AJP: Endocrinology and Metabolism current issue

Adipose tissue function and dysfunction: organ cross talk and metabolic risk

Giorgino, F. — Wed, 21 Oct 2009 14:10:13 PDT

Transdifferentiation properties of adipocytes in the adipose organ

Cinti, S. — Wed, 21 Oct 2009 14:10:13 PDT

Mammals have two types of adipocytes, white and brown, but their anatomy and physiology is different. White adipocytes store lipids, and brown adipocytes burn them to produce heat. Previous descriptions implied their localization in distinct sites, but we demonstrated that they are mixed in many depots, raising the concept of adipose organ. We explain the reason for their cohabitation with the hypothesis of reversible physiological transdifferentiation; they are able to convert one into each other. If needed, the brown component of the organ could increase at the expense of the white component and vice versa. This plasticity is important because the brown phenotype of the organ associates with resistance to obesity and related disorders. Another example of physiological transdifferetiation of adipocytes is offered by the mammary gland; the pregnancy hormonal stimuli seems to trigger a reversible transdifferentiation of adipocytes into milk-secreting epithelial glands. The obese adipose organ is infiltrated by macrophages inducing chronic inflamation that is widely considered as a causative factor for insulin resistance. We showed that the vast majority of macrophages infiltrating the obese organ are arranged around dead adipocytes, forming characteristic crown-like structures. We recently found that visceral fat is more infiltrated than the subcutaneous fat despite a smaller size of visceral adipocytes. This suggests a different susceptibility of visceral and subcutaneous adipocytes to death, raising the concept of smaller critical death size that could be important to explain the key role of visceral fat for the metabolic disorders associated with obesity.

The origin of intermuscular adipose tissue and its pathophysiological implications

Wed, 21 Oct 2009 14:10:13 PDT

The intermuscular adipose tissue (IMAT) is a depot of adipocytes located between muscle bundles. Several investigations have recently been carried out to define the phenotype, the functional characteristics, and the origin of the adipocytes present in this depot. Among the different mechanisms that could be responsible for the accumulation of fat in this site, the dysdifferentiation of muscle-derived stem cells or other mesenchymal progenitors has been postulated, turning them into cells with an adipocyte phenotype. In particular, muscle satellite cells (SCs), a heterogeneous stem cell population characterized by plasticity and self-renewal that allow muscular growth and regeneration, can acquire features of adipocytes, including the abilities to express adipocyte-specific genes and accumulate lipids. Failure to express the transcription factors that direct mesenchymal precursors into fully differentiated functionally specialized cells may be responsible for their phenotypic switch into the adipogenic lineage. We proved that human SCs also possess a clear adipogenic potential that could explain the presence of mature adipocytes within skeletal muscle. This occurs under some pathological conditions (i.e., primary myodystrophies, obesity, hyperglycemia, high plasma free fatty acids, hypoxia, etc.) or as a consequence of thiazolidinedione treatment or simply because of a sedentary lifestyle or during aging. Several pathways and factors (PPARs, WNT growth factors, myokines, GEF-GAP-Rho, p66^shc, mitochondrial ROS production, PKCβ) could be implicated in the adipogenic conversion of SCs. The understanding of the molecular pathways that regulate muscle-to-fat conversion and SC behavior could explain the increase in IMAT depots that characterize many metabolic diseases and age-related sarcopenia.

Inflammation and impaired adipogenesis in hypertrophic obesity in man

Wed, 21 Oct 2009 14:10:13 PDT

Obesity is associated mainly with adipose cell enlargement in adult man (hypertrophic obesity), whereas the formation of new fat cells (hyperplastic obesity) predominates in the prepubertal age. Adipose cell size, independent of body mass index, is negatively correlated with whole body insulin sensitivity. Here, we review recent findings linking hypertrophic obesity with inflammation and a dysregulated adipose tissue, including local cellular insulin resistance with reduced IRS-1 and GLUT4 protein content. In addition, the number of preadipocytes in the abdominal subcutaneous adipose tissue capable of undergoing differentiation to adipose cells is reduced in hypertrophic obesity. This is likely to promote ectopic lipid accumulation, a well-known finding in these individuals and one that promotes insulin resistance and cardiometabolic risk. We also review recent results showing that TNF, but not MCP-1, resistin, or IL-6, completely prevents normal adipogenesis in preadipocytes, activates Wnt signaling, and induces a macrophage-like phenotype in the preadipocytes. In fact, activated preadipocytes, rather than macrophages, may completely account for the increased release of chemokines and cytokines by the adipose tissue in obesity. Understanding the molecular mechanisms for the impaired preadipocyte differentiation in the subcutaneous adipose tissue in hypertrophic obesity is a priority since it may lead to new ways of treating obesity and its associated metabolic complications.

Role of lipid-derived mediators in skeletal muscle insulin resistance

Taube, A., Eckardt, K., Eckel, J. — Wed, 21 Oct 2009 14:10:13 PDT

Imbalance between nutritional intake and energy expenditure has been described to culminate in obesity, which predisposes to insulin resistance and type 2 diabetes mellitus. In such states of energy oversupply, excess amounts of lipids are available in tissues and circulation. Over the past years, an increasingly important role in development of skeletal muscle (SkM) insulin resistance has been attributed to lipids and impaired fatty acid metabolism. In this review, we reflect the current state of knowledge about the effects of various lipid-derived mediators on SkM insulin sensitivity. Furthermore, potential mechanisms underlying the biogenesis of intramyocellular ectopic lipid stores are discussed. Previously, a pivotal role was attributed to mitochondrial dysfunction. However, results of recent studies have suggested an important role for exercise deficiency, accompanied by decreased expression levels of peroxisome proliferator-activated receptor- coactivator-1 and subsequent, incomplete β-oxidation. Additionally, we summarize the implications of increased levels of lipid-derived endocannabinoids (ECs) for metabolic control in peripheral tissue and highlight the benefits of targeting the EC system.

Critical roles for the TSC-mTOR pathway in {beta}-cell function

Wed, 21 Oct 2009 14:10:13 PDT

TSC1 is a tumor suppressor that associates with TSC2 to inactivate Rheb, thereby inhibiting signaling by the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). mTORC1 stimulates cell growth by promoting anabolic cellular processes, such as translation, in response to growth factors and nutrient signals. To test roles for TSC1 and mTORC1 in β-cell function, we utilized Rip2/Cre to generate mice lacking Tsc1 in pancreatic β-cells (Rip-Tsc1cKO mice). Although obesity developed due to hypothalamic Tsc1 excision in older Rip-Tsc1cKO animals, young animals displayed a prominent gain-of-function β-cell phenotype prior to the onset of obesity. The young Rip-Tsc1cKO animals displayed improved glycemic control due to mTOR-mediated enhancement of β-cell size, mass, and insulin production but not determinants of β-cell number (proliferation and apoptosis), consistent with an important anabolic role for mTOR in β-cell function. Furthermore, mTOR mediated these effects in the face of impaired Akt signaling in β-cells. Thus, mTOR promulgates a dominant signal to promote β-cell/islet size and insulin production, and this pathway is crucial for β-cell function and glycemic control.

Comparison between surrogate indexes of insulin sensitivity/resistance and hyperinsulinemic euglycemic clamp estimates in rats

Wed, 21 Oct 2009 14:10:13 PDT

Assessing insulin resistance in rodent models gives insight into mechanisms that cause type 2 diabetes and the metabolic syndrome. The hyperinsulinemic euglycemic glucose clamp, the reference standard for measuring insulin sensitivity in humans and animals, is labor intensive and technically demanding. A number of simple surrogate indexes of insulin sensitivity/resistance have been developed and validated primarily for use in large human studies. These same surrogates are also frequently used in rodent studies. However, in general, these indexes have not been rigorously evaluated in animals. In a recent validation study in mice, we demonstrated that surrogates have a weaker correlation with glucose clamp estimates of insulin sensitivity/resistance than in humans. This may be due to increased technical difficulties in mice and/or intrinsic differences between human and rodent physiology. To help distinguish among these possibilities, in the present study, using data from rats substantially larger than mice, we compared the clamp glucose infusion rate (GIR) with surrogate indexes, including QUICKI, HOMA, 1/HOMA, log (HOMA), and 1/fasting insulin. All surrogates were modestly correlated with GIR (r = 0.34–0.40). Calibration analyses of surrogates adjusted for body weight demonstrated similar predictive accuracy for GIR among all surrogates. We conclude that linear correlations of surrogate indexes with clamp estimates and predictive accuracy of surrogate indexes in rats are similar to those in mice (but not as substantial as in humans). This additional rat study (taken with the previous mouse study) suggests that application of surrogate insulin sensitivity indexes developed for humans may not be appropriate for determining primary outcomes in rodent studies due to intrinsic differences in metabolic physiology. However, use of surrogates may be appropriate in rodents, where feasibility of clamps is an obstacle and measurement of insulin sensitivity is a secondary outcome.

Inhibition of hepatic Niemann-Pick C1-like 1 improves hepatic insulin resistance

Nomura, M., Ishii, H., Kawakami, A., Yoshida, M. — Wed, 21 Oct 2009 14:10:13 PDT

The present study attempted to define the role of hepatic Niemann-Pick C1-like 1 (NPC1L1), a cholesterol transporter, in hepatic insulin resistance as well as hepatic steatosis. The inhibition of NPC1L1 and its molecular consequences were examined in Zucker obese fatty (ZOF) rats and cultured steatotic hepatocytes using ezetimibe, a pharmacoloigcal inhibitor of NPC1L1, and short hairpin RNA (shRNA) of NPC1L1. Ezetimibe improved hepatic insulin signaling as well as hepatic steatosis in ZOF rats. It also restored insulin sensitivity in steatotic hepatocytes in vitro through a reduction in hepatic reactive oxygen species (ROS) generation, JNK activation, and ER stress. In addition, ezetimibe recovered insulin-induced Akt activation and reduced gluconeogenic genes in the liver of ZOF rats and cultured steatotic hepatocytes. Transfection of NPC1L1 shRNA into hepatocytes also reduced ROS generation and ER stress. These results indicate that NPC1L1 contributes to hepatic insulin resistance through cholesterol accumulation, and its inhibition could be a potential therapeutic target of hepatic insulin resistance.

Effects of hypoxia on testosterone release in rat Leydig cells

Hwang, G.-S., Chen, S.-T., Chen, T.-J., Wang, S.-W. — Wed, 21 Oct 2009 14:10:13 PDT

The aim of this study was to explore the effect and action mechanisms of intermittent hypoxia on the production of testosterone both in vivo and in vitro. Male rats were housed in a hypoxic chamber (12% O₂ + 88% N₂, 1.5 l/ml) 8 h/day for 4 days. Normoxic rats were used as control. In an in vivo experiment, hypoxic and normoxic rats were euthanized and the blood samples collected. In the in vitro experiment, the enzymatically dispersed rat Leydig cells were prepared and challenged with forskolin (an adenylyl cyclase activator, 10^–4 M), 8-Br-cAMP (a membrane-permeable analog of cAMP, 10^–4 M), hCG (0.05 IU), the precursors of the biosynthesis testosterone, including 25-OH-C (10^–5 M), pregnenolone (10^–7 M), progesterone (10^–7 M), 17-OH-progesterone (10^–7 M), and androstendione (10^–7-10^–5 M), nifedipine (L-type Ca²⁺ channel blocker, 10^–6-10^–4 M), nimodipine (L-type Ca²⁺ channel blocker, 10^–5 M), tetrandrine (L-type Ca²⁺ channel blocker, 10^–5 M), and NAADP (calcium-signaling messenger causing release of calcium from intracellular stores, 10^–6-10^–4 M). The concentrations of testosterone in plasma and medium were measured by radioimmunoassay. The level of plasma testosterone in hypoxic rats was higher than that in normoxic rats. Enhanced testosterone production was observed in rat Leydig cells treated with hCG, 8-Br-cAMP, or forskolin in both normoxic and hypoxic conditions. Intermittent hypoxia resulted in a further increase of testosterone production in response to the testosterone precursors. The activity of 17β-hydroxysteroid dehydrogenase was stimulated by the treatment of intermittent hypoxia in vitro. The intermittent hypoxia-induced higher production of testosterone was accompanied with the influx of calcium via L-type calcium channel and the increase of intracellular calcium via the mechanism of calcium mobilization. These results suggested that the intermittent hypoxia stimulated the secretion of testosterone at least in part via stimulatory actions on the activities of adenylyl cyclase, cAMP, L-type calcium channel, and steroidogenic enzymes.

Ontogeny of methionine utilization and splanchnic uptake in critically ill children

Wed, 21 Oct 2009 14:10:13 PDT

To determine the rates of methionine splanchnic uptake and utilization in critically ill pediatric patients we used two kinetic models: the plasma methionine enrichment and the "intracellular" homocysteine enrichment. Twenty four patients, eight infants, eight children, and eight adolescents, were studied. They received simultaneous, primed, constant, intravenous infusions of l-[²H₃]methylmethionine and enteral l-[1-¹³C]methionine. The ratio of [¹³C]homocysteine to [¹³C]methionine enrichment was 1.0 ± 0.15, 0.80 ± 0.20, and 0.66 ± 0.10, respectively, for the infants, children, and adolescents, and it was different between the infants and adolescents (P < 0.01). Methionine splanchnic uptake was 63, 45, and 36%, respectively, in the infants, children, and adolescents, and it was higher (P < 0.01) in the infants compared with the adolescents. The infants utilized 73% of methionine flux for nonoxidative disposal, while 27% was used for transulfuration (P < 0.001). Conversely, in the adolescents, 40% was utilized for nonoxidative disposal, while 60% was used for transulfuration. There is ontogeny on the rates of methionine splanchnic uptake and on the fate of methionine utilization in critically ill children, with greater methionine utilization for synthesis of proteins and methionine-derived compounds (P < 0.01) and decreased transulfuration rates in the infants (P < 0.01), while the opposite was observed in the adolescents. The plasma model underestimated methionine kinetics in children and adolescents but not in the infants, suggesting lesser dilution and greater compartmentation of methionine metabolism in the infant population. All patients were in negative methionine balance, indicating that the current enteral nutritional support is inadequate in these patients.

Restoring AS160 phosphorylation rescues skeletal muscle insulin resistance and fatty acid oxidation while not reducing intramuscular lipids

Wed, 21 Oct 2009 14:10:13 PDT

We examined whether AICAR or leptin rapidly rescued skeletal muscle insulin resistance via increased palmitate oxidation, reductions in intramuscular lipids, and/or restoration of insulin-stimulated AS60 phosphorylation. Incubation with palmitate (2 mM, 0–18 h) induced insulin resistance in soleus muscle. From 12–18 h, palmitate was removed or AICAR or leptin was provided while 2 mM palmitate was maintained. Palmitate oxidation, intramuscular triacylglycerol, diacylglycerol, ceramide, AMPK phosphorylation, basal and insulin-stimulated glucose transport, plasmalemmal GLUT4, and Akt and AS160 phosphorylation were examined at 0, 6, 12, and 18 h. Palmitate treatment (12 h) increased intramuscular lipids (triacylglycerol +54%, diacylglycerol +11%, total ceramide +18%, C16:0 ceramide +60%) and AMPK phosphorylation (+118%), whereas it reduced fatty acid oxidation (–60%) and insulin-stimulated glucose transport (–70%), GLUT4 translocation (–50%), and AS160 phosphorylation (–40%). Palmitate removal did not rescue insulin resistance or associated parameters. The AICAR and leptin treatments did not consistently reduce intramuscular lipids, but they did rescue palmitate oxidation and insulin-stimulated glucose transport, GLUT4 translocation, and AS160 phosphorylation. Increased AMPK phosphorylation was associated with these improvements only when AICAR and leptin were present. Hence, across all experiments, AMPK phosphorylation did not correlate with any parameters. In contrast, palmitate oxidation and insulin-stimulated AS160 phosphorylation were highly correlated (r = 0.83). We speculate that AICAR and leptin activate both of these processes concomitantly, involving activation of unknown kinases in addition to AMPK. In conclusion, despite the maintenance of high concentrations of palmitate (2 mM), as well as increased concentrations of intramuscular lipids (triacylglycerol, diacylglycerol, and ceramide), the rapid AICAR- and leptin-mediated rescue of palmitate-induced insulin resistance is attributable to the restoration of insulin-stimulated AS160 phosphorylation and GLUT4 translocation.

Overexpression of the nuclear factor-{kappa}B subunit c-Rel protects against human islet cell death in vitro

Mokhtari, D., Barbu, A., Mehmeti, I., Vercamer, C., Welsh, N. — Wed, 21 Oct 2009 14:10:13 PDT

The transcription factor nuclear factor (NF)-B is known to modulate rates of apoptosis and may therefore play a role in the increased β-cell death that occurs in type 1 and type 2 diabetes. The aim of the present investigation was to study the expression of NF-B subunits in human islet cells and whether overexpression of the NF-B subunit c-Rel affects islet cell survival. We detected expression of p65, Rel-B, p50, p105, p52, and the ribosomal protein S3 (rpS3) in human islet cells. Among these, only p65 and rpS3 were translocated from the cytosolic to the nuclear fraction in response to cytokines. Interestingly, rpS3 participated in p65 binding to the B-element in gel shift analysis experiments. We observed cytoplasmic c-Rel expression in vivo in 6J mice, and signs of nuclear translocation in β-cells of infiltrated nonobese diabetic islets. Human islet cells were also dispersed by trypsin treatment and transduced with a c-Rel adenoviral vector. This resulted in increased expression of c-Rel and inhibitory factor B, increased B-binding activity, and augmented protein levels of Bcl-X_L, c-IAP2, and heat shock protein 72. c-Rel expression in human islet cells protected against cytokine-induced caspase 3 activation and cell death. c-Rel protected also against streptozotocin- and H₂O₂-induced cell death, in both intact rat islets and human islet cells. We conclude that rpS3 participates in NF-B signaling and that a genetic increase in the activity of the NF-B subunit c-Rel results in protection against cell death in human islets.

Differential efficacy of SSTR1, -2, and -5 agonists in the inhibition of C6 glioma growth in nude mice

Wed, 21 Oct 2009 14:10:13 PDT

Somatostatin receptors (SSTR1–5) mediate antiproliferative effects. In C6 rat glioma cells, somatostatin is cytostatic in vitro via phosphotyrosine phosphatase-dependent inhibition of ERK1/2 activity mediated by SSTR1, -2, and -5. Here we analyzed the effects of SSTR activation on C6 glioma growth in vivo and the intracellular mechanisms involved, comparing somatostatin effects with selective agonists for SSTR1, -2, and -5 (BIM-23745, BIM-23120, BIM-23206) or receptor biselective compounds (SSTR1 and -2, BIM-23704; and SSTR2 and -5, BIM-23190). Nude mice subcutaneously xenografted with C6 cells were treated with somatostatin, SSTR agonists (50 µg, twice/day), or vehicle. Tumor growth was evaluated every 3 days for 19 days. The intracellular pathways responsible of SSTR effects in vivo were evaluated measuring Ki-67, phospho-ERK1/2, and p27^kip1 expression by immunohistochemistry in sections from explanted tumors. Somatostatin and SSTR1, -2, and -5 agonists strongly inhibited in vivo C6 tumor growth, intratumoral neovessel formation, Ki-67 expression, and ERK1/2 phosphorylation and induced upregulation of p27^Kip1, whereas only a modest activation of caspase-3 was observed. Somatostatin (acting on SSTR1, -2, and -5) displayed the highest efficacy; SSTR5 selective agonist showed a stronger effect than SSTR1 agonist, and SSTR2 agonist was less effective. On the other hand, SSTR1 and -2 agonists maximally reduced tumor neovascularization. The combined activation of SSTR1 and -2 showed a synergistic activity, reaching a higher efficacy than BIM-23206, whereas the simultaneous activation of SSTR2 and -5 resulted in a response resembling SSTR5 effects. Thus the simultaneous activation of different SSTRs inhibits glioma cell proliferation in vivo through both direct cytotostatic and antiangiogenic effects.

Progesterone stimulates mitochondrial activity with subsequent inhibition of apoptosis in MCF-10A benign breast epithelial cells

Behera, M. A., Dai, Q., Garde, R., Saner, C., Jungheim, E., Price, T. M. — Wed, 21 Oct 2009 14:10:13 PDT

The effects of progesterone on breast epithelial cells remain poorly defined with observations showing both proliferative and antiproliferative effects. As an example, progesterone levels correlate with increased epithelial cell proliferation, but there is discordance between the dividing cells and the cells with nuclear progesterone receptor expression. The release of paracrine growth factors from nuclear receptor-positive cells has been postulated as a mechanism, since in vitro studies show a lack of growth effect by progesterone in breast epithelial cells lacking nuclear receptors. This study examined possible nongenomic effects of progesterone in breast epithelia by using MCF-10A cells known to lack nuclear progesterone receptor expression. Treatment for 30–60 min with progesterone or the progestin, R5020, increased mitochondrial activity as shown by an increase in mitochondrial membrane potential (hyperpolarization) with a concordant increase in total cellular ATP. The reaction was inhibited by a specific progesterone receptor antagonist and not affected by the translation inhibitor cycloheximide. Progestin treatment inhibited apoptosis induced by activation of the FasL pathway, as shown by a decrease in sub-G₁ cell fraction during fluorescence-activated cell sorting and a decrease in caspase 3/7 levels. Progestin treatment did not alter the cell cycle over 48 h. Our study demonstrates a nongenomic action of progesterone on benign breast epithelial cells, resulting in enhanced cellular respiration and protection from apoptosis.

A rosiglitazone-induced increase in adiponectin does not improve glucose metabolism in HIV-infected patients with overt lipoatrophy

Wed, 21 Oct 2009 14:10:13 PDT

HIV-infected patients on antiretroviral therapy frequently develop changes in body fat distribution and disturbances in glucose metabolism, associated with reduced adiponectin levels. Because adiponectin, principally the high-molecular-weight (HMW) form, has insulin-sensitizing properties, we investigated the effects of an increase in adiponectin on glucose metabolism in HIV-lipodystrophy. In this randomized, double-blind, placebo-controlled trial, we included HIV-1-infected patients with severe lipoatrophy, with an undetectable viral load and who had received neither protease inhibitors nor stavudine for ≥6 mo. Patients were randomized to rosiglitazone [8 mg daily (n = 8)] to increase adiponectin levels or placebo (n = 5) for 16 wk. Peripheral glucose disposal, glucose production, and lipolysis were measured after an overnight fast and during a hyperinsulinemic-euglycemic clamp using stable isotopes. Body composition was assessed by computed tomography and dual-energy X-ray absorptiometry. Although body fat distribution was unaffected, rosiglitazone increased total plasma adiponectin levels by 107% (P < 0.02) and the ratio of HMW to total adiponectin by 73% (P < 0.001). In the placebo group, neither total adiponectin levels (P = 0.62) nor the ratio of HMW to total adiponectin changed (P = 0.94). The marked increase in adiponectin induced by rosiglitazone was not associated with significant changes in basal endogenous glucose production (P = 0.90), basal lipolysis (P = 0.90), insulin-mediated suppression of glucose production (P = 0.17) and lipolysis (P = 0.54) nor with changes in peripheral glucose disposal (P = 0.13). Acknowledging the limited statistical power of our small study, these findings, if confirmed by larger studies, could question the importance of adiponectin in regulating glucose metabolism in HIV-lipodystrophy.

Acute glucose-lowering and insulin-sensitizing action of FGF21 in insulin-resistant mouse models--association with liver and adipose tissue effects

Wed, 21 Oct 2009 14:10:13 PDT

Recombinant fibroblast growth factor (FGF)21 has antihyperglycemic, antihyperlipidemic, and antiobesity effects in diabetic rodent and monkey models. Previous studies were confined to measuring steady-state effects of FGF21 following subchronic or chronic administration. The present study focuses on the kinetics of biological actions of FGF21 following a single injection and on the associated physiological and cellular mechanisms underlying FGF21 actions. We show that FGF21 resulted in rapid decline of blood glucose levels and immediate improvement of glucose tolerance and insulin sensitivity in two animal models of insulin resistance (ob/ob and DIO mice). In ob/ob mice, FGF21 led to a 40–60% decrease in blood glucose, insulin, and amylin levels within 1 h after injection, and the maximal effects were sustained for more than 6 h despite the 1- to 2-h half-life of FGF21. In DIO mice, FGF21 reduced fasting blood glucose and insulin levels and improved glucose tolerance and insulin sensitivity within 3 h of treatment. The acute improvement of glucose metabolism was associated with a 30% reduction of hepatic glucose production and an increase in peripheral glucose turnover. FGF21 appeared to have no direct effect on ex vivo pancreatic islet insulin or glucagon secretion. However, it rapidly induced typical FGF signaling in liver and adipose tissues and in several hepatoma-derived cell lines and differentiated adipocytes. FGF21 was able to inhibit glucose release from H4IIE hepatoma cells and stimulate glucose uptake in 3T3-L1 adipocytes. We conclude that the acute glucose-lowering and insulin-sensitizing effects of FGF21 are potentially associated with its metabolic actions in liver and adipose tissues.

Effects of hormone-sensitive lipase disruption on cardiac energy metabolism in response to fasting and refeeding

Wed, 21 Oct 2009 14:10:13 PDT

Increased fatty acid (FA) flux and intracellular lipid accumulation (steatosis) give rise to cardiac lipotoxicity in both pathological and physiological conditions. Since hormone-sensitive lipase (HSL) contributes to intracellular lipolysis in adipose tissue and heart, we investigated the impact of HSL disruption on cardiac energy metabolism in response to fasting and refeeding. HSL-knockout (KO) mice and wild-type (WT) littermates were fasted for 24 h, followed by ~6 h of refeeding. Plasma FA concentration in WT mice was elevated twofold with fasting, whereas KO mice lacked this elevation, resulting in twofold lower cardiac FA uptake compared with WT mice. Echocardiography showed that fractional shortening was 15% decreased during fasting in WT mice and was associated with steatosis, whereas both of these changes were absent in KO mice. Compared with Langendorff-perfused hearts isolated from fasted WT mice, the isolated KO hearts also displayed higher contractile function and a blunted response to FA. Although cardiac glucose uptake in KO mice was comparable with WT mice under all conditions tested, cardiac VLDL uptake and lipoprotein lipase (LPL) activity were twofold higher in KO mice during fasting. The KO hearts showed undetectable activity of neutral cholesteryl esterase and 40% lower non-LPL triglyceride lipase activity compared with WT hearts in refed conditions accompanied by overt steatosis, normal cardiac function, and increased mRNA expression of adipose differentiation-related protein. Thus, the dissociation between cardiac steatosis and functional sequelae observed in HSL-KO mice suggests that excess FA influx, rather than steatosis per se, appears to play an important role in the pathogenesis of cardiac lipotoxicity.

TIP39/parathyroid hormone type 2 receptor signaling is a potent inhibitor of chondrocyte proliferation and differentiation

Panda, D., Goltzman, D., Juppner, H., Karaplis, A. C. — Wed, 21 Oct 2009 14:10:13 PDT

Tuberoinfundibular peptide of 39 residues (TIP39) is a member of the parathyroid hormone (PTH) family of peptide hormones that exerts its function by interacting with the PTH type 2 receptor (PTH2R). Presently, no known function has been attributed to this signaling pathway in the developing skeleton. We observed that TIP39 and PTH2R were present in the newborn mouse growth plate, with the receptor localizing in the resting zone whereas ligand expression was restricted exclusively in prehypertrophic and hypertrophic chondrocytes. By 8 wk of life, PTH2R, and to a lesser degree TIP39, immunoreactivity was present in articular chondrocytes. We therefore sought to investigate the role of TIP39/PTH2R signaling in chondrocytes by generating stably transfected CFK2 chondrocytic cells overexpressing PTH2R (CFK2R). TIP39 treatment of CFK2R clones in culture inhibited their proliferation by restricting cells at the G₀/G₁ phase of the cell cycle, coupled with decreased expression and activity of cyclin-dependent kinases Cdk2 and Cdk4, while p21, an inhibitor of Cdks, was upregulated. In addition, TIP39 treatment decreased expression of differentiation markers in these cells associated with marked alterations in extracellular matrix and metalloproteinase expression. Transcription of Sox9, the master regulator of cartilage differentiation, was reduced in TIP39-treated CFK2R clones. Moreover, Sox9 promoter activity, as measured by luciferase reporter assay, was markedly diminished after TIP39 treatment. In summary, our results show that TIP39/PTH2R signaling inhibits proliferation and alters differentiation of chondrocytes by modulating SOX9 expression, thereby substantiating the functional significance of this signaling pathway in chondrocyte biology.

Novel liver-specific TORC2 siRNA corrects hyperglycemia in rodent models of type 2 diabetes

Wed, 21 Oct 2009 14:10:13 PDT

The transcription factor TORC2 [transducer of regulated cAMP-responsive element-binding protein (CREB) activity 2] is a major regulator of hepatic gluconeogenesis and is increased in hyperglycemic rodent models. Because chronic hyperglycemia and increased hepatic glucose production, via increased gluconeogenesis, is a key feature of type 2 diabetes, an effective in vivo method to efficiently knock down TORC2 could provide a potential therapy for treating hyperglycemia and type 2 diabetes. To assess this, primary mouse hepatocytes, high-fat diet (HFD)-fed mice, and Zucker diabetic fatty (ZDF) rats were treated with a siRNA against TORC2 (siTORC2), which was delivered via a novel lipid nanoparticle system, or control siRNA (siCON). Compared with siCON, administration of siTORC2 resulted in highly efficient, sustained (1–3 wk) knockdown of TORC2 and its gluconeogenic target genes phosphoenolpyruvate carboxykinase and glucose-6-phophatase in primary mouse hepatocytes and in the livers of HFD-fed mice. In mice, this knockdown was specific to the liver and did not occur in kidney, skeletal muscle, or adipose tissue. In HFD-fed mice, siTORC2 reduced in vivo gluconeogenic capacity, fasting hepatic glucose production, and hyperglycemia, and led to improved hepatic and skeletal muscle insulin sensitivity. siTORC2 treatment also improved systemic hyperglycemia in ZDF rats. In conclusion, these results demonstrate the importance of TORC2 in modulating HGP in vivo and highlight a novel, liver-specific siRNA approach for the potential treatment of hyperglycemia and type 2 diabetes.

Impact of type 1 diabetes on cardiac fibroblast activation: enhanced cell cycle progression and reduced myofibroblast content in diabetic myocardium

Wed, 21 Oct 2009 14:10:13 PDT

Diabetic patients are prone to developing myocardial fibrosis and suffer from decreased wound healing capabilities. The purpose of this study was to determine whether diabetes alters cardiac fibroblast activity in the myocardium in a 6-wk streptozotocin-induced type 1 diabetic model. In vivo echocardiography indicated significant dilation of the left ventricle (LV) in the diabetic animals, while cardiac function was comparable to that in the normal group. We isolated cardiac fibroblasts from diabetic and control hearts and observed increased proliferation of the diabetic fibroblasts. Microarray analysis using mRNA collected from whole LVs revealed downregulation of known inhibitors of proliferation, p53 and p21, in the diabetic group, consistent with our proliferation data. Western blot analysis confirmed a reduction in p53 protein expression in the diabetic hearts compared with control. We explored the potential signaling underlying the downregulation of these cell cycle mediators and determined that activated Akt, a signal that inhibits p53, was elevated in the diabetic group. Surprisingly, the hearts from the diabetic group contained lower levels of the myofibroblast marker -smooth muscle actin (-SMA) and higher levels of desmin and platelet endothelial cell adhesion molecule (PECAM). The isolated fibroblasts from the diabetic group also contained significantly less -SMA. These data suggest that early-stage diabetic hearts contain highly proliferative fibroblasts, which predisposes the diabetic myocardium to fibrosis, but have fewer myofibroblasts, which may compromise wound healing.

Effect of hyperinsulinemia and very-low-calorie diet on interstitial cytokine levels in subcutaneous adipose tissue of obese women

Wed, 21 Oct 2009 14:10:13 PDT

Type 2 diabetes and obesity are associated with an enhanced release of a number of adipocytokines. Hyperinsulinemia, frequently present in type 2 diabetes and obesity, might be one of the drivers of the enhanced production of adipocytokines. The aim of this study was to investigate the interstitial levels of cytokines in subcutaneous adipose tissue (SCAT) in response to hyperinsulinemia and the effect of weight-reducing hypocaloric diet on this regulation in obese subjects. Thirteen obese premenopausal women participated in the study. Concentrations of seven cytokines were measured in plasma and in AT interstitial fluid collected by microdialysis during a euglycemic-hyperinsulinemic clamp and during control infusion of physiological saline. A subgroup of six women underwent a 4-wk very-low-calorie diet (VLCD). Microdialysis during the clamp was performed before and at the end of VLCD. Hyperinsulinemia induced an increase of monocyte chemoatractant protein (MCP-1) and IL-6 SCAT interstitial and plasma levels and elevated IL-8 levels in SCAT. The relative changes of IL-6 levels in the dialysate correlated with changes of IL-8 and MCP-1. The interstitial and plasma levels of IL-1β, IL-10, TNF, and plasminogen activator inhibitor (PAI-1) remained unchanged in response to hyperinsulinemia. VLCD resulted in enhancement of the hyperinsulinemia-induced augmentation of MCP-1, IL-6, and IL-8 interstitial levels. In conclusion, hyperinsulinemia upregulates the interstitial levels of MCP-1, IL-6, and IL-8 in SCAT in obese women, whereas it does not affect IL-1β, IL-10, TNF, and PAI-1 levels. Hypocaloric diet associated with weight reduction enhances the hyperinsulinemia-induced upregulation of MCP-1, IL-6, and IL-8 in SCAT.

Effects of the cannabinoid CB1 antagonist rimonabant on hepatic mitochondrial function in rats fed a high-fat diet

Wed, 21 Oct 2009 14:10:13 PDT

The aim of this study was to investigate the effect of rimonabant treatment on hepatic mitochondrial function in rats fed a high-fat diet. Sprague-Dawley rats fed a high-fat diet (35% lard) for 13 wk were treated with rimonabant (10 mg·kg^–1·day^–1) during the last 3 wk and matched with pair-fed controls. Oxygen consumption with various substrates, mitochondrial enzyme activities on isolated liver mitochondria, and mitochondrial DNA quantity were determined. Body weight and fat mass were decreased in rats treated with rimonabant compared with pair-fed controls. Moreover, the serum adiponectin level was increased with rimonabant. Hepatic triglyceride content was increased, while serum triglycerides were decreased. An increase of mitochondrial respiration was observed in rats treated with rimonabant. The increase of mitochondrial respiration with palmitoyl-CoA compared with respiration with palmitoyl-l-carnitine stating that the entry of fatty acids into mitochondria via carnitine palmitoyltransferase I was increased in rats treated with rimonabant. Moreover, rimonabant treatment led to a reduction in the enzymatic activity of ATP synthase, whereas the quantity of mitochondrial DNA and the activity of citrate synthase remained unchanged. To summarize, rimonabant treatment leads to an improvement of hepatic mitochondrial function by increasing substrate oxidation and fatty acid entry into mitochondria for the β-oxidation pathway and by increasing proton leak. However, this increase of mitochondrial oxidation is regulated by a decrease of ATP synthase activity in order to have only ATP required for the cell function.

Fetal liver X receptor activation acutely induces lipogenesis but does not affect plasma lipid response to a high-fat diet in adult mice

Wed, 21 Oct 2009 14:10:14 PDT

There is increasing evidence that the metabolic state of the mother during pregnancy affects long-term glucose and lipid metabolism of the offspring. The liver X receptors (LXR) and -β are key regulators of cholesterol, fatty acid, and glucose metabolism. LXRs are activated by oxysterols and expressed in fetal mouse liver from day 10 of gestation onward. In the present study, we aimed to elucidate whether in utero pharmacological activation of LXR would influence fetal fatty acid and glucose metabolism and whether this would affect lipid homeostasis at adult age. Exposure of pregnant mice to the synthetic LXR agonist T0901317 increased hepatic mRNA expression levels of Lxr target genes and hepatic and plasma triglyceride levels in fetuses and dams. T0901317 treatment increased absolute de novo synthesis and chain elongation of hepatic oleic acid in dams and fetuses. T0901317 exposure in utero influenced lipid metabolism in adulthood in a sex-specific manner; hepatic triglyceride content was increased (+45%) in male offspring and decreased in female offspring (–42%) when they were fed a regular chow diet compared with untreated sex controls. Plasma and hepatic lipid contents and hepatic gene expression patterns in adult male or female mice fed a high-fat diet were not affected by T0901317 pretreatment. We conclude that LXR treatment of pregnant mice induces immediate effects on lipid metabolism in dams and fetuses. Despite the profound changes during fetal life, long-term effects appeared to be rather mild and sex selective without modulating the lipid response to a high-fat diet.

Treatment with SRT1720, a SIRT1 activator, ameliorates fatty liver with reduced expression of lipogenic enzymes in MSG mice

Wed, 21 Oct 2009 14:10:14 PDT

Nonalcoholic fatty liver disease (NAFLD) is an abnormal liver metabolism often observed with insulin resistance and metabolic syndrome. Calorie restriction is a useful treatment for NAFLD and reportedly prolongs the life spans of several species in which sirtuin plays an important role. In this study, we examined whether the activation of SIRT1, a mammalian ortholog of sirtuin, may ameliorate the development of NAFLD. Monosodium glutamate (MSG) mice, which exhibited obesity and insulin resistance, were treated with SRT1720, a specific SIRT1 activator from the age of 6–16 wk. Sixteen-week-old MSG mice exhibited increased liver triglyceride content and elevated levels of aminotransferase. SRT1720 treatment significantly reduced these levels without affecting body weight or food intake. These results suggested that the administration of SRT1720 ameliorated the development of NAFLD in MSG mice. The expressions of lipogenic genes, such as sterol regulatory element-binding protein-1c, acetyl-CoA carboxylase, and fatty acid synthase, and the serum lipid profiles, including free fatty acids, were elevated in MSG mice and were reduced by SRT1720 treatment. SRT1720 treatment also reduced the expressions of lipogenic genes in cultured HepG2 cells. Furthermore, SRT1720 treatment decreased the expressions of marker genes for oxidative stress and inflammatory cytokines in the liver of MSG mice. Taken together, SRT1720 treatment may reduce liver lipid accumulation, at least in part, by directly reducing the expressions of lipogenic genes. The reduction of oxidative stress and inflammation may also be involved in the amelioration of NAFLD.

Nitric oxides mediates a shift from early necrosis to late apoptosis in cytokine-treated {beta}-cells that is associated with irreversible DNA damage

Hughes, K. J., Chambers, K. T., Meares, G. P., Corbett, J. A. — Wed, 21 Oct 2009 14:10:14 PDT

For many cell types, including pancreatic β-cells, nitric oxide is a mediator of cell death; however, it is paradoxical that for a given cell type nitric oxide can induce both necrosis and apoptosis. This report tests the hypothesis that cell death mediated by nitric oxide shifts from an early necrotic to a late apoptotic event. Central to this transition is the ability of β-cells to respond and repair nitric oxide-mediated damage. β-Cells have the ability to repair DNA that is damaged following 24-h incubation with IL-1; however, cytokine-induced DNA damage becomes irreversible following 36-h incubation. This irreversible DNA damage following 36-h incubation with IL-1 correlates with the activation of caspase-3 (cleavage and activity). The increase in caspase activity correlates with reductions in endogenous nitric oxide production, as nitric oxide is an inhibitor of caspase activity. In contrast, caspase cleavage or activation is not observed under conditions in which β-cells are capable of repairing damaged DNA (24-h incubation with cytokines). These findings provide evidence that β-cell death in response to cytokines shifts from an early necrotic process to apoptosis and that this shift is associated with irreversible DNA damage and caspase-3 activation.

A very low carbohydrate ketogenic diet improves glucose tolerance in ob/ob mice independently of weight loss

Badman, M. K., Kennedy, A. R., Adams, A. C., Pissios, P., Maratos-Flier, E. — Wed, 21 Oct 2009 14:10:14 PDT

In mice of normal weight and with diet-induced obesity, a high-fat, low-carbohydrate ketogenic diet (KD) causes weight loss, reduced circulating glucose and lipids, and dramatic changes in hepatic gene expression. Many of the effects of KD are mediated by fibroblast growth factor 21 (FGF21). We tested the effects of KD feeding on ob/ob mice to determine if metabolic effects would occur in obesity secondarily to leptin deficiency. We evaluated the effect of prolonged KD feeding on weight, energy homeostasis, circulating metabolites, glucose homeostasis, and gene expression. Subsequently, we evaluated the effects of leptin and fasting on FGF21 expression in ob/ob mice. KD feeding of ob/ob mice normalized fasting glycemia and substantially reduced insulin and lipid levels in the absence of weight loss. KD feeding was associated with significant increases in lipid oxidative genes and reduced expression of lipid synthetic genes, including stearoyl-coenzyme A desaturase 1, but no change in expression of inflammatory markers. In chow-fed ob/ob mice, FGF21 mRNA was elevated 10-fold compared with wild-type animals, and no increase from this elevated baseline was seen with KD feeding. Administration of leptin to chow-fed ob/ob mice led to a 24-fold induction of FGF21. Fasting also induced hepatic FGF21 in ob/ob mice. Thus, KD feeding improved ob/ob mouse glucose homeostasis without weight loss or altered caloric intake. These data demonstrate that manipulation of dietary macronutrient composition can lead to marked improvements in metabolic profile of leptin-deficient obese mice in the absence of weight loss.

The effect of high-dose sodium salicylate on chronically elevated plasma nonesterified fatty acid-induced insulin resistance and {beta}-cell dysfunction in overweight and obese nondiabetic men

Xiao, C., Giacca, A., Lewis, G. F. — Wed, 21 Oct 2009 14:10:14 PDT

Prolonged elevation of plasma nonesterified fatty acids (NEFA) induces insulin resistance and impairs pancreatic β-cell adaptation to insulin resistance. Studies in rodents suggest that inflammation may play a role in this "lipotoxicity." We studied the effects of sodium salicylate, an anti-inflammatory agent, on lipid-induced alterations in β-cell function and insulin sensitivity in six overweight and obese nondiabetic men. Each subject underwent four separate studies, 4–6 wk apart, in random order: 1) SAL, 1-wk placebo followed by intravenous (iv) infusion of saline for 48 h; 2) IH, 1-wk placebo followed by iv infusion of intralipid plus heparin for 48 h to raise plasma NEFA approximately twofold; 3) IH + SS, 1-wk sodium salicylate (4.5 g/day) followed by 48-h IH infusion; and 4) SS, 1-wk oral sodium salicylate followed by 48-h saline infusion. After 48-h saline or lipid infusion, insulin secretion and sensitivity were assessed by hyperglycemic clamp and euglycemic hyperinsulinemic clamp, respectively, in sequential order. Insulin sensitivity was reduced by lipid infusion (IH = 67% of SAL) and was not improved by salicylate (IH + SS = 56% of SAL). Lipid infusion also reduced the disposition index (P < 0.05), which was not prevented by sodium salicylate. Salicylate reduced insulin clearance. These data suggest that oral sodium salicylate at this dose impairs insulin clearance but does not ameliorate lipid-induced insulin resistance and β-cell dysfunction in overweight and obese nondiabetic men.

Sex differences in the regulation of Kiss1/NKB neurons in juvenile mice: implications for the timing of puberty

Kauffman, A. S., Navarro, V. M., Kim, J., Clifton, D. K., Steiner, R. A. — Wed, 21 Oct 2009 14:10:14 PDT

In mammals, puberty onset typically occurs earlier in females than in males, but the explanation for sexual differentiation in the tempo of pubertal development is unknown. Puberty in both sexes is a brain-dependent phenomenon and involves alterations in the sensitivity of neuronal circuits to gonadal steroid feedback as well as gonadal hormone-independent changes in neuronal circuitry. Kisspeptin, encoded by the Kiss1 gene, plays an essential but ill-defined role in pubertal maturation. Neurokinin B (NKB) is coexpressed with Kiss1 in the arcuate nucleus (ARC) and is also important for puberty. We tested whether sex differences in the timing of pubertal development are attributable to sexual differentiation of gonadal hormone-independent mechanisms regulating hypothalamic Kiss1/NKB gene expression. We found that, in juvenile females, gonadotropin secretion and expression of Kiss1 and NKB in the ARC increased immediately following ovariectomy, suggesting that prepubertal females have negligible gonadal hormone-independent restraint on their reproductive axis. In contrast, in similarly aged juvenile males, no changes occurred in LH levels or Kiss1 or NKB expression following castration, suggesting that gonadal hormone-independent mechanisms restrain kisspeptin/NKB-dependent activation of the male reproductive axis before puberty. Notably, adult mice of both sexes showed comparable rapid increases in Kiss1/NKB expression and LH secretion following gonadectomy, signifying that sex differences in the regulation of ARC Kiss1/NKB neurons are manifest only during peripubertal development. Our findings demonstrate that the mechanisms controlling pubertal activation of reproduction in mice are different between the sexes and suggest that gonadal hormone-independent central restraint on pubertal timing involves Kiss1/NKB neurons in the ARC.

Castration differentially alters basal and leucine-stimulated tissue protein synthesis in skeletal muscle and adipose tissue

Jiao, Q., Pruznak, A. M., Huber, D., Vary, T. C., Lang, C. H. — Wed, 21 Oct 2009 14:10:14 PDT

Reduced testosterone as a result of catabolic illness or aging is associated with loss of muscle and increased adiposity. We hypothesized that these changes in body composition occur because of altered rates of protein synthesis under basal and nutrient-stimulated conditions that are tissue specific. The present study investigated such mechanisms in castrated male rats (75% reduction in testosterone) with demonstrated glucose intolerance. Over 9 wk, castration impaired body weight gain, which resulted from a reduced lean body mass and preferential sparing of adipose tissue. Castration decreased gastrocnemius weight, but this atrophy was not associated with reduced basal muscle protein synthesis or differences in plasma IGF-I, insulin, or individual amino acids. However, oral leucine failed to normally stimulate muscle protein synthesis in castrated rats. In addition, castration-induced atrophy was associated with increased 3-methylhistidine excretion and in vitro-determined ubiquitin proteasome activity in skeletal muscle, changes that were associated with decreased atrogin-1 or MuRF1 mRNA expression. Castration decreased heart and kidney weight without reducing protein synthesis and did not alter either cardiac output or glomerular filtration. In contradistinction, the weight of the retroperitoneal fat depot was increased in castrated rats. This increase was associated with an elevated rate of basal protein synthesis, which was unresponsive to leucine stimulation. Castration also decreased whole body fat oxidation. Castration increased TNF, IL-1, IL-6, and NOS2 mRNA in fat but not muscle. In summary, the castration-induced muscle wasting results from an increased muscle protein breakdown and the inability of leucine to stimulate protein synthesis, whereas the expansion of the retroperitoneal fat depot appears mediated in part by an increased basal rate of protein synthesis-associated increased inflammatory cytokine expression.

Corrigendum

Wed, 21 Oct 2009 14:10:14 PDT