AJP: Endocrinology and Metabolism current issue

Acyl-coenzyme A:cholesterol acyltransferases

Chang, T.-Y., Li, B.-L., Chang, C. C. Y., Urano, Y. — 2009-06-25

The enzymes acyl-coenzyme A (CoA):cholesterol acyltransferases (ACATs) are membrane-bound proteins that utilize long-chain fatty acyl-CoA and cholesterol as substrates to form cholesteryl esters. In mammals, two isoenzymes, ACAT1 and ACAT2, encoded by two different genes, exist. ACATs play important roles in cellular cholesterol homeostasis in various tissues. This chapter summarizes the current knowledge on ACAT-related research in two areas: 1) ACAT genes and proteins and 2) ACAT enzymes as drug targets for atherosclerosis and for Alzheimer's disease.

Beyond triglyceride synthesis: the dynamic functional roles of MGAT and DGAT enzymes in energy metabolism

Shi, Y., Cheng, D. — 2009-06-25

Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.

The genetics of neutral lipid biosynthesis: an evolutionary perspective

Turkish, A. R., Sturley, S. L. — 2009-06-25

The storage of fatty acids and fatty alcohols in the form of neutral lipids such as triacylglycerol (TAG), cholesteryl ester (CE), and wax ester (WE) serves to provide reservoirs for membrane formation and maintenance, lipoprotein trafficking, lipid detoxification, evaporation barriers, and fuel in times of stress or nutrient deprivation. This ancient process likely originated in actinomycetes and has persisted in eukaryotes, albeit by different molecular mechanisms. A surfeit of neutral lipids is strongly, perhaps causally, related to several human diseases such as diabetes mellitus, obesity, atherosclerosis and nonalcoholic fatty liver disease. Therefore, understanding the metabolic pathways of neutral lipid synthesis and the roles of the enzymes involved may facilitate the development of new therapeutic interventions for these syndromes.

Biochemical and physiological function of stearoyl-CoA desaturase

Paton, C. M., Ntambi, J. M. — 2009-06-25

A key and highly regulated enzyme that is required for the biosynthesis of monounsaturated fatty acids is stearoyl-CoA desaturase (SCD), which catalyzes the D⁹-cis desaturation of a range of fatty acyl-CoA substrates. The preferred substrates are palmitoyl- and stearoyl-CoA, which are converted into palmitoleoyl- and oleoyl-CoA respectively. Oleate is the most abundant monounsaturated fatty acid in dietary fat and is therefore readily available. Studies of mice that have a naturally occurring mutation in the SCD-1 gene isoform as well as a mouse model with a targeted disruption of the SCD gene (SCD-1^–/–) have revealed the role of de novo synthesized oleate and thus the physiological importance of SCD-1 expression. SCD-1 deficiency results in reduced body adiposity, increased insulin sensitivity, and resistance to diet-induced obesity. The expression of several genes of lipid oxidation are upregulated, whereas lipid synthesis genes are downregulated. SCD-1 was also found to be a component of the novel metabolic response to the hormone leptin. Therefore, SCD-1 appears to be an important metabolic control point, and inhibition of its expression could be of benefit for the treatment of obesity, diabetes, and other metabolic diseases. In this article, we summarize the recent and timely advances concerning the important role of SCD in the biochemistry and physiology of lipid metabolism.

Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle: effects of high-fat diet and exercise

2009-06-25

Skeletal muscle Na⁺-K⁺-ATPase plays a central role in the clearance of K⁺ from the extracellular fluid, therefore maintaining blood [K⁺]. Na⁺-K⁺-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise training (ET) on skeletal muscle Na⁺-K⁺-ATPase subunit expression and insulin-stimulated translocation. Skeletal muscle expression of Na⁺-K⁺-ATPase isoforms and transcription factor DNA binding was determined before or after 5 days of swim training in Wistar rats fed chow or HFD for 4 or 12 wk. Skeletal muscle insulin resistance was observed after 12 wk of HFD. Na⁺-K⁺-ATPase ₁-subunit protein expression was increased 1.6-fold (P < 0.05), whereas ₂- and β₁-subunits and protein expression were decreased twofold (P < 0.01) in parallel with decrease in plasma membrane Na⁺-K⁺-ATPase activity after 4 wk of HFD. Exercise training restored ₁-, ₂-, and β₁-subunit expression and Na⁺-K⁺-ATPase activity to control levels and reduced β₂-subunit expression 2.2-fold (P < 0.05). DNA binding activity of the ₁-subunit-regulating transcription factor ZEB (AREB6) and ₁ mRNA expression were increased after HFD and restored by ET. DNA binding activity of Sp-1, a transcription factor involved in the regulation of ₂- and β₁-subunit expression, was decreased after HFD. ET increased phosphorylation of the Na⁺-K⁺-ATPase regulatory protein phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the ₂-subunit to plasma membrane was impaired by HFD, whereas ₁-subunit translocation remained unchanged. Alterations in sodium pump function precede the development of skeletal muscle insulin resistance. Disturbances in skeletal muscle Na⁺-K⁺-ATPase regulation, particularly the ₂-subunit, may contribute to impaired ion homeostasis in insulin-resistant states such as obesity and type 2 diabetes.

Central but not systemic lipid infusion augments the counterregulatory response to hypoglycemia

Haywood, S. C., Bree, A. J., Puente, E. C., Daphna-Iken, D., Fisher, S. J. — 2009-06-25

This study tests the hypothesis that lipids could act as an alternative fuel source in the brain during insulin-induced hypoglycemia. Male Sprague-Dawley rats were subjected to hyperinsulinemic (5 mU·kg^–1·min^–1) hypoglycemic (~50 mg/dl) clamps. In protocol 1, intralipid (IL), a fat emulsion, was infused intravenously to prevent the fall in free fatty acid levels that occurs in response to hyperinsulinemic hypoglycemia. Intravenous lipid infusion did not alter the counterregulatory responses to hypoglycemia. To test whether IL could have central effects in mediating the counterregulatory response to hypoglycemia, in protocol 2 the brains of precannulated rats were intracerebroventricularly (icv) infused with IL or artificial cerebrospinal fluid (aCSF) as control. Unexpectedly, the epinephrine and glucagon response to hypoglycemia was significantly augmented with icv IL infusion. To determine whether central IL infusion could restore defective counterregulation, in protocol 3 rats were made recurrently hypoglycemic (RH) for 3 days and on the 4th day underwent hyperinsulinemic hypoglycemic clamps with icv IL or aCSF infusion. RH rats had the expected impaired epinephrine response to hypoglycemia, and icv IL infusion again significantly augmented the epinephrine response in RH rats to normal. With regard to our experimental model of hypoglycemic counterregulation, we conclude that 1) systemic lipid infusion did not alter the counterregulatory response to hypoglycemia, 2) the icv infusion of lipids markedly increased CSF FFA levels and paradoxically augmented the epinephrine and glucagon responses, and 3) the blunted sympathoadrenal response in recurrently hypoglycemic rats was completely normalized with the icv lipid infusion. It is concluded that, in the setting of insulin-induced hypoglycemia, increased brain lipids can enhance the sympathoadrenal response.

Oligomeric resistin impairs insulin and AICAR-stimulated glucose uptake in mouse skeletal muscle by inhibiting GLUT4 translocation

2009-06-25

The hormone resistin is elevated in obesity and impairs glucose homeostasis. Here, we examined the effect of oligomerized human resistin on insulin signaling and glucose metabolism in skeletal muscle and myotubes. This was investigated by incubating mouse extensor digitorum longus (EDL) and soleus muscles and L6 myotubes with physiological concentrations of resistin and assessing insulin-stimulated glucose uptake, cellular signaling, suppressor of cytokine signaling 3 (SOCS-3) mRNA, and GLUT4 translocation. We found that resistin at a concentration of 30 ng/ml decreased insulin-stimulated glucose uptake by 30–40% in soleus muscle and myotubes, whereas in EDL muscle insulin-stimulated glucose uptake was impaired at a resistin concentration of 100 ng/ml. Impaired insulin-stimulated glucose uptake was not associated with reduced Akt phosphorylation or IRS-1 protein or increased SOCS-3 mRNA expression. To further investigate the site(s) at which resistin impairs glucose uptake we treated myotubes and skeletal muscle with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) and found that, although resistin did not impair AMPK activation, it reduced AICAR-stimulated glucose uptake. These data suggested that resistin impairs glucose uptake at a point common to insulin and AMPK signaling pathways, and we thus measured AS160/TBC1D4 Thr⁶⁴² phosphorylation and GLUT4 translocation in myotubes. Resistin did not impair TBC1D4 phosphorylation but did reduce both insulin and AICAR-stimulated GLUT4 plasma membrane translocation. We conclude that resistin impairs insulin-stimulated glucose uptake by mechanisms involving reduced plasma membrane GLUT4 translocation but independent of the proximal insulin-signaling cascade, AMPK, and SOCS-3.

Lipid and insulin infusion-induced skeletal muscle insulin resistance is likely due to metabolic feedback and not changes in IRS-1, Akt, or AS160 phosphorylation

2009-06-25

Type 2 diabetes is characterized by hyperlipidemia, hyperinsulinemia, and insulin resistance. The aim of this study was to investigate whether acute hyperlipidemia-induced insulin resistance in the presence of hyperinsulinemia was due to defective insulin signaling. Hyperinsulinemia (~300 mU/l) with hyperlipidemia or glycerol (control) was produced in cannulated male Wistar rats for 0.5, 1 h, 3 h, or 5 h. The glucose infusion rate required to maintain euglycemia was significantly reduced by 3 h with lipid infusion and was further reduced after 5 h of infusion, with no difference in plasma insulin levels, indicating development of insulin resistance. Consistent with this finding, in vivo skeletal muscle glucose uptake (31%, P < 0.05) and glycogen synthesis rate (38%, P < 0.02) were significantly reduced after 5 h compared with 3 h of lipid infusion. Despite the development of insulin resistance, there was no difference in the phosphorylation state of multiple insulin-signaling intermediates or muscle diacylglyceride and ceramide content over the same time course. However, there was an increase in cumulative exposure to long-chain acyl-CoA (70%) with lipid infusion. Interestingly, although muscle pyruvate dehydrogenase kinase 4 protein content was decreased in hyperinsulinemic glycerol-infused rats, this decrease was blunted in muscle from hyperinsulinemic lipid-infused rats. Decreased pyruvate dehydrogenase complex activity was also observed in lipid- and insulin-infused animals (43%). Overall, these results suggest that acute reductions in muscle glucose metabolism in rats with hyperlipidemia and hyperinsulinemia are more likely a result of substrate competition than a significant early defect in insulin action or signaling.

Hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia in sucrose-fed obese rats via two pathways

2009-06-25

The mechanism by which replacement of some dietary carbohydrates with protein during weight loss favors lipid metabolism remains obscure. In this study, we investigated the effect of an energy-restricted, high-protein/low-carbohydrate diet on lipid metabolism in obese rats. High-sucrose-induced obese rats were assigned randomly to one of two energy-restricted dietary interventions: a carbohydrate-based control diet (CD) or a high-protein diet (HPD). Lean rats of the same age were assigned as normal control. There was significantly greater improvement in fatty liver and hypertriglyceridemia with the HPD diet relative to the CD diet. Expression of genes regulated by fibroblast growth factor-21 (FGF21) and involved in liver lipolysis and lipid utilitization, such as lipase and acyl-CoA oxidase, increased in obese rats fed the HPD. Furthermore, there was an inverse correlation between levels of FGF21 gene expression (regulated by glucagon/insulin balance) and increased triglyceride concentrations in liver from obese rats. Expression of hepatic stearoyl-CoA desaturase-1 (SCD1), regulated primarily by the dietary carbohydrate, was also markedly reduced in the HPD group (similar to plasma triglyceride levels in fasting animals) relative to the CD group. In conclusion, a hypocaloric high-protein diet improves fatty liver and hypertriglyceridemia effectively relative to a carbohydrate diet. The two cellular pathways at work behind these benefits include stimulation of hepatic lipolysis and lipid utilization mediated by FGF21 and reduction of hepatic VLDL-TG production by SCD1 regulation.

Menstrual cycle alters sympathetic neural responses to orthostatic stress in young, eumenorrheic women

Carter, J. R., Lawrence, J. E., Klein, J. C. — 2009-06-25

Sympathetic baroreflex sensitivity (BRS) and muscle sympathetic nerve activity (MSNA) responses during early follicular (EF) and midluteal (ML) phases of the menstrual cycle are controversial. We hypothesize an augmented sympathetic BRS and MSNA response to orthostatic stress during the ML phase of the menstrual cycle. MSNA, mean arterial pressure (MAP), and heart rate (HR) were recorded during progressive lower body negative pressure (LBNP) (–5, –10, –15, –20, –30, and –40 mmHg; 3 min/stage) in 13 healthy, eumenorrheic women (age 21 ± 1 yr). Sympathetic BRS was assessed by examining relations between spontaneous fluctuations of diastolic arterial pressure and MSNA at rest and during progressive LBNP. Plasma estradiol (42 ± 6 vs. 112 ± 12 pg/ml; P < 0.01) and progesterone (2 ± 0 vs. 10 ± 2 ng/ml; P < 0.04) were elevated during the ML phase. Resting MSNA (8 ± 1 vs. 11 ± 1 bursts/min), MAP (79 ± 2 vs. 78 ± 2 mmHg), and HR (58 ± 2 vs. 60 ± 2 beats/min) were not different during EF and ML phases. MSNA and HR increased during progressive LBNP (P < 0.001), and the increases in MSNA burst frequency (bursts/min) and HR were similar during both phases. In contrast, increases in total MSNA (arbitrary units) during progressive LBNP were augmented during the ML phase (P < 0.04), but this response does not appear to be linked to differences in sympathetic BRS. Progressive LBNP did not change MAP during either phase. Our results demonstrate an augmentation of the MSNA response to progressive LBNP during the ML phase of the menstrual cycle. These findings suggest that hormonal fluctuations of eumenorrheic women may influence sympathoexcitation during an orthostatic challenge, but not through sympathetic baroreflex-mediated pathways.

PGC-1{alpha} mediates exercise-induced skeletal muscle VEGF expression in mice

2009-06-25

The aim of the present study was to test the hypothesis that PGC-1 is required for exercise-induced VEGF expression in both young and old mice and that AMPK activation leads to increased VEGF expression through a PGC-1-dependent mechanism. Whole body PGC-1 knockout (KO) and littermate wild-type (WT) mice were submitted to either 1) 5 wk of exercise training, 2) lifelong (from 2 to 13 mo of age) exercise training in activity wheel, 3) a single exercise bout, or 4) 4 wk of daily subcutaneous AICAR or saline injections. In skeletal muscle of PGC-1 KO mice, VEGF protein expression was ~60–80% lower and the capillary-to-fiber ratio ~20% lower than in WT. Basal VEGF mRNA expression was similar in WT and PGC-1 KO mice, but acute exercise and AICAR treatment increased the VEGF mRNA content in WT mice only. Exercise training of young mice increased skeletal muscle VEGF protein expression ~50% in WT mice but with no effect in PGC-1 KO mice. Furthermore, a training-induced prevention of an age-associated decline in VEGF protein content was observed in WT but not in PGC-1 KO muscles. In addition, repeated AICAR treatments increased skeletal muscle VEGF protein expression ~15% in WT but not in PGC-1 KO mice. This study shows that PGC-1 is essential for exercise-induced upregulation of skeletal muscle VEGF expression and for a training-induced prevention of an age-associated decline in VEGF protein content. Furthermore, the findings suggest an AMPK-mediated regulation of VEGF expression through PGC-1.

Involvement of NADPH oxidase in oxidized LDL-induced upregulation of heat shock factor-1 and plasminogen activator inhibitor-1 in vascular endothelial cells

Zhao, R., Ma, X., Xie, X., Shen, G. X. — 2009-06-25

Plasminogen activator inhibitor-1 (PAI-1) is implicated in thrombogenesis, inflammation, and extracellular matrix remodeling. Previous studies indicated that oxidized low-density lipoprotein (LDL) stimulated the generation of PAI-1 in vascular endothelial cells (EC). The present study demonstrated that LDL oxidized by copper, iron, or 3-morpholinosydnonimine increased the expression of NADPH oxidase (NOX) 2, PAI-1, and heat shock factor-1 (HSF1) in human umbilical vein EC or coronary artery EC compared with LDL or vehicle. Diphenyleneiodonium, a NOX inhibitor, prevented the increases of the expression of HSF1 and PAI-1 in EC induced by oxidized LDLs. Small-interference RNA (siRNA) for p22^phox, an essential subunit of NOX, prevented oxidized LDL-induced expression of NOX2, HSF1, and PAI-1 in EC. HSF1 siRNA inhibited oxidized LDL-induced expression of PAI-1 and HSF1, but not NOX2, in EC. The binding of HSF1 to PAI-1 promoter and the activity of PAI-1 promoter in EC were enhanced by oxidized LDL. Butylated hydroxytulene, a potent antioxidant, inhibited oxidized LDL-induced release of hydrogen peroxide (H₂O₂) and the expression of NOX2, HSF1, and PAI-1 in EC. Treatment with H₂O₂ increased the abundance of NOX2, HSF1, and PAI-1 in EC. The results of the present study indicate that oxidized LDL-induced expression of NOX may lead to the elevated release of reactive oxygen species, the activation of HSF1, and the enhancement of the transcription of PAI-1 gene in cultured vascular EC.

Oleic acid-induced ADRP expression requires both AP-1 and PPAR response elements, and is reduced by Pycnogenol through mRNA degradation in NMuLi liver cells

2009-06-25

Fatty acids stimulate lipid accumulation in parallel with increased expression of adipose differentiation-related protein (ADRP) in liver cells. Although it is generally considered that the fatty acid effect on ADRP expression is mediated by peroxisome proliferator-activated receptors (PPARs), we identified here an additional molecular mechanism using the NMuLi mouse liver nonparenchymal cell line, which expresses PPAR and but not . Oleic acid (OA) and specific ligands for PPAR and - stimulated ADRP expression as well as the –2,090-bp ADRP promoter activity which encompasses the PPAR response element (PPRE) adjacent to an Ets/activator protein (AP)-1 site. When the AP-1 site was mutated, OA failed to stimulate the activity despite the presence of the PPRE, whereas ligands for PPAR and - did stimulate it and so did a PPAR ligand under the coexpression of PPAR. DNA binding of AP-1 was stimulated by OA but not by PPAR ligands. Because we previously demonstrated that Pycnogenol (PYC), a French maritime pine bark extract, suppressed ADRP expression in macrophages partly by suppression of AP-1 activity, we tested the effect of PYC on NMuLi cells. PYC reduced the OA-induced ADRP expression along with suppression of lipid droplet formation. However, PYC neither suppressed the OA-stimulated ADRP promoter activity nor DNA binding of AP-1 but, instead, reduced the ADRP mRNA half-life. All these results indicate that the effect of OA on ADRP expression requires AP-1 as well as PPRE, and PYC suppresses the ADRP expression in part by facilitating mRNA degradation. PYC, a widely used dietary supplement, could be beneficial for the prevention of excessive lipid accumulation such as hepatic steatosis.

Participation of ER{alpha} and ER{beta} in glucose homeostasis in skeletal muscle and white adipose tissue

Barros, R. P. A., Gabbi, C., Morani, A., Warner, M., Gustafsson, J.-A. — 2009-06-25

Glucose uptake and homeostasis are regulated mainly by skeletal muscle (SM), white adipose tissue (WAT), pancreas, and the liver. Participation of estradiol in this regulation is still under intense investigation. We have demonstrated that, in SM of male mice, expression of the insulin-regulated glucose transporter (GLUT)4 is reduced by estrogen receptor (ER)β agonists. In the present study, to investigate the relative contributions of ER and ERβ in glucose homeostasis, we examined the effects of tamoxifen (Tam) on GLUT4 expression in SM and WAT in wild-type (WT) and ER–/– mice. ERβ–/– mice were characterized by fasting hypoglycemia, increased levels of SM GLUT4, pancreatic islet hypertrophy, and a belated rise in plasma insulin in response to a glucose challenge. ER–/– mice, on the contrary, were hyperglycemic and glucose intolerant, and expression of SM GLUT4 was markedly lower than in WT mice. Tam had no effect on glucose tolerance or insulin sensitivity in WT mice. In ER–/– mice, Tam increased GLUT4 and improved insulin sensitivity. i.e., it behaved as an ERβ antagonist in SM but had no effect on WAT. In ERβ–/– mice, Tam did not affect GLUT4 in SM but acted as an ER antagonist in WAT, decreasing GLUT4. Thus, in the interplay between ER and ERβ, ERβ-mediated repression of GLUT4 predominates in SM but ER-mediated induction of GLUT4 predominates in WAT. This tissue-specific difference in dominance of one ER over the other is reflected in the ratio of the expression of the two receptors. ER predominates in WAT and ERβ in SM.

Colocalization of ghrelin O-acyltransferase and ghrelin in gastric mucosal cells

2009-06-25

Ghrelin is a peptide hormone with many known functions, including orexigenic, blood glucose-regulatory, and antidepressant actions, among others. Mature ghrelin is unique in that it is the only known naturally occurring peptide to be posttranslationally modified by O-acylation with octanoate. This acylation is required for many of ghrelin's actions, including its effects on promoting increases in food intake and body weight. GOAT (ghrelin O-acyltransferase), one of 16 members of the MBOAT family of membrane-bound O-acyltransferases, has recently been identified as the enzyme responsible for catalyzing the addition of the octanoyl group to ghrelin. Although the initial reports of GOAT have localized its encoding mRNA to tissues known to contain ghrelin, it is as yet unclear whether the octanoylation occurs within ghrelin-producing cells or in neighboring cells. Here, we have performed dual-label histochemical analysis on mouse stomach sections and quantitative PCR on mRNAs from highly enriched pools of mouse gastric ghrelin cells to demonstrate a high degree of GOAT mRNA expression within ghrelin-producing cells of the gastric oxyntic mucosa. We also demonstrate that GOAT is the only member of the MBOAT family whose expression is highly enriched within gastric ghrelin cells and whose whole body distribution mirrors that of ghrelin.

Actions of fibroblast growth factor-8 in bone cells in vitro

2009-06-25

The fibroblast growth factors (FGFs) are a group of at least 25 structurally related peptides that are involved in many biological processes. Some FGFs are active in bone, including FGF-1, FGF-2, and FGF-18, and recent evidence indicates that FGF-8 is osteogenic, particularly in mesenchymal stem cells. In the current study, we found that FGF-8 was expressed in rat primary osteoblasts and in osteoblastic UMR-106 and MC3T3-E1 cells. Both FGF-8a and FGF-8b potently stimulated the proliferation of osteoblastic cells, whereas they inhibited the formation of mineralized bone nodules in long-term cultures of osteoblasts and reduced the levels of osteoblast differentiation markers, osteocalcin, and bone sialoprotein. FGF-8a induced the phosphorylation of p42/p44 mitogen-activated protein kinase (MAPK) in osteoblastic cells; however, its mitogenic actions were not blocked by either the MAPK kinase (MEK) inhibitor U-0126 or the PI 3-kinase (PI3K) inhibitor LY-294002. Interestingly, FGF-8a, unlike FGF-8b and other members of the family, inhibited osteoclastogenesis in mouse bone marrow cultures, and this was via a receptor activator of NF-B ligand (RANKL)/osteoprotegerin (OPG)-independendent manner. However, FGF-8a did not affect osteoclastogenesis in RAW 264.7 cells (a macrophage cell line devoid of stromal cells) exogenously stimulated by RANKL, nor did it affect mature osteoclast function as assessed in rat calvarial organ cultures and isolated mature osteoclasts. In summary, we have demonstrated that FGF-8 is active in bone cells, stimulating osteoblast proliferation in a MAPK-independent pathway and inhibiting osteoclastogenesis via a RANKL/OPG-independent mechanism. These data suggest that FGF-8 may have a physiological role in bone acting in an autocrine/paracrine manner.

Effects of 7 days of exercise training on insulin sensitivity and responsiveness in type 2 diabetes mellitus

Kirwan, J. P., Solomon, T. P. J., Wojta, D. M., Staten, M. A., Holloszy, J. O. — 2009-06-25

The objectives of this study were to determine whether 1) the improvement in insulin action induced by short-term exercise training in patients with type 2 diabetes is due to an improvement in insulin sensitivity, an improvement in insulin responsiveness, or a combination of improved insulin sensitivity and responsiveness and 2) short-term exercise training results in improved suppression of hepatic glucose production by insulin. Fourteen obese patients with type 2 diabetes, age 64 ± 2 yr, underwent a two-stage hyperinsulinemic euglycemic clamp procedure, first stage 40 mU·m^–2·min^–1 insulin infusion, second stage 1,000 mU·m^–2·min^–1 insulin infusion, together with a [3-³H]glucose infusion, before and after 7 days of exercise. The training consisted of 30 min of cycling and 30 min of treadmill walking at ~70% of maximal aerobic capacity daily for 7 days. The exercise program resulted in improvements in insulin action in the absence of weight loss. Glucose disposal rates during the euglycemic clamp were significantly increased at both hyperinsulinemic stages after training (40 mU: 1.84 ± 0.32 to 2.67 ± 0.37 mg·kg^–1·min^–1, P < 0.0001; 1,000 mU: 7.57 ± 0.61 to 8.84 ± 0.56 mg·kg^–1·min^–1, P = 0.008). Hepatic glucose production, both in the basal state (3.17 ± 0.43 vs. 2.54 ± 0.26 mg·kg^–1·min^–1, P = 0.05) and during the 40-mU clamp stage (1.15 ± 0.41 vs. 0.46 ± 0.20 mg·kg^–1·min^–1, P = 0.03), was significantly reduced after training. One week of vigorous exercise training can induce significant improvements in insulin action in type 2 diabetes. These improvements include increased peripheral insulin sensitivity and responsiveness as well as enhanced suppression of hepatic glucose production.

Follistatin induces muscle hypertrophy through satellite cell proliferation and inhibition of both myostatin and activin

Gilson, H., Schakman, O., Kalista, S., Lause, P., Tsuchida, K., Thissen, J.-P. — 2009-06-25

Follistatin (FS) inhibits several members of the TGF-β superfamily, including myostatin (Mstn), a negative regulator of muscle growth. Mstn inhibition by FS represents a potential therapeutic approach of muscle atrophy. The aim of our study was to investigate the mechanisms of the FS-induced muscle hypertrophy. To test the role of satellite cells in the FS effect, we used irradiation to destroy their proliferative capacity. FS overexpression increased the muscle weight by about 37% in control animals, but the increase reached only 20% in irradiated muscle, supporting the role of cell proliferation in the FS-induced hypertrophy. Surprisingly, the muscle hypertrophy caused by FS reached the same magnitude in Mstn-KO as in WT mice, suggesting that Mstn might not be the only ligand of FS involved in the regulation of muscle mass. To assess the role of activin (Act), another FS ligand, in the FS-induced hypertrophy, we electroporated FSI-I, a FS mutant that does not bind Act with high affinity. Whereas FS electroporation increased muscle weight by 32%, the muscle weight gain induced by FSI-I reached only 14%. Furthermore, in Mstn-KO mice, FSI-I overexpression failed to induce hypertrophy, in contrast to FS. Therefore, these results suggest that Act inhibition may contribute to FS-induced hypertrophy. Finally, the role of Act as a regulator of muscle mass was supported by the observation that ActA overexpression induced muscle weight loss (–15%). In conclusion, our results show that satellite cell proliferation and both Mstn and Act inhibition are involved in the FS-induced muscle hypertrophy.

Inhibiting gluconeogenesis prevents fatty acid-induced increases in endogenous glucose production

2009-06-25

Glucose effectiveness, the ability of glucose per se to suppress endogenous glucose production (EGP), is lost in type 2 diabetes mellitus (T2DM). Free fatty acids (FFA) may contribute to this loss of glucose effectiveness in T2DM by increasing gluconeogenesis (GNG) and impairing the response to hyperglycemia. Thus, we first examined the effects of increasing plasma FFA levels for 3, 6, or 16 h on glucose effectiveness in nondiabetic subjects. Under fixed hormonal conditions, hyperglycemia suppressed EGP by 61% in nondiabetic subjects. Raising FFA levels with Liposyn infusion for ≥3 h reduced the normal suppressive effect of glucose by one-half. Second, we hypothesized that inhibiting GNG would prevent the negative impact of FFA on glucose effectiveness. Raising plasma FFA levels increased gluconeogenesis by ~52% during euglycemia and blunted the suppression of EGP by hyperglycemia. Infusion of ethanol rapidly inhibited GNG and doubled the suppression of EGP by hyperglycemia, thereby restoring glucose effectiveness. In conclusion, elevated FFA levels rapidly increased GNG and impaired hepatic glucose effectiveness in nondiabetic subjects. Inhibiting GNG could have therapeutic potential in restoring the regulation of glucose production in type 2 diabetes mellitus.

PPAR{gamma} inhibits NF-{kappa}B-dependent transcriptional activation in skeletal muscle

2009-06-25

Skeletal muscle pathology associated with a chronic inflammatory disease state (e.g., skeletal muscle atrophy and insulin resistance) is a potential consequence of chronic activation of NF-B. It has been demonstrated that peroxisome proliferator-activated receptors (PPARs) can exert anti-inflammatory effects by interfering with transcriptional regulation of inflammatory responses. The goal of the present study, therefore, was to evaluate whether PPAR activation affects cytokine-induced NF-B activity in skeletal muscle. Using C₂C₁₂ myotubes as an in vitro model of myofibers, we demonstrate that PPAR, and specifically PPAR, activation potently inhibits inflammatory mediator-induced NF-B transcriptional activity in a time- and dose-dependent manner. Furthermore, PPAR activation by rosiglitazone strongly suppresses cytokine-induced transcript levels of the NF-B-dependent genes intracellular adhesion molecule 1 (ICAM-1) and CXCL1 (KC), the murine homolog of IL-8, in myotubes. To verify whether muscular NF-B activity in human subjects is suppressed by PPAR activation, we examined the effect of 8 wk of rosiglitazone treatment on muscular gene expression of ICAM-1 and IL-8 in type 2 diabetes mellitus patients. In these subjects, we observed a trend toward decreased basal expression of ICAM-1 mRNA levels. Subsequent analyses in cultured myotubes revealed that the anti-inflammatory effect of PPAR activation is not due to decreased RelA translocation to the nucleus or reduced RelA DNA binding. These findings demonstrate that muscle-specific inhibition of NF-B activation may be an interesting therapeutic avenue for treatment of several inflammation-associated skeletal muscle abnormalities.

Haploinsufficiency of the retinoblastoma protein gene reduces diet-induced obesity, insulin resistance, and hepatosteatosis in mice

2009-06-25

Brown adipose tissue activity dissipates energy as heat, and there is evidence that lack of the retinoblastoma protein (pRb) may favor the development of the brown adipocyte phenotype in adipose cells. In this work we assessed the impact of germ line haploinsufficiency of the pRb gene (Rb) on the response to high-fat diet feeding in mice. Rb^+/– mice had body weight and adiposity indistinguishable from that of wild-type (Rb^+/+) littermates when maintained on a standard diet, yet they gained less body weight and body fat after long-term high-fat diet feeding coupled with reduced feed efficiency and increased rectal temperature. Rb haploinsufficiency ameliorated insulin resistance and hepatosteatosis after high-fat diet in male mice, in which these disturbances were more marked than in females. Compared with wild-type littermates, Rb^+/– mice fed a high-fat diet displayed higher expression of peroxisome proliferator-activated receptor (PPAR) as well as of genes involved in mitochondrial function, cAMP sensitivity, brown adipocyte determination, and tissue vascularization in white adipose tissue depots. Furthermore, Rb^+/– mice exhibited signs of enhanced activation of brown adipose tissue and higher expression levels of PPAR in liver and of PPAR in skeletal muscle, suggestive of an increased capability for fatty acid oxidation in these tissues. These findings support a role for pRb in modulating whole body energy metabolism and the plasticity of the adipose tissues in vivo and constitute first evidence that partial deficiency in the Rb gene protects against the development of obesity and associated metabolic disturbances.

Diabetes increases brain damage caused by severe hypoglycemia

Bree, A. J., Puente, E. C., Daphna-Iken, D., Fisher, S. J. — 2009-06-25

Insulin-induced severe hypoglycemia causes brain damage. The hypothesis to be tested was that diabetes portends to more extensive brain tissue damage following an episode of severe hypoglycemia. Nine-week-old male streptozotocin-diabetic (DIAB; n = 10) or vehicle-injected control (CONT; n = 7) Sprague-Dawley rats were subjected to hyperinsulinemic (0.2 U·kg^–1·min^–1) severe hypoglycemic (10–15 mg/dl) clamps while awake and unrestrained. Groups were precisely matched for depth and duration (1 h) of severe hypoglycemia (CONT 11 ± 0.5 and DIAB 12 ± 0.2 mg/dl, P = not significant). During severe hypoglycemia, an equal number of episodes of seizure-like activity were noted in both groups. One week later, histological analysis demonstrated extensive neuronal damage in regions of the hippocampus, especially in the dentate gyrus and CA1 regions and less so in the CA3 region (P < 0.05), although total hippocampal damage was not different between groups. However, in the cortex, DIAB rats had significantly (2.3-fold) more dead neurons than CONT rats (P < 0.05). There was a strong correlation between neuronal damage and the occurrence of seizure-like activity (r² > 0.9). Separate studies conducted in groups of diabetic (n = 5) and nondiabetic (n = 5) rats not exposed to severe hypoglycemia showed no brain damage. In summary, under the conditions studied, severe hypoglycemia causes brain damage in the cortex and regions within the hippocampus, and the extent of damage is closely correlated to the presence of seizure-like activity in nonanesthetized rats. It is concluded that, in response to insulin-induced severe hypoglycemia, diabetes uniquely increases the vulnerability of specific brain areas to neuronal damage.

The action of leptin in the ventral tegmental area to decrease food intake is dependent on Jak-2 signaling

Morton, G. J., Blevins, J. E., Kim, F., Matsen, M., Figlewicz, D. P. — 2009-06-25

Recent evidence suggests that leptin reduces food intake via actions in the brain circuitry of food reward, such as the ventral tegmental area (VTA), as leptin receptors are present in the VTA, and leptin injection in the VTA reduces food intake. In the hypothalamus, leptin-induced anorexia requires signaling via Janus kinase-signal transducer and activator of transcription (Jak-STAT), insulin receptor substrate (IRS)-phosphatidylinositol 3-kinase (PI 3-kinase), and mammalian target of rapamycin (mTOR). In this study, we determined whether leptin activates each of these signal transduction pathways in the VTA and whether these signaling pathways are required for VTA-leptin induced anorexia. Here, we show that pSTAT3-Tyr⁷⁰⁵, a marker of leptin activation, was induced in a midbrain region containing the VTA and substantia nigra following either intracerebroventricular leptin or direct administration of leptin to the VTA, but these interventions failed to increase levels of either pAKT-Ser⁴⁷³ or phospho-p70S6K-Thr³⁸⁹, markers of IRS-PI 3-kinase and mTOR signaling, respectively. Moreover, the effect of intra-VTA leptin administration to reduce 4- and 20-h food intake and 20-h body weight was blocked by an inhibitor of Jak-2, at a dose that had no effect on food intake or body weight by itself, but not by local inhibition of either PI 3-kinase (LY-294002) or mTOR (rapamycin) in this timeframe. Taken together, these data support the hypothesis that leptin signaling in the VTA is involved in the regulation of energy balance, but, in contrast to the leptin signaling in the hypothalamus, these effects are mediated predominantly via Jak-2 signaling rather than via the IRS-PI 3-kinase or mTOR signaling pathway.

Central role of ceramide biosynthesis in body weight regulation, energy metabolism, and the metabolic syndrome

Yang, G., Badeanlou, L., Bielawski, J., Roberts, A. J., Hannun, Y. A., Samad, F. — 2009-06-25

Although obesity is associated with multiple features of the metabolic syndrome (insulin resistance, leptin resistance, hepatic steatosis, chronic inflammation, etc.), the molecular changes that promote these conditions are not completely understood. Here, we tested the hypothesis that elevated ceramide biosynthesis contributes to the pathogenesis of obesity and the metabolic syndrome. Chronic treatment for 8 wk of genetically obese (ob/ob), and, high-fat diet-induced obese (DIO) mice with myriocin, an inhibitor of de novo ceramide synthesis, decreased circulating ceramides. Decreased ceramide was associated with reduced weight, enhanced metabolism and energy expenditure, decreased hepatic steatosis, and improved glucose hemostasis via enhancement of insulin signaling in the liver and muscle. Inhibition of de novo ceramide biosynthesis decreased adipose expression of suppressor of cytokine signaling-3 (SOCS-3) and induced adipose uncoupling protein-3 (UCP3). Moreover, ceramide directly induced SOCS-3 and inhibited UCP3 mRNA in cultured adipocytes suggesting a direct role for ceramide in regulation of metabolism and energy expenditure. Inhibition of de novo ceramide synthesis had no effect on adipose tumor necrosis factor- (TNF-) expression but dramatically reduced adipose plasminogen activator inhibitor-1 (PAI-1) and monocyte chemoattactant protein-1 (MCP-1). This study highlights a novel role for ceramide biosynthesis in body weight regulation, energy expenditure, and the metabolic syndrome.

Decreased whole body lipolysis as a mechanism of the lipid-lowering effect of pioglitazone in type 2 diabetic patients

2009-06-25

Pioglitazone has been shown to reduce fasting triglyceride levels. The mechanisms of this effect have not been fully elucidated, but decreased lipolysis may contribute to blunt the hypertriglyceridemic response to a meal. To test this hypothesis, we studied 27 type 2 diabetes mellitus (T2DM) patients and 7 sex-, age-, and body mass index-matched nondiabetic controls. Patients were randomized to pioglitazone (45 mg/day) or placebo for 16 wk. Whole body lipolysis was measured [as the [²H₅]glycerol rate of appearance (R_a)] in the fasting state and for 6 h following a mixed meal. Compared with controls, T2DM had higher postprandial profiles of plasma triglycerides, free fatty acid (FFA), and β-hydroxybutyrate, and a decreased suppression of glycerol R_a (P < 0.04) despite higher insulin levels [268 (156) vs. 190 (123) pmol/l, median (interquartile range)]. Following pioglitazone, triglycerides and FFA were reduced (P = 0.05 and P < 0.04, respectively), and glycerol R_a was more suppressed [–40 (137) vs. +7 (202) µmol/min of placebo, P < 0.05] despite a greater fall in insulin [–85 (176) vs. –20 (58) pmol/l, P = 0.05]. We conclude that, in well-controlled T2DM patients, whole body lipolysis is insulin resistant, and pioglitazone improves the insulin sensitivity of lipolysis.

Regulation of net hepatic glycogenolysis and gluconeogenesis by epinephrine in humans

Dufour, S., Lebon, V., Shulman, G. I., Petersen, K. F. — 2009-06-25

The relative contributions of net hepatic glycogenolysis (NHG) and gluconeogenesis to rates of glucose production during a physiological increment in plasma epinephrine concentrations, independent of changes in plasma insulin concentrations, were determined in seven fasting, healthy young subjects. Plasma insulin concentrations were kept constant by infusing somatostatin (0.1 µg·kg^–1·min^–1) and replacing basal insulin (24 pmol·m^–2·min^–1). Epinephrine (1.2 µg·m^–2·min^–1) was infused for 90 min while NHG was assessed directly by ¹³C magnetic resonance spectroscopy. The rate of glucose production was assessed using [6,6-²H₂]glucose, and gluconeogenesis was calculated as the difference between the rate of glucose production and NHG. Plasma epinephrine concentrations increased rapidly from ~100 to ~2,000 pmol/l (P < 0.00001) accompanied by an increase in plasma glucose concentrations from 4.3 ± 0.2 to 13.3 ± 0.3 mmol/l at 90 min (P = 0.00001). This increase in plasma epinephrine concentration resulted in a 2.5-fold increase in glucose production (from 14.4 ± 1.0 µmol·kg^–1·min^–1 to 35.7 ± 2.0 µmol·kg^–1·min^–1, P < 0.0001), which lasted for ~60 min (phase 1), after which glucose production decreased to 31.2 ± 1.9 µmol·kg^–1·min^–1 (P < 0.0001 vs. basal) during the last 30 min of the epinephrine infusion (phase 2). Hepatic glycogen concentrations decreased almost linearly during phase 1, and rates of NHG were 19.9 ± 3.0 µmol·kg^–1·min^–1 (P = 0.005 vs. basal), which could account for ~60% of glucose production. During phase 2, NHG decreased to 7.3 ± 2.8 µmol·kg^–1·min^–1 (P = 0.02 vs. peak), accounting for only ~20% of glucose production. In conclusion, in the presence of basal plasma insulin and glucagon concentrations, a physiological increase in plasma epinephrine concentrations stimulates glucose production with an initial, 60-min transient phase caused by stimulation of NHG and a second phase that can mostly be attributed to a twofold increase in rates of gluconeogenesis.

Is "fat-induced" muscle insulin resistance rapidly reversible?

Han, D.-H., Hancock, C., Jung, S.-R., Holloszy, J. O. — 2009-06-25

Elevated plasma free fatty acids (FFA) cause insulin resistance and are thought to play a key role in mediating insulin resistance in patients with the metabolic syndrome (MTS) and type 2 diabetes mellitus (DM). Two experimental models used to study the mechanisms responsible for insulin resistance in patients are high-fat diet-fed rodents and administration of triglycerides and heparin to raise plasma FFA. As evidence that insulin resistance in high-fat diet-fed rats is due to high FFA, it has been reported that the insulin resistance is rapidly reversed by an overnight fast, a high-glucose meal, and an exercise bout. If true, these findings would invalidate the high-fat diet-fed rodent as a model for MTS or type 2 DM, because insulin resistance is not rapidly reversed by these treatments in patients. The purpose of this study was to determine whether diet-induced insulin resistance is, in fact, rapidly reversible. Incubation of muscles in vitro rapidly reversed insulin resistance induced by administration of triglycerides and heparin, but not by a high-fat diet. An overnight fast and a high-glucose meal were followed by a large increase in insulin-stimulated muscle glucose transport. However, these are adaptive responses, rather than reversals of insulin resistance, because they also occurred in muscles of insulin-sensitive, chow-fed control rats. Our results show that insulin resistance induced by high FFA, i.e., Randle glucose-fatty acid cycle, is transient. In contrast, the insulin resistance induced by a high-fat diet does not reverse rapidly.

Increased AS160 phosphorylation, but not TBC1D1 phosphorylation, with increased postexercise insulin sensitivity in rat skeletal muscle

Funai, K., Schweitzer, G. G., Sharma, N., Kanzaki, M., Cartee, G. D. — 2009-06-25

A single exercise bout can increase insulin-independent glucose transport immediately postexercise and insulin-dependent glucose transport (GT) for several hours postexercise. Akt substrate of 160 kDa (AS160) and TBC1D1 are paralog Rab GTPase-activating proteins that have been proposed to contribute to these exercise effects. Previous research demonstrated greater AS160 and Akt threonine phosphorylation in rat skeletal muscle at 3–4 h postexercise concomitant with enhanced insulin-stimulated GT. To further probe whether these signaling events or TBC1D1 phosphorylation were important for the enhanced postexercise insulin-stimulated GT, male Wistar rats were studied using four experimental protocols (2-h swim exercise, differing with regard to timing of muscle sampling and whether food was provided postexercise) that were known to vary in their influence of insulin-independent and insulin-dependent GT postexercise. The results indicated that, in isolated rat epitrochlearis muscle, 1) elevated phosphorylation of AS160 (measured using anti-phospho-Akt substrate, PAS-AS160, and phosphospecific anti-Thr⁶⁴²-AS160, pThr⁶⁴²-AS160) consistently tracked with elevated insulin-stimulated GT; 2) PAS-TBC1D1 was not different from sedentary values at 3 or 27 h postexercise, when insulin sensitivity was increased; 3) insulin-stimulated Akt activity was not increased postexercise in muscles with increased insulin sensitivity; 4) PAS-TBC1D1 was increased immediately postexercise, when insulin-independent GT was elevated, and reversed at 3 and 27 h postexercise, when insulin-independent GT was also reversed; and 5) there was no significant effect of exercise or insulin on total abundance of AS160, TBC1D1, Akt, or GLUT4 protein with any of the protocols. The results are consistent with increased AS160 phosphorylation (PAS-AS160 or pThr⁶⁴²-AS160) but not increased PAS-TBC1D1 or Akt activity, which is important for increased postexercise insulin-stimulated GT in rat skeletal muscle. They also support the idea that increased TBC1D1 phosphorylation may play a role in the insulin-independent increase in GT postexercise.

A comparison of 2H2O and phenylalanine flooding dose to investigate muscle protein synthesis with acute exercise in rats

Gasier, H. G., Riechman, S. E., Wiggs, M. P., Previs, S. F., Fluckey, J. D. — 2009-06-25

The primary objective of this investigation was to determine whether ²H₂O and phenylalanine (Phe) flooding dose methods yield comparable fractional rates of protein synthesis (FSR) in skeletal muscle following a single bout of high-intensity resistance exercise (RE). Sprague-Dawley rats were assigned by body mass to either 4-h control (CON 4 h; n = 6), 4-h resistance exercise (RE 4 h; n = 6), 24-h control (CON 24 h; n = 6), or 24-h resistance exercise (RE 24 h; n = 6). The RE groups were operantly conditioned to engage in a single bout of high-intensity, "squat-like" RE. All rats were given an intraperitoneal injection of 99.9% ²H₂O and provided 4.0% ²H₂O drinking water for either 24 (n = 12) or 4 h (n = 12) prior to receiving a flooding dose of l-[2,3,4,5,6-³H]Phe 16 h post-RE. Neither method detected an effect of RE on FSR in the mixed gastrocnemius, plantaris, or soleus muscle. Aside from the qualitative similarities between methods, the 4-h ²H₂O FSR measurements, when expressed in percent per hour, were quantitatively greater than the 24-h ²H₂O and Phe flooding in all muscles (P < 0.001), and the 24-h ²H₂O was greater than the Phe flooding dose in the mixed gastrocnemius and plantaris (P < 0.05). In contrast, the actual percentage of newly synthesized protein was significantly higher in the 24- vs. 4-h ²H₂O and Phe flooding dose groups (P < 0.001). These results suggest that the methodologies provide "qualitatively" similar results when a perturbation such as RE is studied. However, due to potential quantitative differences between methods, the experimental question should determine what approach should be used.

Dynamics of glutathione and ophthalmate traced with 2H-enriched body water in rats and humans

2009-06-25

We developed a LC-MS-MS assay of the ²H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from ²H-enriched body water. In rats whose body water was 2.5% ²H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [¹³C₂,¹⁵N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 µmol·min^–1·kg^–1, and the half-life of plasma GSH/GSSR was 6–8 min. In healthy humans whose body fluids were 0.5% ²H enriched, the ²H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.

Corrigendum

2009-06-25