To explore the requirement for a functional insulin receptor/PI3K/AKT/NOS signalling pathway, we repeated PIV and Ach-stimulated tests after administering pathway selective inhibitors in 4-HCD mice. pressure-induced (PIV) and Ach-induced (endothelium-dependent) vasodilation occurred early, in overweight-metabolically healthy mice. Residual vasodilatory responses were NOS-independent but sensitive to COX inhibition. These changes were associated with reductions in NO and adiponectin bioavailability, and rescued by exogenous adiponectin or hyperinsulinemia. Obese-prediabetic mice continued to exhibit impaired Ach-dependent vasodilation but PIV appeared normalized. This normalization coincided with elevated endogenous adiponectin and insulin levels, and was sensitive to NOS, COX and PI3K, inhibition. In obese-type 2 diabetic mice, both Ach-stimulated and pressure-induced vasodilatory responses were increased through enhanced COX-2-dependent prostaglandin response. Conclusions We demonstrate that the development of obesity, metabolic dysfunction and type 2 diabetes, in HCD-fed mice, is accompanied by increased dermal adiposity and associated metaflammation in dWAT. Importantly, these temporal changes are also linked to disease stage-specific dermal microvascular reactivity, which may reflect adaptive mechanisms driven by metaflammation. strong class=”kwd-title” Subject terms: Obesity, Obesity Introduction With the increased prevalence of obesity and type 2 diabetes, and limited success in preventative approaches, there is an urgent need to better understand and manage the long-term consequences of metabolic disease . Obesity complications include skin disorders that may increase the prevalence of more NK-252 severe pressure ulcers (PU) [2, 3]. For example, obesity is associated with decreased tensile strength  and dermal elasticity in mice  and humans . However, an obesity paradox has also been reported wherein people with a body mass index (BMI) between 25 and 40 appear to be protected from the development of PU . Indeed, we have recently found that in a murine model of diet-induced obesity, pressure-induced ischaemia and skin lesions are reduced with increasing obesity . This suggests that pressure-induced regulation of cutaneous blood flow may be altered by changes in dermal adiposity. However, this and the underlying mechanisms currently remain unclear. In addition, none of the clinical studies focused on PU incidence have assessed the metabolic status of the obese subjects under investigation. Hence, the impact of increased dermal adiposity per se, or that of the metabolic deregulation that accompanies obesity-linked type 2 diabetes, on vascular fragility of the skin remains unclear. Mechanistically, numerous features of obesity-associated metabolic deregulation could impact dermal microvascular functionality NK-252 through local paracrine interactions with expanding adipose tissue. These include obesity-associated impaired metabolic functionality of adipose tissue, altered adipokine production  and low-grade chronic inflammation (metaflammation) [9C11]. Some of these have been implicated in perivascular adipose tissue-mediated, endothelial cell dysfunction in arteries and arterioles [12, 13]. Another major causal feature of obesity-linked type 2 diabetes is insulin resistance, which induces endothelial dysfunction in vascular disease via an inadequate production of endothelial NO and endothelin-1 [14, 15]. Among the dermal changes linked to diabetes , the disruption of microvascular adjustment to pressure, as revealed by pressure-induced vasodilation (PIV), correlates with increased vascular fragility of the skin [16C19]. IB2 Type 2 diabetic patients also exhibit a range of vascular, oxidative stress and inflammatory changes  that may affect skin and neurovascular quality [21, 22]. The potential impact of obesity-linked type 2 diabetes on NK-252 the arterial microenvironment  could affect microvascular adjustment to pressure in a context-dependent manner, by changes in adiposity, followed by progressive changes in metabolic dysfunction prior to the establishment of NK-252 type 2 diabetes. In this study, we investigate the temporal changes in dermal adiposity, dermal microvascular functionality and in endothelial function during the development of obesity and type 2 diabetes. We hypothesize that remodelling of dermal adipose layer and the development of type 2 diabetes are linked to changes in dermal microvascular NK-252 reactivity to pressure. Our findings suggest that initially, at the onset of increased adiposity, alterations in neurovascular and endothelial function are associated with altered adipokine production. However, as obesity progresses to pre-diabetic and diabetic states, additional adaptions occur to normalize and then enhance dermal vascular reactivity to pressure. Mechanistically, these adaptive changes involve a shift in key vasodilatory signalling pathways from a NO-dependent to pro-inflammatory COX-2/PG-driven programmes. Research design and methods Animals Male C57Bl/6J mice (aged.