The results of the present study indicate that in rats with T1D, 3-4 weeks of ExT significantly improved the blunted erectile response to either NMDA or SNP microinjection within the PVN of the hypothalamus. Consistent with the erectile response, other behavior responses including yawning and stretching, induced by central NMDA and SNP microinjection were also significantly increased in T1D rats after ExT. Furthermore, we found ExT restored the nNOS expression in the PVN in T1D rats by using real time PCR, Western blot and histochemistry, approaches. Taken together, these data suggest that ExT improves the central component of the erectile response in type I diabetes.
In the present study, administration of NMDA within the PVN demonstrated a decreased response in penile erection, yawning, and stretching in T1D rats confirming our previous observations 13
. That these responses were mediated by altered levels of nNOS are supported by the observation that the levels of nNOS mRNA and protein are decreased in rats with T1D compared with control rats. Administration of NMDA within the PVN did not show a significantly decreased response in grooming and chewing in T1D rats. This may imply a different activation of neural circuit controlling different behavioral responses 29-31
The presence of the penile erectile responses to SNP administration in the PVN in diabetic rats, albeit smaller, implies that the peripheral mechanisms are involved in the overall response. However, because the response to SNP was blunted in diabetic rats, these results could not differentiate between an additional peripheral abnormality and a central abnormality in the diabetic rats. In the present study, ExT improved the nNOS within the PVN with concomitant improvement in SNP response within the PVN in diabetic rats. It is of interest to note that adenoviral mediated up-regulation of nNOS within the PVN was able restore the NMDA responses in diabetic rat. Presumably there was no peripheral component improvement in that study 13
. Taken together, we suggest that of the central restoration of nNOS within the PVN in the ExT diabetic rats was crucial for the improvement of the central component of the erectile response. Furthermore, ExT improved the blunted responses to SNP suggesting that the peripheral mechanisms may also be improved as suggested by others 20
In the present study, we have further shown that ExT improved the blunted erectile, yawning and stretching responses to either NMDA or SNP microinjection within the PVN. We also found ExT restored the nNOS expression in the PVN in T1D rats. The results of these studies may have a more general implication of a central NO mechanism within the PVN involved in erectile dysfunction.. This is consistent with the observation that mice that lack nNOS display less erectile responses to the electrical stimulation around the cavernous nerve 32
. This implies that nNOS may be a major mediator of sexual behavior that may be affected in various disease states that are associated with erectile dysfunction reported in various disease conditions.
The mechanism by which central NMDA induces penile erectile function appears via the release of NO, which causes in turn the activation of oxytocinergic neurons in the PVN. Pharmacological, electrophysiological and immunocytochemical studies have identified oxytocinergic neurons, which regulated penile erection, projecting from the PVN to the brain stem and finally to the spinal cord 33, 34
. In addition to this well documented peripheral action, evidence is accumulating which indicates that NO may also function as a neurotransmitter in the central nervous system to modulate sexual behavior and penile erection 35
. The PVN is a primary site within the forebrain that has been implicated in NO-mediated penile erection 33, 34
. Delivering NO or NO donors to the PVN of conscious rats elicits episodes of penile erection, and an increase in intracavernous pressure (ICP), consistent with the observations in this study. Interestingly, we have observed a marked reduction in the central NO mechanism in the PVN, which, as least in part, leads to a lack of NMDA-mediated penile erection in T1D rats 13
The mechanisms responsible for the ExT-induced increase in nNOS within the PVN remain unknown. However, it is well known that the PVN receives information from various cardiopulmonary receptors including myocardial vagal afferents 36, 37
. Some studies suggest that ExT increases various hemodynamic parameters within the heart that dictate cardiac vagal activity 38, 39
. Accordingly, the activation of cardiac vagal activity during ExT may mediate an increase of nNOS within the PVN. This is consistent with the enhanced inhibitory influence of cardiac vagal afferents on directly measured sympathetic activity and regional vascular resistance after ExT 40
Modulation of NO synthesis and the interaction of NO with angiotensin II (AngII) in the central nervous system may be another important mechanism for the regulation of central NMDA-induced erectile function during ExT in T1D. AngII has been implicated as a contributor in the erectile dysfunction observed in diabetes. Plasma levels of AngII are increased in diabetes 41
, while angiotensin-converting enzyme (ACE) inhibitors and AngII type 1 (AT1
) receptor antagonists improve erectile function in diabetic patients 42
. Increased AngII may enhance oxidative stress and decrease NO bioavailability in diabetes. Interestingly, it has been shown that ExT reverses the increased oxidative stress in NO-deficient rats and ameliorating the erectile dysfunction. So it is possible that ExT may be reducing elevated levels of AngII in diabetes 43
and thus improving or modulating the central levels of nNOS within the PVN of diabetic rats. This possibility remains to be examined more thoroughly in the future.
The pathophysiology of diabetes-related erectile dysfunction is well known to be multifactorial 44
, including neuropathy, vascular disease, metabolic abnormalities, endocrine disorders, and psychogenic factors. NO plays key role in regulating erectile function by balancing vasodilation, which leads to the rigid state, and vasoconstriction, which results in detumescence of the penis. Although our results show ExT improving centrally mediated erectile function in T1D, the impact of ExT on systemic endothelial dysfunction and corpus cavernosum responses is also confirmed by other studies 19-21
. Claudino et al showed that running exercise for 8 weeks in rats increases the relaxation responses of corpus cavernosum through the NO-cGMP signaling pathway activation 19, 20
. Recently, they have shown that the physical preconditioning markedly restores the reduced relaxation response of corpus cavernosum for the muscarinic agonist acetylcholine and electrical field stimulation in rats with diabetes 21
. Regular ExT up-regulates both eNOS and nNOS expressions in the aged and young rat penis to improve penile erection 45
. Since eNOS expression has been previously documented to be reduced in the penis of T1D rats 46
, we are assuming that ExT may prevent erectile dysfunction associated with diabetes by improving both nNOS and eNOS function at the level of the penis. In the case of the central nervous system, eNOS is seen in blood vessel-like structures in the PVN, while nNOS is localized in the PVN neurons 47
. Although, in the PVN neurons, nNOS is predominant NOS, the details of central eNOS within the PVN involvement in erectile response and dysfunction in diabetes remain to be examined.
Furthermore, the endocrine disorders (such as reduced testosterone levels, hypothyroidism, or hyperthyroidism) associated with diabetes may compound the problem, making the etiology of erectile dysfunction in men with diabetes a complex issue. Sex steroid hormones play an important role in maintaining endothelial health and sex steroid deficiency is associated with endothelial dysfunction, vascular disease and erectile dysfunction 48
. STZ-induced T1D has been previously described to alter erectile function due to an underlying condition of hypogonadotropic hypogonadism 5, 49
. These observations suggest that, at least in the male, consolidation of a state of diabetes is linked to suppression of the functionality of hypothalamic system. Reported histomorphological analysis of penile tissue shows that neuronal damage is associated with penile hypoxia and tissue alteration from diabetic and hypogonadic condition 50, 51
. Our result shows the plasma testosterone is significantly lower in T1D rats. This is consistent with other reports from patients and animal experiments 52, 53
. Testosterone replacement therapy may lead to symptomatic improvement in diabetes with erectile dysfunction 5, 54
. The contractile RhoA/Rho-kinase (ROCK) signaling pathway is upregulated in penile tissue in animal models of experimental diabetes and has been proposed to contribute to diabetes-related erectile dysfunction. Treating hypogonadism in course of diabetes may maintain erectile function also by normalizing RhoA/ROCK pathway upregulation 55
. However, our data shows that despite the lack of change in plasma testosterone to ExT in rats with T1D, there was restoration of centrally mediated erectile responses suggesting that altered levels of plasma testosterone level in T1D, do not contribute to the centrally mediated erectile response in this paradigm. The effects of ExT on the centrally involved sex steroid levels in controlling erectile function remains to be investigated.
STZ-induced diabetic rats provide an interesting and relevant model to study the effects of diabetes on male sexual dysfunction, since they exhibit several deficits in copulatory behavior similar to those in diabetic men 56, 57
. Four weeks-STZ-induced diabetic rats have shown an endocrine and metabolic disorder often associated with erectile dysfunction and peripheral neuropathy 58
. Diabetic rats show significant deficits in mount, intromission, and ejaculatory behaviors, suggesting that both the sexual arousal (libido) and potency components of male sexual behavior are adversely affected by diabetes 56, 59
. Examination of these responses early in the diabetic condition, as done in this experiment (7-8 weeks after STZ injection), also further emphasizes the importance of these observations since minimal/none of the chronic functional effects of metabolic dysfunction or complications are evident at this early stage. Time-dependent change in erectile function and responsiveness to PDE5 inhibitor were investigated in STZ-induced diabetic rats 60
. Erectile responses were significantly decreased in 10 weeks diabetic rats, and administration of PDE5 inhibitor resulted in partial recovery of normal responses. At more than 12 weeks, rats demonstrated severe deterioration of erectile function, which did not fully respond to PDE5 inhibitor. It is possible that the impairment of centrally medicated erectile function in the present study might be the early pathophysiology of diabetes-related erectile dysfunction. The results are compatible with those of previous cohort studies showing that risk of erectile dysfunction increased with duration of diabetes 61-63
. Perhaps restoration of this central component with ExT may represent a critical early window for therapeutic intervention for this disease process.