We demonstrate here that infusion of HN into the third ventricle of rats, and peripheral infusion of the potent HN analog HNGF6A, improves both hepatic and peripheral insulin sensitivity. We show that, similar to effects on apoptosis, dimerization of HN is essential for its actions on glucose metabolism. We also demonstrate that hypothalamic STAT-3 activation is required for the insulin-sensitizing action of HN. Furthermore, we show that the effects of HN on glucose metabolism are tempered by binding of HN to IGFBP-3 in the hypothalamus, as central infusion of a non-IGFBP-3 binding analog, F6AHN, resulted in a more potent effect on insulin action than HN. We also show that the effect of peripherally administered HN analog on the liver is entirely through the hypothalamus, while the effect on the skeletal muscle may involve both direct and central effects.
The critical role of the MBH in mediating hepatic glucose metabolism has been unraveled by prior studies with leptin, insulin and IGF-1. In the studies presented here, the hypothalamus appears to play a key role in mediating the effects on hepatic insulin sensitivity in response to the administration of both central and peripheral HN and its analogs. Remarkably, centrally-administered HN dramatically increased GIR not only due to effects on HGP, but also through an increase in peripheral glucose uptake, demonstrating that HN is the first centrally-acting peptide to favorably modulate glucose uptake in skeletal muscle. The autonomic nervous system may serve as the efferent in mediating the effects of HN on peripheral glucose metabolism. Hypothalamic nuclei have been shown to influence HGP through the vagus nerve 
, while leptin and IGF-1 have been shown to effect output through the sympathetic nervous system 
. More studies are needed to specifically evaluate the role of autonomic nervous system in mediating the effects of HN.
This study also provides evidence that HN and IGFBP-3 not only have opposing roles on apoptosis but also on peripheral insulin action via the hypothalamus. The opposing roles of these two peptides on glucose homeostasis and apoptosis are especially interesting when one considers the close relationship between insulin resistance and cell survival in AD. In fact, defects in glucose utilization, a striking reduction in insulin receptor mRNA levels, and attenuated insulin signaling, as evidenced by decreased IRS-1, PI3K, and pAKT have been demonstrated in AD 
. On the other hand, insulin sensitizers such as thiazolinediones improve cognitive function in mouse models as well as humans with early AD, highlighting the role of insulin resistance in this condition 
. While IGFBP-3 production is increased in AD brain 
, HN is expressed in the non-apoptotic hippocampal regions of Alzheimer's patients' brains and is absent in senile plaques 
. Taken together, the improvement in insulin sensitivity demonstrated with central HN may be one of the primary mechanisms by which HN regulates cell survival and, therefore, may provide an additional potential mechanism by which HN is protective against AD.
Molecular manipulations of HN at key amino acids have been shown to offer significant increases in its potency or chemical characteristics. For instance, a change at position 14, [Gly14
]-HN (S14G, HNG), has been shown to enhance neurosurvival activity and also offers added protection against memory impairment 
and stroke 
. A substitution of phenylalanine in the 6th
position with alanine (F6A, F6AHN) alters the binding of HN to IGFBP-3 and we show here that this substitution significantly enhances its central effect on glucose metabolism. Interestingly a novel HN analog termed HNGF6A, which we created by combining both changes at position 6 and 14, dramatically increases its ability to modulate insulin action and offers a mode of administration that is more clinically applicable. We also show here that HNGF6A not only improves insulin sensitivity during a clamp, but also significantly improves blood glucose levels in ZDF rats with a single dose. The potential of HNGF6A as a treatment for T2DM is an exciting observation which warrants further investigation, including chronic treatment of diabetic models with this peptide.
Another important observation from this study is that intact STAT-3 signaling in the hypothalamus is necessary for the effects of HN on glucose metabolism. This is in partial agreement with prior studies which have pinpointed the mechanism by which HN confers neuronal protection to the activation of tyrosine kinases and STAT-3 phosphorylation 
. STAT-3 phosphorylation and activation in the hypothalamus is well known to play a pivotal role in the regulation of energy homeostasis. For example, neuronal STAT-3 KO mice are morbidly obese, hyperphagic and diabetic 
. This effect is due at least in part to attenuated leptin signaling in the hypothalamus. This has been directly demonstrated in rats by co-infusion of a STAT-3 inhibitor with leptin which resulted in attenuation of the actions of leptin on food intake and glucose metabolism 
. Likewise, we show that co-infusion of a STAT-3 inhibitor with HN into the third ventricle completely attenuates the ability of HN to modulate peripheral insulin action. Indeed, when STAT-3 signaling was inhibited in the hypothalamus, the effects of peripherally administered HN analog on liver was completely abolished, once again emphasizing the role of the hypothalamus in mediating the hepatic effects of HN and its analogues. Furthermore, the absence of any effect on phosphorylation levels of other signaling kinases, including AKT, demonstrates that the effects of HN are independent of hypo thalamic insulin signaling.
The potential of anti-apoptotic therapy for neurodegeneration has been gaining attention 
, and growth factors of various types including IGF-1 and other neuro-survival peptides 
have been considered. While the role of HN in neuroprotection has been substantially characterized and HN has been proposed to have enormous therapeutic potential for neurodegenerative diseases, the studies described here comprise the first evidence demonstrating a role for HN in glucose metabolism. Considering that diseases associated with aging such as T2DM and AD have been proposed to be associated with mitochondrial dysfunction 
, it is especially interesting that the mitochondrial-derived peptide HN modulates them. In summary, these data demonstrate for the first time that HN regulates peripheral insulin action. The link between the decrease in circulating levels of HN as well as levels in hypothalamus and skeletal muscle with age, in parallel with the increase in age- associated diseases such as AD and T2DM is extremely intriguing and warrants further investigation. HN or its non-IGFBP-3 binding analogues may provide potential therapeutic options for prevention or treatment of at least two age-related diseases, namely impaired carbohydrate metabolism/T2DM and neurodegeneration.