Andrew Butler

The arcuate nucleus as a conduit for diverse signals relevant to energy homeostasis

Arcuate nucleus neurons are known to be responsive to a wide array of hormones and nutrients, including leptin, insulin, gonadal steroids and glucose. In addition to potential transport mechanisms, peripheral substances may access these neurons via arcuate cell bodies in and projections to the median eminence, a region considered to be a circumventricular organ. The arcuate is a potent site of leptin action, probably mediating a component of leptins effects via arcuate neuropeptide Y/agouti-related peptide (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons, and implicating this structure in the long-term control of energy stores. However, ghrelin, the endogenous ligand of the growth hormone secretagogue receptor, may also stimulate feeding and weight gain, in part through action on receptors in arcuate NPY neurons. Since ghrelin is secreted by the stomach upon content depletion, with a half-life of no more than an hour, the arcuate nucleus may also be important in sensing and responding to acute changes in nutrients. We have developed a system for recording from arcuate POMC neurons using a mouse containing a transgene in which the POMC promoter is driving expression of the green fluorescent protein (GFP). In these mice, 99% of the β-endorphin positive neurons express GFP, making whole cell patch clamp recordings from the sparsely distributed POMC neurons facile. All of the POMC neurons appear to be activated by leptin, via two different mechanisms, while approximately 30–50% of the neurons appear to be inhibited by a gamma-melanocyte stimulating hormone (MSH) specific agonist. The latter result suggests that the melanocortin-3 receptor (MC3-R) may act as an autoinhibitory receptor on some POMC neurons. This hypothalamic slice preparation also confirms the responsiveness of arcuate POMC neurons to a wide variety of nutrients and hormones. Thus the arcuate melanocortin system is best described as a conduit of many diverse signals involved in energy homeostasis, with leptin acting tonically to regulate the responsiveness of the circuit to a wide variety of hormones and nutrients.

Melanocortin-4 receptor is required for acute homeostatic responses to increased dietary fat.

In response to moderately increased dietary fat content, melanocortin-4 receptor-null mutant (MC4R−/−) mice exhibit hyperphagia and accelerated weight gain compared to wild-type mice. An increased feed efficiency (weight gain/kcal consumed) argues that mechanisms in addition to hyperphagia are instrumental in causing weight gain. We report two specific defects in coordinating energy expenditure with food intake in MC4R−/− mice. Wild-type mice respond to an increase in the fat content of the diet by rapidly increasing diet-induced thermogenesis and by increasing physical activity, neither of which are observed in MC4R−/− mice. Leptin-deficient and MC3R−/− mice regulate metabolic rate similarly to wild-type mice in this protocol. Melanocortinergic pathways involving MC4-R-regulated neurons, which rapidly respond to signals not requiring changes in leptin, thus seem to be important in regulating metabolic and behavioral responses to dietary fat.

INSULIN-LIKE GROWTH FACTOR-I RECEPTOR SIGNAL TRANSDUCTION : AT THE INTERFACE BETWEEN PHYSIOLOGY AND CELL BIOLOGY

The insulin-like growth factor-I receptor (IGF-IR) mediates the biological actions of IGF-I and IGF-II. The IGFs play a critical role in promoting development, stimulating growth and organogenesis via mitogenic, antiapoptotic and chemotactic activity. Recent research has focused on the events that occur intracellularly upon receptor activation. Several pathways have been shown to be important. The insulin-receptor substrate (IRS), SHC, GRB2, CRKII and CRKL adaptor proteins have all been implicated in transmitting signals to the nucleus of the cell. This review outlines some of the signalling pathways believed to be important in converting IGF-IR activation into changes in cell behavior and metabolism.

What is the role of circulating IGF-I?

Postnatal growth and development are coordinated by genetic and environmental influences and numerous growth factors. The growth hormone-insulin-like growth factor-I (GH-IGF-I) axis plays an essential role in these processes. Although the GH-IGF-I axis is a closely coordinated system, both GH and IGF-I have independent actions, many of which have become apparent more recently following the characterization of clinical syndromes and the development of mouse models. Genetic manipulation of mice has enabled investigators to re-examine many of the established hypotheses regarding the GH-IGF-I axis. Results gleaned from a mouse model created by tissue-specific gene deletion of liver IGF-I has enabled investigators to re-evaluate the original somatomedin hypothesis.

Knockout models resulting in the development of obesity.

Our understanding of body weight regulation has been greatly advanced by the characterization of previously existing mutations in mice that cause obesity. Subsequent analysis of a number of mouse knockout models has greatly expanded the number of genes known to influence adiposity by affecting metabolic rate, physical activity, and/or appetite.

Disproportionate Inhibition of Feeding in Ay Mice by Certain Stressors: A Cautionary Note

A study of the effects of insulin-induced hypoglycemia in the obese yellow agouti Ay mouse was initiated to test the hypothesis that the central melanocortin pathways are required for a normal sympathetic response to hypoglycemia. An experimental protocol was performed in which young nonobese male mice were isolated and fasted beginning on day 1, then tested for glucose responses to insulin-induced hypoglycemia on day 2. Normal mice demonstrated the expected glucose rebound to hypoglycemia, exceeding baseline glucose levels by 2–3 times as a consequence of increased gluconeogenesis and glycogenolysis before returning to baseline levels. Ay animals lacked the rebound, exhibiting instead a gradual restoration of baseline glucose levels. The results suggested a defective sympathetic response to hypoglycemia in the Ay mouse. However, a more detailed analysis demonstrated that the lack of a hyperglycemic rebound was due to an acute inhibition of feeding specifically in the Ay mouse, which resulted not from the hypoglycemia stressor, but rather from the stress of isolation. Handling and intraperitoneal administration of saline also specifically inhibited food intake in the Ay but not the wild-type mouse, while restraint stress had an equivalent inhibitory effect on food intake on wild-type and Ay mice. Since the Ay mouse has defective hypothalamic melanocortin-4 receptor (MC4-R) signaling, these data imply that the central melanocortin pathway is necessary for regulating the effects of stress on feeding behavior. Furthermore, these data demonstrate the need for exercising extreme caution in designing experiments to analyze feeding behavior and metabolism in genetic or pharmacological models involving perturbation of the melanocortin system.

In Vivo Regulation of CrkII and CrkL Proto-oncogenes in the Uterus by Insulin-like Growth Factor-I DIFFERENTIAL EFFECTS ON TYROSINE PHOSPHORYLATION AND ASSOCIATION WITH PAXILLIN

Changes in CrkII and CrkL phosphorylation are associated with insulin-like growth factor receptor activation in cultured cells. We examined whether similar changes also occur following administration of recombinant human insulin-like growth factor-I to the intact animal. In female rats starved overnight, CrkL phosphorylation was significantly increased 12 min after insulin-like growth factor-I administration. Tyrosine phosphorylation of CrkII was not detectable in either control or treated animals. Paxillin, a 65–70-kDa phosphoprotein containing high affinity binding sites common for the Src homology 2 (SH2) domains of CrkII and CrkL, was observed in both CrkII and CrkL immunoprecipitates. Insulin-like growth factor-I treatment stimulated the association of CrkII with paxillin. In contrast, the same treatment resulted in the dissociation of the CrkL-paxillin complex. Similar effects of insulin-like growth factor-I treatment on the association of CrkL with tyrosine phosphorylated paxillin were observed in fibroblasts overexpressing CrkL. This study demonstrates that the activation of the insulin-like growth factor-I receptor induces changes in the tyrosine phosphorylation and protein-protein interactions of the Crk proteins in vivo. The different responses of CrkL and CrkII to insulin-like growth factor-I receptor activation suggest distinct roles for these two adapter proteins in signal transduction.

IGF-I Receptor Function

The insulin-like growth factor (IGF)-I receptor, or type 1 IGF receptor, is a transmembrane tyrosine kinase receptor that mediates the majority of the biological actions of IGF-I and IGF-II (1,2). The ligands, IGF-I or IGF-II, bind to the extracellular domain of the receptor and initiate a conformational change that is transmitted to the intracellular domain. The receptor is then autophosphorylated on several intracellular tyrosine residues. The tyrosine-phosphorylated receptor is then fully active as a tyrosine kinase toward endogenous substrates. Both IGF-I and IGF-II circulate bound to IGF binding proteins (IGFBPs) (3). The IGF—IGFBP complexes either enhance or inhibit IGF action in a tissue-specific manner The known roles of the IGFBPs are presented in other chapters. Whereas the IGFBPs regulate the activation of the receptor by the binding of its cognate ligands, IGF-I receptor activation constitutes the ultimate requisite for the transduction of IGF-mediated signals.