Adenoviral (Ad)-mediated in vivo gene transfer and expression are limited in part by cellular immune responses to viral-encoded proteins and/or transgene immunogenicity. In an attempt to diminish the former responses, we have previously developed and described helper-dependent (HD) Ad vectors in which the viral protein coding sequences are completely eliminated. These HD vectors have up to 37 kb insert capacity, are easily propagated in a Cre recombinase-based system, and can be produced to high concentration and purity (>99.9% helper-free vector). In this study, we compared safety and efficacy of leptin gene delivery mediated by an HD vector (HD-leptin) and a first-generation E1-deleted Ad vector (Ad-leptin) in normal lean and ob/ob (leptin-deficient) mice. In contrast to evidence of liver toxicity, inflammation, and cellular infiltration observed with Ad-leptin delivery in mice, HD-leptin delivery was associated with a significant improvement in associated safety/toxicity and resulted in efficient gene delivery, prolonged elevation of serum leptin levels, and associated weight loss. The greater safety, efficient gene delivery, and increased insert capacity of HD vectors are significant improvements over current Ad vectors and represent favorable features especially for clinical gene therapy applications.

The repertoire of approximately 1200 odorant receptors (ORs) is mapped onto the array of approximately 1800 glomeruli in the mouse olfactory bulb (OB). The spatial organization of this array is influenced by the ORs. Here we show that glomerular mapping to broad domains in the dorsal OB is determined by two types of olfactory sensory neurons (OSNs), which reside in the dorsal olfactory epithelium. The OSN types express either class I or class II OR genes. Axons from the two OSN types segregate already within the olfactory nerve and form distinct domains of glomeruli in the OB. These class-specific anatomical domains correlate with known functional odorant response domains. However, axonal segregation and domain formation are not determined by the class of the expressed OR protein. Thus, the two OSN types are determinants of axonal wiring, operate at a higher level than ORs, and contribute to the functional organization of the glomerular array.

Nonalcoholic steatohepatitis (NASH) is a progressive form of nonalcoholic fatty liver disease that can develop into cirrhosis, hepatic failure, and hepatocellular carcinoma. Although several metabolic pathways are disrupted and endogenous metabolites may change in NASH, the alterations in serum metabolites during NASH development remain unclear. To gain insight into the disease mechanism, serum metabolite changes were assessed using metabolomics with ultraperformance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry and a conventional mouse NASH model induced by a methionine- and choline-deficient (MCD) diet. Significant decreases in serum palmitoyl-, stearoyl-, and oleoyl-lysophosphatidylcholine (LPC) and marked increases in tauro-β-muricholate, taurocholate and 12-hydroxyeicosatetraenoic acid (12-HETE) were detected in mice with NASH. In agreement with these metabolite changes, hepatic mRNAs encoding enzymes and proteins involved in LPC degradation (lysophosphatidylcholine acyltransferase [Lpcat] 1-4), basolateral bile acid excretion (ATP-binding cassette subfamily C member [Abcc] 1/4/5 and organic solute transporter β), and 12-HETE synthesis (arachidonate 12-lipoxygenase) were significantly up-regulated. In contrast, the expression of solute carrier family 10 member 1 (Slc10a1) and solute carrier organic anion transporter family member (Slco) 1a1 and 1b2, responsible for transporting bile acids into hepatocytes, were markedly suppressed. Supplementation of the MCD diet with methionine revealed that the changes in serum metabolites and the related gene expression were derived from steatohepatitis, but not dietary choline deficiency or steatosis. Furthermore, tumor necrosis factor-α and transforming growth factor-β1 induced the expression of Lpcat2/4 and Abcc1/4 and down-regulated Slc10a1 and Slco1a1 in primary hepatocytes, suggesting an association between the changes in serum LPC and bile acids and proinflammatory cytokines. Finally, induction of hepatitis in ob/ob mice by D-galactosamine injection led to similar changes in serum metabolites and related gene expression.

CONCLUSIONS

Phospholipid and bile acid metabolism is disrupted in NASH, likely due to enhanced hepatic inflammatory signaling.

Leptin, a hormone secreted from white adipose tissue, has been shown to normalize the body weight of ob/ob but not db/db mice as postulated by Coleman in his classical parabiosis experiments. The major effect of leptin is therefore to decrease food intake, thus resulting in a breakdown of fat stores. Recently, we have suggested that leptin plays a role in reproductive physiology based on the observation that leptin treatment but not food restriction rescues the sterility of ob/ob females. In the present communication, we treated sterile ob/ob males with leptin and asked whether fertility could be induced, thus selecting their reproductive ability as the endpoint of the experiment. Our results show that all food-restricted ob/ob males are unable to impregnate normal C57BL/6J females. However, all leptin-treated ob/ob males fertilized normal females mice that carried out normal pregnancies and deliveries, demonstrating that the reproductive capacity of ob/ob males was corrected only with leptin treatment. Furthermore, reproductive indices such as testicular weight and histology are normalized in leptin-treated animals. Therefore, as in ob/ob females, leptin plays a significant role in the male mouse reproductive pathways.

The possible role of astrocytes in the regulation of feeding has been overlooked. It is well-established that the endothelial cells constituting the blood-brain barrier transport leptin from blood to brain and that hypothalamic neurons respond to leptin to induce anorexic signaling. However, few studies have addressed the role of astrocytes in either leptin transport or cellular activation. We recently showed that the obese agouti viable yellow mouse has prominent astrocytic expression of the leptin receptor. In this study, we test the hypothesis that diet-induced obesity increases astrocytic leptin receptor expression and function in the hypothalamus. Double-labelling immunohistochemistry and confocal microscopic analysis showed that all astrocytes in the hypothalamus express leptin receptors. In adult obese mice, 2 months after being placed on a high-fat diet, there was a striking increase of leptin receptor (+) astrocytes, most prominent in the dorsomedial hypothalamus and arcuate nucleus. Agouti viable yellow mice with their adult-onset obesity showed similar changes, but the increase of leptin receptor (+) astrocytes was barely seen in ob/ob or db/db mice with their early-onset obesity and defective leptin systems. The marked leptin receptor protein expression in the astrocytes, shown with several antibodies against different receptor epitopes, was supported by RT-PCR detection of leptin receptor-a and -b mRNAs in primary hypothalamic astrocytes. Unexpectedly, the protein expression of GFAP, a marker of astrocytes, was also increased in adult-onset obesity. Real-time confocal imaging showed that leptin caused a robust increase of calcium signalling in primary astrocytes from the hypothalamus, confirming their functionality. The results indicate that metabolic changes in obese mice can rapidly alter leptin receptor expression and astrocytic activity, and that leptin receptor is responsible for leptin-induced calcium signalling in astrocytes. This novel and clinically relevant finding opens new avenues in astrocyte biology.

Obstructive sleep apnea (OSA), a condition tightly linked to obesity, leads to chronic intermittent hypoxia (CIH) during sleep. There is emerging evidence that OSA is independently associated with insulin resistance and fatty liver disease, suggesting that OSA may affect hepatic lipid metabolism. To test this hypothesis, leptin-deficient obese (ob/ob) mice were exposed to CIH during the light phase (9 AM-9 PM) for 12 wk. Liver lipid content and gene expression profile in the liver (Affymetrix 430 GeneChip with real-time PCR validation) were determined on completion of the exposure. CIH caused a 30% increase in triglyceride and phospholipid liver content (P < 0.05), whereas liver cholesterol content was unchanged. Gene expression analysis showed that CIH upregulated multiple genes controlling 1) cholesterol and fatty acid biosynthesis [malic enzyme and acetyl coenzyme A (CoA) synthetase], 2) predominantly fatty acid biosynthesis (acetyl-CoA carboxylase and stearoyl-CoA desaturases 1 and 2), and 3) triglyceride and phospholipid biosynthesis (mitochondrial glycerol-3-phosphate acyltransferase). A majority of overexpressed genes were transcriptionally regulated by sterol regulatory element-binding protein (SREBP) 1, a master regulator of lipogenesis. A 2.8-fold increase in SREBP-1 gene expression in CIH was confirmed by real-time PCR (P = 0.001). Expression of major genes of cholesterol biosynthesis, SREBP-2 and 3-hydroxy-3-methylglutaryl-CoA reductase, was unchanged. In conclusion, we have shown that CIH may exacerbate preexisting fatty liver of obesity via upregulation of the pathways of lipid biosynthesis in the liver.

Distinct olfactory bulb (OB) interneurons are thought to become specified depending on from which of the different subregions lining the lateral ventricle wall they originate, but the role of region-specific transcription factors (TFs) in the generation of OB interneurons diversity is still poorly understood. Despite the crucial roles of the Dlx family of TFs for patterning and neurogenesis in the ventral telencephalon during embryonic development, their role in adult neurogenesis has not yet been addressed. Here we show that in the adult brain, Dlx 1 and Dlx2 are expressed in progenitors of the lateral but not the dorsal subependymal zone (SEZ), thus exhibiting a striking regional specificity. Using retroviral vectors to examine the function of Dlx2 in a cell-autonomous manner, we demonstrate that this TF is necessary for neurogenesis of virtually all OB interneurons arising from the lateral SEZ. Beyond its function in generic neurogenesis, Dlx2 also plays a crucial role in neuronal subtype specification in the OB, promoting specification of adult-born periglomerular neurons (PGNs) toward a dopaminergic fate. Strikingly, Dlx2 requires interaction with Pax6, because Pax6 deletion blocks Dlx2-mediated PGN specification. Thus, Dlx2 wields a dual function by first instructing generic neurogenesis from adult precursors and subsequently specifying PGN subtypes in conjunction with Pax6.

The olfactory epithelium of fish contains two major types of olfactory sensory neurons (OSNs) that are distinct morphologically (ciliated vs microvillous) and possibly functionally. Here, we found that these OSNs express different sets of signal transduction machineries: the ciliated OSNs express OR-type odorant receptors, cyclic nucleotide-gated channel A2 subunit, and olfactory marker protein (OMP), whereas the microvillous OSNs express V2R-type receptors and transient receptor potential channel C2 (TRPC2). To visualize patterns of axonal projection from the two types of OSNs to the olfactory bulb (OB), we generated transgenic zebrafish in which spectrally distinct fluorescent proteins are expressed in the ciliated and microvillous OSNs under the control of OMP and TRPC2 gene promoters, respectively. An observation of whole-mount OB in adult double-transgenic zebrafish revealed that the ciliated OSNs project axons mostly to the dorsal and medial regions of the OB, whereas the microvillous OSNs project axons to the lateral region of the OB. A careful histological examination of OB sections clarified that the axons from the two distinct types of OSNs target different glomeruli in a mutually exclusive manner. This segregation is already established at very early developmental stages in zebrafish embryos. These findings clearly demonstrate the relationships among cell morphology, molecular signatures, and axonal terminations of the two distinct types of OSNs and suggest that the two segregated neural pathways are responsible for coding and processing of different types of odor information in the zebrafish olfactory system.

BACKGROUND

Incretin hormones, such as glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in meal-related insulin secretion. We previously demonstrated that glutamine is a potent stimulus of GLP-1 secretion in vitro.

OBJECTIVE

Our objective was to determine whether glutamine increases circulating GLP-1 and GIP concentrations in vivo and, if so, whether this is associated with an increase in plasma insulin.

METHODS

We recruited 8 healthy normal-weight volunteers (LEAN), 8 obese individuals with type 2 diabetes or impaired glucose tolerance (OB-DIAB) and 8 obese nondiabetic control subjects (OB-CON). Oral glucose (75 g), glutamine (30 g), and water were administered on 3 separate days in random order, and plasma concentrations of GLP-1, GIP, insulin, glucagon, and glucose were measured over 120 min.

RESULTS

Oral glucose led to increases in circulating GLP-1 concentrations, which peaked at 30 min in LEAN (31.9 +/- 5.7 pmol/L) and OB-CON (24.3 +/- 2.1 pmol/L) subjects and at 45 min in OB-DIAB subjects (19.5 +/- 1.8 pmol/L). Circulating GLP-1 concentrations increased in all study groups after glutamine ingestion, with peak concentrations at 30 min of 22.5 +/- 3.4, 17.9 +/- 1.1, and 17.3 +/- 3.4 pmol/L in LEAN, OB-CON, and OB-DIAB subjects, respectively. Glutamine also increased plasma GIP concentrations but less effectively than glucose. Consistent with the increases in GLP-1 and GIP, glutamine significantly increased circulating plasma insulin concentrations. Glutamine stimulated glucagon secretion in all 3 study groups.

CONCLUSIONS

Glutamine effectively increases circulating GLP-1, GIP, and insulin concentrations in vivo and may represent a novel therapeutic approach to stimulating insulin secretion in obesity and type 2 diabetes.

The hormone leptin acts on the brain to regulate feeding, metabolism, and reproduction; however, its cellular targets and molecular mechanisms of action remain to be fully elucidated. The melanocortins, which are derived from the precursor proopiomelanocortin (POMC), are also implicated in the physiological regulation of body weight. POMC-containing neurons express the leptin receptor, and thus it is conceivable that the POMC gene itself may be part of the signaling pathway involved in leptin's action on the brain. Using in situ hybridization and computerized image analysis, we tested the hypothesis that the POMC gene is a target for regulation by leptin by comparing cellular levels of POMC mRNA in the hypothalamus among groups of leptin-deficient (ob/ob) mice, leptin-treated ob/ob mice, and wild-type controls. POMC mRNA levels were significantly reduced throughout the arcuate nucleus in vehicle-treated ob/ob mice relative to wild-type controls, whereas POMC mRNA levels in leptin-treated ob/ob mice were indistinguishable from wild-type controls. These observations suggest that one or more products of POMC serve as an integrative link between leptin and the central mechanisms governing body weight regulation and reproduction.

Myostatin (MSTN) is a secreted growth inhibitor expressed in muscle and adipose. We sought to determine whether expression of MSTN, its receptor activin RIIb (ActRIIb), or its binding protein follistatin-like-3 (FSTL3) are altered in subcutaneous or visceral adipose or in skeletal muscle in response to obesity. MSTN and ActRIIb mRNA levels were low in subcutaneous (SQF) and visceral fat (VF) from wild-type mice but were 50- to 100-fold higher in both SQF and VF from ob/ob compared with wild-type mice. FSTL3 mRNA levels were increased in SQF but decreased in VF in ob/ob compared with wild-type mice. Moreover, MSTN mRNA levels were twofold greater in tibialis anterior (TA) from ob/ob mice, whereas ActRIIb and FSTL3 mRNA levels were unchanged. MSTN mRNA levels were also increased in TA and SQF from mice on a high-fat diet. Injection of ob/ob mice with recombinant leptin caused FSTL3 mRNA levels to decrease in both VF and SQF in ob/ob mice; MSTN and ActRIIb mRNA levels tended to decrease only in VF. Finally, MSTN mRNA levels and promoter activity were low in adipogenic 3T3-L1 cells, but an MSTN promoter-reporter construct was activated in 3T3-L1 cells by cotransfection with the adipogenic transcription factors SREBP-1c, C/EBPalpha, and PPARgamma. These results demonstrate that expression of MSTN and its associated binding proteins can be modulated in adipose tissue and skeletal muscle by chronic obesity and suggest that alterations in their expression may contribute to the changes in growth and metabolism of lean and fat tissues occurring during obesity.

Insulin-producing cells normally occur only in the pancreas and thymus. Surprisingly, we found widespread insulin mRNA and protein expression in different diabetic mouse and rat models, including streptozotocin-treated mice and rats, ob/ob mice, and mice fed high-fat diets. We detected in diabetic mice proinsulin- and insulin-positive cells in the liver, adipose tissue, spleen, bone marrow, and thymus; many cells also produced glucagon, somatostatin, and pancreatic polypeptide. By in situ nucleic acid hybridization, diabetic, but not nondiabetic, mouse liver exhibited insulin transcript-positive cells, indicating that insulin was synthesized by these cells. In transgenic mice that express GFP driven by the mouse insulin promoter, streptozotocin-induced diabetes led to the appearance of GFP-positive cells in liver, adipose tissue, and bone marrow; the fluorescent signals showed complete concordance with the presence of immunoreactive proinsulin. Hyperglycemia produced by glucose injections in nondiabetic mice led to the appearance of proinsulin- and insulin-positive cells within 3 days. Bone marrow transplantation experiments showed that most of the extrapancreatic proinsulin-producing cells originated from the bone marrow. Immunoreactive proinsulin- and insulin-positive cells were also detected in the liver, adipose tissue, and bone marrow of diabetic rats, indicating that extrapancreatic, extrathymic insulin production occurs in more than one species. These observations have implications for the regulation of insulin gene expression, modulation of self-tolerance by insulin gene expression, and strategies for the generation of insulin-producing cells for the treatment of diabetes.

Fatty liver, formerly associated predominantly with excessive alcohol intake, is now also recognized as a complication of obesity and an important precursor state to more severe forms of liver pathology including ischemia/reperfusion injury. No standard protocol for treating fatty liver exists at this time. We therefore examined the effects of 10 days of interleukin 6 (IL-6) injection in 3 murine models of fatty liver: leptin deficient ob/ob mice, ethanol-fed mice, and mice fed a high-fat diet. In all 3 models, IL-6 injection decreased steatosis and normalized serum aminotransferase. The beneficial effects of IL-6 treatment in vivo resulted in part from an increase in mitochondrial beta oxidation of fatty acid and an increase in hepatic export of triglyceride and cholesterol. However, administration of IL-6 to isolated cultured steatotic hepatocytes failed to decrease lipid contents, suggesting that the beneficial effects of IL-6 in vivo do not result from its effects on hepatocytes alone. IL-6 treatment increased hepatic peroxisome proliferator-activated receptor (PPAR) alpha and decreased liver and serum tumor necrosis factor (TNF) alpha. Finally, 10 days of treatment with IL-6 prevented the susceptibility of fatty livers to warm ischemia/reperfusion injury. In conclusion, long-term IL-6 administration ameliorates fatty livers and protects against warm ischemia/reperfusion fatty liver injury, suggesting the therapeutic potential of IL-6 in treating human fatty liver disease.

OBJECTIVE

Nonalcoholic fatty liver disease (NAFLD) is characterized by excessive triglyceride accumulation in hepatocytes. Expression of the lipid droplet protein adipose differentiation-related protein (ADRP) is increased in NAFLD, but whether this is causally linked to hepatic lipid metabolism is unclear. We postulated that a reduction in ADRP would ameliorate hepatic steatosis and improve insulin action.

METHODS

Leptin deficient Lep(ob/ob) and diet-induced obese (DIO) mice were treated with antisense oligonucleotide (ASO) against ADRP, and effects on hepatic and serum lipids and glucose homeostasis were examined.

RESULTS

ADRP ASO specifically decreased ADRP mRNA and protein levels in the livers of Lep(ob/ob) and DIO mice, without altering the levels of other lipid droplet proteins, that is, S3-12 and TIP47. ADRP ASO suppressed expression of lipogenic genes, reduced liver triglyceride content without affecting cholesterol, attenuated triglyceride secretion, and decreased serum triglyceride and alanine aminotransaminase levels. The reduction in hepatic steatosis by ADRP ASO was associated with improvement in glucose homeostasis in both Lep(ob/ob) and DIO mice.

CONCLUSIONS

This study demonstrates a crucial role for the lipid droplet protein ADRP in regulation of lipid metabolism. Reduction in hepatic ADRP level using an antisense oligonucleotide reverses hepatic steatosis, hypertriglyceridemia, and insulin resistance in obese mice, suggesting that ADRP may be targeted for the treatment of NAFLD and associated lipid and glucose abnormalities.

NN2211 is a long-acting, metabolically stable glucagon-like peptide-1 (GLP-1) derivative designed for once daily administration in humans. NN2211 dose dependently reduced the glycemic levels in ob/ob mice, with antihyperglycemic activity still evident 24 h postdose. Apart from an initial reduction in food intake, there were no significant differences between NN2211 and vehicle treatment, and body weight was not affected. Histological examination revealed that beta-cell proliferation and mass were not increased significantly in ob/ob mice with NN2211, although there was a strong tendency for increased proliferation. In db/db mice, exendin-4 and NN2211 decreased blood glucose compared with vehicle, but NN2211 had a longer duration of action. Food intake was lowered only on day 1 with both compounds, and body weight was unaffected. beta-Cell proliferation rate and mass were significantly increased with NN2211, but with exendin-4, only the beta-cell proliferation rate was significantly increased. In conclusion, NN2211 reduced blood glucose after acute and chronic treatment in ob/ob and db/db mice and was associated with increased beta-cell mass and proliferation in db/db mice. NN2211 is currently in phase 2 clinical development.

The peroxisome proliferator-activated receptor-gamma (PPARgamma) binds diverse ligands to transcriptionally regulate metabolism and inflammation. Activators of PPARgamma include lipids and anti-hyperglycemic drugs such as thiazolidinediones (TZDs). Recently, TZDs have raised concern after being linked with increased risk of peripheral edema, weight gain, and adverse cardiovascular events. Most reported endogenous PPARgamma ligands are intermediates of lipid metabolism and oxidation that bind PPARgamma with very low affinity. In contrast, nitro derivatives of unsaturated fatty acids (NO(2)-FA) are endogenous products of nitric oxide ((*)NO) and nitrite (NO(2)(-))-mediated redox reactions that activate PPARgamma at nanomolar concentrations. We report that NO(2)-FA act as partial agonists of PPARgamma and covalently bind PPARgamma at Cys-285 via Michael addition. NO(2)-FA show selective PPARgamma modulator characteristics by inducing coregulator protein interactions, PPARgamma-dependent expression of key target genes, and lipid accumulation is distinctively different from responses induced by the TZD rosiglitazone. Administration of this class of signaling mediators to ob/ob mice revealed that NO(2)-FA lower insulin and glucose levels without inducing adverse side effects such as the increased weight gain induced by TZDs.

Epidemiological studies indicate the incidence of asthma is increased in obese and overweight humans. Responses to ozone (O(3)), an asthma trigger, are increased in obese (ob/ob) mice lacking the satiety hormone leptin. The long form of leptin receptor (Ob-R(b)) is required for satiety; mice lacking this receptor (db/db mice) are also substantially obese. Here, wild-type (WT) and db/db mice were exposed to air or O(3) (2 ppm) for 3 h. Airway responsiveness, measured by the forced oscillation technique, was greater in db/db than WT mice after air exposure. O(3)-induced increases in pulmonary resistance and airway responsiveness were also greater in db/db mice. BALF eotaxin, IL-6, KC, and MIP-2 increased 4 h after O(3) exposure and subsided by 24 h, whereas protein and neutrophils continued to increase through 24 h. For each outcome, the effect of O(3) was significantly greater in db/db than WT mice. Previously published results obtained in ob/ob mice were similar except for O(3)-induced neutrophils and MIP-2, which were not different from WT mice. O(3) also induced pulmonary IL-1beta and TNF-alpha mRNA expression in db/db but not ob/ob mice. Leptin was increased in serum of db/db mice, and pulmonary mRNA expression of short form of leptin receptor (Ob-R(a)) was similar in db/db and WT mice. These data confirm obese mice have innate airway hyperresponsiveness and increased pulmonary responses to O(3). Differences between ob/ob mice, which lack leptin, and db/db mice, which lack Ob-R(b) but not Ob-R(a), suggest leptin, acting through Ob-R(a), can modify some pulmonary responses to O(3).

Intracellular insulin signaling involves a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (IRS) and the various isoforms of SH2 domain signaling molecules that can interact with these substrates. In this study, we have evaluated the roles of IRS-1 and IRS-2 in signaling to the phosphatidylinositol (PI) 3-kinase pathway in the ob/ob mouse, a model of the insulin resistance of obesity and non-insulin-dependent diabetes mellitus. We find that the levels of expression of both IRS-1 and IRS-2 are decreased approximately 50% in muscle, whereas in liver the decrease is significantly greater for IRS-2 (72%) than for IRS-1 (29%). This results in differential decreases in IRS-1 and IRS-2 phosphorylation, docking of the p85alpha regulatory subunit of PI 3-kinase, and activation of this enzyme in these two insulin target tissues. In ob/ob liver there is also a change in expression of the alternatively spliced isoforms of the regulatory subunits for PI 3-kinase that was detected at the protein and mRNA level. This resulted in a 45% decrease in the p85alpha form of PI 3-kinase, a ninefold increase in the AS53/p55alpha, and a twofold increase in p50alpha isoforms. Thus, there are multiple alterations in the early steps of insulin signaling in the ob/ob mouse, with differential regulation of IRS-1 and IRS-2, various PI 3-kinase regulatory isoforms, and a lack of compensation for the decrease in insulin signaling by any of the known alternative pathways at these levels.

Although obesity-related fatty livers are vulnerable to damage from endotoxin, the mechanisms involved remain obscure. The purpose of this study was to determine if immunologic priming might be involved by determining if fatty livers resemble normal livers that have been sensitized to endotoxin damage by Propionibacterium acnes infection. The latter induces interleukin (IL)-12 and -18, causing a selective reduction of CD4+NK T cells, diminished IL-4 production, deficient production of T-helper type 2 (Th-2) cytokines (e.g., IL-10), and excessive production of Th-1 cytokines (e.g., interferon gamma [IFN-gamma]). Liver and spleen lymphocyte populations and hepatic cytokine production were compared in genetically obese, ob/ob mice (a model for obesity-related fatty liver) and lean mice. Obese mice have a selective reduction of hepatic CD4+NK T cells. Serum IL-18 is also increased basally, and the hepatic mRNA levels of IL-18 and -12 are greater after endotoxin challenge. Thus, up-regulation of IL-18 and IL-12 in fatty livers may reduce hepatic CD4+NK T cells. In addition, mononuclear cells from fatty livers have decreased expression of the adhesion molecule, leukocyte factor antigen-1 (LFA-1), which is necessary for the hepatic accumulation of CD4+NK T cells. Consistent with reduced numbers of hepatic CD4+NK T cells, mononuclear cells from fatty livers produce less IL-4. Furthermore, after endotoxin treatment, hepatic induction of IL-10 is inhibited, while that of IFN-gamma is enhanced. Thus, fatty livers have inherent immunologic alterations that may predispose them to damage from endotoxin and other insults that induce a proinflammatory cytokine response.

OBJECTIVE

To examine the effects of dietary protein and obesity classification on energy-restriction-induced changes in weight, body composition, appetite, mood, and cardiovascular and kidney health.

METHODS

Forty-six women, ages 28 to 80, BMI 26 to 37 kg/m(2), followed a 12-week 750-kcal/d energy-deficit diet containing higher protein (HP, 30% protein) or normal protein (NP, 18% protein) and were retrospectively subgrouped according to obesity classification [pre-obese (POB), BMI = 26 to 29.9 kg/m(2); obese (OB), BMI = 30 to 37 kg/m(2)).

RESULTS

All subjects lost weight, fat mass, and lean body mass (LBM; p < 0.001). With comparable weight loss, LBM losses were less in HP vs. NP (-1.5 +/- 0.3 vs. -2.8 +/- 0.5 kg; p < 0.05) and POB vs. OB (-1.2 +/- 0.3 vs. -2.9 +/- 0.4 kg; p < 0.005). The main effects of protein and obesity on LBM changes were independent and additive; POB-HP lost less LBM vs. OB-NP (p < 0.05). The energy-restriction-induced decline in satiety was less pronounced in HP vs. NP (p < 0.005). Perceived pleasure increased with HP and decreased with NP (p < 0.05). Lipid-lipoprotein profile and blood pressure improved and kidney function minimally changed with energy restriction (p < 0.05), independently of protein intake.

CONCLUSIONS

Consuming a higher-protein diet and accomplishing weight loss before becoming obese help women preserve LBM. Use of a higher-protein diet also improves perceptions of satiety and pleasure during energy restriction.

Leptin deficiency causes obesity in rodents and humans, but circulating levels of leptin are paradoxically elevated in obesity. The mechanisms underlying this leptin resistance are unknown, but may involve reduced leptin transport across the blood-brain barrier via short isoforms of the leptin receptor (Ob-R). Here, we first quantified short Ob-R mRNA expression in isolated rat cerebral microvessels constituting the blood-brain barrier and found that Ob-Ra and Ob-Rc mRNA were abundantly expressed in similar amounts. Second, brain uptake of leptin was reduced in mice lacking Ob-R. Third, brain uptake of leptin in New Zealand Obese mice, a strain that responds to central, but not peripheral, leptin, was reduced, suggesting that their obesity is at least partly due to deficient leptin transport into the brain. Fourth, brain uptake of leptin was significantly reduced in diet-induced obese mice. Neither New Zealand Obese mice nor diet-induced obese mice exhibited significant decreases in Ob-R mRNA expression in isolated cerebral microvessels. These data support the ideas that short isoforms of Ob-R are involved in brain uptake of leptin and that impaired blood-brain barrier function contributes to the pathogenesis of obesity. However, the mechanisms by which obesity-related deficits in brain uptake of leptin occur remain to be defined.

The orphan receptor Small Heterodimer Partner (SHP, NROBOB(-/-) mice increases insulin sensitivity, and prevents the development of fatty liver. The prevention of steatosis in OB(-/-)/SHP(-/-) double mutants is not due to decreased body weight but is associated with increased hepatic very-low-density lipoprotein (VLDL) secretion and elevated microsomal triglyceride transfer protein (MTP) mRNA and protein levels. SHP represses the transactivation of the MTP promoter and the induction of MTP mRNA by LRH-1 in hepatocytes. Adenoviral overexpression of SHP inhibits MTP activity as well as VLDL-apoB protein secretion, and RNAi knockdown of SHP exhibits opposite effects. The expression of SHP in induced in fatty livers of OB(-/-) mice and other genetic or dietary models of steatosis, and acute overexpression of SHP by adenovirus, result in rapid accumulation of neutral lipids in hepatocytes. In addition, the pathways for hepatic lipid uptake and lipogenic program are also downregulated in OB(-/-)/SHP(-/-) mice, which may contribute to the decreased hepatic lipid content.

CONCLUSIONS

These studies demonstrate that SHP regulates the development of fatty liver by modulating hepatic lipid export, uptake, and synthesis, and that the improved peripheral insulin sensitivity in OB(-/-)/SHP(-/-) mice is associated with decreased hepatic steatosis.

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(d,l-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760+/-210 nm. The average Young's modulus of electrospun PLGA nanofibers was 42+/-26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1-4 weeks at a density of 2 x 10(6)cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells.

The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.