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Increased expression and activity of the intracellular glucocorticoid-reactivating enzyme 11[beta]-hydroxysteroid dehydrogenase type 1 (11[beta]-HSD1) contribute to dysfunction of adipose tissue. Although the pathophysiological role of 11[beta]-HSD1 in mature adipocytes has long been investigated, its potential role in preadipocytes still remains obscure. The present study demonstrates that the expression of 11[beta]-HSD1 in preadipocyte-rich stromal vascular fraction (SVF) cells in fat depots from ob/ob and diet-induced obese mice was markedly elevated compared with lean control. In 3T3-L1 preadipocytes, the level of mRNA and reductase activity of 11[beta]-HSD1 was augmented by TNF-[alpha], IL-1[beta], and LPS, with a concomitant increase in inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), or IL-6 secretion. Pharmacological inhibition of 11[beta]-HSD1 and RNA interference against 11[beta]-HSD1 reduced the mRNA and protein levels of iNOS, MCP-1, and IL-6. In contrast, overexpression of 11[beta]-HSD1 further augmented TNF-[alpha]-induced iNOS, IL-6, and MCP-1 expression. Moreover, 11[beta]-HSD1 inhibitors attenuated TNF-[alpha]-induced phosphorylation of NF-[kappa]B p65 and p38-, JNK-, and ERK1/2-MAPK. Collectively, the present study provides novel evidence that inflammatory stimuli-induced 11[beta]-HSD1 in activated preadipocytes intensifies NF-[kappa]B and MAPK signaling pathways and results in further induction of proinflammatory molecules. Not limited to 3T3-L1 preadipocytes, we also demonstrated that the notion was reproducible in the primary SVF cells from obese mice. These findings highlight an unexpected, proinflammatory role of reamplified glucocorticoids within preadipocytes in obese adipose tissue. 11[beta]-hydroxysteroid dehydrogenase type 1; preadipocyte; nuclear factor-[kappa]B; mitogen-activated protein kinase; adipose inflammation doi: 10.1152/ajpendo.00320.2009.

Ghrelin is a stomach-derived peptide that has growth hormone-stimulating and orexigenic activities. Although there have been several reports of ghrelinoma cases, only a few cases have elevated circulating ghrelin levels, hampering the investigation of pathophysiological features of ghrelinoma and chronic effects of ghrelin excess. Furthermore, standard transgenic technique has resulted in desacyl ghrelin production only because of the limited tissue expression of ghrelin O-acyltransferase, which mediates acylation of ghrelin. Accordingly, we attempted to create ghrelin promoter SV40 T-antigen transgenic (GP-Tag Tg) mice, in which ghrelin-producing cells continued to proliferate and finally developed into ghrelinoma. Adult GP-Tag Tg mice showed elevated plasma ghrelin levels with preserved physiological regulation. Adult GP-Tag Tg mice with increased plasma ghrelin levels exhibited elevated IGF-I levels despite poor nutrition. Although basal growth hormone levels were not changed, those after growth hormone-releasing hormone injection tended to be higher. These results indicate that chronic elevation of ghrelin activates GH-IGF-I axis. In addition, GP-Tag Tg mice demonstrated glucose intolerance. Insulin secretion by glucose tolerance tests was significantly attenuated in GP-Tag Tg, whereas insulin sensitivity determined by insulin tolerance tests was preserved, indicating that chronic elevation of ghrelin suppresses insulin secretion and leads to glucose intorelance. Thus, we successfully generated a Tg model of ghrelinoma, which is a good tool to investigate chronic effects of ghrelin excess. Moreover, their characteristic features could be a hint on ghrelinoma. ghrelin; glucose metabolism

Obesity is characterized by markedly decreased ghrelin and growth hormone (GH) secretion. Ghrelin is a GH-stimulating, stomach-derived peptide that also has orexigenic action. Ghrelin supplement may restore decreased GH secretion in obesity, but it may worsen obesity by its orexigenic action. To reveal effects of ghrelin administration on obese animals, we first examined acute GH and orexigenic responses to ghrelin in three different obese and/or diabetic mouse models: db/db mice, mice on a high-fat diet (HFD mice), and Akita mice for comparison. GH responses to ghrelin were significantly suppressed in db/db, HFD, and Akita mice. Food intake of db/db and Akita mice were basally higher, and further stimulation of food intake by ghrelin was suppressed. Pituitary GH secretagogue receptor mRNA levels in db/db and HFD mice were significantly decreased, which may partly contribute to decreased GH response to ghrelin in these mice. In Akita mice for comparison, decreased hypothalamic GH-releasing hormone (GHRH) mRNA levels may be responsible for decreased GH response, since maximum GH response to ghrelin needs GHRH. When ghrelin was injected into HFD mice with GHRH coadministrated, GH responses to ghrelin were significantly emphasized. HFD mice injected with low-dose ghrelin and GHRH for 10 days did not show weight gain. These results indicate that low-dose ghrelin and GHRH treatment may restore decreased GH secretion in obesity without worsening obesity. growth hormone secretagogue receptor; obesity; diabetes

A clinically employed antihyperlipidemic drug, bezafibrate, has been characterized as a PPAR([alpha], -[gamma] and -[delta]) pan-agonist in vitro. Recent extended trials have highlighted its antidiabetic properties in humans. However, the underlying molecular mechanism is not fully elucidated. The present study was designed to explore potential regulatory mechanisms of intracellular glucocorticoid reactivating enzyme, 11[beta]-HSD1 and anti-diabetic hormone, adiponectin by bezafibrate in murine adipose tissue, and cultured adipocytes. Treatment of db/db mice with bezafibrate significantly ameliorated hyperglycemia and insulin resistance, accompanied by a marked reduction of triglyceride and nonesterified fatty acids. Despite equipotent in lipid-lowering effects, another fibrate, fenofibrate, did not show such beneficial effects on glycemic control. Treatment of bezafibrate caused a marked decrease in the mRNA level of 11[beta]-HSD1 preferentially in adipose tissue of db/db mice (-47%, P < 0.05), concomitant with a significant increase in plasma adiponectin level (+37%, P < 0.01). Notably, treatment of bezafibrate caused a marked decrease in the mRNA level (-34%, P < 0.01) and enzyme activity (-32%, P < 0.01) of 11[3-HSD1, whereas the treatment substantially augmented the expression (+71%, P < 0.01) and secretion (+27%, P < 0.01) of adiponectin in 3T3-L1 adipocytes. Knockdown of 11[beta]-HSD1 by siRNA confirmed that 11[beta]-HSD1 acts as a distinct oxoreductase in adipocytes and validated the enzyme activity assays in the present study. Effects of bezafibrate on regulation of 11[beta]-HSD1 and adiponectin in murine adipocytes were comparable with those in thiazolidinediones. This is the first demonstration that bezafibrate directly regulates 11[beta]-HSD 1 and adiponectin in murine adipocytes, both of which may contribute to metabolicallybeneficial effects by bezafibrate. metabolic syndrome; adiponectin

Skeletal muscle expresses two catalytic subunits, [alpha]1 and [alpha]2, of the 5'-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the [alpha]2-containing AMPK complex (AMPK[alpha]2), but this activation may occur with or without activation of the [alpha]1-containing AMPK complex (AMPK[alpha]1), suggesting that AMPK[alpha]2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPK[alpha]1, but not AMPK[alpha]2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPK[alpha]1 activity increased twofold and AMPK[alpha]2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of [Thr.sup.172] of the [alpha]-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-D-glucose transport. In contrast, increasing the frequency ([greater than or equal to] 5 Hz) or duration ([greater than or equal to] 5 min) of contraction activated AMPK[alpha]1 and AMPK[alpha]2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPK[alpha]1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPK[alpha]2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle. exercise; twitch; glucose transport; acetyl-coenzyme A carboxylase; [beta]-oxidation

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent [H.sub.2][O.sub.2], AMPK[alpha]1 activity increased in a time- and dose-dependent manner, whereas AMPK[alpha]2 activity remained unchanged. The activation of AMPK[alpha]1 was associated with phosphorylation of AMPK [Thr.sup.172], suggesting that an upstream kinase is involved in the activation process. [H.sub.2][O.sub.2]-induced AMPK[alpha]1 activation was blocked in the presence of the antioxidant N-acetyl-L-cysteine (NAC), and [H.sub.2][O.sub.2] significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. [H.sub.2][O.sub.2] did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. [H.sub.2][O.sub.2] increased 3-O-methyl-D-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPK[alpha]1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPK[alpha]1-mediated mechanism. contraction; epitrochlearis muscle; hydrogen peroxide; hypoxanthine: xanthine oxidase

The expression of the obese (ob) gene in the adipose tissue of KK mice and congenic lethal yellow obese KKA(super y) mice is dependent on the nutritional status of the mice. The ob mRNA levels in epididymal, mesenteric and subcutaneous white adipose tissue (WAT) are equal in four wks old KK and KKA(super y) mice. However, it increases in the mesenteric and subcutaneous WAT of KKA(super y) mice at 12 wks and the mice develop obesity. Fasting decreases ob gene expression in the epididymal and mesenteric WAT but fails to affect ob gene expression in subcutaneous WAT.

Skeletal muscle expresses two catalytic subunits, α1 and α2, of the 5′-AMP-activated protein kinase (AMPK), which has been implicated in contraction-stimulated glucose transport and fatty acid oxidation. Muscle contraction activates the α2-containing AMPK complex (AMPKα2), but this activation may occur with or without activation of the α1-containing AMPK complex (AMPKα1), suggesting that AMPKα2 is the major isoform responsible for contraction-induced metabolic events in skeletal muscle. We report for the first time that AMPKα1, but not AMPKα2, can be activated in contracting skeletal muscle. Rat epitrochlearis muscles were isolated and incubated in Krebs-Ringer bicarbonate buffer containing pyruvate. In muscles stimulated to contract at a frequency of 1 and 2 Hz during the last 2 min of incubation, AMPKα1 activity increased twofold and AMPKα2 activity remained unchanged. Muscle stimulation did not change the muscle AMP concentration or the AMP-to-ATP ratio. AMPK activation was associated with increased phosphorylation of Thr172 of the α-subunit, the primary activation site. Muscle stimulation increased the phosphorylation of acetyl-CoA carboxylase (ACC), a downstream target of AMPK, and the rate of 3-O-methyl-D-glucose transport. In contrast, increasing the frequency (≥5 Hz) or duration (≥5 min) of contraction activated AMPKα1 and AMPKα2 and increased AMP concentration and the AMP/ATP ratio. These results suggest that 1) AMPKα1 is the predominant isoform activated by AMP-independent phosphorylation in low-intensity contracting muscle, 2) AMPKα2 is activated by an AMP-dependent mechanism in high-intensity contracting muscle, and 3) activation of each isoform enhances glucose transport and ACC phosphorylation in skeletal muscle. [ABSTRACT FROM AUTHOR]

Recent studies have suggested that 5′AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H2O2, AMPKα1 activity increased in a time- and dose-dependent manner, whereas AMPKα2 activity remained unchanged. The activation of AMPKα1 was associated with phosphorylation of AMPK Thr172, suggesting that an upstream kinase is involved in the activation process. H2O2-induced AMPKα1 activation was blocked in the presence of the antioxidant N-acetyl-L-cysteine (NAC), and H2O2 significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H2O2 did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H2O2 increased 3-O-methyl-D-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKα1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKα1- mediated mechanism. [ABSTRACT FROM AUTHOR]

Leptin may reduce pancreatic lipid deposition, which increases with progression of obesity and can impair β-cell function. The insulinotropic effect of glucagon-like peptide-1 (GLP-1) and the efficacy of GLP-1 receptor agonist are reduced associated with impaired β-cell function. In this study, we examined whether leptin could restore the efficacy of exenatide, a GLP-1 receptor agonist, in type 2 diabetes with increased adiposity. We chronically administered leptin (500 μg·kg⁻¹·day⁻¹) and/or exenatide (20 μg·kg⁻¹·day⁻¹) for 2 wk in a mouse model of type 2 diabetes with increased adiposity induced by streptozotocin and high-fat diet (STZ/HFD mice). The STZ/HFD mice exhibited hyperglycemia, overweight, increased pancreatic triglyceride level, and reduced glucose-stimulated insulin secretion (GSIS); moreover, the insulinotropic effect of exenatide was reduced. However, leptin significantly reduced pancreatic triglyceride level, and adding leptin to exenatide (LEP/EX) remarkably enhanced GSIS. These results suggested that the leptin treatment restored the insulinotropic effect of exenatide in the mice. In addition, LEP/EX reduced food intake, body weight, and triglyceride levels in the skeletal muscle and liver, and corrected hyperglycemia to a greater extent than either monotherapy. The pair-feeding experiment indicated that the marked reduction of pancreatic triglyceride level and enhancement of GSIS by LEP/EX occurred via mechanisms other than calorie restriction. These results suggest that leptin treatment may restore the insulinotropic effect of exenatide associated with the reduction of pancreatic lipid deposition in type 2 diabetes with increased adiposity. Combination therapy with leptin and exenatide could be an effective treatment for patients with type 2 diabetes with increased adiposity., (Copyright © 2014 the American Physiological Society.)

Previously, we reported that exogenous administration of ghrelin ameliorates glucose metabolism in a neonate streptozotocin (STZ)-induced diabetic rat model through enhancement of β-cell proliferation. However, it was not clear whether the observed β-cell proliferation was a direct or indirect effect (e.g., via orexigenic or growth hormone-stimulated pathways) of ghrelin activity. Here, we aimed to investigate whether ghrelin directly impacts β-cell proliferation after STZ-induced injury in mice. Seven-week-old male rat insulin II promoter-ghrelin internal ribosomal sequence ghrelin O-acyltransferase transgenic (RIP-GG Tg) mice, which have elevated pancreatic ghrelin levels, but only minor changes in plasma ghrelin levels when fed a medium-chain triglyceride-rich diet, were treated with STZ. Then, serum insulin, pancreatic insulin mRNA expression, and islet histology were evaluated. We found that the serum insulin levels, but not blood glucose levels, of RIP-GG Tg mice were significantly ameliorated 14 days post-STZ treatment. Pancreatic insulin mRNA expression was significantly elevated in RIP-GG Tg mice, and β-cell numbers in islets were increased. Furthermore, the number of phospho-histone H3⁺ or Ki67⁺ proliferating β-cells was significantly elevated in RIP-GG Tg mice, whereas the apoptotic indexes within the islets, as determined by TUNEL assay, were not changed. These results indicate that ghrelin can directly stimulate β-cell proliferation in vivo after β-cell injury even without its orexigenic or GH-stimulating activities, although it did not have enough impact to normalize the glucose tolerance in adult mice.

Leptin enhances insulin sensitivity in addition to reducing food intake and body weight. Recently, amylin, a pancreatic β-cell-derived hormone, was shown to restore a weight-reducing effect of leptin in leptin-resistant diet-induced obesity. However, whether amylin improves the effect of leptin on insulin sensitivity in diet-induced obesity is unclear. Diet-induced obese (DIO) mice were infused with either saline (S), leptin (L; 500 μg·kg⁻¹·day⁻¹), amylin (A; 100 μg·kg⁻¹·day⁻¹), or leptin plus amylin (L/A) for 14 days using osmotic minipumps. Food intake, body weight, metabolic parameters, tissue triglyceride content, and AMP-activated protein kinase (AMPK) activity were examined. Pair-feeding and weight-matched calorie restriction experiments were performed to assess the influence of food intake and body weight reduction. Continuous L/A coadministration significantly reduced food intake, increased energy expenditure, and reduced body weight, whereas administration of L or A alone had no effects. L/A coadministration did not affect blood glucose levels during ad libitum feeding but decreased plasma insulin levels significantly (by 48%), suggesting the enhancement of insulin sensitivity. Insulin tolerance test actually showed the increased effect of insulin in L/A-treated mice. In addition, L/A coadministration significantly decreased tissue triglyceride content and increased AMPKα2 activity in skeletal muscle (by 67%). L/A coadministration enhanced insulin sensitivity more than pair-feeding and weight-matched calorie restriction. In conclusion, this study demonstrates the beneficial effect of L/A coadministration on glucose and lipid metabolism in DIO mice, indicating the possible clinical usefulness of L/A coadministration as a new antidiabetic treatment in obesity-associated diabetes.

Whereas ghrelin is produced primarily in the stomach, a small amount of it is produced in pancreatic islets. Although exogenous administration of ghrelin suppresses insulin secretion in vitro or in vivo, the role of intraislet ghrelin in the regulation of insulin secretion in vivo remains unclear. To understand the physiological role of intraislet ghrelin in insulin secretion and glucose metabolism, we developed a transgenic (Tg) mouse model, rat insulin II promoter ghrelin-internal ribosomal entry site-ghrelin O-acyl transferase (RIP-GG) Tg mice, in which mouse ghrelin cDNA and ghrelin O-acyltransferase are overexpressed under the control of the rat insulin II promoter. Although pancreatic desacyl ghrelin levels were elevated in RIP-GG Tg mice, pancreatic ghrelin levels were not altered in animals on a standard diet. However, when Tg mice were fed a medium-chain triglyceride-rich diet (MCTD), pancreatic ghrelin levels were elevated to ∼16 times that seen in control animals. It seems likely that the gastric ghrelin cells possess specific machinery to provide the octanoyl acid necessary for ghrelin acylation but that this machinery is absent from pancreatic β-cells. Despite the overexpression of ghrelin, plasma ghrelin levels in the portal veins of RIP-GG Tg mice were unchanged from control levels. Glucose tolerance, insulin secretion, and islet architecture in RIP-GG Tg mice were not significantly different even when the mice were fed a MCTD. These results indicate that intraislet ghrelin does not play a major role in the regulation of insulin secretion in vivo.