Thesis (doctoral)--Münster in Westfalen, 1933.

A recently discovered peptide hormone, pituitary adenylate cyclase-activating polypeptide (PACAP) regulates several endocrine systems affecting essential physiological processes such as metabolism, growth, reproduction, and the stress response. PACAP acts as a hypophysiotropic factor, is a potent secretogogue of insulin, regulates production and release of catecholamines from the adrenal medulla and acts as a neuromodulator in the sympathetic and parasympathetic nervous system. The primary structure of PACAP has been highly conserved during the evolution of chordates suggesting it plays an important physiological role. The objective of my thesis was to identify PACAP’s primary physiological function and to determine if it is essential for survival by generating a mouse line deficient in PACAP through targeted disruption of the PACAP gene locus. Postnatal PACAP expression was examined to determine sites of peripheral PACAP production. In addition, several splice variants of the PACAP gene with alternate 5’untranslated regions were identified suggesting a complex system for regulating expression of the mouse PACAP gene. A targeting vector that allows tissue specific or developmental stage specific knockout of the PACAP gene was constructed in the event that PACAP gene deletion resulted in embryonic lethality. PACAP null mice were generated from homologously recombined embryonic stem cells. Initial characterization of the PACAP null mice determined that in the absence of PACAP, mice died within the first two postnatal weeks with abnormal lipid metabolism. Lipid accumulation was present in liver, heart and skeletal muscle and serum lipids were high. Mitochondrial dysfunction in the liver was not the cause of the lipid accumulation, as P-oxidative function was normal. I conclude that PACAP null mice are unable to regulate lipid release from white adipose tissue stores, resulting in a flood of lipids to non-adipose tissues. The abnormal distribution of lipids observed in the PACAP null mice is characteristic of diabetes type 2, yet classical insulin resistance is not observed. Thus, elevated insulin levels were accompanied by low blood glucose levels and the response to a glucose challenge was normal. The uncontrolled release of free fatty acids may result if glucose that is taken up by cells can not be utilized and an alternate energy source is required or if white adipocytes only are insulin resistant. The PACAP null mice were temperature sensitive, in that when raised at 21“C they exhibited metabolic dysfunction and died by two weeks of age. At 24°C most (85%) of the mice survived to adulthood with no obvious signs of metabolic dysfunction. We have determined that the inability of the PACAP null pups to thermoregulate normally when exposed to a lower environmental temperature may be associated with decreased norepinephrine levels to the brown adipose tissue. PACAP may be important for the production and release of catecholamines in the adrenal gland or within the sympathetic nervous system in times of prolonged stress. A mechanistic connection between the lipid abnormalities and the temperature sensitivity in the PACAP null pups has yet to be made. Catecholamines affect a wide range of tissues and the problems associated with insulin regulation within the PACAP null mice may be due to the imbalance in catecholamine production. As one of two main stress response systems, the sympathetic nervous system elicits a vital coping mechanism in times of stress and PACAP’s ability to regulate this system may explain why the primary structure of PACAP has remained so highly conserved. PACAP is a wide acting hormone and therefore the metabolic problems seen in the PACAP null mice may result from altered regulation of several endocrine systems at once. Targeted disruption of the PACAP gene in mouse has revealed a role for PACAP in the regulation of lipid metabolism and in the sympathetic control of thermoregulation. / Graduate

An overview of the noradrenergic system, including the identification of norepinephrine (NE) in animal tissue, its synthesis and metabolism, adrenoceptor classification, peripheral and central actions, uptake and storage, and mechanisms of NE release are presented. After characterizing the kinetic, ion dependence and inhibitor sensitivity of the norepinephrine transporter (NET) expressed in a recombinant cell line (LLC-NET cells), the influence of catecholamine (CA) metabolizing enzymes on studies of transport was assessed. Inhibitors of catechol-O-methyltransferase (COMT) potentiated the apparent uptake and retention of [3H]NE and [3H]DA. COMT inhibition had a greater influence on [3H]DA than [3H]NE uptake and retention, which corresponds to the higher spontaneous loss of radiolabel from cells exposed to [3H]DA than [3H]NE ([3H]methoxytyramine, is more lipophilic than [3H]normetanephrine). The monoamine oxidase inhibitor, pargyline, had no augmentary action on [3H]CA uptake, but actually inhibited substrate influx by blocking the NET. [3H]substrate specific differences were demonstrated for [3H]NE, [3H]DA and [3H]MPP+. For a given length of exposure to low Na+ or tyramine, [3H]NE release was the lowest, but most sensitive to NET inhibitors. Disparities in the kinetics of each [3H]substrate for the inwardly facing NET may account for this. Inhibitors of the NET were found to stimulate the efflux of [3H]substrates from preloaded cells incubated in a physiological HEPES buffer. Efflux was NET-dependent and differed greatly for each [3H]substrate. Inhibitor-induced release was greatest for [3H]MPP+ and least for [3H]NE. Finally, a functional model of carrier-mediated NE release in myocardial ischemia, was developed in this study. Release of [3H]MPP+ was stimulated by Na+-H+ exchanger (NHE) activation and modulated by inhibitors of the NET, NHE, Na+,K+-ATPase, and via a receptor-operated pathway. Excessive NE release contributes to severe myocardial arrhythmias, therefore an improved understanding of the carrier-mediated NE release process will ultimately enhance our ability to intervene and prevent the deleterious effects of excessive NE release.

Through multiple complex mechanisms, the endocrine system plays a vital regulatory role in virtually every organ system including the skin. Cutaneous lesions may be seen incidentally in patients with endocrine disorders. Alternatively, certain skin conditions are seen exclusively in the setting of endocrine disorders and may rarely be the first clinical symptom of the underlying endocrinopathy. Although clinical examination is often sufficient, a tissue biopsy may still be needed to arrive at or confirm the diagnosis and to direct therapy. In many cases, the cutaneous condition might improve or resolve completely after treatment of the underlying endocrine disorder. In this review, we summarize the most common skin conditions associated with endocrine disorders, uncovering their relationship to the underlying endocrinopathy, demonstrating their clinicopathologic presentation, and highlighting their typical modalities of treatment.

Introduction New onset hyperandrogenism in postmenopausal female is a rare occurrence, presenting with hirsutism or signs and symptoms of virilization. The causes of hyperandrogenism in postmenopausal female can be categorized into tumorous (androgen-secreting ovarian or adrenal tumors) and non-tumorous in origin (Hyperthecosis, Cushing’s syndrome, Acromegaly, Congenital Adrenal Hyperplasia, and iatrogenic). Here we present a case of severe hyperandrogenism of ovarian origin in a postmenopausal female where definite etiology could not be ascertained either by imaging or pathology. Case A 72-year-old female was referred to endocrinology clinic for complaints of worsening alopecia and hirsutism for the past 4 years. History was positive for weight gain of 80 lbs in the last ten years, menstrual irregularities since menarche, and recent deepening of voice. She denied exposure to exogenous androgenic steroid. Physical examination was remarkable for android obesity, severe male pattern alopecia, hirsutism involving face and deepening of voice, no clitoromegaly was noted. Lab evaluation showed elevated total testosterone level 261 ng/dl (normal value: 2-45 ng/dL) and free testosterone 14.1 pg/mL (normal value: 0.2-3.7 pg/mL ). Estradiol level was elevated and FSH and LH were low for a post-menopausal state. 17 hydroxy progesterone, TSH, and 1 mg overnight dexamethasone suppression test were normal ruling out congenital adrenal hyperplasia, thyroid dysfunction and Cushing’s syndrome. IGF-1 was not elevated ruling out acromegaly. DHEAS level was normal and CT abdomen and pelvis showed no evidence of an adrenal tumor, excluding adrenal source of androgen excess. Transvaginal Ultrasound showed normal volume of the ovaries, thickened endometrium and uterine myomas. Given markedly elevated testosterone levels and exclusion of adrenal tumor, suspicion for an ovarian source remained high. An MRI of the pelvis was done that showed 1.9 cm left adnexal cyst. She was then referred to Gynecology and underwent total hysterectomy with bilateral salpingo-oophorectomy. Interestingly surgical pathology was negative for tumor, showed unremarkable ovaries and right fallopian tube, left fallopian tube with hydrosalpinx, and showed atypical hyperplasia of the endometrium. However, testosterone levels decreased to normal two months after surgery; Free testosterone 1.8 pg/ml (normal values: 0.2-3.7 pg/mL), total testosterone 31 ng/dl (normal value: 2-45 ng/dL) indicating removal of ovarian source of testosterone production. Discussion Although relatively rare, severe hyperandrogenism (total testosterone >150 ng/dL, DHEAS >700 mcg/dL, signs of virilization) in postmenopausal women is caused either by adrenal or ovarian androgen secreting tumor or ovarian hyperthecosis, which is characterized by a hyperplastic ovarian stroma. Severity of symptoms, degree of androgen excess followed by imaging studies lead to identification of source of excessive androgen secretion in most cases. Diagnosis of ovarian virilizing tumors can be difficult since size of such tumors is often too small to allow detection on imaging studies, but are generally detected on surgical pathology and therefore bilateral salpingo-oophorectomy is recommended after exclusion of adrenal cause. However, rarely etiology may remain undetermined in some cases with conventional histology as in our patient.

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It is estimated that 366 million people were living with diabetes in 2011, and this is predicted to rise to 522 million by 2030. One of the most common complications of diabetes is diabetic neuropathy, where patients experience various symptoms of neuropathic pain including mechanical allodynia. Using a high fat diet (HFD) in combination with streptozotocin (STZ) produces a model of diabetes which mimics aspects of type 2 diabetes. The aim of this thesis was to characterise pain responses in the HFD/STZ model and to explore some of the peripheral and spinal mechanisms associated with the changes in somatosensory processing. The effectiveness of a variety of drugs in alleviating/preventing neuropathic pain was also investigated in this model. The effects of the HFD/STZ model on mechanical sensitivity, and changes in metabolic parameters were investigated, and it was found to cause a robust development of mechanical hypersensitivity and a large increase in plasma glucose and a contrasting decrease in plasma insulin. The impacts of the HFD/STZ model on peripheral nerve function and pathology, and spinal mechanisms of central sensitisation, were explored to help identify the mechanisms underpinning the behavioural pain phenotype in these rats. No neuronal degeneration was detected in DRGs or the spinal cord at the timepoints investigated, and a decrease in microglial activation and GFAP immunoreactivity was observed at later timepoints (day 50). Changes in neuronal responses in the dorsal horn of the spinal cord were then investigated, and there was a trend towards a decrease in mechanically evoked responses of spinal neurones in the HFD/STZ group, but no changes in the threshold for electrical activation of C-fibres, nor any significant changes to electrically evoked responses, were observed. There was no change in spontaneous firing, possibly due to the search criteria used. The effects of different types of interventions on aberrant pain responses were also investigated. As neuropathic pain often proves intractable, one of the key objectives is to develop new drugs, or to find alternative uses of current drugs, that are able to provide symptomatic relief of pain. The gold standard treatment for pain in diabetic neuropathy, pregabalin (10mgkg-1, p.o.), was effective at alleviating established mechanical hypersensitivity at day 37 in the HFD/STZ model. A novel MAGL inhibitor, MJN110 (5mgkg-1, i.p.), was found to be as effective as pregabalin in this model, highlighting a possible role for endocannabinoid modulators in providing pain relief in diabetes. The antidiabetic pioglitazone (10mgkg-1, p.o.), however, was unable to alleviate mechanical hypersensitivity when administered at day 21 for 28 days. Two other antidiabetic drugs, linagliptin (3mgkg-1, p.o.) and metformin (200mgkg-1, p.o.), did show promise in preventing the development of mechanical hypersensitivity in this model when administered from day 4, independent of glycemic control. It is worth investigating these findings further since both of these drugs are already licensed and have undergone all necessary safety testing, and so could rapidly be put to use if effective. In conclusion, this thesis has highlighted the role that the HFD/STZ model can play in investigating underlying mechanisms of diabetic peripheral neuropathic pain, and its use in exploring potential new therapeutic options for alleviating this pain.

published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

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