Chronic hyperinsulinemia contributes to insulin resistance under dietary restriction in association with altered lipid metabolism in Zucker diabetic fatty rats.

Hyperinsulinemia is widely thought to be a compensatory response to insulin resistance, whereas its potentially causal role in the progression of insulin resistance remains to be established. Here, we aimed to examine whether hyperinsulinemia could affect the progression of insulin resistance in Zucker fatty diabetic (ZDF) rats. Male ZDF rats at 8 wk of age were fed a diet ad libitum (AL) or dietary restriction (DR) of either 15 or 30% from AL feeding over 6 wk. Insulin sensitivity was determined by hyperinsulinemic euglycemic clamp. ZDF rats in the AL group progressively developed hyperglycemia and hyperinsulinemia by 10 wk of age, and then plasma insulin rapidly declined to nearly normal levels by 12 wk of age. Compared with AL group, DR groups showed delayed onset of hyperglycemia and persistent hyperinsulinemia, leading to weight gain and raised plasma triglycerides and free fatty acids by 14 wk of age. Notably, insulin sensitivity was significantly reduced in the DR group rather than the AL group and inversely correlated with plasma levels of insulin and triglyceride but not glucose. Moreover, enhanced lipid deposition and upregulation of genes involved in lipogenesis were detected in liver, skeletal muscle, and adipose tissues of the DR group rather than the AL group. Alternatively, continuous hyperinsulinemia induced by insulin pellet implantation produced a decrease in insulin sensitivity in ZDF rats. These results suggest that chronic hyperinsulinemia may lead to the progression of insulin resistance under DR conditions in association with altered lipid metabolism in peripheral tissues in ZDF rats.

The role of acylated-ghrelin in the regulation of sucrose intake.

The octanoyl modification of ghrelin by ghrelin O-acyltransferase (GOAT) is essential for exerting its physiologic actions. Since exogenous acylated-ghrelin has shown to stimulate food intake in humans and rodents, GOAT has been regarded as a promising target for modulating appetite, thereby treating obesity and diabetes. However, GOAT-knockout (KO) mice have been reported to show no meaningful body weight reduction, when fed a high-fat diet. In this study, we sought to determine whether GOAT has a role in the regulation of body weight and food intake when fed a dietary sucrose. We found that GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, when fed a high-fat + high-sucrose diet. In addition, GOAT KO mice fed a medium-chain triglyceride (MCT) + high-sucrose diet showed a marked resistance to obesity and reduced feed efficiency. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity caused by overconsumption of palatable food.

Pharmacological evaluation of adipose dysfunction via 11ß-hydroxysteroid dehydrogenase type 1 in the development of diabetes in diet-induced obese mice with cortisone pellet implantation.

Signals from intracellular glucocorticoids (GCs) via 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) in adipose tissues have been reported to serve as amplifiers leading to deterioration of glucose metabolism associated with obesity. To elucidate adipose dysfunction via 11ß-HSD1 activation in the development of obesity-related diabetes, we established novel diabetic mice by implanting a cortisone pellet (CP) in diet-induced obesity (DIO) mice. Cortisone pellet-implanted DIO mice (DIO/CP mice) showed hyperglycemia, insulin resistance, hyperlipidemia, and ectopic fat accumulation, whereas cortisone pellet implantation in lean mice did not induce hyperglycemia. In DIO/CP mice, indexes of lipolysis such as plasma glycerol and nonesterified fatty acids (NEFAs) increased before hyperglycemia appeared. Furthermore, the adipose mRNA level of 11ß-HSD1 was up-regulated in DIO/CP mice compared with sham-operated DIO mice. RU486 (mifepristone, 11ß-[p-(dimethylamino)phenyl]-17ß-hydroxy-17-(1-propynyl)estra-4,9-dien-3-one), a glucocorticoid receptor antagonist, decreased adipose mRNA levels of 11ß-HSD1 as well as adipose triglyceride lipase. RU486 also improved plasma NEFA, glycerol, and glucose levels in DIO/CP mice. These results demonstrate that lipolysis in adipose tissues caused by GC activation via 11ß-HSD1 serves as a trigger for diabetes with ectopic fat accumulation. Our findings also indicate the possibility of a vicious circle of GC signals via 11ß-HSD1 up-regulation in adipose tissues, contributing to deterioration of glucose metabolism to result in diabetes. Our DIO/CP mouse could be a suitable model of type 2 diabetes to evaluate adipose dysfunction via 11ß-HSD1.

Decrease in cytosolic phospholipase A2alpha mRNA levels by reactive oxygen species via MAP kinase pathways in PC12 cells: effects of dopaminergic neurotoxins.

Excess production of reactive oxygen species (ROS), including H2O2, leads to neuronal death in pathological conditions. Although ROS stimulates alpha-type cytosolic phospholipase A2 (cPLA2alpha) activity, their role in cPLA2alpha expression has not been elucidated. We investigated the effect of ROS on cPLA2alpha mRNA levels and signaling pathways in rat pheochromocytoma PC12 cells. Treatment with H2O2 and xanthine-xanthine oxidase (X/XO) for 4 h decreased cPLA2alpha mRNA levels without changing the mRNA levels of other tested proteins. H2O2 and X/XO caused cell toxicity not after 4 h but 24 h after their addition. The H2O2-induced decrease in cPLA2alpha mRNA levels was inhibited in cells treated with N-acetyl-cysteine and selective inhibitors of mitogen-activated protein kinase (MAPK) pathways (extracellular signal-regulated kinase and p38 MAPK). Treatment with dopaminergic neurotoxins, including 1,2,3,4-tetrahydroisoquinoline (TIQ)-inducing ROS formation, decreased cPLA2alpha mRNA levels. These findings suggest that ROS decreases cPLA2alpha mRNA levels via MAPK pathways in PC12 cells.

Ghrelin O-acyltransferase knockout mice show resistance to obesity when fed high-sucrose diet.

Ghrelin is an appetite-stimulating hormone secreted from stomach. Since the discovery that acylation of the serine-3 residue by ghrelin O-acyltransferase (GOAT) is essential for exerting its functions, GOAT has been regarded as an therapeutic target for attenuating appetite, and thus for the treatment of obesity and diabetes. However, contrary to the expectations, GOAT-knockout (KO) mice have not shown meaningful body weight reduction, under high-fat diet. Here, in this study, we sought to determine whether GOAT has a role in body weight regulation and glucose metabolism with a focus on dietary sucrose, because macronutrient composition of diet is important for appetite regulation. We found that peripherally administered acylated-ghrelin, but not unacylated one, stimulated sucrose consumption in a two-bottle-drinking test. The role of acylated-ghrelin in sucrose preference was further supported by the finding that GOAT KO mice consumed less sucrose solution compared with WT littermates. Then, we investigated the effect of dietary composition of sucrose on food intake and body weight in GOAT KO and WT mice. As a result, when fed on high-fat diet, food intake and body weight were similar between GOAT KO and WT mice. However, when fed on high-fat, high-sucrose diet, GOAT KO mice showed significantly reduced food intake and marked resistance to obesity, leading to amelioration of glucose metabolism. These results suggest that blockade of acylated-ghrelin production offers therapeutic potential for obesity and metabolic disorders caused by overeating of palatable food.

Reduced intake of carbohydrate prevents the development of obesity and impaired glucose metabolism in ghrelin O-acyltransferase knockout mice.

A close relationship between acylated-ghrelin and sucrose intake has been reported. However, little has been examined about the physiological action of ghrelin on preference for different types of carbohydrate such as glucose, fructose, and starch. The current study was aimed to investigate the role of acylated-ghrelin in the determinants of the choice of carbohydrates, and pathogenesis of chronic disorders, including obesity and insulin resistance. In a two-bottle-drinking test, ghrelin O-acyltransferase (GOAT) knockout (KO) mice consumed a less amount of glucose and maltodextrin, and almost the same amount of fructose and saccharin solution compared to WT littermates. The increased consumption of glucose and maltodextrin was observed when acylated-ghrelin, but not unacylated-ghrelin, was exogeneously administered in normal C57BL/6J mice, suggesting an association of acylated-ghrelin with glucose-containing carbohydrate intake. When fed a diet rich in maltodextrin, starch and fat for 12 weeks, GOAT KO mice showed less food intake and weight gain, as well as improved glucose tolerance and insulin sensitivity than WT mice. Our data suggests that blockade of GOAT activity may offer a therapeutic option for treatment of obesity and its associated metabolic syndrome by preventing from overconsumption of carbohydrate-rich food.

Expression of vanilloid VR1 receptor in PC12 cells.

Capsaicin, a pungent ingredient of chili pepper, activates vanilloid receptor subtype 1 (VR1), which is a nonselective cation channel with high Ca(2+) permeability. Although VR1 and its splice variant are highly expressed in sensory neurons, they are expressed in neuronal cells in brain and peripheral non-neuronal cells. In this study, we investigated whether VR1 is expressed in PC12 cells, rat pheochromocytoma. Capsaicin at concentrations above 100 microM induced an increase in intracellular free Ca(2+) concentrations by influx from extracellular spaces, and the effect was blocked by capsazepine, a selective antagonist of VR1. VR1 transcript and protein were detected by reverse transcription-polymerase chain reaction and Western blotting analysis, respectively. Immunocytochemical analysis revealed that VR1 protein was expressed in the cytosol and the plasma membrane of PC12 cells, and treatment with the antisense oligonucleotide for VR1 decreased the expression. VR1 in PC12 cells showed different characters from that in sensory neurons; capsaicin concentration-dependency and heat- and nerve growth factor-sensitivities. These results suggested that VR1 was functionally expressed in PC12 cells. The usefulness of PC12 cell line for studying functions and/or expression of VR1 is discussed.

Expression of adhesion molecules by sphingosine 1-phosphate and histamine in endothelial cells.

We investigated the effects of sphingosine 1-phosphate and histamine on the expression of vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and E-selectin, and their signaling pathways in human umbilical vein endothelial cells. Sphingosine 1-phosphate increased the mRNA and protein level of VCAM-1, and the mRNAs of E-selectin and ICAM-1. The effects of sphingosine 1-phosphate were inhibited by the pertussis toxin and the respective inhibitors (10 microM 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione (U73122) for phosphoinositide-specific phospholipase C; 10 microM 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole (SB203580) for p38 mitogen-activated protein kinase (MAPK); 1 microM 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(3,4-c)-carbazole (Gö6976) for the alpha form of protein kinase C (PKC-alpha)), but not by a PKC-delta inhibitor (1 microM rottlerin). Histamine, which alone showed no effect, enhanced the sphingosine 1-phosphate-induced expressions via histamine H(1) receptor. The histamine response decreased by U73122 and rottlerin, but not by SB203580 and Gö6976. The effects of sphingosine 1-phosphate with and without histamine were abolished by the higher concentrations of PKC inhibitors and in the PKC-depleted cells. Sphingosine 1-phosphate and histamine alone stimulated phosphorylation of p38 MAPK in a phosphoinositide-specific phospholipase C-dependent but not in a PKCs-independent manner. These findings suggest that sphingosine 1-phosphate-induced expression of adhesion molecules was mediated by phosphoinositide-specific phospholipase C and preferentially by PKC-alpha and p38 MAPK, and the histamine response was mediated by phosphoinositide-specific phospholipase C and PKC-delta in human umbilical vein endothelial cells.

Nerve growth factor-induced up-regulation of cytosolic phospholipase A2alpha level in rat PC12 cells.

Nerve growth factor (NGF) regulates various types of gene transcription in neurons. One of the cytosolic phospholipase A(2)s, cPLA(2)alpha, which preferentially cleaves phospholipids at the sn-2 position to arachidonic acid (AA), is involved in neuronal responses including survival. We investigated the effect of NGF on cPLA(2)alpha expression and its signaling pathways in PC12 cells, which differentiate into neuronal-like cells with neurites by NGF treatment. Treatment with NGF increased cPLA(2)alpha mRNA level after 4h and its protein level 24h after NGF addition. The NGF-induced increase in cPLA(2)alpha mRNA was inhibited by actinomycin D. NGF caused phosphorylation of mitogen-activated protein kinases (MAPKs); sustained phosphorylation of extracellular-regulated kinases (ERK1/2) and transient phosphorylation of p38 MAPK. NGF responses (cPLA(2)alpha mRNA and its protein) were inhibited by selective inhibitors for the ERK1/2 pathway, p38 MAPK and c-Jun NH(2)-terminal kinase. Epidermal growth factor, which transiently activates ERK1/2, did not modify cPLA(2)alpha expression. Although phorbol 12-myristate 13-acetate, an activator of protein kinase C (PKC), alone showed no effect, NGF-induced cPLA(2)alpha mRNA expression decreased due to the inhibition of PKC. These findings suggest that NGF-induced cPLA(2)alpha expression is regulated by gene transcription via the ERK1/2, p38 MAPK and PKC pathways in PC12 cells.

Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement.

Up-regulation of cytosolic phospholipase A2alpha expression by N,N-diethyldithiocarbamate in PC12 cells; involvement of reactive oxygen species and nitric oxide.

Disulfiram (an alcohol-aversive drug) and related compounds are known to provoke several side effects involving behavioral and neurological complications. N,N-diethyldithiocarbamate (DDC) is considered as one of the main toxic species of disulfiram and acts as an inhibitor of superoxide dismutase. Since arachidonic acid (AA) formation is regulated by reactive oxygen species (ROS) and related to toxicity in neuronal cells, we investigated the effects of DDC on AA release and expression of the alpha type of cytosolic phospholipase A(2) (cPLA(2)alpha) in PC12 cells. Treatment with 80-120 microM DDC that causes a moderate increase in ROS levels without cell toxicity stimulated cPLA(2)alpha mRNA and its protein expression. The expression was mediated by extracellular-signal-regulated kinase (ERK1/2), one of the mitogen-activated protein kinases. Treatment with N(G) nitro-L-arginine methyl ester (an inhibitor of nitric oxide synthase, 1 mM) and oxy-hemoglobin (a scavenger of nitric oxide, 2 mg/mL) abolished the DDC-induced responses (ERK1/2 phosphorylation and cPLA(2)alpha expression). We also showed DDC-induced up-regulation of the mRNA expression of lipocortin 1, an inhibitor of PLA(2). Furthermore, DDC treatment of the cells enhanced Ca(2+)-ionophore-induced AA release in 30 min, although the effect was limited. Changes in AA metabolism in DDC-treated cells may have a potential role in mediating neurotoxic actions of disulfiram. In this study, we show the first to demonstrate the up-regulation of cPLA(2)alpha expression by DDC treatment in neuronal cells.

Possible role of interleukin-6 in PC12 cell death induced by MPP+ and tetrahydroisoquinoline.

Interleukin (IL)-6 has been shown to protect neuronal cells from cell death induced by various stimulants. Although neuronal cells including PC12 cells were shown to produce IL-6, little is known about the effects of dopaminergic neurotoxins, 1,2,3,4-tetrahydroisoquinoline (TIQ) and 1-methyl-4-phenylpyridinium ion (MPP(+)), on IL-6 expression in PC12 cells. In the present study, we investigated the role of IL-6 in the TIQ- and MPP(+)-induced cell death in PC12 cells. Treatment with 3.2 mM TIQ for 24 h caused a delayed cell death (lactate dehydrogenase (LDH) leakage and nuclear DNA fragmentation) markedly 72 h after the addition. Addition of 0.4 mM MPP(+) caused LDH leakage and nuclear DNA fragmentation 24 h after the addition. The cell death induced by MPP(+) was inhibited by an inhibitor of caspases, z-Val-Ala-Asp(OMe)-fluoromethylketone. The cell death induced by TIQ or MPP(+) was inhibited by nerve growth factor and 10% serum and significantly enhanced by the treatment with anti-IL-6 antibody. Both neurotoxins decreased the IL-6 mRNA level in PC12 cells without changing the other tested mRNA levels (IL-1 alpha, beta-actin, etc.). These findings suggest that dopaminergic neurotoxins cause cell death in PC12 cells at least partially by changing IL-6 expression.