Long-term Over-expression Of Neuropeptide Y In Hypothalamic Paraventricular Nucleus Contributes To Peripheral Insulin Resistance

1. Background and Objective:Neuropeptide Y(NPY) is one of the most common peptides in the brain has been shown to play a role in energy metabolism, appetite regulation, cardiac rhythm, blood pressure, smooth muscle contraction and relaxation. Cumulative evidence also suggests that NPY acts as a metabolic signal that may contribute to obesity, hyperinsulinemia, and hyperglycemia. Intracerebroventricular injection of NPY and overexpression of NPY in the paraventricular nucleus(PVN) of the hypothalamus, for example, have been shown to temporarily increase food intake and promote insulin release. Recent study has indicated that intracerebroventricular administration of NPY induced hepatic insulin resistance by sympathetic innervations. In the periphery, NPY is widely distributed in the sympathetic nerves, the adrenal medulla, platelets and various cell types within WAT which modulate angiogenesis, adipogenesis, lipolysis and hypertrophy of adipocytes via NPY receptors. On the other hand, it is well known that the hypothalamus is a major source of forebrain input into the SNS. Immunostaining after pseudorabies virus(a transneuronal tract tracer) injection into fat revealed that PVN was one of the sites that modulated SNS outflow to the WAT. The hypothalamus-SNS-adipose tissue axis provides a plausible mechanism to explain how central NPY exerts a function on WAT. Moreover, there is data that demonstrated that the release of NPY as a sympathetic neurotransmitter directly into the WAT leads to abdominal obesity. Those cues suggest NPY in central nervous system might be contribute to peripheral insulin resistance, especially in adipose tissue.The activity of NPY in cellular metabolism appears to be mediated through its ability to bind the transmembrane domain G protein coupled receptors NPY Y1- Y5 These receptors are found in a broad array of tissues including those involved in metabolism, like adipose tissue and liver. There is evidence suggesting that NPY influences metabolic function in peripheral tissue mostly via Y1 and Y5 receptor signaling. Further study need to be performed to indentify Y1/Y5 receptor contributes to insulin resistance in adipose tissue induced by NPY.In this study, our hypothesis was that NPY contributes to peripheral insulin resistance in adipose tissue via the Y5 receptor. To test this hypothesis, obesity and diabetic animal model were performed by high fat diet and high fat diet combined with STZ injection, then NPY and Cocaine-and amphetamine regulated transcript(CART) protein expression in PVN as well as PI3K/Akt/GSK3 signaling pathway were detected. Moreover, we also created an insulin resistant in vivo model by injecting recombinant NPY lentiviral particles in the PVN of the hypothalamus in rats. Eight weeks after injection, euglycemic-hyperinsulinemic clamp and intravenous glucose tolerance tests confirmed that the rats had developed insulin resistance in adipose tissue. PI3K/Akt/GSK3 signaling pathway and Y5 receptor in adipose tissue were assessed. In intro experiment indicated that NPY inhibited glucose consumption and uptake in 3T3-L1 adipocytes via the NPY Y5 receptor. Taken together, this study demonstrates that central NPY contribute to insulin resistance of adipose tissue by Y5 receptor. And this study might provide more direct pharmacological evidence that using NPY receptors anta gonists could prove beneficial in the treatment of diseases induced by NPY disorders.2. Methods2.1 The expression of NPY in PVN and PI3K/Akt/GSK3 signaling pathway in obesity and diabetic rats.Obesity and diabetic animal model were performed by high fat diet(HFD) for 10 weeks with or without STZ injection. 4 weeks later, those rats whose fasting blood glucose(FBG)≥16.7 mmol/L were used as diabetic animals. Body weight, FBG, plasma Triglyceride(TG), l cholesterol(CHO), fasting insulin levels(FINS), homeostasis model of assessment for insulin resistance index(HOMA-IR) were measured. NPY and CART expression in PVN were detected by immunofluorescence, as well as PI3K/Akt/GSK3 signaling pathway and Y5 receptor in adipose tissue were assessed by Western blotting.2.2 Evaluation of transduction efficiency of lentiviral particles in PVN.Lentiviral particles marked with green fluorescent protein(GFP) were injected into PVN of rats. The location of lentiviral particles injection as well as the GFP expression(efficiency of transduction) were detected by confocal scanning laser microscope.2.3 Assessment of relationship of NPY in PVN and peripheral insulin resistance.Recombinant NPY lentiviral particles was injected into PVN of rats with or without high fat diet intervene for 8 weeks. The transduction efficiency of lentiviral particles and NPY expression were detected, and BW, body temperature, food intake per day, plasma TG, CHO, relative adipose tissue weight, Lee index were measured. Insulin resistance was evaluated by FBG, FINS, glycated hemoglobin A1c(Hb A1c), glucose infusion rate(GIR), Ho MA-IR, Intravenous glucose tolerance test(IVGTT), glucose utilization index(Rg). Moreover, PI3K/Akt/GSK3 signaling pathway and Y5 receptor in adipose tissue were assessed by Western blotting.2.4 The effect of NPY on glucose consumption and glucose uptake in 3T3-L1 adipocytes.Oil O staining and special molecular marker was detected in differentiated 3T3-L1 adipocytes. Glucose consumption and glucose uptake of 3T3-L1 adipocytes intervened with NPY combined with Y1/Y5 receptor antagonists were measured. And Western blotting was used to assess PI3K/Akt/GSK3 signaling pathway and Y5 receptor in 3T3-L1 adipocytes intervened with NPY combined with Y1/Y5 receptor antagonists.3. Results3.1 The expression of NPY and CART increased in PVN of obesity and diabetic rat.The levels of BW, FBG, CHO, TC and FINS in HFD rats were significantly higher than those in low fat diet(LFD) rats. Lee index indicated that obesity was induced by long tern HFD. HFD and HFD combined with STZ injection obviously increased HOMA-IR which is an insulin resistance index. The co-localization of NPY and CART in neurons of PVN was confirmed by immunefluorescence. The expression levels of NPY and CART in HFD rats were higher than those in LFD rats, but the diabetic rats have highest NPY and CART expression. To our surprise, the increase of NPY expression was obviously higher than CART.3.2 Constitutive transduction of lentiviral particles in situ.The transduction of lentiviral particles with GFP marker into neurons was shown. GFP protein over-expressed in the cytoplasm of neurons in situ. Constitutive expression of GFP protein was displayed in 1, 2 and 4 week after lentiviral particles injection.3.3 Over-expression of NPY induces peripheral insulin resistance.3.3.1 Long-term over-expression of NPY in PVN.The LV-Cherry(vehicle control) or LV-NPY-Cherry was injected into the PVN of rats. Eight weeks after injection, the expression of the vectors was measure d using immunofluorescence. Rats were only included in the study when the reporter protein(Cherry) expression was located exactly in the PVN. Additionally, the expression of NPY and Cherry co-localized. Comparing with vehicle control groups, LV-NPY-Cherry injection in PVN induced an over 3 fold increase of NPY protein expression in LFD+NPY and HFD+NPY groups. These findings suggest that we were successfully able to overexpress NPY in the PVN of rats, and overexpression of NPY can be sustained for more than 8 weeks in the hypothalamic PVN of rats by injection of recombinant lentivirus-mediated NPY particles in situ.3.3.2 Over-expression of NPY contributes to insulin resistance in ratsThe euglycemic-hyperinsulinemic clamp assay showed that HFD and NPY overexpressing rats had lower GIR60-120 than LFD rats, which indicated that peripheral insulin resistance was induced in rats fed with HFD or rats over-expressing NPY. Otherwise, even though the plasma glucose levels did not increase, the insulin levels measured during a 120 min intravenous glucose tolerance test(IVGTT) increased in the three groups. Moreover, HOMA-IR test confirmed those results, demonstrating that rats overexpressing NPY develop insulin resistance.3.3.3 Overexpression of NPY induces obesity and insulin resistance in adipose tissueThe WB of HFD, LFD+NPY and HFD+NPY rats were significantly higher than those of LFD rats, especially during the last period of the experiment. Noticeably, however, the food intake per day of LFD+NPY and HFD+NPY groups was greater than the LFD and HFD groups at the beginning of the experiment but leveled out by the week 4. Overexpression of NPY did not alter the rats’ body temperature. The serum Hb A1 c, triglyceride, and cholesterol concentrations were not different in the control versus NPY-overexpressing rats; however, they were significantly elevated in rats fed a HFD, and in rats overexpressing NPY who were fed a HFD. This finding correlates with the previous results showing that a HFD also increases fasting glucose levels. A HFD and chronic overexpression of NPY in the hypothalamic PVN of rats increased the raltive adipose tissue weight, which is consistent with the Lee index, the indicator of obesity. These results indicated that both HFD and NPY overexpression induced obesity, but the latter didn’t increase glucose, Hb A1 c, or blood lipid levels. On the other hand, peripheral insulin resistance had been confirmed by the euglycemic-hyperinsulinemic clamp test and IVGTT. To further reveal the contribution of individual tissues to peripheral insulin resistance, the glucose utilization index(Rg’) indicated that the glucose uptake of adipose tissue decreased in HFD, LFD+NPY and HFD+NPY groups, while there were not obvious differences in glucose uptake in muscle among all groups.3.3.4 NPY modulates the PI3K-Akt and GSK signaling pathways and Y5 receptor expressionCompared with rats fed a LFD, the PI3 K protein levels in the adipose tissue of rats overexpressing NPY fed a LFD or HFD decreased slightly, although the Akt, GSK3β, and GSK3α protein levels did not change. Specifically, constitutive overexpression of NPY in PVN of rats dramatically decreased the phosphorylation of GSK3β, PI3 K and Akt independently of the diet they were fed. However, the decrease of phosphorylation of GSK3α and increase of Y5 receptor expression in the HFD, LFD+NPY and HFD+NPY groups were shown.3.4 NPY effects glucose metabolism in 3T3-L1 adipocytes.3.4.1 The differentiation of 3T3-L1 cellsThe differentiated 3T3-L1 adipocytes show mature adipocytes characters, such as positive oil red O staining, αP2 and PPARγ2 expression.3.4.2 NPY inhibits glucose consumption and uptake in 3T3-L1 adipocytes via the NPY Y5 receptorThe results showed NPY can directly decrease the glucose uptake in 3T3-L1 adipocytes. High dose NPY educed basal glucose consumption, whereas lower dose NPY failed to affect the basal glucose consumption in 3T3-L1 adipocytes. Although NPY can also reduce the insulin-simulated glucose consumption, BIBP-3226, a NPY Y1 receptor antagonist, did not reverse the effect of NPY treatment in the adipocytes. NPY Y5 receptor antagonist L-152,804 obviously reversed the restrain of NPY on insulin-simulated glucose consumption. Furthermore, 2-[3H] DG uptake experiments demonstrated that the NPY Y1 receptor antagonist did not contribute to the insulin-simulated 2-[3H] DG uptake of 3T3-L1 adipocytes incubated with NPY, whereas the NPY Y5 receptor antagonist does, suggesting that the NPY Y5 receptor is responsible for the observed NPY effects.3.4.3 NPY changes the PI3K-Akt and GSK signaling pathways in 3T3-L1 adipocytes via the NPY Y5 receptorNPY inhibited the phosphorylation of GSK3α, GSK3β, PI3 K and Akt significantly, although NPY did not change the Akt, PI3 K, GSK3α and GSK3β total protein levels in 3T3-L1 adipocytes. Treatment with the NPY Y5 receptor antagonist reversed the suppression, whereas the NPY Y1 receptor antagonist did not, corroborating the previous findings that the NPY Y5 receptor is responsible for the effects of NPY in adipocytes.4. Conclusion4.1 Peripheral insulin resistance was identified in obesity rats induced by HFD and diabetic rats induced by HFD combined with STZ injection. NPY and CART partly co-localized in neurons of PVN. HFD induced higher expression of NPY and CART than LFD. However, the diabetic rats show highest NPY and CART expression with a shift towards NPY predominance.4.2 Exogenous injection with GFP Lentivirus carrier particles can infect neurons. GFP widely expressed in the cytoplasm of neurons, stably and constitutively. Even in 4 weeks after injection, GFP over-expression is still clearly visible in PVN.4.3 Insulin resistance of adipose tissue were induced by NPY over-expression in PVN with or without HFD. NPY increased food intake, but did not change blood glucose, Hb A1 c or lipid levels. However, NPY decreased the expression of pGSK3β, PI3 K p85 and p Akt Ser473 as well as increase Y5 receptor expression in adipose tissue of rats.4.4 In vitro, glucose consumption and glucose uptake were inhibited by NPY, while a decrease in PI3K-Akt pathway signaling and a decreased expression of pGSK3α and pGSK3β were observed. Nevertheless, a Y5 receptor antagonist(L-152,804) reversed the effects of NPY on glucose uptake and consumption, but a NPY Y1 receptor antagonist did not reverse the effect, suggesting that the NPY Y5 receptor is responsible for the observed NPY effects.