Photoperiodic and Diurnal Regulation of WNT Signaling in the Arcuate Nucleus of the Female Djungarian Hamster, Phodopus sungorus.

The WNT pathway was shown to play an important role in the adult central nervous system. We previously identified the WNT pathway as a novel integration site of the adipokine leptin in mediating its neuroendocrine control of metabolism in obese mice. Here we investigated the implication of WNT signaling in seasonal body weight regulation exhibited by the Djungarian hamster (Phodopus sungorus), a seasonal mammal that exhibits profound annual changes in leptin sensitivity. We furthermore investigated whether crucial components of the WNT pathway are regulated in a diurnal manner. Gene expression of key components of the WNT pathway in the hypothalamus of hamsters acclimated to either long day (LD) or short day (SD) photoperiod was analyzed by in situ hybridization. We detected elevated expression of the genes WNT-4, Axin-2, Cyclin-D1, and SFRP-2, in the hypothalamic arcuate nucleus, a key energy balance integration site, during LD compared with SD as well as a diurnal regulation of Axin-2, Cyclin-D1, and DKK-3. Investigating the effect of photoperiod as well as leptin on the activation (phosphorylation) of the WNT coreceptor LRP-6-(Ser1490) by immunohistochemistry, we found elevated activity in the arcuate nucleus during LD relative to SD as well as after leptin treatment (2 mg/kg body weight). These findings indicate that differential WNT signaling may be associated with seasonal body weight regulation and is partially regulated in a diurnal manner in the adult brain. Furthermore, they suggest that this pathway plays a key role in the neuroendocrine regulation of body weight and integration of the leptin signal.

Hypothalamic WNT signalling is impaired during obesity and reinstated by leptin treatment in male mice.

The WNT pathway has been well characterized in embryogenesis and tumorigenesis. In humans, specific polymorphisms in the T cell-specific transcription factor 7 and the WNT coreceptor, low-density lipoprotein receptor-related protein-6 (LRP-6), both prominent components of this pathway, correlate with a higher incidence of type 2 diabetes, suggesting that the WNT pathway might be involved in the control of adult glucose homeostasis. We previously demonstrated that glycogen-synthase-kinase-3ß (GSK-3ß), the key enzyme of the WNT pathway, is increased in the hypothalamus during obesity and exacerbates high-fat diet-induced weight gain as well as glucose intolerance. These data suggest that WNT action in the hypothalamus might be required for normal glucose homeostasis. Here we characterized whether WNT signaling in general is altered in the hypothalamus of adult obese mice relative to controls. First we identified expression of multiple components of this pathway in the murine arcuate nucleus by in situ hybridization. In this region mRNA of ligands and target genes of the WNT pathway were down-regulated in obese and glucose-intolerant leptin-deficient mice. Similarly, the number of cells immunoreactive for the phosphorylated (active) form of the WNT-coreceptor LRP-6 was also decreased in leptin-deficient mice. Leptin treatment normalized expression of the WNT-target genes Axin-2 and Cylin-D1 and increased the number of phospho-LRP-6-immunoreactive cells reaching levels of lean controls. Leptin also increased the levels of phosphorylated (inactive) GSK-3ß in the arcuate nucleus, and this effect was colocalized to neuropeptide Y neurons, suggesting that inactivation of GSK-3ß may contribute to the neuroendocrine control of energy homeostasis. Taken together our findings identify hypothalamic WNT signaling as an important novel pathway that integrates peripheral information of the body's energy status encoded by leptin.

The dietary flavonoids naringenin and quercetin acutely impair glucose metabolism in rodents possibly via inhibition of hypothalamic insulin signalling.

Secondary metabolites of herbs and spices are widely used as an alternative strategy in the therapy of various diseases. The polyphenols naringenin, quercetin and curcumin have been characterised as anti-diabetic agents. Conversely, in vitro, naringenin and quercetin are described to inhibit phosphoinositide-3-kinase (PI3K), an enzyme that is essential for the neuronal control of whole body glucose homoeostasis. Using both in vitro and in vivo experiments, we tested whether the inhibitory effect on PI3K occurs in neurons and if it might affect whole body glucose homoeostasis. Quercetin was found to inhibit basal and insulin-induced phosphorylation of Akt (Ser473), a downstream target of PI3K, in HT-22 cells, whereas naringenin and curcumin had no effect. In Djungarian hamsters (Phodopus sungorus) naringenin and quercetin (10 mg/kg administered orally) diminished insulin-induced phosphorylation of Akt (Ser473) in the arcuate nucleus, indicating a reduction in hypothalamic PI3K activity. In agreement with this finding, glucose tolerance in naringenin-treated hamsters (oral) and mice (oral and intracerebroventricular) was reduced compared with controls. Dietary quercetin also impaired glucose tolerance, whereas curcumin was ineffective. Circulating levels of insulin and insulin-like growth factor-binding protein were not affected by the polyphenols. Oral quercetin reduced the respiratory quotient, suggesting that glucose utilisation was impaired after treatment. These data demonstrate that low doses of naringenin and quercetin acutely and potently impair glucose homoeostasis. This effect may be mediated by inhibition of hypothalamic PI3K signalling. Whether chronic impairments in glucose homoeostasis occur after long-term application remains to be identified.

Hypothalamic glycogen synthase kinase 3ß has a central role in the regulation of food intake and glucose metabolism.

GSK3ß (glycogen synthase kinase 3ß) is a ubiquitous kinase that plays a key role in multiple intracellular signalling pathways, and increased GSK3ß activity is implicated in disorders ranging from cancer to Alzheimer's disease. In the present study, we provide the first evidence of increased hypothalamic signalling via GSK3ß in leptin-deficient Lep(ob/ob) mice and show that intracerebroventricular injection of a GSK3ß inhibitor acutely improves glucose tolerance in these mice. The beneficial effect of the GSK3ß inhibitor was dependent on hypothalamic signalling via PI3K (phosphoinositide 3-kinase), a key intracellular mediator of both leptin and insulin action. Conversely, neuron-specific overexpression of GSK3ß in the mediobasal hypothalamus exacerbated the hyperphagia, obesity and impairment of glucose tolerance induced by a high-fat diet, while having little effect in controls fed standard chow. These results demonstrate that increased hypothalamic GSK3ß signalling contributes to deleterious effects of leptin deficiency and exacerbates high-fat diet-induced weight gain and glucose intolerance.

Seasonal leptin resistance is associated with impaired signalling via JAK2-STAT3 but not ERK, possibly mediated by reduced hypothalamic GRB2 protein.

The Siberian hamster, Phodopus sungorus, undergoes a striking seasonal cycle of leptin sensitivity and body weight regulation, but the molecular mechanism and relevance to human leptin insensitivity are unknown. Here we show that nuclear translocation of phospho-STAT3 in the hypothalamus is rapidly stimulated by leptin to a greater extent in hamsters held in short-day length (SD) as compared to long-day length (LD). Intriguingly, effects of leptin on STAT3 appeared to be in part limited to nuclear translocation of phospho-STAT3 associated with the cell surface rather than phosphorylation of STAT3. The number of phospho-ERK cells within the hypothalamus was unaffected by either photoperiod or leptin. However, proximal to ERK phosphorylation, hypothalamic SH2-containing tyrosine phosphatase (SHP2) and the small growth factor receptor-binding protein (GRB2), which act as competitive negative modulators on binding of SOCS3 to leptin receptor (LRb)-associated Tyr985, were increased in SD compared to LD. Our findings suggest that activation of STAT3 by leptin may be dependent on interaction of stimulatory SHP2/GRB2 as well as inhibitory SOCS3 on the level of competitive binding to LRb-associated Tyr985. This hypothetical mechanism may represent the molecular identity of seasonally induced adjustments in leptin sensitivity and may be applied to investigating leptin sensitivity in other rodent models.