Although the mechanisms underlying salt-induced hypertension have been investigated for a long time, they remained unclear until recently. The main reasons were that the sensors detecting increases in [Na+] in body fluids had not been identified and the mechanisms responsible for sensing and transmitting signals to the center controlling sympathetic nerve activity (SNA) were unknown 1) . We, for the first time, elucidated the brain mechanisms underlying salt-induced elevations in blood pressure (BP) 2) (Fig. 1). Briefly, increases in [Na+] in body fluids activate Nax channels in the organum vasculosum lamina terminalis (OVLT). The Nax signal in glial cells is transferred to the central nuclei [paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM)] controlling SNA. This increase in SNA elevates BP. On the other hand, obesity and psychological stress also elevate BP by activating SNA.

Figure 1. Central mechanisms responsible for salt-induced BP elevations

　Under obese or stress conditions, blood levels of leptin, angiotensin II, or aldosterone increase. The sensing loci of leptin, angiotensin II, and aldosterone in the brain as well as the cellular mechanisms underlying their signal transmission remain unclear. After these factors are received by specific brain loci, the signals are eventually transferred to the central nuclei controlling SNA. We hypothesized that the sensing receptive loci of these factors are the circumventricular organs (CVOs), which lack a blood-brain barrier in the brain (Fig. 2). We will integratively elucidate the brain mechanisms responsible for BP elevations caused by these endogenous pressor factors.

Figure 2. Circumventricular organs (CVOs)

　In the present study, we will promote the following projects: The elucidation of: i) receptive loci for the respective factors, ii) signaling pathways to the PVN or RVLM, and iii) the integration mechanism of these signals in some central nuclei. To achieve these goals, we will employ modern research techniques, such as tracing methods using multiple viral vectors, optogenetics to reveal the functions of specific neural pathways, and Ca++ imaging of a nucleus at the single neuronal level (Movie 1.), in conjunction with the telemetry system of BP.

Figure 3. A mouse in a Ca++ imaging experiment


Movie 1. Ca++ imaging data in a mouse nucleus

(References)
1. Noda, M. & Sakuta, H. (2013) Central regulation of body-fluid homeostasis. Trends Neurosci. 36, 661-673.
2. Nomura, K., Hiyama, T.Y., Sakuta, H., Matsuda, T., Lin, C.-H., Kobayashi, K., Kobayashi, K., Kuwaki, T., Takahashi, K., Matsui, S., & Noda, M. (2018) [Na+] increases in body fluids sensed by central Nax induce sympathetically mediated blood pressure elevations via H+ -dependent activation of ASIC1a. Neuron, 101, 60-75.

　 Terrestrial animals, including humans, have mechanisms that maintain body fluid homeostasis under various body fluid conditions. For example, thirst and the avoidance of salty food are associated with dehydration. To achieve this, the brain continuously monitors body fluid conditions and controls thirst and salt appetite. The monitoring loci responsible have been suggested to be at sensory circumventricular organs (sCVOs) because they lack a blood-brain barrier, but contain neurons. sCVOs consist of the subfornical organ (SFO), organum vasculosum of the lamina terminalis (OVLT), and area postrema (AP). We almost revealed the neural mechanisms responsible for the regulation of water and salt intakes that originate from these organs 1-8).

　 The peptide hormone, angiotensin II (ang II), the levels of which increases in blood under water- as well as sodium (Na)-depleted conditions, stimulates the intake of water and salt. We previously identified a subset of neurons that drive thirst and salt appetite in the SFO, referred to as “water neurons” and “salt neurons”, respectively 4) . Both of these neurons expressed Ang II receptors (AT1a). Under water-depleted conditions, glial cells expressing Nax channels in the SFO detected an increase in Na+ levels ([Na+]) in body fluids, and activated GABAergic inhibitory neurons to suppress “salt neurons” through the secretion of lactate. As a result, water-intake behavior was selectively induced under water-depleted conditions 4) (Figs. 1, 2). In contrast, under Na-depleted conditions, the activation of other GABAergic neurons suppressed “water neurons” in response to cholecystokinin (CCK), and, thus, selectively induced salt-intake behavior 3) .

　 We also demonstrated that Nax channels in the OVLT detected an increase in [Na+] in body fluids and induced the secretion of epoxyeicosatrienoic acids (EETs) from Nax-positive glial cells 5) . EETs then activate TRPV4-positive neurons to induce water-intake behavior. In the OVLT, there exist SLC9A4-expressing neurons, which also sense increases in [Na+] in body fluids. This pathway also induces water intake independently 2) (Fig. 3). In these neurons, Ang II receptors are coexpressed, and may induce water intake Ang II-dependently. In the course of these studies, we identified a syndrome with hypernatremia caused by autoimmunity to Nax 6).

Fig. 1: Sensing mechanism of a [Na+] increase in body fluids by Nax in the SFO



Fig. 2. Neural mechanisms controlling Ang II-induced thirst and salt appetite by bodyfluid conditions



Fig. 3. Thirst-inducing mechanisms by Nax, SLC9A4, and Ang II in the OVLT

　 However, the osmosensor or neural mechanisms responsible for the detection of osmolality in the brain remain unclear. We are now attempting to elucidate the whole picture of brain mechanisms underlying the sensing of body fluid conditions , as well as the integration mechanisms of neural information downstream of the SFO and OVLT. To achieve this, we are conducting research using advanced neuroscientific techniques, such as optogenetics (Movie 2) to artificially control neural activities using light, and in vivo calcium imaging, a technique to observe neuronal activities at the single cell level.

Movie 2. Drinking behavior induced by an optical stimulation

(References)
1. Noda M and Matsuda T. (2022) Central regulation of body fluid homeostasis. Proceedings of the Japan Academy. Series B, Physical and biological sciences 98, 283-324.
2. Sakuta H, Lin C-H, Hiyama TY, Matsuda T, Yamaguchi K, Shigenobu S, Kobayashi K and Noda M. (2020) SLC9A4 in the organum vasculosum of the lamina terminalis is a [Na+] sensor for the control of water intake. Eur. J. Phys. 472, 609-624.
3. Matsuda T., Hiyama TY, Kobayashi K, Kobayashi K and Noda M. (2020) Distinct CCK-positive SFO neurons are involved in persistent or transient suppression of water intake. Nature Communications 11, 5692.
4. Matsuda T., Hiyama TY, Niimuara F, Matsusaka T, Fukamizu A, Kobayashi K, Kobayashi K and Noda M. (2017) Distinct neural mechanisms for the control of thirst and salt appetite in the subfornical organ. Nature Neurosci. 20, 230-241.
5. Sakuta H, Nishihara E, Hiyama TY, Lin CH and Noda M. (2016) Nax signaling evoked by an increase in [Na+] inCSF induces water intake via EET-mediated TRPV4 activation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 311, R299-306.
6. Hiyama TY, Matsuda S, Fujikawa A, Matsumoto M, Watanabe E, Kajiwara H, Niimura F, and Noda M. (2010) Autoimmunity to the sodium-level sensor in the brain causes essential hypernatremia. Neuron 66, 508-522.
7. Shimizu H, Watanabe E, Hiyama TY, Nagakura A, Fujikawa A, Okado H, Yanagawa Y, Obata K and Noda M. (2007) Glial Nax channels control lactate signaling to neurons for brain [Na+] sensing. Neuron 54, 59- 72.
8. Hiyama TY, Watanabe E, Ono K, Inenaga K, Tamkun MM, Yoshida S, and Noda M. (2002) Nax is involved in the sodium level sensing in the CNS. Nature Neruosci. 5, 511-2.

　Obesity is a major risk factor for type 2 diabetes, hypertension, dyslipidemia, and cardiovascular diseases. It is now an important public health issue worldwide because obesity rates are increasing at an explosive rate. The balance between food intake and energy expenditure has a direct influence on body weight, and the disruption of this energy homeostasis causes obesity. However, the regulatory mechanisms underlying obesity currently remain unclear.
　Signal transduction pathways involving tyrosine phosphorylation play pivotal roles in the regulation of food intake and metabolism. Insulin and leptin, which are important for glucose homeostasis and appetite regulation, respectively, activate tyrosine phosphorylation signals. Insulin is secreted from the pancreas and reduces blood glucose levels by promoting the absorption of carbohydrates into skeletal muscle, fat, and liver cells. However, obesity induces ‘insulin resistance’, a pathological condition in which the target cells fail to respond normally to insulin. On the other hand, leptin, an adipocyte-derived hormone, acts on the hypothalamus to strongly suppress food intake. However, most obese individuals show an increased food intake despite high circulating leptin levels, and this is referred to as ‘leptin resistance’. High circulating leptin levels also induce hypertension in obesity.
　We have been investigating the functional roles of receptor-like protein tyrosine phosphatases (RPTPs). We previously revealed that insulin and leptin signals were both suppressed by PTPRJ, a RPTP that is expressed in insulin-target tissues and the hypothalamus 1,2) (Fig. 1). Ptprj-KO mice showed a lean phenotype. Insulin and leptin signals were both increased in Ptprj-KO mice, and, thus, they exhibited high blood glucose-lowering and anti-obesity activities. Moreover, we found that the induction of PTPRJ in the hypothalamus contributed to ‘leptin resistance’ in obesity (Figs. 2, 3).
　One of the mechanisms by which obesity causes metabolic complications is the accumulation of fatty acids in non-adipose tissues, such as the liver, skeletal muscle, and pancreas. The accumulation of ectopic fat is induced by free fatty acids that leak from adipose tissue when storage exceeds the capacity limitation, resulting in dysfunctional adipose and non-adipose tissues.
　We recently reported that Ptpro-KO mice fed a high-fat/high-sucrose diet (HFHSD) exhibited a markedly larger body mass than wild-type (WT) mice, but higher insulin sensitivity. In these Ptpro-KO mice, the accumulation of ectopic fat in the liver was very small (Fig. 4), and inflammation in the liver and adipose tissue was significantly suppressed. Moreover, the administration of a specific inhibitor of PTPRO to ob/ob mice induced a decrease in ectopic fat accumulation in the liver together with adipose tissue expansion, reduced systemic inflammation, and improved insulin sensitivity, as observed in Pipro-KO mice (Fig. 5). Therefore, PTPRO appears to be involved in the control of insulin signaling, the limitation of fat accumulation in adipose tissue, and the induction of inflammation in obesity.

Figure 1. Suppression of insulin and leptin signals by PTPRJ


Figure 2. Injection of PTPRJ-expressing viral vectors in the arcuate nucleus.


Figure 3. The induction of leptin resistance by PTPRJ overexpression in the arcuate nucleus. Daily changes in body weights were examined.


Figure 4. Accumulation of ectopic fat is suppressed in the liver of Ptpro-KO mice (upper). On the other hand, in the adipose tissue, the growth of lipid droplets in adipocytes increases fat accumulation (lower).


Figure 5. PTPRO is involved in the regulation of insulin sensitivity, the induction of lipogenic genes, the growth of lipid droplets in adipocytes, and the induction of inflammation. Therefore, PTPRO may be a hub regulator for the development of chronic inflammation, insulin resistance, dyslipidemia, diabetes mellitus, and fatty liver, which are all associated with obesity.

(References)
1. Shintani T, Suzuki R, Takeuchi Y, Shirasawa T, and Noda M. (2023) Deletion or inhibition of PTPRO prevents ectopic fat accumulation and induces healthy obesity with markedly reduced systemic inflammation. Life Sciences 313, 121292.
2. Shintani T, Higashi S, Suzuki R, Takeuchi Y, Ikaga R, Yamazaki T, Kobayashi K and Noda M. (2017) PTPRJ inhibits leptin signaling, and induction of PTPRJ in the hypothalamus is a cause of the development of leptin resistance. Sci. Reports 7, 11627.
3. Shintani T, Higashi S, Takeuchi Y, Gaudio E, Trapasso F, Fusco A and Noda M. (2015) The R3 receptor-like protein-tyrosine phosphatase subfamily inhibits insulin signaling by dephosphorylating the insulin receptor at specific sites. J. Biochem. 158, 235-248.
4. Sakuraba J, Shintani T, Tani S, and Noda M. (2013) Substrate specificity of R3 receptor-like protein-tyrosine phosphatase subfamily toward receptor protein-tyrosine kinases. J. Biol. Chem. 288, 23421-23431.