Temporal pattern of neuronal insulin release during Caenorhabditis elegans aging: Role of redox homeostasis

The insulin‐IGF‐1/DAF‐2 pathway has a central role in the determination of aging and longevity in Caenorhabditis elegans and other organisms. In this paper, we measured neuronal insulin secretion (using INS‐22::Venus) during C. elegans lifespan and monitored how this secretion is modified by redox homeostasis. We showed that INS‐22::Venus secretion fluctuates during the organism lifetime reaching maximum levels in the active reproductive stage. We also demonstrate that long‐lived daf‐2 insulin receptor mutants show remarkable low levels of INS‐22::Venus secretion. In contrast, we found that short‐lived mutant worms that lack the oxidation repair enzyme MSRA‐1 show increased levels of INS‐22::Venus secretion, specifically during the reproductive stage. MSRA‐1 is a target of the insulin‐IGF‐1/DAF‐2 pathway, and the expression of this antioxidant enzyme exclusively in the nervous system rescues the mutant insulin release phenotype and longevity. The msra‐1 mutant phenotype can also be reverted by antioxidant treatment during the active reproductive stage. We showed for the first time that there is a pattern of neuronal insulin release with a noticeable increment during the peak of reproduction. Our results suggest that redox homeostasis can modulate longevity through the regulation of insulin secretion, and that the insulin‐IGF‐1/DAF‐2 pathway could be regulated, at least in part, by a feedback loop. These findings highlight the importance of timing for therapeutic interventions aimed at improving health span.     

Insulin secretion impairment induced by rosuvastatin partly though autophagy in INS‐1E cells

Statins are used extensively for the clinical treatment of cardiovascular diseases. Recent studies suggest that statins increase the risk of new‐onset diabetes mellitus (NODM). However, the mechanisms of statin‐induced NODM remain unclear. The present study investigated the effects of autophagy on insulin secretion impairment induced by rosuvastatin (RS) in rat insulinoma cells (INS‐1E) cells. INS‐1E cells were cultured and treated with RS at different concentrations (0.2–20 μM) for 24 h. Insulin secretion in INS‐1E cells was detected by enzyme‐linked immunosorbent assay, and the co‐localization of microtubule‐associated protein light chain 3 (LC3) and lysosome‐associated membrane protein 2 (LAMP‐2) was observed by immunofluorescence staining. Western blotting was used to assess the conversion of LC3 and p62. The results showed that the insulin secretion and cell viability decrease induced by RS treatment for 24 h occurred in a dose‐dependent manner in INS‐1E cells. RS significantly inhibited the expression of LC3‐II but increased the protein expression of p62. Simultaneously, RS diminished the co‐localization of LC3‐II and LAMP‐2 fluorescence signals. These results suggested that RS‐inhibited autophagy in INS‐1E cells. Rapamycin, an autophagy agonist, reversed the insulin secretion and cell viability suppression induced by RS in INS‐1E cells. RS also decreased the phosphorylation of the mammalian target of rapamycin (mTOR). The results indicated that RS impairs insulin secretion in INS‐1E cells, which may be partly due to the inhibition of autophagy via an mTOR‐dependent pathway.     

A 3D cell culture system: Separation distance between INS‐1 cell and endothelial cell monolayers co‐cultured in fibrin influences INS‐1 cells insulin secretion

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS‐1) and human umbilical vein endothelial cells (HUVEC) co‐cultured in fibrin over INS‐1 cell insulin secretion. For this purpose, a three‐dimensional (3D) cell culture chamber was designed, built using micro‐fabrication techniques and validated. The co‐culture was successfully carried out and the effect on INS‐1 cell insulin secretion was investigated. After 48 and 72 h, INS‐1 cells co‐cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS‐1 cells cultured alone or co‐cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Biotechnol. Bioeng. 2013; 110: 619–627. © 2012 Wiley Periodicals, Inc.     

An Actin Filament Population Defined by the Tropomyosin Tpm3.1 Regulates Glucose Uptake

Actin has an ill‐defined role in the trafficking of GLUT4 glucose transporter vesicles to the plasma membrane (PM). We have identified novel actin filaments defined by the tropomyosin Tpm3.1 at glucose uptake sites in white adipose tissue (WAT) and skeletal muscle. In Tpm 3.1‐overexpressing mice, insulin‐stimulated glucose uptake was increased; while Tpm3.1‐null mice they were more sensitive to the impact of high‐fat diet on glucose uptake. Inhibition of Tpm3.1 function in 3T3‐L1 adipocytes abrogates insulin‐stimulated GLUT4 translocation and glucose uptake. In WAT, the amount of filamentous actin is determined by Tpm3.1 levels and is paralleled by changes in exocyst component (sec8) and Myo1c levels. In adipocytes, Tpm3.1 localizes with MyoIIA, but not Myo1c, and it inhibits Myo1c binding to actin. We propose that Tpm3.1 determines the amount of cortical actin that can engage MyoIIA and generate contractile force, and in parallel limits the interaction of Myo1c with actin filaments. The balance between these actin filament populations may determine the efficiency of movement and/or fusion of GLUT4 vesicles with the PM.      

ENPP1 affects insulin action and secretion: evidences from in vitro studies

The aim of this study was to deeper investigate the mechanisms through which ENPP1, a negative modulator of insulin receptor (IR) activation, plays a role on insulin signaling, insulin secretion and eventually glucose metabolism. ENPP1 cDNA (carrying either K121 or Q121 variant) was transfected in HepG2 liver-, L6 skeletal muscle- and INS1E beta-cells. Insulin-induced IR-autophosphorylation (HepG2, L6, INS1E), Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) phosphorylation (HepG2, L6), PEPCK mRNA levels (HepG2) and 2-deoxy-D-glucose uptake (L6) was studied. GLUT 4 mRNA (L6), insulin secretion and caspase-3 activation (INS1E) were also investigated. Insulin-induced IR-autophosphorylation was decreased in HepG2-K, L6-K, INS1E-K (20%, 52% and 11% reduction vs. untransfected cells) and twice as much in HepG2-Q, L6-Q, INS1E-Q (44%, 92% and 30%). Similar data were obtained with Akt-Ser(473), ERK1/2-Thr(202)/Tyr(204) and GSK3-beta Ser(9) in HepG2 and L6. Insulin-induced reduction of PEPCK mRNA was progressively lower in untransfected, HepG2-K and HepG2-Q cells (65%, 54%, 23%). Insulin-induced glucose uptake in untransfected L6 (60% increase over basal), was totally abolished in L6-K and L6-Q cells. GLUT 4 mRNA was slightly reduced in L6-K and twice as much in L6-Q (13% and 25% reduction vs. untransfected cells). Glucose-induced insulin secretion was 60% reduced in INS1E-K and almost abolished in INS1E-Q. Serum deficiency activated caspase-3 by two, three and four folds in untransfected INS1E, INS1E-K and INS1E-Q. Glyburide-induced insulin secretion was reduced by 50% in isolated human islets from homozygous QQ donors as compared to those from KK and KQ individuals. Our data clearly indicate that ENPP1, especially when the Q121 variant is operating, affects insulin signaling and glucose metabolism in skeletal muscle- and liver-cells and both function and survival of insulin secreting beta-cells, thus representing a strong pathogenic factor predisposing to insulin resistance, defective insulin secretion and glucose metabolism abnormalities.     

Adiponectin Improves Cardiomyocyte Contractile Function in db/db Diabetic Obese Mice

Low levels of adiponectin, a fat‐derived hormone, are found to be correlated with coronary heart disease, type 2 diabetes, obesity, and insulin resistance. Conversely, high adiponectin levels are predictive of reduced coronary risk in long‐term epidemiologic studies. However, the precise role of adiponectin in cardiomyocyte function is still not clear. This study was designed to examine the role of adiponectin in cardiac contractile function in the db/db model of diabetic obesity. Mechanical properties and intracellular Ca²⁺ transients were evaluated in cardiomyocytes from lean control and db/db mice with or without adiponectin (10 µg/ml) treatment. Expression and phosphorylation of IRS‐1, Akt, c‐Jun, and c‐Jun N terminal kinase (JNK) as well as markers of endoplasmic reticulum (ER) stress were evaluated using western blotting. Cardiomyocytes from db/db mice exhibited greater cross‐sectional area, depressed peak shortening (PS), and maximal velocity of shortening/re‐lengthening as well as prolonged duration of re‐lengthening. Consistently, myocytes from db/db mice displayed a reduced electrically stimulated rise in intracellular Ca²⁺ and prolonged intracellular Ca²⁺ decay, which were abrogated by adiponectin treatment. Ratios between phosphorylated c‐Jun and c‐Jun as well as phosphorylated IRS‐1 and IRS‐1 were increased in db/db mice, the effect of which was attenuated by adiponectin. Levels of the phosphorylated ER stress makers PERK (Thr980), IRE‐1, and eIF2α were significantly elevated in db/db mice compared with lean controls, although the effect was unaffected by adiponectin. Collectively, our data suggest that adiponectin improves cardiomyocyte dysfunction in db/db diabetic obese mice through a mechanism possibly related to c‐Jun and IRS‐1.     

Cooperative effects of galanin and leptin on alleviation of insulin resistance in adipose tissue of diabetic rats

It was reported that either orexigenic neuropeptide galanin or anorexigenic hormone leptin caught benefit insulin sensitivity through increasing the translocation of glucose transporter 4 (GLUT4) in patients with diabetes. To date, it is unknown whether galanin can potentiate the effect of leptin on alleviation of insulin resistance. Therefore, in the current study we sought to assess the combined effect of central leptin and galanin on insulin resistance in the adipose tissues of type 2 diabetic rats. Galanin and leptin were injected into the intracerebroventricle of the diabetic rats, respectively, or cooperatively once a day for 2 weeks. Then, several indexes of insulin resistance were examined. The results showed that glucose infusion rates in the hyperinsulinaemic‐euglycaemic clamp test, plasma adiponectin content and GLUT4 translocation, as well as Akt phosphorylation in fat cells, were higher, not GLUT4 protein and GLUT4 mRNA expression, but HOMA index was lower in the galanin + leptin group than either one of them. Furthermore, treatment with MK‐2206, an Akt inhibitor, blocked the combined effects of galanin + leptin on alleviation of insulin resistance. These results suggest that galanin can improve the leptin‐induced mitigative effects on insulin resistance in the fat cells, and those provided new insights into the potential tactics for prevention and remedy of insulin resistance.     

Activation of the small GTPase Rac1 by a specific guanine-nucleotide-exchange factor suffices to induce glucose uptake into skeletal-muscle cells

Background information. Insulin‐stimulated glucose uptake into skeletal muscle is crucial for glucose homoeostasis, and depends on the recruitment of GLUT4 (glucose transporter 4) to the plasma membrane. Mechanisms underlying insulin‐dependent GLUT4 translocation, particularly the role of Rho family GTPases, remain controversial. Results. In the present study, we show that constitutively active Rac1, but not other Rho family GTPases tested, induced GLUT4 translocation in the absence of insulin, suggesting that Rac1 activation is sufficient for GLUT4 translocation in muscle cells. Rac1 activation occurred in dorsal membrane ruffles of insulin‐stimulated cells as revealed by a novel method to visualize activated Rac1 in situ. We further identified FLJ00068 as a GEF (guanine‐nucleotide‐exchange factor) responsible for this Rac1 activation. Indeed, constitutively active FLJ00068 caused Rac1 activation in dorsal membrane ruffles and GLUT4 translocation without insulin stimulation. Down‐regulation of Rac1 or FLJ00068 by RNA interference, on the other hand, abrogated insulin‐induced GLUT4 translocation. Basal, but not insulin‐stimulated, activity of the serine/threonine kinase Akt was required for the induction of GLUT4 translocation by constitutively active Rac1 or FLJ00068. Conclusion. Collectively, Rac1 activation specifically in membrane ruffles by the GEF FLJ00068 is sufficient for insulin induction of glucose uptake into skeletal‐muscle cells.     

Abnormal glucose metabolism in heterozygous mutant mice for a type I receptor required for BMP signaling

BMPRIA and its high‐affinity ligand BMP4 have recently been shown to be expressed in the β‐cells of the pancreas. Here, we report the abnormalities of heterozygous mice for Bmpr1a in glucose metabolism during the course of intraperitoneal glucose tolerance test. The heterozygous mice had increased blood glucose levels throughout the first 2.5 h after the administration of glucose. Analysis of glucose‐stimulated insulin secretion (GSIS) indicates that insulin secretion in the heterozygous mice is compromised, and induction of secreted insulin by stimulation is substantially lower compared with the wild‐type controls. No apparent abnormalities in pancreas, thyroid, and liver were seen upon histological examination. Real‐time PCR results of selected genes showed an increase in the mRNA level of Ins1 and Ins2 in the heterozygous group. These results indicate that the glucose‐sensing pathway in these heterozygous mice is altered because of the heterozygosity in Bmpr1a. Together, our data suggest that BMP signaling through BMPRIA plays an important role in glucose metabolism and possibly working through the GSIS pathway. genesis 47:385–391, 2009. © 2009 Wiley‐Liss, Inc.     

Nonobese, insulin-deficient Ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling

Although insulin resistance has been traditionally associated with type 2 diabetes, recent evidence in humans and animal models indicates that insulin resistance may also develop in type 1 diabetes. A point mutation of insulin 2 gene in Ins2(Akita) mice leads to pancreatic beta-cell apoptosis and hyperglycemia, and these mice are commonly used to investigate type 1 diabetes and complications. Since insulin resistance plays an important role in diabetic complications, we performed hyperinsulinemic-euglycemic clamps in awake Ins2(Akita) and wild-type mice to measure insulin action and glucose metabolism in vivo. Nonobese Ins2(Akita) mice developed insulin resistance, as indicated by an approximately 80% reduction in glucose infusion rate during clamps. Insulin resistance was due to approximately 50% decreases in glucose uptake in skeletal muscle and brown adipose tissue as well as hepatic insulin action. Skeletal muscle insulin resistance was associated with a 40% reduction in total GLUT4 and a threefold increase in PKCepsilon levels in Ins2(Akita) mice. Chronic phloridzin treatment lowered systemic glucose levels and normalized muscle insulin action, GLUT4 and PKCepsilon levels in Ins2(Akita) mice, indicating that hyperglycemia plays a role in insulin resistance. Echocardiography showed significant cardiac remodeling with ventricular hypertrophy that was ameliorated following chronic phloridzin treatment in Ins2(Akita) mice. Overall, we report for the first time that nonobese, insulin-deficient Ins2(Akita) mice develop type 2 diabetes phenotypes including peripheral and hepatic insulin resistance and cardiac remodeling. Our findings provide important insights into the pathogenesis of metabolic abnormalities and complications affecting type 1 diabetes and lean type 2 diabetes subjects.     

The caveolin‐3 P104L mutation in LGMD‐1C patients inhibits non‐insulin‐stimulated glucose metabolism and growth but promotes myocyte proliferation

The caveolin‐3 (CAV3) protein is known to be specifically expressed in various myocytes, and skeletal muscle consumes most of the blood glucose as an energy source to maintain normal cell metabolism and function. The P104L mutation in the coding sequence of the human CAV3 gene leads to autosomal dominant disease limb‐girdle muscular dystrophy type 1C (LGMD‐1C). We previously reported that C2C12 cells transiently transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis after insulin stimulation. The present study aimed to examine whether the P104L mutation affects C2C12 cell glucose metabolism, growth, and proliferation without insulin stimulation. C2C12 cells stably transfected with CAV3‐P104L were established, and biochemical assays, western blot analysis and confocal microscopy were used to observe glucose metabolism as well as cell growth and proliferation and to determine the effect of the P104L mutation on the PI3K/Akt signaling pathway. Without insulin stimulation, C2C12 cells stably transfected with the P104L CAV3 mutant exhibited decreased glucose uptake and glycogen synthesis, decreased CAV3 expression and reduced localization of CAV3 and GLUT4 on the cell membrane. The P104L mutant significantly reduced the cell diameters, but accelerated cell proliferation. Akt phosphorylation was inhibited, and protein expression of GLUT4, p‐GSK3β, and p‐p70s6K, which are molecules downstream of Akt, was significantly decreased. The CAV3‐P104L mutation inhibits glycometabolism and cell growth but accelerates C2C12 cell proliferation by reducing CAV3 protein expression and cell membrane localization, which may contribute to the pathogenesis of LGMD‐1C.     

Targeted inhibition of KCa3.1 attenuates TGF‐β‐induced reactive astrogliosis through the Smad2/3 signaling pathway

Reactive astrogliosis, characterized by cellular hypertrophy and various alterations in gene expression and proliferative phenotypes, is considered to contribute to brain injuries and diseases as diverse as trauma, neurodegeneration, and ischemia. KCa3.1 (intermediate‐conductance calcium‐activated potassium channel), a potassium channel protein, has been reported to be up‐regulated in reactive astrocytes after spinal cord injury in vivo. However, little is known regarding the exact role of KCa3.1 in reactive astrogliosis. To elucidate the role of KCa3.1 in regulating reactive astrogliosis, we investigated the effects of either blocking or knockout of KCa3.1 channels on the production of astrogliosis and astrocytic proliferation in response to transforming growth factor (TGF)‐β in primary cultures of mouse astrocytes. We found that TGF‐β increased KCa3.1 protein expression in astrocytes, with a concomitant marked increase in the expression of reactive astrogliosis, including glial fibrillary acidic protein and chondroitin sulfate proteoglycans. These changes were significantly attenuated by the KCa3.1 inhibitor 1‐((2‐chlorophenyl) (diphenyl)methyl)‐1H‐pyrazole (TRAM‐34). Similarly, the increase in glial fibrillary acidic protein and chondroitin sulfate proteoglycans in response to TGF‐β was attenuated in KCa3.1^?/? astrocytes. TRAM‐34 also suppressed astrocytic proliferation. In addition, the TGF‐β‐induced phosphorylation of Smad2 and Smad3 proteins was reduced with either inhibition of KCa3.1 with TRAM‐34 or in KCa3.1^?/? astrocytes. These findings highlight a novel role for the KCa3.1 channel in reactive astrogliosis phenotypic modulation and provide a potential target for therapeutic intervention for brain injuries.

     

High Expression of KCa3.1 in Patients with Clear Cell Renal Carcinoma Predicts High Metastatic Risk and Poor Survival

Background

Ca²⁺-activated K⁺ channels have been implicated in cancer cell growth, metastasis, and tumor angiogenesis. Here we hypothesized that high mRNA and protein expression of the intermediate-conductance Ca²⁺-activated K⁺ channel, KCa3.1, is a molecular marker of clear cell Renal Cell Carcinoma (ccRCC) and metastatic potential and survival.

Methodology/Principal Findings

We analyzed channel expression by qRT-PCR, immunohistochemistry, and patch-clamp in ccRCC and benign oncocytoma specimens, in primary ccRCC and oncocytoma cell lines, as well as in two ccRCC cell lines (Caki-1 and Caki-2). CcRCC specimens contained 12-fold higher mRNA levels of KCa3.1 than oncocytoma specimens. The large-conductance channel, KCa1.1, was 3-fold more highly expressed in ccRCC than in oncocytoma. KCa3.1 mRNA expression in ccRCC was 2-fold higher than in the healthy cortex of the same kidney. Disease specific survival trended towards reduction in the subgroup of high-KCa3.1-expressing tumors (p<0.08 vs. low-KCa3.1-expressing tumors). Progression-free survival (time to metastasis/recurrence) was reduced significantly in the subgroup of high-KCa3.1-expressing tumors (p<0.02, vs. low-KCa3.1-expressing tumors). Immunohistochemistry revealed high protein expression of KCa3.1 in tumor vessels of ccRCC and oncocytoma and in a subset of ccRCC cells. Oncocytoma cells were devoid of KCa3.1 protein. In a primary ccRCC cell line and Caki-1/2-ccRCC cells, we found KCa3.1-protein as well as TRAM-34-sensitive KCa3.1-currents in a subset of cells. Furthermore, Caki-1/2-ccRCC cells displayed functional Paxilline-sensitive KCa1.1 currents. Neither KCa3.1 nor KCa1.1 were found in a primary oncocytoma cell line. Yet KCa-blockers, like TRAM-34 (KCa3.1) and Paxilline (KCa1.1), had no appreciable effects on Caki-1 proliferation in-vitro.

Conclusions/Significance

Our study demonstrated expression of KCa3.1 in ccRCC but not in benign oncocytoma. Moreover, high KCa3.1-mRNA expression levels were indicative of low disease specific survival of ccRCC patients, short progression-free survival, and a high metastatic potential. Therefore, KCa3.1 is of prognostic value in ccRCC.     

The isoflavonoid aglycone-rich fractions of Chungkookjang, fermented unsalted soybeans, enhance insulin signaling and peroxisome proliferator-activated receptor-gamma activity in vitro

We investigated anti-diabetic candidates and their mechanisms from the fractions of Chungkookjang (CKJ), a traditional fermented unsalted soybean, by investigating insulin signaling, peroxisome proliferator-activated receptor (PPAR)-gamma activity and glucose-stimulated insulin secretion, in vitro. Cooked soybeans (CSB) and CKJ, fermented predominantly with Bacillus subtilis, were extracted by 70% EtOH followed by an XAD-4 column chromatography with a serial mixture of solvents comprised of MeOH and water. During fermentation, the contents of isoflavonoid aglycones were elevated, and the fractions enriched with aglycones enhanced insulin-stimulated glucose uptake in 3T3-L1 adipocytes. This increase in glucose uptake resulted from stimulating a translocation of the glucose transporter (GLUT)-4 into the plasma membrane through the phosphorylation of insulin receptor substrate (IRS)-1 and Akt. Especially, daidzein enriched fractions elevated insulin-stimulated glucose uptake by acting as PPAR-gamma agonist up to levels exhibited when 10 nM insulin is administered. Fractions containing small peptides with low polarity in CKJ slightly increased glucose-stimulated insulin secretion. The data suggest that an increase in isoflavonoid aglycones in CKJ, in comparison to CSB, enhances glucose utilization via activating insulin signaling and stimulates PPAR-gamma activity in adipocytes. In addition, CKJ contains small peptides improving glucose-stimulated insulin secretion in insulinoma cells. Overall, CKJ is superior to CSB in anti-diabetic action.