Hypothalamic control of bone formation: Distinct actions of leptin and Y2 receptor pathways

Leptin and Y2 receptors on hypothalamic NPY neurons mediate leptin effects on energy homeostasis; however, their interaction in modulating osteoblast activity is not established. Here, direct testing of this possibility indicates distinct mechanisms of action for leptin anti-osteogenic and Y2(-/-) anabolic pathways in modulating bone formation. Introduction: Central enhancement of bone formation by hypothalamic neurons is observed in leptin-deficient oblob and Y2 receptor null mice. Similar elevation in central neuropeptide Y (NPY) expression and effects on osteoblast activity in these two models suggest a shared pathway between leptin and Y2 receptors in the central control of bone physiology. The aim of this study was to test whether the leptin and Y2 receptor pathways regulate bone by the same or distinct mechanisms. Materials and Methods: The interaction of concomitant leptin and Y2 receptor deficiency in controlling bone was examined in Y2(-/-) oblob double mutant mice, to determine whether leptin and Y2 receptor deficiency have additive effects. Interaction between leptin excess and Y2 receptor deletion was examined using recombinant adeno-associated viral vector overproduction of NPY (AAV-NPY) to produce weight gain and thus leptin excess in adult Y2(-/-) mice. Cancellous bone volume and bone cell function were assessed. Results: Osteoblast activity was comparably elevated in oblob, Y2(-/-), and Y2(-/-) oblob mice. However, greater bone resorption in oblob and Y2(-/-) oblob mice reduced cancellous bone volume compared with Y2(-/-). Both wildtype and Y2(-/-) AAV-NPY mice exhibited marked elevation of white adipose tissue accumulation and hence leptin expression, thereby reducing osteoblast activity. Despite this anti-osteogenic leptin effect in the obese AAV-NPY model, osteoblast activity in Y2(-/-) AAV-NPY mice remained significantly greater than in wildtype AAV-NPY mice. Conclusions: This study suggests that NPY is not a key regulator of the leptin-dependent osteoblast activity, because both the leptin-deficient stimulation of bone formation and the excess leptin inhibition of bone formation can occur in the presence of high hypothalamic NPY. The Y2(-/-) pathway acts consistently to stimulate bone formation; in contrast, leptin continues to suppress bone formation as circulating levels increase. As a result, they act increasingly in opposition as obesity becomes more marked. Thus, in the absence of leptin, the cancellous bone response to loss of Y2 receptor and leptin activity can not be distinguished. However, as leptin levels increase to physiological levels, distinct signaling pathways are revealed.

Cholesterol-induced caveolin targeting to lipid droplets in adipocytes: A role for caveolar endocytosis

We have investigated the targeting of caveolin to lipid bodies in adipocytes that express high levels of caveolins and contain well-developed lipid droplets. We observed that the lipid droplets isolated from adipocytes of caveolin-1 knock out mice contained dramatically reduced levels of cholesterol, indicating that caveolin is required for maintaining the cholesterol content of this organelle. Analysis of caveolin distribution by cell fractionation and fluorescent light microscopy in 3T3-L1 adipocytes indicated that addition of cholesterol rapidly stimulated translocation of caveolin to lipid droplets. The cholesterol-induced trafficking of caveolins to lipid droplets was shown to be dynamin- and protein kinase C (PKC)-dependent and modulated by src tyrosine kinase activation, suggesting a role for caveolar endocytosis in this novel trafficking pathway. Consistent with this, caveolae budding was stimulated by cholesterol addition. The present data identify lipid droplets as potential target organelles for caveolar endocytosis and demonstrate a role for caveolin-1 in the maintenance of free cholesterol levels in adipocyte lipid droplets.

Clinically active Crohn's disease in the presence of a low C-reactive protein

Objective. Clinical interest in C-reactive protein (CRP) - a component of the innate immune system - has focused mainly on its worth as an indicator of disease activity. There has been a resurgence of interest in CRP in the Crohn's disease ( CD) literature because several trials of new treatments for active CD have been characterized by both a large proportion of patients with low CRP ( < 10 mg/l) at entry to the trials and by a negative therapeutic outcome. It is therefore of interest to study the clinical characteristics of patients who are thought to have at the same time both active CD and a low CRP. Material and methods. Patients were prospectively recruited as part of the Brisbane IBD clinical and research programme. Subjects were included in the low CRP group only if there were complete datasets for CRP on all occasions of active CD, and CRP was < 10 mg/l. Active disease was defined as CD activity index (CDAI) > 200. The low CRP group was compared with patients in the raised CRP group for a range of clinical variables as well as the major NOD2 variants. Results. There were data sets for 223 CD patients, with a mean disease duration of 12 years. Of these, 22 patients fulfilled the criteria for low CRP. The low CRP group ( group 1) showed significant differences for disease site (p < 0.01) and for BMI (p = 0.006) compared to the raised CRP group ( group 2). Specifically, group 1 had a predominance of pure ileal disease (95% versus 53%) and lack of pure colonic disease (0% versus 24%) compared to group 2, and their BMI was significantly lower (20.3 kg/m(2) versus 25.0 kg/m(2)). Groups 1 and 2 did not differ with respect to Vienna behaviour at diagnosis, smoking, appendicectomy, extra-intestinal manifestations of CD, or NOD2 SNP variants. There was a trend for low CRP patients with previous ileal resection to evolve to a stricturing phenotype. Fat wrapping was noted in 11/13 (85%) of low CRP patients undergoing ileal resections. Conclusions. Patients with CD and a persistently low CRP in the face of active disease were characterized by an almost exclusive ileal disease distribution and a low BMI, compared to those with a raised CRP. These patients had a similar frequency and distribution of NOD2/CARD15 variants. Stricturing ( v inflammatory or penetrating) behaviour may explain some low CRP. Despite the abnormally low BMI, fat wrapping was noted in the majority of low CRP patients undergoing ileal resection.

Lipid droplets: a unified view of a dynamic organelle

Lipid droplets form the main lipid store in eukaryotic cells. Although all cells seem to be able to generate lipid droplets, their biogenesis, regulatory mechanisms and interactions with other organelles remain largely elusive. In this article, we outline some of the recent developments in lipid droplet cell biology. We show the mobile and dynamic nature of this organelle, and advocate the adoption of a unified nomenclature to consolidate terminology in this emerging field.

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth

Prolactin and the expression of suppressor of cytokine signaling-3 in the sheep adrenal gland before birth. Am J Physiol Regul Integr Comp Physiol 291: R1399-R1405, 2006. First published June 29, 2006; doi: 10.1152/ajpregu.00252.2006.-The fetal pituitary-adrenal axis plays a key role in the fetal response to intrauterine stress and in the timing of parturition. The fetal sheep adrenal gland is relatively refractory to stimulation in midgestation (90-120 days) before the prepartum activation, which occurs around 135 days gestation (term = 147 +/- 3 days). The mechanisms underlying the switch from adrenal quiescence to activation are unclear. Therefore, we have investigated the expression of suppressor of cytokine signaling-3 (SOCS-3), a putative inhibitor of tissue growth in the fetal sheep adrenal between 50 and 145 days gestation and in the adrenal of the growth-restricted fetal sheep in late gestation. SOCS-3 is activated by a range of cytokines, including prolactin (PRL), and we have, therefore, determined whether PRL administered in vivo or in vitro stimulates SOCS-3 mRNA expression in the fetal adrenal in late gestation. There was a decrease (P < 0.005) in SOCS-3 expression in the fetal adrenal between 54 and 133 days and between 141 and 144 days gestation. Infusion of the dopaminergic agonist, bromocriptine, which suppressed fetal PRL concentrations but did not decrease adrenal SOCS-3 mRNA expression. PRL administration, however, significantly increased adrenal SOCS-3 mRNA expression (P < 0.05). Similarly, there was an increase (P < 0.05) in SOCS-3 mRNA expression in adrenocortical cells in vitro after exposure to PRL (50 ng/ml). Placental and fetal growth restriction had no effect on SOCS-3 expression in the adrenal during late gestation. In summary, the decrease in the expression of the inhibitor SOCS-3 after 133 days gestation may be permissive for a subsequent increase in fetal adrenal growth before birth. We conclude that factors other than PRL act to maintain adrenal SOCS-3 mRNA expression before 133 days gestation but that acute elevations of PRL can act to upregulate adrenal SOCS-3 expression in the sheep fetus during late gestation.

Rapid Atrophy of the Lumbar Multifidus Follows Experimental Disc or Nerve Root Injury.

Study Design. Experimental study of muscle changes after lumbar spinal injury. Objectives. To investigate effects of intervertebral disc and nerve root lesions on cross-sectional area, histology and chemistry of porcine lumbar multifidus. Summary of Background Data. The multifidus cross-sectional area is reduced in acute and chronic low back pain. Although chronic changes are widespread, acute changes at 1 segment are identified within days of injury. It is uncertain whether changes precede or follow injury, or what is the mechanism. Methods. The multifidus cross-sectional area was measured in 21 pigs from L1 to S1 with ultrasound before and 3 or 6 days after lesions: incision into L3 - L4 disc, medial branch transection of the L3 dorsal ramus, and a sham procedure. Samples from L3 to L5 were studied histologically and chemically. Results. The multifidus cross-sectional area was reduced at L4 ipsilateral to disc lesion but at L4 - L6 after nerve lesion. There was no change after sham or on the opposite side. Water and lactate were reduced bilaterally after disc lesion and ipsilateral to nerve lesion. Histology revealed enlargement of adipocytes and clustering of myofibers at multiple levels after disc and nerve lesions. Conclusions. These data resolve the controversy that the multifidus cross-sectional area reduces rapidly after lumbar injury. Changes after disc lesion affect 1 level with a different distribution to denervation. Such changes may be due to disuse following reflex inhibitory mechanisms.

Conditional deletion of hypothalamic Y2 receptors reverts gonadectomy-induced bone loss in adult mice

Reduction in levels of sex hormones at menopause in women is associated with two common, major outcomes, the accumulation of white adipose tissue, and the progressive loss of bone because of excess osteoclastic bone resorption exceeding osteoblastic bone formation. Current antiresorptive therapies can reduce osteoclastic activity but have only limited capacity to stimulate osteoblastic bone formation and restore lost skeletal mass. Likewise, the availability of effective pharmacological weight loss treatments is currently limited. Here we demonstrate that conditional deletion of hypothalamic neuropeptide Y2 receptors can prevent ongoing bone loss in sex hormone-deficient adult male and female mice. This benefit is attributable solely to activation of an anabolic osteoblastic bone formation response that counterbalances persistent elevation of bone resorption, suggesting the Y2-mediated anabolic pathway to be independent of sex hormones. Furthermore, the increase in fat mass that typically occurs after ovariectomy is prevented by germ line deletion of Y2 receptors, whereas in male mice body weight and fat mass were consistently lower than wild-type regardless of sex hormone status. Therefore, this study indicates a role for Y2 receptors in the accumulation of adipose tissue in the hypogonadal state and demonstrates that hypothalamic Y2 receptors constitutively restrain osteoblastic activity even in the absence of sex hormones. The increase in bone formation after release of this tonic inhibition suggests a promising new avenue for osteoporosis treatment.

Degradable poly(ethylene glycol)-based hydrogels:synthesis, physico-chemical properties and in vitro characterization

Designing degradable hydrogels is complicated by the structural and temporal complexities of the gel and evolving tissue. A major challenge is to create scaffolds with sufficient mechanical properties to restore initial function while simultaneously controlling temporal changes in the gel structure to facilitate tissue formation. Poly(ethylene glycol) was used in this work, to form biodegradable poly(ethylene glycol)-based hydrogels with hydrolyzable poly-l-lactide segments in the backbone. Non-degradable poly(ethylene glycol) was also introduced in the formulation to obtain control of the degradation profile that encompasses cell growth and new tissue formation. The dependence on polymer composition was observed by higher degradation profiles and decreased mechanical properties as the content of degradable segments was increased in the formulation. Based on in vitro tests, no toxicity of extracts or biomaterial in direct contact with human adipose tissue stem cells was observed, and the ultraviolet light treatment did not affect the proliferation capacity of the cells.

Mechanisms of Cancer Cachexia

Up to 50% of cancer patients suffer from a progressive atrophy of adipose tissue and skeletal muscle, called cachexia, resulting in weight loss, a reduced quality of life, and a shortened survival time. Anorexia often accompanies cachexia, but appears not to be responsible for the tissue loss, particularly lean body mass. An increased resting energy expenditure is seen, possibly arising from an increased thermogenesis in skeletal muscle due to an increased expression of uncoupling protein, and increased operation of the Cori cycle. Loss of adipose tissue is due to an increased lipolysis by tumor or host products. Loss of skeletal muscle in cachexia results from a depression in protein synthesis combined with an increase in protein degradation. The increase in protein degradation may include both increased activity of the ubiquitin-proteasome pathway and lysosomes. The decrease in protein synthesis is due to a reduced level of the initiation factor 4F, decreased elongation, and decreased binding of methionyl-tRNA to the 40S ribosomal subunit through increased phosphorylation of eIF2 on the a-subunit by activation of the dsRNA-dependent protein kinase, which also increases expression of the ubiquitin-proteasome pathway through activation of NF?B. Tumor factors such as proteolysis-inducing factor and host factors such as tumor necrosis factor-a, angiotensin II, and glucocorticoids can all induce muscle atrophy. Knowledge of the mechanisms of tissue destruction in cachexia should improve methods of treatment. Copyright © 2009 the American Physiological Society

Molecular pathways leading to cancer cachexia

Loss of body weight in cancer patients strongly influences morbidity and mortality. Recent studies have suggested that both tumor and host factors play a major role in tissue catabolism in cachexia, leading to upregulation of degradative pathways in both skeletal muscle and adipose tissue. ©2005 Int. Union Physiol. Sci./Am. Physiol. Soc.

Tumor-host interactions

A number of malignant tumors interact with the host to cause a syndrome of cachexia, characterized by extensive loss of adipose tissue and skeletal muscle mass, but with preservation of proteins in visceral tissues. Although anorexia is frequently present, the body composition changes in cancer cachexia cannot be explained by nutritional deprivation alone. Loss of skeletal muscle mass is a result of depression in protein synthesis and an increase in protein degradation. The main degradative pathway that has been found to have increased expression and activity in the skeletal muscle of cachectic patients is the ubiquitin-proteasome proteolytic pathway. Cachexia-inducing tumors produce catabolic factors such as proteolysis-inducing factor (PIF), a 24 kDa sulfated glycoprotein, which inhibit protein synthesis and stimulate degradation of intracellular proteins in skeletal muscle by inducing an increased expression of regulatory components of the ubiquitin-proteasome proteolytic pathway. While the oligosaccharide chains in PIF are required to initiate protein degradation the central polypeptide core may act as a growth and survival factor. Only cachexia-inducing tumors are capable of elaborating fully glycosylated PIF, and the selectivity of production possibly rests with the acquisition of the necessary glycosylating enzymes, rather than expressing the gene for the polypeptide core. Loss of adipose tissue is probably the result of an increase in catabolism rather than a defect in anabolism. A lipid mobilizing factor (LMF), identical with the plasma protein Zn-α2-glycoprotein (ZAG) is found in the urine of cachectic cancer patients and is produced by tumors causing a decrease in carcass lipid. LMF causes triglyceride hydrolysis in adipose tissue through a cyclic AMP-mediated process by interaction with a β3-adrenoreceptor. Thus, by producing circulating factors certain malignant tumors are able to interfere with host metabolism even without metastasis to that particular site. © 2004 Wiley-Liss, Inc.

Role of lipid-mobilising factor (LMF) in protecting tumour cells from oxidative damage

Lipid-mobilising factor (LMF) is produced by cachexia-inducing tumours and is involved in the degradation of adipose tissue, with increased oxidation of the released fatty acids through an induction of uncoupling protein (UCP) expression. Since UCP-2 is thought to be involved in the detoxification of free radicals if LMF induced UCP-2 expression in tumour cells, it might attenuate free radical toxicity. As a model system we have used MAC13 tumour cells, which do not produce LMF. Addition of LMF caused a concentration-dependent increase in UCP-2 expression, as determined by immunoblotting. This effect was attenuated by the β3 antagonist SR59230A, suggesting that it was mediated through a β3 adrenoreceptor. Co-incubation of LMF with MAC13 cells reduced the growth-inhibitory effects of bleomycin, paraquat and hydrogen peroxide, known to be free radical generators, but not chlorambucil, an alkylating agent. There was no effect of LMF alone on cellular proliferation. These results indicate that LMF antagonises the antiproliferative effect of agents working through a free radical mechanism, and may partly explain the unresponsiveness to the chemotherapy of cachexia-inducing tumours. © 2004 Cancer Research UK.

Cachexia in cancer patients

Cachexia — the massive (up to 80%) loss of both adipose tissue and skeletal muscle mass — is a significant factor in the poor performance status and high mortality rate of cancer patients. Although this metabolic defect has been known since cancer was first studied, it is only recently that major advances have been made in the identification of catabolic factors that act to destroy host tissues during the cachectic process. Although anorexia is frequently present, depression of food intake alone seems not to be responsible for the wasting of body tissues, as nutritional supplementation or pharmacological manipulation of appetite is unable to reverse the catabolic process — particularly with respect to skeletal muscle. However, recent clinical studies in cancer patients have shown that nutritional supplementation can be effective when combined with agents that attenuate the action of tumour factors and modifiers of the central effects on appetite might also show promise.

Modulation of adipocyte G-protein expression in cancer cachexia by a lipid-mobilizing factor (LMF)

Adipocytes isolated from cachectic mice bearing the MAC 16 tumour showed over a 3-fold increase in lipolytic response to both low concentrations of isoprenaline and a tumour-derived lipid mobilizing factor (LMF). This was reflected by an enhanced stimulation of adenylate cyclase in plasma membrane fractions of adipocytes in the presence of both factors. There was no up-regulation of adenylate cyclase in response to forskolin, suggesting that the effect arose from a change in receptor number or G-protein expression. Immunoblotting of adipocyte membranes from mice bearing the MAC16 tumour showed an increased expression of Gαs up to 10% weight loss and a reciprocal decrease in Gα. There was also an increased expression of Gαs and a decrease in Gα in adipose tissue from a patient with cancer-associated weight loss compared with a non-cachectic cancer patient. The changes in G-protein expression were also seen in adipose tissue of normal mice administered pure LMF as well as in 3T3L1 adipocytes in vitro. The changes in G-protein expression induced by LMF were attenuated by the polyunsaturated fatty acid, eicosapentaenoic acid (EPA). This suggests that this tumour-derived lipolytic factor acts to sensitize adipose tissue to lipolytic stimuli, and that this effect is attenuated by EPA, which is known to preserve adipose tissue in cancer cachexia. © 2001 Cancer Research Campaign.

Treating type 2 diabetes through insulin resistance

Type 2 diabetes is an insidious disorder, with micro and/or macrovascular and nervous damage occurring in many patients before diagnosis. This damage is caused by hyperglycaemia and the diverse effects of insulin resistance. Obesity, in particular central obesity, is a strong pre-disposing factor for type 2 diabetes. Skeletal muscle is the main site of insulin-stimulated glucose disposal and appears to be the first organ that becomes insulin resistant in the diabetic state, with later involvement of adipose tissue and the liver. This study has investigated the use of novel agents to ameliorate insulin-resistance in skeletal muscle as a means of identifying intervention sites against insulin resistance and of improving glucose uptake and metabolism by skeletal muscle. Glucose uptake was measured in vitro by cultured L6 myocytes and isolated muscles from normal and obese diabetic ob/ob mice, using either the tritiated non-metabolised glucose analogue 2-deoxy-D-glucose or by glucose disposal. Agents studied included lipoic acid, isoferulic acid, bradykinin, lipid mobilising factor (provisionally synonymous with Zinca2 glycoprotein) and the trace elements lithium, selenium and chromium. The putative role of TNFa in insulin resistance was also investigated. Lipoic acid improved insulin-stimulated glucose uptake in normal and insulin resistance murine muscles, as well as cultured myocytes. Isoferulic acid, bradykinin and LMF also produced a transient increase in glucose uptake in cultured myocytes. Physiological concentrations of TNFa were found to cause insulin resistance in cultured, but no in excised murine muscles. The effect of the M2 metabolite of the satiety-inducing agent sibutramine on lipolysis in excised murine and human adipocytes was also investigated. M2 increased lipolysis from normal lean and obese ob/ob mouse adipocytes. Arguably the most important observation was that M2 also increased the lipolytic rate in adipocytes from catecholamine resistant obese subjects. The studies reported in this thesis indicate that a diversity of agents can improve glucose uptake and ameliorate insulin resistance. It is likely that these agents are acting via different pathways. This thesis has also shown that M2 can induce lipolysis in both rodent and human adipocytes. M2 hence has potential to directly reduce adiposity, in addition to well documented effects via the central nervous system.