OBJECTIVE

Antioxidants protect an organism from the detrimental effects of free radicals via scavenging or inhibiting their formation. Alterations in the levels of antioxidants and several essential trace elements in the plasma and various tissues of ob/ob mice have been reported previously. The aim of this study was to investigate oxidative status and trace elements in obese individuals.

METHODS

Seventy-six obese men (body mass index (BMI)>> 30 kg/m(2)) and 24 healthy, age-matched male control volunteers were enrolled in the study. Fasting plasma insulin, glucose, triglyceride (TG), total cholesterol, VLDL, and HDL levels, erythrocyte glutathione peroxidase (GSH-Px) and copper zinc-superoxide dismutase (CuZn-SOD) activities, and erythrocyte thiobarbituric acid reactive substances (TBARS) levels were measured in both groups. Erythrocyte copper (Cu), zinc (Zn) and iron (Fe) levels were also measured.

RESULTS

We found that the mean Cu and Fe levels in obese individuals were not significantly different than those in the control group, whereas the mean Zn levels were significantly lower than those of the control group (p = 0.023). The mean erythrocyte CuZn-SOD and GSH-Px levels in obese individuals were significantly lower than those in controls (p = 0.001) whereas erythrocyte TBARS levels were significantly higher (p = 0.001) than those of the control group.

CONCLUSIONS

We conclude that male obesity is associated with defective antioxidant status and hypozincemia, which may have implications in the development of obesity related health problems.

Insulin release and the content of cAMP were studied in microdissected pancreatic islets of noninbred ob/ob (obese) mice. In the absence of 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, 20 mM glucose had no effect on cAMP save a very small initial rise detectable by a freeze-stop perifusion technique only. However, combined with this methylxanthine, 20 mM glucose produced significant increases of cAMP both in perifused islets and in islets conventionally incubated in closed vials. Glucose shared this capacity to raise the cAMP level with D-glyceraldehyde and 1,3-dihydroxyacetone. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin, an activator of adenylate cyclase, also increased the islet cAMP level; the effects of the methylxanthine, whether or not combined with cholera toxin, were potentiated by glucose. Isobutylmethylxanthine (0.05-1.0 mM) or 5 mug/ml of cholera toxin potentiated insulin release in response to 20 mM glucose. However, only 0.5-1.0 mM isobutylmethylxanthine stimulated insulin release in the presence of 3 mM glucose, whereas 0.05-0.1 mM isobutylmethylxanthine or 5 mug/ml of cholera toxin had no effect on secretion at the low glucose concentration. These discrepancies between cAMP-promoting and insulin-releasing activities suggest that glucose does not initiate insulin release by activating the beta-cell adenylate cyclase. By being metabolized in the beta-cells, glucose may both create a release-initiating signal not identical with cAMP and enhance cAMP formation, leading to potentiation of the effect of the initiator signal.

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM.

Performance and cognitive effort in humans have recently been related to amplitude and multisite coherence of alpha (7-12 Hz) and theta (4-7 Hz) band electroencephalogram oscillations. I examined this phenomenon in rats by using theta band oscillations of the local field potential to signify sniffing as a sensorimotor process. Olfactory bulb (OB) theta oscillations are coherent with those in the dorsal hippocampus (HPC) during odor sniffing in a two-odor olfactory discrimination task. Coherence is restricted to the high-frequency theta band (6-12 Hz) associated with directed sniffing in the OB and type 1 theta in the HPC. Coherence and performance fluctuate on a time scale of several minutes. Coherence magnitude is positively correlated with performance in the two-odor condition but not in extended runs of single odor conditional-stimulus-positive trials. Simultaneous with enhanced OB-HPC theta band coherence during odor sniffing is a significant decrease in lateral entorhinal cortex (EC)-HPC and OB-EC coherence, suggesting that linkage of the olfactory and hippocampal theta rhythms is not through the synaptic relay from OB to HPC in the lateral EC. OB-HPC coupling at the sniffing frequency is proposed as a mechanism underlying olfactory sensorimotor effort as a cognitive process.

Gamma oscillations (40-100 Hz), originally seen in the olfactory bulb (OB), have long been a defining characteristic of sensory coding in the olfactory system. This study proposes that gamma oscillations are of two types, associated with different behavioral features and synaptic origins within the OB. Local field potentials were recorded from rat and mouse OBs during various behavioral periods (immobility, alert motionlessness, exploration and odor discrimination). High frequency gamma activity (65-100 Hz) is shown to be correlated with the sniff cycle, initiated at the peak of inhalation and is called type 1 gamma. It is prominent during exploratory behavior, but also present during resting and trained odor discrimination. Low frequency gamma activity (35-65 Hz), called type 2 gamma, is not strongly correlated with the sniff cycle, is inhibited by the sniff onset and is prominent during alert immobility. Rest and alert immobility are characterized by alternating type 1 and type 2 gamma rhythms, while exploratory sniffing and odor discrimination show a dramatic decrease in type 2 gamma with a broadband increase in the power of type 1 gamma. Periods of alert immobility prior to odor discrimination in trained animals show dominance of type 2 gamma, with episodes lasting up to 0.5 second. Data from mice with selective deletion of granule cell inhibition in the OB show a selective loss of type 2 gamma with type 1 gamma dramatically enhanced during exploratory behavior, suggesting that mutual inhibition between granule cells or centrifugal inhibitory input drives type 2 gamma, and that the excitatory-inhibitory connections between mitral and granule cells likely drive type 1 gamma. Gamma activity is not a single type of oscillation, and the largest amplitude gamma bursts are often those associated with an attentive cognitive state rather than odor sniffing.

There is a negative relationship between obesity and GH. However, it is not known how metabolic changes, associated with obesity, lead to a reduction in GH output. This study examined the GH axis of two mouse models of obesity, the leptin-deficient (ob/ob) mouse and the diet-induced obese (DIO; high-fat fed) mouse. Both models displayed hyperglycemia and hyperinsulinemia with reduced expression of GH as well as reduced expression of pituitary receptors important for GH synthesis and release [GHRH receptor (DIO only) and the ghrelin receptor (ob/ob and DIO)]. These pituitary changes were not accompanied by changes in hypothalamic expression of GHRH or somatostatin; suggesting that alterations in pituitary function may be precipitated in part by direct effects of systemic signals. Of the metabolic and hormonal parameters examined (insulin, glucose, corticosterone, free fatty acids, ghrelin, and IGF-I), only insulin/glucose showed a significant, and negative, correlation with pituitary expression. Pituitaries of DIO mice remained responsive to the acute in vivo actions of insulin, as assessed by phosphorylation of Akt, despite systemic (skeletal muscle and fat) insulin resistance. In addition, treating primary pituitary cell cultures from lean mice with insulin reduced GH release as well as GH, GHRH receptor, and ghrelin receptor mRNA levels compared with vehicle-treated controls, where the magnitude of suppression of pituitary mRNA levels was similar to that observed in the DIO mouse. These results coupled with the fact that the pituitary expresses the insulin receptor at levels comparable to tissues classically considered insulin sensitive, indicates high circulating insulin levels can directly contribute to the suppression of GH synthesis and release in the obese state.

Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot experiments showed no acute effect of leptin on cytochrome P450-17alpha messenger RNA accumulation in rat Leydig cells in basal and hCG-stimulated conditions, excluding that the rapid changes observed were caused by messenger RNA degradation. In conclusion, these findings, for the first time, show that leptin has direct, receptor-mediated actions on rodent Leydig cells in culture, at concentrations within the range of obese men.

The recruitment of mesenchymal progenitor cells (MPCs) and their subsequent differentiation to osteoblasts is mandatory for bone development, remodeling, and repair. To study the possible involvement of platelet-derived growth factor (PDGF) isoforms, primary human MPCs and osteogenic differentiated progenitor cells (dOB) were examined for chemotaxic response to homodimeric human platelet-derived growth factor AA, -BB, and heterodimeric PDGF-AB. The role of PDGF receptors was addressed by preincubation with PDGF receptor alpha and beta chain specific antibodies. Migration of MPCs, dOB, and primary osteoblasts (OB) was stimulated by the addition of rhPDGF-AA, rhPDGF-BB, and rhPDGF-AB. The effect was highest in MPCs and for rhPDGF-BB, and declining with osteogenic differentiation. Preincubation with the receptor alpha specific antibody decreased the CI to borderline values while pretreatment with the receptor beta specific antibody led to a complete loss of chemotactic response to PDGF isoforms. In control experiments, basal migration values and rhBMP-2 as well as rxBMP-4 induced chemotaxis of MPC were not influenced by the addition of receptor alpha or beta antibodies. Interestingly, without preincubation the parallel exposure of MPC to rhTGF-beta1 instantaneously leads to a selective loss of migratory stimulation by rhPDGF-AA. The chemotactic effect of PDGF isoforms for primary human MPCs and the influence of osteogenic differentiation suggest a functional role for recruitment of MPCs during bone development and remodeling. Moreover, these observations may be useful for novel approaches towards guided tissue regeneration or tissue engineering of bone.

OBJECTIVE

Although epidemiological studies reveal an increased incidence of obesity and an association between obesity and the prevalence/severity of ischemic stroke, little is known about the mechanisms that link obesity to ischemic stroke. This study tested the hypothesis that obesity exacerbates the cerebrovascular dysfunction and tissue injury induced by brain ischemia and reperfusion.

METHODS

The adhesion of leukocytes and platelets in cerebral venules, blood-brain barrier permeability, brain water content, and infarct volume were measured in wild-type, obese (ob/ob), and leptin-reconstituted ob/ob mice subjected to 30 minutes middle cerebral artery occlusion and reperfusion. Tissue and plasma cytokine levels were determined by cytometric bead array, and a role for monocyte chemoattractant protein-1 and interleukin-6 was assessed using blocking antibodies.

RESULTS

Compared with wild-type mice, ob/ob exhibited larger increases in leukocyte and platelet adhesion, blood-brain barrier permeability, water content, and infarct volume after middle cerebral artery occlusion-reperfusion. Reconstitution of leptin in ob/ob mice tended to further enhance all reperfusion-induced responses. Ob/ob mice also exhibited higher plasma levels of monocyte chemoattractant protein-1 and interleukin-6 than wild-type mice. Immunoneutralization of monocyte chemoattractant protein-1, but not interleukin-6, reduced infarct volume in ob/ob mice.

CONCLUSIONS

Obesity worsens the inflammatory and injury responses to middle cerebral artery occlusion and reperfusion by a mechanism independent of leptin deficiency. monocyte chemoattractant protein-1 appears to contribute to the exaggerated responses to ischemic stroke in obese mice.

Skeletal muscle is one of the primary tissues responsible for insulin resistance and type 2 diabetes (T2D). The fetal stage is crucial for skeletal muscle development. Obesity induces inflammatory responses, which might regulate myogenesis through Wnt/beta-catenin signaling. This study evaluated the effects of maternal obesity (>30% increase in body mass index) during pregnancy on myogenesis and the Wnt/beta-catenin and IKK/NF-kappaB pathways in fetal skeletal muscle using an obese pregnant sheep model. Nonpregnant ewes were assigned to a control group (C; fed 100% of National Research Council recommendations; n=5) or obesogenic (OB; fed 150% of National Research Council recommendations; n=5) diet from 60 days before to 75 days after conception (term approximately 148 days) when fetal semitendenosus skeletal muscle was sampled for analyses. Myogenic markers including MyoD, myogenin, and desmin contents were reduced in OB compared with C fetal semitendenosus, indicating the downregulation of myogenesis. The diameter of primary muscle fibers was smaller in OB fetal muscle. Phosphorylation of GSK3beta was reduced in OB compared with C fetal semitendenosus. Although the beta-catenin level was lower in OB than C fetal muscle, more beta-catenin was associated with FOXO3a in the OB fetuses. Moreover, we found phosphorylation levels of IKKbeta and RelA/p65 were both increased in OB fetal muscle. In conclusion, our data showed that myogenesis and the Wnt/beta-catenin signaling pathway were downregulated, which might be due to the upregulation of inflammatory IKK/NF-kappaB signaling pathways in fetal muscle of obese mothers.

After menopause, increased tumor necrosis factor-alpha (TNF-alpha) stimulates bone resorption while inhibiting differentiation of new bone-forming osteoblasts (OB). TNF receptors, p55 and p75, signal similar intracellular pathways, but only p55 activates apoptosis. To evaluate the relationship between the TNF receptor mediating inhibition of OB differentiation and the role of apoptosis, marrow stromal cells (MSC) were cultured from mice deficient in either or both receptors. Cells grown in ascorbate and beta-glycerophosphate produce alkaline phosphatase and osteocalcin and mineralize matrix. Treatment of wild-type or p55(+/+)/p75(-/-) MSC with murine TNF (binds p55 and p75) or human TNF (binds only p55) inhibited OB differentiation. TNF did not inhibit OB differentiation in p55(-/-) MSC. Expression of p75 modestly attenuated sensitivity to TNF. To determine the role of apoptosis, changes in total DNA, cell viability, caspase 3, and percentage of annexin V-positive cells were measured in MSC and preosteoblastic MC3T3 cells. TNF treatment that reduced differentiation by 50% did not decrease cell viability or increase apoptosis, as determined by alamar blue reduction, trypan blue exclusion, and percentage of annexin V-positive cells. TNF increased caspase 3 activity 1.5-fold in MC3T3 and insignificantly in MSC cells compared with>> 4-fold after 4 h actinomycin D. Treatment of MSC or MC3T3 cells with three caspase inhibitors failed to reverse the inhibitory effect of TNF on OB differentiation despite inhibition of caspase activity. These results suggest that the p55 receptor is essential, and p75 dispensable, for TNF inhibition of OB differentiation through a mechanism that does not require apoptosis.

Understanding central processing requires precise monitoring of neural activity across populations of identified neurons in the intact brain. In the present study, we used recently optimized variants of the genetically encoded calcium sensor GCaMP (GCaMP3 and GCaMPG5G) to image activity among genetically and anatomically defined neuronal populations in the olfactory bulb (OB), including two types of GABAergic interneurons (periglomerular [PG] and short axon [SA] cells) and OB output neurons (mitral/tufted [MT] cells) projecting to the piriform cortex. We first established that changes in neuronal spiking can be related accurately to GCaMP fluorescence changes via a simple quantitative relationship over a large dynamic range. We next used in vivo two-photon imaging from individual neurons and epifluorescence signals reflecting population-level activity to investigate the spatiotemporal representation of odorants across these neuron types in anesthetized and awake mice. Under anesthesia, individual PG and SA cells showed temporally simple responses and little spontaneous activity, whereas MT cells were spontaneously active and showed diverse temporal responses. At the population level, response patterns of PG, SA, and MT cells were surprisingly similar to those imaged from sensory inputs, with shared odorant-specific topography across the dorsal OB and inhalation-coupled temporal dynamics. During wakefulness, PG and SA cell responses increased in magnitude but remained temporally simple, whereas those of MT cells changed to complex spatiotemporal patterns reflecting restricted excitation and widespread inhibition. These results suggest multiple circuit elements with distinct roles in transforming odor representations in the OB and provide a framework for further study of early olfactory processing using optical and genetic tools.

Deficits in emotion regulation processes are a common and widely used explanation for the development and maintenance of binge eating disorder (BED). It is assumed that BED patients - as they have difficulty regulating their negative emotions - use binge eating to cope with these emotions and to find relief. However, the number of experimental studies investigating this assumption is scarce and the differentiation of obese individuals with and without BED regarding the emotion regulation model is not verified. We reviewed literature for experimental studies investigating the emotion regulation model in obese patients (OB) with and without BED. Our search resulted in 18 experimental studies examining the triggering effect of negative emotions for binge eating or its effects on subsequent relief. We found evidence indicating that negative emotion serves as a trigger for binge eating in the BED group unlike the obese group without BED. Considering the small number of studies, we found evidence for a (short-term) improvement of mood through food intake, irrespective of group.

IkappaBalpha is an ankyrin repeat protein that inhibits NF-kappaB transcriptional activity by sequestering NF-kappaB outside of the nucleus in resting cells. We have characterized the binding thermodynamics and kinetics of the IkappaBalpha ankyrin repeat domain to NF-kappaB(p50/p65) using surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). SPR data showed that the IkappaBalpha and NF-kappaB associate rapidly but dissociate very slowly, leading to an extremely stable complex with a K(D,obs) of approximately 40 pM at 37 degrees C. As reported previously, the amino-terminal DNA-binding domain of p65 contributes little to the overall binding affinity. Conversely, helix four of p65, which forms part of the nuclear localization sequence, was essential for high-affinity binding. This was surprising, given the small size of the binding interface formed by this part of p65. The NF-kappaB(p50/p65) heterodimer and p65 homodimer bound IkappaBalpha with almost indistinguishable thermodynamics, except that the NF-kappaB p65 homodimer was characterized by a more favorable DeltaH(obs) relative to the NF-kappaB(p50/p65) heterodimer. Both interactions were characterized by a large negative heat capacity change (DeltaC(P,obs)), approximately half of which was contributed by the p65 helix four that was necessary for tight binding. This could not be accounted for readily by the small loss of buried non-polar surface area and we hypothesize that the observed effect is due to additional folding of some regions of the complex.

Obesity and metabolic syndrome result from excess calorie intake and genetic predisposition and are mechanistically linked to type II diabetes and accelerated body aging; abnormal nutrient and insulin signaling participate in this pathologic process, yet the underlying molecular mechanisms are incompletely understood. Mice lacking the p66 kDa isoform of the Shc adaptor molecule live longer and are leaner than wild-type animals, suggesting that this molecule may have a role in metabolic derangement and premature senescence by overnutrition. We found that p66 deficiency exerts a modest but significant protective effect on fat accumulation and premature death in lepOb/Ob mice, an established genetic model of obesity and insulin resistance; strikingly, however, p66 inactivation improved glucose tolerance in these animals, without affecting (hyper)insulinaemia and independent of body weight. Protection from insulin resistance was cell autonomous, because isolated p66KO preadipocytes were relatively resistant to insulin desensitization by free fatty acids in vitro. Biochemical studies revealed that p66shc promotes the signal-inhibitory phosphorylation of the major insulin transducer IRS-1, by bridging IRS-1 and the mTOR effector p70S6 kinase, a molecule previously linked to obesity-induced insulin resistance. Importantly, IRS-1 was strongly up-regulated in the adipose tissue of p66KO lepOb/Ob mice, confirming that effects of p66 on tissue responsiveness to insulin are largely mediated by this molecule. Taken together, these findings identify p66shc as a major mediator of insulin resistance by excess nutrients, and by extension, as a potential molecular target against the spreading epidemic of obesity and type II diabetes.

Leptin is involved in the hypothalamic control of food intake and body weight. Fos immunohistochemistry has been used to functionally map leptin target neurons involved in these regulatory processes. However, only a subset of hypothalamic neurons expressing the long form of the leptin receptor (Ob-Rb) also coexpress the neuronal activation marker Fos after leptin stimulation. To functionally map all leptin target neurons, regardless of whether leptin-mediated neuronal activation or inhibition occurs, we immunohistochemically investigated the leptin-induced nuclear translocation of the signal transducer and activator of transcription molecule STAT3, which represents a crucial step in the regulation of leptin-dependent gene expression. As proven by colocalization studies with the nuclear 4',6-diamidino-2-phenylindole dilactate stain, intracerebroventricular leptin treatment, but not intracerebroventricular application of pyrogen-free saline, induced a time-dependent nuclear translocation of STAT3 immunoreactivity in hypothalamic nuclei, with strong nuclear STAT3 signals detectable in the arcuate nucleus, the lateral hypothalamus, and the ventromedial and dorsomedial hypothalamic nuclei. This leptin-induced STAT3 translocation pattern proved to be distinct from that induced by interleukin-6, another cytokine using STAT3 in its signaling pathway. Combined immunohistochemical STAT3 and Fos detection after leptin treatment revealed a higher number of STAT3-positive than Fos-positive cell nuclei in the aforementioned hypothalamic structures and showed that Fos immunoreactivity colocalized only in a subset of all leptin-responsive STAT3 nuclei. These results suggest that the detection of nuclear STAT3 immunoreactivity represents a new neuroanatomical tool to functionally map central leptin actions. They further support the importance of ventrally located caudal hypothalamic structures representing the main leptin targets involved in body weight regulation.

Christakis and colleagues have shown that health behaviors cluster in social networks and suggest social norms may account for the clustering. This study examined: (i) whether obesity clusters among young adults and whether social norms do in fact account for the clustering, and (ii) among overweight/obese (OW/OB) young adults, whether number of social contacts trying to lose weight is associated with weight loss intentions and whether social norms for weight loss account for this effect. Normal weight (NW) and OW/OB young adults (N = 288; 66% female; 75% white) completed measures assessing number of OW social contacts and social norms for obesity. OW/OB young adults also indicated number of OW social contacts currently trying to lose weight, social norms for weight loss, and weight loss intentions. Compared to NW, OW/OB young adults were more likely to have OW romantic partners and best friends and had more OW casual friends and family members (Ps < 0.05), but social norms for obesity did not differ between groups, and social norms did not mediate the relationship between OW social contacts and participants' weight status. However, among OW/OB young adults, having more social contacts trying to lose weight was associated with greater intention to lose weight (r = 0.20, P = 0.02) and social norms for weight loss fully mediated this effect (P < 0.01). This study is the first to show that social contacts and normative beliefs influence weight status and intentions for weight control in young adults. Findings underscore the importance of targeting social influence in the treatment and prevention of obesity in this high-risk age group.

Animal models of hepatic steatosis and steatohepatitis have improved our understanding of the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Three models, genetically obese ob/ob mice, lipoatrophic mice and normal rats fed choline-deficient, methionine-restricted diets, have been particularly informative. All support the multiple 'hit' hypothesis for NAFLD pathogenesis that suggests that fatty livers are unusually vulnerable to oxidants and develop steatohepatitis when secondary insults generate sufficient oxidants to cause liver cell death and inflammation. Steatohepatitis, in turn, increases sensitivity to other insults that induce hepatic fibrosis, promoting the evolution of cirrhosis. Early during NAFLD pathogenesis, inhibitor kappa kinase beta (IKKbeta), an enzyme that induces tumour necrosis factor alpha (TNFalpha) and other proinflammatory cytokines, is activated and this causes insulin resistance. Inhibition of IKKbeta or TNFalpha improves insulin sensitivity, steatosis and steatohepatitis in animals, suggesting novel strategies to prevent and treat early NAFLD in humans.

We continue our development of a kinetic model of bursting electrical activity in the pancreatic beta-cell (J. Keizer and G. Magnus. Biophys. J. 56: 229-242, 1989), including the influence of Ca2+ handling by the mitochondria. Our minimal model of mitochondrial Ca2+ handling [G. Magnus and J. Keizer. Am. J. Physiol. 273 (Cell Physiol. 42): C717-C733, 1997] is expanded to include the D-glucose dependence of the rate of production of mitochondrial reducing equivalents. The Ca2+ dependence of the mitochondrial dehydrogenases, which is also included in the model, plays only a small role in the simulations, since the dehydrogenases appear to be maximally activated when D-glucose concentrations are sufficient to produce bursting. A previous model of ionic currents in the plasma membrane is updated using a recent experimental characterization of the dependence of the conductance of the ATP-sensitive K+ (KATP) current on adenine nucleotides. The resulting whole cell model is complex, involving 12 dynamic variables that couple Ca2+ handling in the cytoplasm and the mitochondria with electrical activity in the plasma and inner mitochondrial membranes. Simulations with the whole cell model give rise to bursting electrical activity similar to that seen in pancreatic islets and clusters of pancreatic beta-cells. The full D-glucose dose response of electrical activity is obtained if the cytosolic rate of ATP hydrolysis is a sigmoidal function of glucose. The simulations give the correct shape, period, and phase of the associated oscillations in cytosolic Ca2+, predict that the conductance of the KATP current oscillates out of phase with electrical activity [as recently observed in ob/ob mice (O. Larsson, H. Kindmark, R. Bränstrom, B. Fredholm, and P.-O. Berggren. Proc. Natl. Acad. Sci. USA 93: 5161-5165, 1996)], and make other novel predictions. In this model, bursting results because Ca2+ uptake into mitochondria during the active phase reduces the mitochondrial inner membrane potential, reducing the rate of production of ATP, which in turn activates the KATP current and repolarizes the plasma membrane.

The kinetics of folding of a tryptophan containing mutant of the IgG binding domain of protein L were characterized using stopped-flow circular dichroism, stopped-flow fluorescence, and HD exchange coupled with high-resolution mass spectrometry. Both the thermodynamics and kinetics of folding fit well to a simple two-state model: (1) Guanidine induced equilibrium denaturation transitions measured by fluorescence and circular dichroism were virtually superimposable. (2) The kinetics of folding/unfolding were single exponential under all conditions examined, and the rate constants obtained using all probes were similar. (3) Mass spectra from pulsed HD exchange refolding experiments showed that a species with very little protection from exchange is converted to a fully protected species (the native state) at a rate very similar to that of the overall change in tryptophan fluorescence; no intervening partially protected species were observed. (4) Rate constants (in H2O) and m values for folding and unfolding determined by fitting observed relaxation rates obtained over a broad range of denaturant concentrations to a two-state model were consistent with the equilibrium parameters deltaG and m: -RT ln(k(u)/k(f))/deltaG(U)H2O = 1.02; (m(u) + m(f))/m = 1.08. In contrast to results with a number of other proteins, there was no deviation from linearity in plots of ln k(obs) versus guanidine at low guanidine concentrations, both in the presence and absence of 0.4 M Na2SO4, suggesting that significantly stabilized intermediates do not accumulate during folding. Although all of the change in fluorescence signal during folding in phosphate buffer was accounted for by the simple exponential describing the overall folding reaction, fluorescence-quenching experiments using sodium iodide revealed a small reduction in the extent of quenching of the protein within the first two milliseconds after initiation of refolding in low concentrations of guanidine, suggesting a partial collapse of the unfolded chain may occur under these conditions. Comparison with results on the structurally and functionally similar IgG binding domain of streptococcal protein G show intriguing differences in the folding of the two proteins.

OBJECTIVE

Human obesity is associated with elevated leptin levels and a high risk of death from cardiovascular disease. In the present study, we investigated the effects of leptin on vascular wound healing and arterial lesion growth in mice.

RESULTS

Wild-type mice placed on an atherogenic, high-fat diet had elevated (9-fold) leptin levels compared with their counterparts maintained on normal chow, and the former demonstrated significantly enhanced neointimal thickening after carotid artery injury with ferric chloride. The lesions forming in response to injury strongly expressed leptin receptor mRNA and protein. Unexpectedly, the atherogenic diet had no effect on injured vessels from leptin-deficient ob/ob mice despite aggravating obesity, diabetes, and hyperlipidemia in these animals. Daily administration of leptin to ob/ob mice during the 3-week period after injury reversed this phenotype, dramatically increasing neointimal thickness and the severity of luminal stenosis. Exogenous leptin also enhanced lesion growth and increased cellular proliferation in injured arteries from wild-type mice but had no effect on vessels from leptin receptor-deficient db/db mice.

CONCLUSIONS

Our results raise the possibility that there might be a direct, leptin receptor-mediated link between the hyperleptinemia in human obesity and the increased risk for cardiovascular complications associated with this condition.

Leptin, the product of the ob gene, plays a key role in the regulation of food intake via a cross-talk between hypothalamic leptin receptors and neuropeptides that affect feeding behaviour. Recent studies have shown a synergistic interaction between leptin and cholecystokinin (CCK) leading to suppression of food intake, which involves CCK-1 receptors and capsaicin-sensitive vagal fibres. In this study, we have investigated the presence of leptin receptors in afferent and efferent neurons of the vagus nerve. By using reverse transcription-polymerase chain reaction, mRNAs encoding long (Ob-Rb) and short (Ob-Ra) leptin receptor isoforms were detected in the rat nodose ganglion, which contains the cell bodies of the vagal afferent neurons. Western blot analysis confirmed the presence of leptin receptor-immunoreactive proteins in extracts from the vagal trunk. Immunohistochemistry showed the presence of leptin receptors and the leptin-induced transcription factor STAT3 in the cytoplasm of nodose ganglion cells. In cervical vagal segments, levels of leptin receptor protein displayed physiological regulation, with decreased amounts after feeding and increased levels after food restriction. In addition, leptin receptor and STAT3 immunoreactivities were detected in neurons of the nucleus of tractus solitarius (NTS) and the dorsal motor nucleus of the vagus nerve (DMNX) by immunofluorescence histochemistry. Furthermore, direct double-labelling demonstrated colocalization of Ob-Rb and STAT3 immunoreactivities in cholinergic vagal efferent cell bodies of the DMNX. It is speculated that vagal leptin receptors, apart from being activated by adipocyte-derived leptin, may also be influenced by leptin produced by the stomach. This may explain the synergistic action of leptin and CCK on neuronal activity in the NTS and on food intake.

Osteoblasts are derived from precursor cells present in low frequency in the stromal element of bone marrow. Because of the lack of a practical procedure to isolate osteoblast precursors from early cultures of plastic adherent cells from bone marrow, previous studies of marrow stromal cells have been made in confluent cultures of bone marrow when the osteoblast (OB) precursors are already differentiated. Also these studies utilized cultures containing mixed populations of cells including hematopoietic cells. Thus we have employed a negative immunoselection procedure to remove contaminating hematopoietic cells and to isolate nearly homogeneous populations of early human stromal cells derived from the plastic-adherent mononuclear marrow cells cultured in the presence of serum. By reverse transcriptase polymerase chain reaction (RT-PCR) analysis for mRNA, and by immunocytochemical study for protein, we studied the sequential expression in culture of multiple markers of the osteoblast phenotype--alkaline phosphatase, osteopontin, parathyroid hormone receptor, types I and III procollagen, and osteocalcin--as well as lipoprotein lipase (LPL), a marker of the adipocyte phenotype. At an early stage of culture (7-9 days), human OB precursors formed colonies of variable sizes that expressed low levels of mRNA and protein concentrations of OB markers, and their concentration increased on growth to a confluent monolayer (approximately 14 days). LPL mRNA was expressed at high levels in the colony stage, and its level decreased upon confluency, suggesting a loss of potential for commitment to the adipocyte lineage. Interestingly, treatment with dexamethasone at 10(-8) M increased the expression for some of the osteoblast markers and for the LPL gene and was required for the deposition of mineralized matrix and for the formation of adipocytes containing cytoplasmic lipid droplets in confluent cultures. Cloned single early colonies were able to coexpress the osteoblast and adipocyte markers (as assessed by RT-PCR). Thus these immunoselected marrow stromal cells have the characteristics of authentic human osteoblast precursor cells which also are capable of differentiating into adipocytes.