Ryoichi Banno

PTP1B and SHP2 in POMC neurons reciprocally regulate energy balance in mice

Protein tyrosine phosphatase 1B (PTP1B) and SH2 domain-containing protein tyrosine phosphatase-2 (SHP2) have been shown in mice to regulate metabolism via the central nervous system, but the specific neurons mediating these effects are unknown. Here, we have shown that proopiomelanocortin (POMC) neuron-specific deficiency in PTP1B or SHP2 in mice results in reciprocal effects on weight gain, adiposity, and energy balance induced by high-fat diet. Mice with POMC neuron-specific deletion of the gene encoding PTP1B (referred to herein as POMC-Ptp1b-/- mice) had reduced adiposity, improved leptin sensitivity, and increased energy expenditure compared with wild-type mice, whereas mice with POMC neuron-specific deletion of the gene encoding SHP2 (referred to herein as POMC-Shp2-/- mice) had elevated adiposity, decreased leptin sensitivity, and reduced energy expenditure. POMC-Ptp1b-/- mice showed substantially improved glucose homeostasis on a high-fat diet, and hyperinsulinemic-euglycemic clamp studies revealed that insulin sensitivity in these mice was improved on a standard chow diet in the absence of any weight difference. In contrast, POMC-Shp2-/- mice displayed impaired glucose tolerance only secondary to their increased weight gain. Interestingly, hypothalamic Pomc mRNA and alpha-melanocyte-stimulating hormone (alphaMSH) peptide levels were markedly reduced in POMC-Shp2-/- mice. These studies implicate PTP1B and SHP2 as important components of POMC neuron regulation of energy balance and point to what we believe to be a novel role for SHP2 in the normal function of the melanocortin system.

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.

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.

TNFα increases hypothalamic PTP1B activity via the NFκB pathway in rat hypothalamic organotypic cultures.

In obesity, levels of tumor necrosis-factor α (TNFα) are well known to be elevated in adipose tissues or serum, and a high-fat diet (HFD) reportedly increases TNFα expression in the hypothalamus. The expression levels of hypothalamic protein tyrosine phosphatase 1B (PTP1B), a negative regulator of leptin and insulin signaling, are also elevated by HFD, and several lines of evidence support a relationship between TNFα and PTP1B. It remains unclear however how TNFα acts locally in the hypothalamus to regulate hypothalamic PTP1B expression and activity. In this study, we examined whether TNFα can regulate PTP1B expression and activity using rat hypothalamic organotypic cultures. Incubation of cultures with TNFα resulted in increases in mRNA expression, protein levels and activity of PTP1B in a dose- and time-dependent manner, respectively compared with controls. TNFα-induced PTP1B protein levels were not influenced by co-incubation with the sodium channel blocker tetrodotoxin, indicating that the action of TNFα is independent of action potentials. TNFα also increased phosphorylation of p65, a subunit of nuclear factor-κB (NFκB), in a dose- and time-dependent manner. While incubation with inhibitors of NFκB did not affect basal levels of either p65 phosphorylation or PTP1B expression, it markedly suppressed both TNFα-induced p65 phosphorylation and PTP1B expression to almost basal levels. These data suggest that TNFα acts on the hypothalamus to increase hypothalamic PTP1B expression and activity via the NFκB pathway, and that TNFα-mediated induction of NFκB in the hypothalamus may cause leptin and insulin resistance in the hypothalamus by increasing hypothalamic PTP1B activity.

GABA Type B Receptor Signaling in Proopiomelanocortin Neurons Protects Against Obesity, Insulin Resistance, and Hypothalamic Inflammation in Male Mice on a High-Fat Diet

There is evidence suggesting that the GABA system in the arcuate nucleus, where orexigenic neuropeptide Y and agouti-related peptide as well as anorexigenic proopiomelanocortin (POMC) are expressed, plays an important role in energy balance. In this study, we generated POMC-specific GABAB receptor-deficient [knock-out (KO)] mice. Male KO mice on a high-fat diet (HFD) showed mild increases in body weight (BW) at the age of 9 weeks compared to wild-type (WT) mice, and the differences remained significant until 16 weeks old. However, there was no difference in BW in females between genotypes. While food intake was similar between genotypes, oxygen consumption was significantly decreased in the male KO mice. The insulin tolerance test revealed that the male KO mice were less insulin sensitive compared to WT mice at the age of 8 weeks, when there was no significant difference in BW between genotypes. Despite increased BW, POMC mRNA expression in the arcuate nucleus was significantly decreased in the KO mice compared to WT mice at the age of 16 weeks. Furthermore, the expression of TNFα as well as IL-6, proinflammatory markers in the hypothalamus, was significantly increased in the KO mice on a HFD compared to WT mice. This demonstrates that the deletion of GABAB receptors in POMC neurons in the male mice on a HFD results in obesity, insulin resistance, and hypothalamic inflammation. Furthermore, the decreased POMC expression in the obese KO mice suggests that the regulation of POMC expression through GABAB receptors is essential for proper energy balance.

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.

Arginine vasopressin neuronal loss results from autophagy-associated cell death in a mouse model for familial neurohypophysial diabetes insipidus

Familial neurohypophysial diabetes insipidus (FNDI) characterized by progressive polyuria is mostly caused by mutations in the gene encoding neurophysin II (NPII), which is the carrier protein of the antidiuretic hormone, arginine vasopressin (AVP). Although accumulation of mutant NPII in the endoplasmic reticulum (ER) could be toxic for AVP neurons, the precise mechanisms of cell death of AVP neurons, reported in autopsy studies, remain unclear. Here, we subjected FNDI model mice to intermittent water deprivation (WD) in order to promote the phenotypes. Electron microscopic analyses demonstrated that, while aggregates are confined to a certain compartment of the ER in the AVP neurons of FNDI mice with water access ad libitum, they were scattered throughout the dilated ER lumen in the FNDI mice subjected to WD for 4 weeks. It is also demonstrated that phagophores, the autophagosome precursors, emerged in the vicinity of aggregates and engulfed the ER containing scattered aggregates. Immunohistochemical analyses revealed that expression of p62, an adapter protein between ubiquitin and autophagosome, was elicited on autophagosomal membranes in the AVP neurons, suggesting selective autophagy induction at this time point. Treatment of hypothalamic explants of green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) transgenic mice with an ER stressor thapsigargin increased the number of GFP-LC3 puncta, suggesting that ER stress could induce autophagosome formation in the hypothalamus of wild-type mice as well. The cytoplasm of AVP neurons in FNDI mice was occupied with vacuoles in the mice subjected to WD for 12 weeks, when 30–40% of AVP neurons are lost. Our data thus demonstrated that autophagy was induced in the AVP neurons subjected to ER stress in FNDI mice. Although autophagy should primarily be protective for neurons, it is suggested that the organelles including ER were lost over time through autophagy, leading to autophagy-associated cell death of AVP neurons.

Activating Transcription Factor 6α Is Required for the Vasopressin Neuron System to Maintain Water Balance Under Dehydration in Male Mice

Activating transcription factor 6α (ATF6α) is a sensor of endoplasmic reticulum (ER) stress and increases the expression of ER chaperones and molecules related to the ER-associated degradation of unfolded/misfolded proteins. In this study, we used ATF6α knockout (ATF6α(-/-)) mice to clarify the role of ATF6α in the arginine vasopressin (AVP) neuron system. Although urine volumes were not different between ATF6α(-/-) and wild-type (ATF6α(+/+)) mice with access to water ad libitum, they were increased in ATF6α(-/-) mice compared with those in ATF6α(+/+) mice under intermittent water deprivation (WD) and accompanied by less urine AVP in ATF6α(-/-) mice. The mRNA expression of immunoglobulin heavy chain binding protein, an ER chaperone, was significantly increased in the supraoptic nucleus in ATF6α(+/+) but not ATF6α(-/-) mice after WD. Electron microscopic analyses demonstrated that the ER lumen of AVP neurons was more dilated in ATF6α(-/-) mice than in ATF6α(+/+) mice after WD. ATF6α(-/-) mice that were mated with mice possessing a mutation causing familial neurohypophysial diabetes insipidus (FNDI), which is characterized by progressive polyuria and AVP neuronal loss due to the accumulation of mutant AVP precursor in the ER, manifested increased urine volume under intermittent WD. The aggregate formation in the ER of AVP neurons was further impaired in FNDI/ATF6α(-/-) mice compared with that in FNDI mice, and AVP neuronal loss was accelerated in FNDI/ATF6α(-/-) mice under WD. These data suggest that ATF6α is required for the AVP neuron system to maintain water balance under dehydration.

Minocycline prevents osmotic demyelination associated with aquaresis

Overly rapid correction of chronic hyponatremia can cause osmotic demyelination syndrome (ODS). Minocycline protects ODS associated with overly rapid correction of chronic hyponatremia with hypertonic saline infusion in rats. In clinical practice, inadvertent rapid correction frequently occurs due to water diuresis, when vasopressin action suddenly ceases. In addition, vasopressin receptor antagonists have been applied to treat hyponatremia. Here the susceptibility to and pathology of ODS were evaluated using rat models developed to represent rapid correction of chronic hyponatremia in the clinical setting. The protective effect of minocycline against ODS was assessed. Chronic hyponatremia was rapidly corrected by 1 (T1) or 10 mg/kg (T10) of tolvaptan, removal of desmopressin infusion pumps (RP), or administration of hypertonic saline. The severity of neurological impairment in the T1 group was significantly milder than in other groups and brain hemorrhage was found only in the T10 and desmopressin infusion removal groups. Minocycline inhibited demyelination in the T1 group. Further, immunohistochemistry showed loss of aquaporin-4 (AQP4) in astrocytes before demyelination developed. Interestingly, serum AQP4 levels were associated with neurological impairments. Thus, minocycline can prevent ODS caused by overly rapid correction of hyponatremia due to water diuresis associated with vasopressin action suppression. Increased serum AQP4 levels may be a predictive marker for ODS.

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.