Glucose intolerance and pancreatic ß-cell dysfunction in the anorectic anx/anx mouse.

Inflammation and impaired mitochondrial oxidative phosphorylation are considered key players in the development of several metabolic disorders, including diabetes. We have previously shown inflammation and mitochondrial dysfunction in the hypothalamus of an animal model for anorexia, the anx/anx mouse. Moreover, increased incidence of eating disorders, e.g., anorexia nervosa, has been observed in diabetic individuals. In the present investigation we evaluated whether impaired mitochondrial phosphorylation and inflammation also occur in endocrine pancreas of anorectic mice, and if glucose homeostasis is disturbed. We show that anx/anx mice exhibit marked glucose intolerance associated with reduced insulin release following an intraperitoneal injection of glucose. In contrast, insulin release from isolated anx/anx islets is increased after stimulation with glucose or KCl. In isolated anx/anx islets there is a strong downregulation of the mitochondrial complex I (CI) assembly factor, NADH dehydrogenase (ubiquinone) 1α subcomplex, assembly factor 1 (Ndufaf1), and a reduced CI activity. In addition, we show elevated concentrations of free fatty acids (FFAs) in anx/anx serum and increased macrophage infiltration (indicative of inflammation) in anx/anx islets. However, isolated islets from anx/anx mice cultured in the absence of FFAs do not exhibit increased inflammation. We conclude that the phenotype of the endocrine pancreas of the anx/anx mouse is characterized by increased levels of circulating FFAs, as well as inflammation, which can inhibit insulin secretion in vivo. The anx/anx mouse may represent a useful tool for studying molecular mechanisms underlying the association between diabetes and eating disorders.

Hypothalamic mitochondrial dysfunction associated with anorexia in the anx/anx mouse.

The anorectic anx/anx mouse exhibits disturbed feeding behavior and aberrances, including neurodegeneration, in peptidergic neurons in the appetite regulating hypothalamic arcuate nucleus. Poor feeding in infants, as well as neurodegeneration, are common phenotypes in human disorders caused by dysfunction of the mitochondrial oxidative phosphorylation system (OXPHOS). We therefore hypothesized that the anorexia and degenerative phenotypes in the anx/anx mouse could be related to defects in the OXPHOS. In this study, we found reduced efficiency of hypothalamic OXPHOS complex I assembly and activity in the anx/anx mouse. We also recorded signs of increased oxidative stress in anx/anx hypothalamus, possibly as an effect of the decreased hypothalamic levels of fully assembled complex I, that were demonstrated by native Western blots. Furthermore, the Ndufaf1 gene, encoding a complex I assembly factor, was genetically mapped to the anx interval and found to be down-regulated in anx/anx mice. These results suggest that the anorexia and hypothalamic neurodegeneration of the anx/anx mouse are associated with dysfunction of mitochondrial complex I.

Evidence of hypothalamic degeneration in the anorectic anx/anx mouse.

Mice homozygous for the anorexia (anx) mutation are characterized by poor food intake and death by three to five weeks after birth. By P21 these mice display lower density of hypothalamic neuropeptides, including Agouti gene-related protein (AGRP). The AGRP/neuropeptide Y (NPY) system of the anx/anx mice develops normally until postnatal day (P) 12, then the normal increase in fiber density ceases, in some areas even distinctly decreases. This overlaps with activation of microglia, indicating an inflammatory and/or degenerative process. Here we studied, by in situ hybridization and immunohistochemistry (IHC), the expression of major histocompatibility complex (MHC) class I-related molecules and markers for cellular reactivity in hypothalamus of anx/anx mice. MHC class I transcript and -related proteins were found in arcuate nucleus (Arc), presumably both in neurons and glia, the latter also in areas innervated by AGRP (NPY) neurons. In the anx/anx hypothalamus, using TUNEL labeling, significantly higher number of apoptotic cells were found compared with +/+ mice, and active caspase 6 immunoreactivity was detected in degenerating NPY-fibers as well as signs of "microglia-associated cell death". In addition, Y1 receptor-labeled processes and soma of pro-opiomelanocortin (POMC) neurons, were markedly decreased at P21. These results support the hypothesis of degeneration of hypothalamic arcuate neuron populations in the anx/anx mice, whereby the AGRP system may be first affected, the changes in the POMC system being secondary in this process.

NPY and its involvement in axon guidance, neurogenesis, and feeding.

OBJECTIVES: The role of neuropeptides in nervous system function is still in many cases undefined. In the present study we examined a possible role of the 36-amino acid neuropeptide Y (NPY) with regard to three functions: axon guidance and attraction/repulsion, adult neurogenesis, and control of food intake. METHODS: Growth cones from embryonic dorsal root ganglion neurons were studied in culture during asymmetrical gradient application of NPY. Growth cones were monitored over a 60-min period, and final turning angle and growth rate were recorded. In the second part the NPY Y(1) and Y(2) receptors were studied in the subventricular zone, the rostral migratory stream, and the olfactory bulb in normal mice and mice with genetically deleted NPY Y(1) or Y(2) receptors. In the third part an anorectic mouse was analyzed with immunohistochemistry. RESULTS: 1) NPY elicited an attractive turning response and an increase in growth rate, effects exerted via the NPY Y(1) receptor. 2) The NPY Y(1) receptor was expressed in neuroblasts in the anterior rostral migratory stream. Mice deficient in the Y(1) or Y(2) receptor had fewer proliferating precursor cells and neuroblasts in the subventricular zone and rostral migratory stream and fewer neurons in the olfactory bulb expressing calbindin, calretinin or tyrosine hydroxylase. 3) In the anorectic mouse markers for microglia were strongly upregulated in the arcuate nucleus and in projection areas of the NPY/agouti gene-related protein arcuate system. CONCLUSION: NPY participates in several mechanisms involved in the development of the nervous system and is of importance in the control of food intake.

Reduced metabolism in the hypothalamus of the anorectic anx/anx mouse.

The anorectic anx/anx mouse exhibits a mitochondrial complex I dysfunction that is related to aberrant expression of hypothalamic neuropeptides and transmitters regulating food intake. Hypothalamic activity, i.e. neuronal firing and transmitter release, is dependent on glucose utilization and energy metabolism. To better understand the role of hypothalamic activity in anorexia, we assessed carbohydrate and high-energy phosphate metabolism, in vivo and in vitro, in the anx/anx hypothalamus. In the fasted state, hypothalamic glucose uptake in the anx/anx mouse was reduced by ~50% of that seen in wild-type (wt) mice (P < 0.05). Under basal conditions, anx/anx hypothalamus ATP and glucose 6-P contents were similar to those in wt hypothalamus, whereas phosphocreatine was elevated (~2-fold; P < 0.001) and lactate was reduced (~35%; P < 0.001). The anx/anx hypothalamus had elevated total AMPK (~25%; P < 0.05) and GLUT4 (~60%; P < 0.01) protein contents, whereas GLUT1 and GLUT3 were similar to that of wt hypothalamus. Interestingly, the activation state of AMPK (ratio of phosphorylated AMPK/total AMPK) was significantly decreased in hypothalamus of the anx/anx mouse (~60% of that in wt; P < 0.05). Finally, during metabolic stress (ischemia), accumulation of lactate (measure of glycolysis) and IMP and AMP (breakdown products of ATP) were ~50% lower in anx/anx vs wt hypothalamus. These data demonstrate that carbohydrate and high-energy phosphate utilization in the anx/anx hypothalamus are diminished under basal and stress conditions. The decrease in hypothalamic metabolism may contribute to the anorectic behavior of the anx/anx mouse, i.e. its inability to regulate food intake in accordance with energy status.

Anorexia and hypothalamic degeneration.

Anorexia, meaning poor appetite, occurs in many human conditions, for example, anorexia nervosa, cachexia, and failure to thrive in infants. A key player in the regulation of appetite/food intake in general, as well as conditions of anorexia, is the hypothalamus, in particular, the AGRP/NPY and POMC/CART neurons in the arcuate nucleus. In this chapter, we review the hypothalamic aberrances seen in the anorectic anx/anx mouse. This mouse displays deviations in neuropeptidergic/-transmitter systems, including selective hypothalamic degeneration and inflammation that have been associated with mitochondrial dysfunction. In addition, we discuss data from other animal models, as well as clinical data relating hypothalamic inflammation/degeneration, neurogenesis, and mitochondrial dysfunction to conditions of disturbed regulation of food intake.

Aberrant agouti-related protein system in the hypothalamus of the anx/anx mouse is associated with activation of microglia.

Agouti-related protein (AgRP) is a key orexigenic neuropeptide expressed in the hypothalamic arcuate nucleus and a marker for neurons conveying hormonal signals of hunger to the brain. Mice homozygous for the anorexia (anx) mutation are characterized by decreased food intake, starvation, and death by 3-5 weeks of age. At this stage immunoreactivity for AgRP is increased in cell bodies but decreased in the nerve terminals. We studied when during early postnatal development the aberrant phenotype of the AgRP system becomes apparent in anx/anx mice and possible underlying mechanisms. AgRP and ionized calcium binding adapter molecule (Iba1), a marker for activated microglia, as well as Toll-like receptor 2 (TLR-2), were studied by immunohistochemistry at postnatal days P1, P5, P10, P12, P15 and P21 in anx/anx and wild-type mice. We found that the AgRP system in the anx/anx mouse develops similarly to the wild type until P12, when AgRP fibers in anx/anx mice cease to increase in density in the main projection areas. At P21, AgRP fiber density in anx/anx mice was significantly reduced vs. P15, in certain regions. At P21, many strongly AgRP-positive cell bodies were observed in the anx/anx arcuate nucleus vs. only few and weakly fluorescent ones in the wild type. The decrease in AgRP fiber density in anx/anx mice overlapped with an increase in Iba1 and TLR-2 immunoreactivities. Thus, the aberrant appearance of the AgRP system in the anx/anx mouse in the early postnatal development could involve a microglia-associated process and the innate immune system.