific Znt8 knockout (Ins2Cre:Znt8loxP/loxP) and whole body Znt8 knockout (Cre-:Znt8(-/-)) mice placed on a HFHC diet for 16 wk. Ins2Cre:Znt8loxP/loxP mice on HFHC diet had similar body weights throughout the study but displayed impaired insulin biosynthesis and secretion and were glucose intolerant compared with littermate control Ins2Cre mice. In contrast, Cre-:Znt8(-/-) mice became remarkably obese, hyperglycemic, hyperinsulinemic, insulin resistant, and glucose intolerant compared with littermate control Cre- mice. These data show that β-cell Znt8 alone does not considerably aggravate weight gain and glucose intolerance during metabolic stress imposed by an HFHC diet. However, global loss of Znt8 is involved in exacerbating diet-induced obesity and resulting insulin resistance, and this may be due to the loss of Znt8 activity in a tissue other than the β-cell. Thus, our data suggest that Znt8 contributes to the risk of developing type 2 diabetes through β-cell- and non-β-cell-specific effects.     

Simultaneous Treatment of Dioxins and Heavy Metals in Tagonoura Harbor Sediment by Two-Step Pyrolysis

This paper describes the simultaneous treatment of PCDD/PCDF and toxic heavy metals contaminated sediment by pyrolysis with emphasis on complete removal and destruction of PCDD/PCDF in Tagonoura harbor sediment during a two-step pyrolysis process. Pyrolysis was conducted at different temperatures and N ₂ gas flow rates. Results showed that almost all PCDD/PCDF (99.998%) can be removed from the sediment by treatment at 800°C for 30 minutes in single-step pyrolysis; however, PCDD/PCDF remained in the gas phase without destruction. Analysis also revealed that some PCDD/PCDF formation occurred during the single-step pyrolysis. However, by the use of two-step pyrolysis, where off-gases from the first furnace are heated at 1000°C, PCDD/PCDF in the final gas effluent was dramatically reduced by a thermal degradation mechanism. Stabilization of toxic heavy metals was also observed. Specifically, their leaching activity drastically decreased with increasing pyrolysis temperature. Significant metal volatilization did not occur. Though the low temperature pyrolysis was effective in removing PCDD/PCDF, the leachability of toxic heavy metals such as Cr and Ni was higher that of 800°C; however, lower than untreated. Two-step pyrolysis at 800°C for 30 minutes in the single-step and 1000°C in the two-step pyrolyzer would be effective for the treatment of sediments contaminated with PCDD/PCDF, toxic heavy metals and organic matters simultaneously.     

Free radical scavenging and angiotensin-I converting enzyme inhibitory peptides from Pacific cod (Gadus macrocephalus) skin gelatin

Potent antioxidative peptides were purified from Pacific cod (Gadus macrocephalus) skin gelatin using alcalase, neutrase, papain, trypsin, pepsin, and α-chymotrypsin. Among them, the papain hydrolysate exhibited the highest antioxidant activity. Therefore, it was further purified and obtained two peptides with amino acid sequences of Thr-Cys-Ser-Pro (388 Da) and Thr-Gly-Gly-Gly-Asn-Val (485.5 Da). The antioxidant activity of the purified peptides was performed by electron spin resonance technique. Moreover, their intracellular free radical scavenging activity using 2′,7′-dichlorofluorescin diacetate and the protective effect against oxidation-induced DNA damage were evaluated in mouse macrophages (RAW 264.7 cells). Furthermore, both peptides have shown potential angiotensin-I converting enzyme inhibitory effect. The present study demonstrated that the peptides derived from Pacific cod (G. macrocephalus) skin gelatin could be used in the food industry as functional ingredients with potent antioxidative and antihypertensive benefits.     

Characterization of Erg K+ Channels in ��- and ��-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K⁺ channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in α-TC6 and Min6 cells α- and β-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in α- and β-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca²⁺-imaging experiments were performed on isolated α- and β-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca²⁺ increase in both α- and β-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of α- and β-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.Voltage-gated eag-related gene (Erg)² potassium (K⁺) channels are part of the larger family of voltage dependent K⁺ (Kv) channels (1). Three channel isoforms Erg1, Erg2, and Erg3 have been discovered (2, 3), and they differ by their activation and inactivation voltage dependence, gating properties, and pharmacological profile (4–7). Erg channels control cellular activity by controlling the repolarization of the action potential (AP). In atrial cells and ventricular myocytes, Erg regulates plateau formation and AP repolarization, as blocking Erg channels increases AP length (8, 9). These channels are also strongly involved in the pacemaking activity of cardiac cells (10, 11). Interestingly, a rare congenital heart condition, the inherited form of long QT syndrome is caused by mutations of Erg channel genes (9, 12). Erg channels also control the resting membrane potential in various cell types. For example, in neurons of the medial vestibular nucleus, blocking Erg channels produce an increase in AP discharge or in smooth muscle cells, blocking Erg channels mediates depolarization up to 20 mV (13–15). Hormone secretion studies also demonstrated the involvement of Erg channels in the secretion of prolactin from neurons of the anterior pituitary. Thyrotropin-releasing factor decreases Erg current, which depolarizes neurons and thereby stimulates prolactin secretion (16, 17).In the pancreas, Kv channels and more specifically Kv2.1, regulate insulin secretion by controlling the repolarization of β-cell membrane potential (18–20), although the contribution of this isoform in humans has recently been questioned (21). In α-cells, Kv2.1 and Kv1.4 channels repolarize the membrane potential (22, 23); however, the involvement of Kv channels in the secretion of glucagon is yet to be investigated. One study showed that Erg1, -2, and -3 are expressed in rat α- and β-cells and the rat insulinoma cell line, INS-1, and that they are involved in decreasing membrane potential. Blocking Erg channels with the channel antagonist E4031 increases insulin secretion from INS1 cells (24); however, definitive data regarding the role of Erg channels in insulin and glucagon secretion is limited.Therefore this study aimed to define the functions of Erg channels in α- and β-cells. We found that Erg1 channels are strongly expressed in pancreatic α- and β-cells. Pharmacological and genetic manipulation combined with whole cell recordings in pancreatic cell lines and primary islet cells determined that Erg1 produces a functional current in α- and β-cells. Blocking Erg1 increased intracellular calcium ([Ca²⁺]_i) in mouse β-cells, but only in a minority of mouse and human α-cells. Secretion studies using isolated mouse islets demonstrated that Erg1 are negative regulators of insulin secretion, but positive regulators of glucagon secretion, suggesting distinct roles for Erg1 in β- and α-cells.