Biochemical studies of cutaneous damage in mouse models of insulin resistance and obesity

Alhabian, Amgad (2014) Biochemical studies of cutaneous damage in mouse models of insulin resistance and obesity. Doctoral thesis, University of Buckingham.

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Abstract

Mammalian skin is a highly-specialised organ, providing a robust barrier against environmental challenges. In order to maintain functional integrity and a healthy state, the skin’s constituent tissues undergo continuous renewal, controlled by finely-controlled homeostatic mechanisms. Skin disease is incredibly common, ranging from debilitating (but not life threatening) disorders such as acne or eczema to increasingly aggressive, pernicious disorders like psoriasis, through to neoplasms such as carcinomas and melanoma. Importantly, the skin may present manifestations of systemic disease such as in diabetes, obesity, cardiovascular diseases (CVD) and metabolic syndrome (Azfar and Gelfand, 2008, Van Hattem et al., 2008, Nawale et al., 2006). Such cutaneous alterations could be the first signs of the disease, and may precede diagnosis by many years. The integrity of the skin degrades with age, and numerous metabolic disorders lead to pathological conditions that exacerbate this process (Nikolakis et al., 2013). For example, the vast majority of individuals suffering from type 2 diabetes will experience skin complications during the natural history of their disease, especially with years of poorly- controlled plasma glucose (Romano et al., 1998, Levy and Zeichner, 2012). Problems range from the sub-clinical, such as neuropathy, chronic inflammation micro- and macrovascular damage and collagen disorganisation, which lead to potentially catastrophic events such as delayed wound healing, ulceration, infection and gangrene. Proactive control of the glycaemic state may prevent or delay many of these associated complications, but it may not reverse pathological changes once established. Obesity and related risk factors in humans are associated with increased fat deposition, with many studies documenting the abnormal structure and function of dermal adipose tissue. Impaired fibroblasts function may contribute to many dermatological changes, such as the loss of dermis or fibrosis, via mechanisms related to insulin signalling (Rivera-Gonzalez et al., 2014). However, the dysfunction of dermal adipocyte which is characterised by adipocyte hyperplasia and hypertrophy, hypoxia, elevated inflammation, and adipokines signalling are now emerging as a potential mediator of insulin-resistance (Klöting and Blüher, 2014). Thus it is possible that the chronic exposure of fibroblasts to adipokines underlies their impaired function. Although many reports describe the use of mouse skin to reveal insights into aspects of human disease pathology and aetiology, it remains a challenge to find a standard protocol that can create optimal sections, retaining the intrinsic structure of mouse skin effectively, and researchers tend to use human skin histology protocols. This is not necessarily acceptable when one considers the anatomical differences between human and mice (Treuting and Dintzis, 2011). Mouse skin is thinner and more fragile than human skin, and so loss of architecture during processing has implications for downstream applications. Obtaining good histology from diabetic mouse skin, and maintaining subcutaneous adipocytes architecture and the discrimination of components such as fine elastic fibres, present a considerable challenge. Therefore, great care must be taken when selecting the conditions, particularly fixative type, to ensure that meaningful conclusions may be drawn (Al-Habian et al., 2014). The skin sections from mouse models used in chapter 3 were prepared using best practice at the time for a retrospective study, but I was able to better histology at the cost of time and materials in sectioning and staining by using tissue macroarrays techniques. Thus, for prospective analysis of mouse skin histology I revised some histology methods. By evaluating the processing protocol and then four commonly used fixatives I enhanced IHC, and histomorphological analysis of normal mouse skin. Moreover, these histopathology techniques are applicable to different mouse tissues, and I recommend using these findings as a guideline not only for diabetic mouse skin histology but for many tissues derived from a variety of species. The dermatologic sequelae of type 2 diabetes mellitus are manifold. Murine models of insulin resistance are useful in elucidating molecular mechanisms of impaired wound healing, but little is known of other pathophysiological changes in these models. A variety of histological and in vitro techniques were used in this thesis to study skin organisation in environmental (diet-induced obesity) and genetic (C57BL/6 Lepob/Lepob and C57BLKS/J Leprdb/Leprdb) models of insulin resistance, type 2 diabetes mellitus, and in chronological age. An expanded subcutis was accompanied by progressive dermal erosion in which the papillary dermis was spared at the expense of the deeper reticular layer as insulin resistance increased. Elastosis was also observed in our models, but damage was not accompanied by an increase in immune infiltration, nor an increase in advanced glycation end products. Altered epidermal differentiation was associated only with the most extreme obese phenotype. Moreover, compromised fibroblast function was maintained in vitro, with C57BL/6 Lepob/Lepob cells displaying compromised growth and reduced collagen synthesis. While mouse skin does not necessarily model the range of cutaneous sequelae observed in human diabetic subjects, it is likely that underlying cellular mechanisms are shared. An improved understanding of the contribution of the layers of the skin, particularly the dermis during insulin resistance, and ageing and its pathological processes may provide new insights into the mitigation of damage. While impaired insulin signalling in skin is associated with disrupted skin homeostasis, and extra cellular matrix remodelling in ageing and type 2 diabetes mellitus (Nikolakis et al., 2013), a recent study has linked these changes to dermal fibroblast , and adipocyte dysfunction (Rivera-Gonzalez et al., 2014). Interestingly, our observation showed that both aged and Lepob/Lepob murine fibroblasts show lowered insulin receptor expression suggesting that this will be a fruitful area for future investigation for improving insulin sensitivity and glucose utilisation in the dermal layers could have important consequences for cutaneous health. This thesis reports a specific pattern of cutaneous damage that is initiated before the onset of frank diabetes and is exacerbated with increasing insulin resistance. This work provides a new insight into the consequences of metabolic disease on skin structure. Finally, further insights into how dermal damage in genetically modified and environmentally adapted diabetic mouse models and vice versa are likely to subserve in detecting early signs of cutaneous insulin resistance and may likely offer better approaches to prevent or at least cure the dermatological sequelae of type 2 diabetes mellitus.

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