Minemori Watanabe

Glucocorticoids Increase Neuropeptide Y and Agouti-Related Peptide Gene Expression via Adenosine Monophosphate-Activated Protein Kinase Signaling in the Arcuate Nucleus of Rats

Recent studies suggest that the AMP-activated protein kinase (AMPK) signaling in the hypothalamus is the master regulator of energy balance. We reported in previous studies that glucocorticoids play a permissive role in the regulation of orexigenic neuropeptide Y (Npy) gene expression in the arcuate nucleus. In this study, we examined whether any cross talk occurs between glucocorticoids and AMPK signaling in the hypothalamus to regulate Npy as well as agouti-related peptide (Agrp) gene expression in the arcuate nucleus. In the hypothalamic organotypic cultures, the addition to the medium of the AMPK activator, 5-aminoimidazole-4-carboxamide-1-b-d-ribofuranoside, increased phosphorylated AMPK (p-AMPK) as well as phosphorylated acetyl-coenzyme A carboxylase (p-ACC) in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The incubation with dexamethasone (DEX) also activated AMPK signaling in the explants, accompanied by significant increases in Npy and Agrp gene expression in the arcuate nucleus. The addition of the AMPK inhibitor compound C to the medium, which blocked increases of p-AMPK and p-ACC by DEX, significantly attenuated Npy and Agrp gene expression stimulated by DEX. Furthermore, p-AMPK and p-ACC levels in the arcuate nucleus were significantly decreased in adrenalectomized rats compared with sham-operated rats, and a replacement of glucocorticoids reversed the AMPK signaling in adrenalectomized rats. Thus, our data demonstrated that glucocorticoids up-regulate the Npy and Agrp gene expression in the arcuate nucleus through AMPK signaling, suggesting that the activation of the hypothalamic APMK signaling by glucocorticoids might be essential to the energy homeostasis.

Insulin Inhibits Neuropeptide Y Gene Expression in the Arcuate Nucleus through GABAergic Systems

Neuropeptide Y (NPY) in the arcuate nucleus is an orexigenic hormone of which levels are regulated by humoral as well as neural signals. In this study, we examined the regulation of NPY gene expression in the arcuate nucleus in hypothalamic organotypic cultures. Dexamethasone (DEX) (10–9 to 10–7 m) significantly increased NPY mRNA expression, and the effects were not influenced by coincubation with the sodium channel blocker tetrodotoxin (TTX), indicating that the action of DEX is independent of action potentials. Conversely, insulin (10–11 to 10–9 m) significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was abolished in the presence of TTX. Because GABA and its receptors are expressed in the arcuate nucleus in vivo, we examined whether GABAergic systems were involved in the insulin action. The GABAB agonist baclofen significantly inhibited NPY expression stimulated by DEX, and the inhibitory action of insulin was completely abolished in the presence of either the GABAA antagonist bicuculline or the GABAB antagonist CGP35348 (p-3-aminopropyl-p-diethoxymethyl phosphoric acid). Furthermore, increases in the GABA-synthesizing enzyme glutamic acid decarboxylase 65 (GAD65) mRNA expression preceded decreases in NPY mRNA expression in the arcuate nucleus in the cultures. Experiments in vivo also demonstrated that increases in GAD65 mRNA expression in the arcuate nucleus preceded decreases in the NPY mRNA expression in a fasting-refeeding paradigm and that intracerebroventricular injection of insulin increased GAD65 mRNA expression in the arcuate nucleus in fasted rats. These data suggest that insulin inhibits NPY gene expression in the arcuate nucleus through GABAergic systems.

Central administration of melanocortin agonist increased insulin sensitivity in diet-induced obese rats

In this study, we examined the effects of intracerebroventricular administration of melanotan II (MTII), a melanocortin agonist, on insulin sensitivity in diet‐induced obese (DIO) rats. Although MTII treatment significantly decreased food intake and body weight for 10 days, there was no significant difference in body weight between MTII and pair‐fed groups. The insulin tolerance test showed that insulin sensitivity was significantly improved in the MTII group compared to the pair‐fed group. Furthermore, MTII treatment increased the number of small‐sized adipocytes in epididymal white adipose tissues, suggesting that MTII increased insulin sensitivity through action on the white adipose tissues in DIO rats.

Peripherally administered baclofen reduced food intake and body weight in db/db as well as diet-induced obese mice.

Peripheral administration of baclofen significantly reduced food intake and body weight increase in both diabetic (db/db) and diet‐induced obese mice for 5 weeks, whereas it had no significant effects on energy balance in their lean control mice. Despite the decreased body weight, neuropeptide Y expression in the arcuate nucleus was significantly decreased, whereas pro‐opiomelanocortin expression was significantly increased by baclofen treatment. These data demonstrate that the inhibitory effects of baclofen on body weight in the obese mice were mediated via the arcuate nucleus at least partially, and suggest that GABAB agonists could be a new therapeutic reagent for obesity.

Glucocorticoids increase NPY gene expression in the arcuate nucleus by inhibiting mTOR signaling in rat hypothalamic organotypic cultures.

The mammalian target of rapamycin (mTOR) has been implicated in the regulation of physiological functions such as cell growth and proliferation, and glucocorticoids reportedly inhibit mTOR signaling in peripheral tissues. Recent studies suggest that the mTOR signaling in the hypothalamus plays a critical role in maintaining energy homeostasis. In this study, we examined whether the mTOR signaling in the hypothalamus is involved in the regulation of neuropeptide Y (Npy) gene expression in the arcuate nucleus by glucocorticoids. In the hypothalamic organotypic cultures, the incubation with rapamycin significantly inhibited the mTOR signaling which was shown by decreases in the levels of phosphorylated p70S6K1 and S6. Similar to the action of the mTOR inhibitor rapamycin, dexamethasone (DEX), a synthetic glucocorticoid, also inhibited the mTOR signaling in the hypothalamic explants. Analyses of the explants with in situ hybridization demonstrated that the DEX or rapamycin alone significantly increased Npy gene expression in the arcuate nucleus, but that there were no additive effects of DEX and rapamycin on the expression. These data suggest that glucocorticoids upregulate the Npy gene expression in the arcuate nucleus by inhibiting mTOR signaling, at least in part.

Direct and indirect modulation of neuropeptide Y gene expression in response to hypoglycemia in rat arcuate nucleus

Expression of neuropeptide Y (Npy) heteronuclear (hn) RNA, an indicator of gene transcription, was significantly increased in the arcuate nucleus of rats 30 min after insulin injection. Npy hnRNA levels were also increased significantly in response to hypoglycemia in rats in which the hypothalamus was deafferentated, although the absolute levels were significantly lower than in sham‐operated rats. Direct effects of lowering glucose levels on Npy gene expression were also confirmed in hypothalamic organotypic cultures. Thus, Npy gene transcription in the arcuate nucleus increases rapidly in response to hypoglycemia, and both direct and indirect inputs are involved in the rapid upregulation.

Insulin is not a prerequisite for rapid regulation of neuropeptide Y gene transcription in the arcuate nucleus in food-restricted rats

Neuropeptide Y (NPY), synthesized in the arcuate nucleus of the hypothalamus, is one of the most potent orexigenic neuropeptides in the brain. The NPY neurons project to other hypothalamic nuclei, such as paraventricular nucleus (PVN), and it is reported that NPY contents in the PVN, but not NPY mRNA levels in the arcuate nucleus, decreased rapidly after food consumption. While many signals reflecting energy balance in the periphery are integrated at the NPY neurons, insulin has been implicated as one of the key regulators for NPY neurons. In the present study, we first examined whether there exist dynamic changes in NPY gene transcription in the arcuate nucleus in association with food intake in rats which had access to food only 4h a day. To detect possible changes in NPY gene transcription, we measured the expression levels of NPY heteronuclear (hn) RNA, a sensitive indicator of gene transcription, with intronic in situ hybridization. Our data showed that NPY hnRNA levels in the arcuate nucleus decreased rapidly after food consumption. We next examined whether postprandial increases in insulin release might contribute to the rapid downregulation of NPY gene transcription. To do so, insulin-deficient rats by streptozotocin injection were subjected to the same paradigm. Our data showed that NPY hnRNA levels also decreased rapidly after food consumption, suggesting that the postprandial increase in insulin release is not a prerequisite for the rapid downregulation of NPY gene transcription in the arcuate nucleus.

Copper Accumulates in Hemosiderins in Livers of Patients with Iron Overload Syndromes.

In biology, redox reactions are essential and sometimes harmful, and therefore, iron metabolism is tightly regulated by cuproproteins. Since the state of copper in iron overload syndromes remains unclear, we investigated whether copper metabolism is altered in these syndromes. Eleven patients with iron overload syndromes participated in this study. The clinical diagnoses were aceruloplasminemia (n=2), hemochromatosis (n=5), ferroportin disease (n=2), and receiving excess intravenous iron supplementation (n=2). Liver specimens were analyzed using a light microscope and transmission electron microscope equipped with an X-ray analyzer. In addition to a large amount of iron associated with oxygen and phosphorus, the iron-rich hemosiderins of hepatocytes and Kupffer cells contained small amounts of copper and sulfur, regardless of disease etiology. Two-dimensional imaging clearly showed that cuproproteins were distributed homogenously with iron complexes within hemosiderins. Copper stasis was unlikely in noncirrhotic patients. The enhanced induction of cuproproteins by excess iron may contribute to copper accumulation in hemosiderins. In conclusion, we have demonstrated that copper accumulates in hemosiderins in iron overload conditions, perhaps due to alterations in copper metabolism.

Repeated glucoprivation delayed hyperphagic responses while activating neuropeptide Y neurons in rats

It is well known that glucoprivation induces the release of counterregulatory hormones such as glucagon, and that the response is attenuated when the stimuli are repeated. Glucoprivation also activates orexigenic neurons and induces hyperphagic responses, although it remains unclear whether these responses are attenuated in repeated glucoprivation. In this study, we examined time course changes in feeding as well as activities of orexigenic neuropeptide Y (NPY) neurons in repeated glucoprivation in rats. Either 2-deoxy-d-glucose (2DG), which blocks glucose utilization, or isotonic saline (control) was injected subcutaneously to rats for 14 days, and food consumption for 1 and 2h after injection was monitored throughout the experiment. While 2DG injection induced robust feeding responses during the first 1h after injection, the response was gradually attenuated and the food consumption was significantly less on days 12-14 compared to that on day 1. On the other hand, food consumption during 2h after 2DG injection was not changed significantly for 14 days. The transcriptional activities of NPY neurons in the arcuate nucleus and C1/A1 region of the hindbrain, measured by intronic in situ hybridization, were significantly enhanced after repeated 2DG injection for 14 days, while the feeding responses to intracerebroventricular injection of NPY were significantly less in the 2DG-repeated group compared to the saline-repeated group. It is thus demonstrated that repeated glucoprivation delayed hyperphagic responses while activating NPY neurons in rats. Our data also suggest that decreased feeding responses to NPY might be at least partially responsible for the delayed response.

The medial hypothalamus is required for the feeding response to glucoprivation but not to food deprivation.

While the hypothalamus has been implicated in the regulation of energy balance, the central mechanisms and neural circuit that coordinate the feeding response to energy deficit have not been fully clarified. To better understand the role of the hypothalamus in mediating hyperphagic responses to food deprivation or glucoprivation, we examined the feeding responses in rats in which the medial hypothalamus (MH) was isolated from the rest of the brain. The isolation of the MH was performed with a Halaszs knife cut, and experiments were performed 7 days after the operation. Food consumption between 9:00 a.m. and 11:00 a.m. in rats which had been fasted overnight was significantly increased compared to that in rats which had access to food ad libitum before the measurement in both the sham and MH-isolated groups, and the absolute values of food consumption in fasted rats were not significantly different between the groups. On the other hand, while an injection of 2-deoxy-d-glucose, which blocks glucose utilization, significantly increased food consumption for 2h after injection compared to a saline injection in the sham group, it did not increase food intake compared to saline injection in the MH-isolated groups. Thus, it is demonstrated that glucoprivation is not an effective stimulus to induce feeding in MH-isolated rats.