Limb haemodynamic responses during heat-stress and the importance of local vs. central temperature-sensitive mechanisms towards their regulation remain poorly understood, both at a whole-limb level and within individual tissues (i.e. skeletal muscle and skin). The aims of this thesis were to 1) investigate the haemodynamic responses at rest to direct thermal challenges both at a local level and during progressive elevations in systemic heat stress, 2) to ascertain the contribution of local vs. systemic mechanisms towards this regulation, and 3) to investigate the same responses during single-legged small-muscle mass exercise to near maximal levels. Results from Chapters 4 and 5 characterised the haemodynamic responses during isolated cooling and heating of the arm and leg, and provided evidence of alterations in both skin and skeletal muscle blood flow controlled solely through local temperature-sensitive mechanisms. While local cooling led to modest decreases in limb blood flow due to decreases in mean blood velocity alone, increases during heating occurred as a result of an increased antegrade flow, a diminished retrograde flow, and a reduction in the potentially pro-atherogenic oscillatory shear index. In Chapter 6, whole-body heating with isolated single leg cooling displayed the continued control of limb blood flow via local thermosensitive mechanisms alone, as cooled leg blood flow remained unchanged despite significant elevations in core temperature, cardiac output, and opposing heated leg blood flow. Furthermore, elevations in heated leg V̇O2 suggested a possible metabolic contribution to the observed skeletal muscle hyperaemic response. During incremental single-legged knee-extensor exercise to near maximal levels, blood flow was determined by a combination of metabolic workload and local tissue temperatures, regardless of whether systemic heat stress was present. Chapter 7 revealed that whilst skin and muscle blood flow in the leg continued to increase in line with local temperatures to levels of severe heat stress, rapid cooling of the leg when hyperthermic resulted in a similar reverse response in muscle tissues only, as skin blood flow remained elevated despite the abolition of high skin and subcutaneous temperatures. In addition, evidence was provided that moderate levels of whole-body heat stress provided little additional benefit to anti-atherogenic shear profiles than that experienced during isolated limb heating alone. Taken together, these findings suggest that local thermosensitive mechanisms dominate limb blood flow control during direct rapid heating in humans both at rest and during small muscle mass exercise, but that underlying central mechanisms may act to maintain flow when local temperatures are reduced in the face of high core temperatures.

The overall aim of this thesis was to combine optical methods into a system with the ability to simultaneously measure blood flow changes at different tissue depths. The goal of such a system was to reveal vascular mechanisms relevant to pressure ulcer etiology under clinically relevant conditions and in relation to the evaluation of pressure-redistribution support surfaces. This thesis consists of four quantitative, cross-sectional studies measuring blood flow responses before, during, and after pressure exposure of the sacral tissue. Two optical methods – photoplethysmography and laser Doppler flowmetry – were combined in a newly developed system that has the ability to discriminate blood flows at different tissue depths. Studies I and II explored blood flow responses at different depths in 17 individuals. In Study I the blood flow was related to tissue thickness and tissue compression during pressure exposure of ≥ 220 mmHg. In Study II, the sacral tissue was loaded with 37.5 mmHg and 50.0 mmHg, and the variation in blood flow was measured. Studies III and IV included 42 healthy individuals < 65 years, 38 healthy individuals ≥ 65 years, and 35 patients ≥ 65 years. Study III included between-subject comparisons of blood flow and pressure between individuals in the three study groups lying in supine positions on a standard hospital mattress. Study IV added within-subject comparisons while the individual was lying on four different types of mattress. The studies explored the vascular phenomena pressure-induced vasodilation (PIV) and reactive hyperemia (RH). The most common blood flow response to tissue exposure in this thesis was PIV, although a decrease in blood flow (a lack of PIV) was observed in some individuals. The patients tended to have higher interface pressure during pressure exposure than the healthy groups but no differences in blood flow responses were seen. Our results showed that pressure levels that are normally considered to be harmless could have a significant effect on the microcirculation in different tissue structures. Differences in individual blood flow responses in terms of PIV and RH were seen, and a larger proportion of individuals lacked these responses in the deeper tissue structures compared to more superficial tissue structures. This thesis identified PIV and RH that are important vascular mechanisms for pressure ulcer development and revealed for the first time that PIV and RH are present at different depths under clinically relevant conditions. The thesis also identified a population of individuals not previously identified who lack both PIV and RH and seem to be particularly vulnerable to pressure exposure. Further, this thesis has added a new perspective to the microcirculation in pressure ulcer etiology in terms of blood flow regulation and endothelial function that are anchored in clinically relevant studies. Finally, the evaluation of pressureredistribution support surfaces in terms of mean blood flow during and after tissue exposure was shown to be unfeasible, but the assessment of PIV and RH could provide a new possibility for measuring individual physiological responses that are known to be related to pressure ulcer development.

Le tissu adipeux sous-cutané (TAsc) est le site préférentiel du stockage postprandial des triglycérides (TG). Quand les capacités d’accrétion sont dépassées, le stockage des TG se fait dans des sites ectopiques du TA et dans des tissus non adipocytaires, par exemple foie et muscles, ce qui entraine de multiples dysfonctionnements dans ces organes et tissus, et permet le développement du syndrome d’insulinorésistance. Chez les sujets obèses, la période postprandiale est caractérisée par des anomalies métaboliques, immunitaires, hormonales, et également par une diminution importante du flot sanguin dans le tissu adipeux (FSTA) sous-cutané. Ce blocage de la perfusion postprandiale du TA a aussi été montrée chez des individus minces qui avaient de très lourds antécédents familiaux de maladies cardiométabolique (obésité, diabète de type 2, maladies cardiovasculaires). Dans cette thèse, on classifiera ces individus comme « non-répondeurs ». À ce jour, peu d’attention a été accordée à ce phénomène. L’hypothèse qui sous-tend cette thèse est que les anomalies du FSTA sont innées ou primaires et sont impliquées très tôt dans le développement de la résistance à l’insuline (RI), du diabète de type 2 et du syndrome métabolique. Le but de notre recherche était donc de vérifier si les altérations du FSTA sont présentes chez les personnes saines et minces, mais à très haut risque de développer une RI ou une maladie cardiométabolique. Nous avons aussi cherché à déterminer les facteurs liés à la non-réponse. Pour cela il nous a fallu explorer certains facteurs hormonaux impliqués dans la régulation du FSTA. Nos résultats montrent que le FSTA est très diminué, à jeun et en postprandial, chez les sujets à haut risque de maladies cardiométaboliques mais encore minces et métaboliquement sains, sans RI. Nous avons aussi montré, pour la première fois, l’effet vasodilatateur du peptide intestinal vasoactif (VIP) dans le TAsc, tout comme le rôle stimulant du système cholinergique dans la régulation postprandiale du FSTA. Cependant, aucun de ces facteurs ne participe au dysfonctionnement du FSTA postprandial chez les non-répondeurs. Des taux répétés de TG plus élevés chez les non-répondeurs et l’association du FSTA avec certains indices de la RI décrits dans la littérature suggèrent que l’altération du métabolisme lipidique suite à la diminution du FSTA puisse servir de médiateur à la détérioration de la sensibilité à l’insuline. / Abstract : Subcutaneous adipose tissue (SCAT) is the preferential site of triacylglycerols (TAG) postprandial disposal. When the buffering capacity of SCAT for lipids is exceeded, TAG are disposed in ectopic adipose tissue depots and in non-adipose tissues, such as liver and muscles. Consequently, multiple dysfunctions of these organs and tissues develop including insulin resistance (IR). In obese people, the postprandial period is characterized by metabolic, immune and hormonal alterations, but also by severely altered adipose tissue blood flow (ATBF). Nevertheless, significant alteration of postprandial ATBF was also found in lean individuals with highly positive familiar history of cardiometabolic diseases (obesity, type 2 diabetes, cardiovascular diseases). In the thesis, we term them as "non-responders". Up to date, little attention has been payed to this phenomenon. The underlying hypothesis of this thesis is that alterations in ATBF are inborne or very early and that they participate on the development of IR, type 2 diabetes and metabolic syndrome. Consequently, the aim of our research was to verify if the alterations in ATBF are present in healthy, normal-weight subjects, but at very high risk for development of IR or cardiometabolic diseases. Simultaneously, we searched for factors linked with nonresponsiveness phenomenon. To do this, we examined some hormonal factors in ATBF regulation. Our results confirm the presence of altered fasting and postprandial ATBF in at highrisk subjects for cardiometabolic diseases, but still lean and metabolically healthy, without IR. For the first time, we have also demonstrated the role of cholinergic system in postprandial ATBF regulation, and vasodilatory effect of vasoactive intestinal peptide (VIP) in SCAT. However, none of these factors takes part in postprandial ATBF dysfunction in non-responders. Higher TAG levels repeatedly found in non-responders and the association of ATBF with some indices of insulin sensitivity described in the literature suggest that alteration of lipid metabolism as a result of low ATBF may mediate deterioration of insulin sensitivity.

Résumé: Comparées aux classiques insulines humaines régulières (IHR), les insulines analogues ultrarapides (IAUR) ont été conçues pour mieux synchroniser le pic insulinémique avec l’absorption du repas. Le progrès a été démontré chez les patients diabétiques de type 1, mais le contrôle glycémique s’est peu ou pas amélioré chez les patients diabétiques de type 2 (DT2), qu’ils soient sous IAUR ou IHR. Or ces patients constituent 75 % des utilisateurs d’insuline. L’utilité des IAUR est donc toujours débattue. La dose (donc le volume) injectée et le flot sanguin dans le tissu adipeux sous-cutané (FSTA) sont les facteurs majeurs de l’absorption de l’insuline. Les patients DT2, résistants à l’insuline, s’injectent des doses importantes et leur FSTA est de 50 à 70 % plus faible que celui des sujets sains de poids normal (PN). Nous avons montré que l’absorption sous-cutanée des IAUR est diminuée chez les sujets obèses et DT2 (ODT2) par rapport aux sujets PN, que le volume injecté avait un effet délétère additionnel et que le FSTA peut être augmenté de façon pharmacologique avec un agent vasoactif (AV) chez des sujets résistants à l’insuline. Nous suggérons que l’ajout d’un AV à une IAUR va augmenter le FSTA au site d’injection et donc améliorer sa pharmacocinétique (PK) et sa pharmacodynamie (PD). Pour vérifier cette hypothèse, nous avons 1) évalué la réponse du FSTA à 4 AV chez des sujets PN, obèses non-diabétiques et ODT2; 2) évalué la PK/PD et la biodisponibilité de l’IAUR lispro ± AV chez des sujets ODT2; et 3) caractérisé l’expression des cibles des AV dans le tissu adipeux sous-cutané chez les sujets énumérés en 1). Les 4 AV ont augmenté le FSTA des sujets ODT2, mais moins que celui des autres sujets. L’occurrence de la raréfaction et/ou dysfonction microvasculaire chez les sujets ODT2 pourrait expliquer l’hyporéactivité vasculaire aux AV testés. Le plus actif des AV chez les sujets ODT2 a été ajouté à l’IAUR lispro pour améliorer sont absorption sc. Les PK/PD ont été améliorées seulement chez les sujets ODT2 avec une hémoglobine glycosylée A1c ≥ 8 %; c’est-à-dire 4 sujets sur 8. Chez ces derniers, l’absorption de 30 U + AV a été plus rapide de 14 et 71 min à 20 et 80 % de l’aire sous la courbe totale de la lispro plasmatique, respectivement. Chez les 4 autres sujets ODT2, l’absorption de la lispro semble s’être détériorée avec l’AV. Une interaction chimique a peut-être eu lieu entre l’AV et la lispro, ce qui aurait perturbé son absorption. Selon nos résultats, le niveau de contrôle du diabète, le volume d’injection et les caractéristiques chimiques de l’AV seraient des modulateurs de l’efficacité du concept IAUR + AV. Il nous faut maintenant déterminer l’impact de ces facteurs sur la capacité d’un AV à améliorer l’absorption sc de l’IAUR chez les sujets ODT2. / Abstract: Compared to classic regular human insulin (RHI), short-acting insulin analogues were designed to better synchronize plasma insulin increase to food absorption. Although improvements were noted in subjects with type 1 diabetes, slight to no improvement in glycemic control were observed in subjects with type 2 diabetes (T2D) using SAIA instead of RHI. Nevertheless, they represent 75 % of all insulin users. Consequently, the relative useful-ness of SAIA in T2D patients is currently hotly debated. Injected volume and subcutaneous (sc) adipose tissue blood flow (ATBF) are two main factors involved in insulin absorption. In fact, T2D patients use large doses of insulin because of their resistance to insulin and have an ATBF 50 to 70 % lower than lean healthy subjects. We already showed that SAIA absorption is decreased in obese T2D (OT2D) subjects compared to normal weight healthy subjects and that volume has additional detrimental effects. We also showed that ATBF can be increased pharmacologically with vasoactive agents (VA) in healthy and insulin-resistant subjects. Then we suggest that in OT2D subjects, addition of VA to SAIA preparations will locally increase ATBF, improve insulin sc absorption (Pharmacokinetic - PK) and bioavailability, thus insulin hypoglycemic effect (Pharmacodynamic - PD). To test this hypothesis, we 1) assessed ATBF response of 4 selected VA within three experimental groups (normal weight, obese non-diabetic and OT2D subjects); 2) evaluated insulin PK/PD and bioavailability improvement in OT2D subjects after the addition of the best VA to SAIA lispro and 3) characterized expression of selected VA targets in sc adipose tissue biopsies, within equivalent experimental groups, and compared results with ATBF responses. All 4 VA were able to increase ATBF of OT2D subjects but in a less extend than other subjects. The occurrence of microvascular rarefaction and/or dysfunction in OT2D subjects can explain the hyporeactivity to tested VA. Nevertheless, one VA among others was shown more effective to increase ATBF in OT2D subjects and was then tested (mixed) with SAIA lispro. With the AV, PK/PD were improved only in OT2D subjects with A1c glycated hemoglobin ≥ 8 %; 4 subjects on 8. The sc absorption of 30 U + VA was faster by 14 and 71 min for respectively 20 and 80 % of the total area under the lispro plasmatic curve. But the sc absorption with VA appeared blunted with the other subjects. Maybe detrimental chemical interactions occurred between the VA and lispro, which could impede absorption. Our results suggest that diabetes control state, injection volume, and VA chemical characteristics influence the efficacy of our SAIA + VA concept. Further tests are needed to seize the impact of these factors on VA effectiveness in sc absorption improvement of SAIA in OT2D subjects.

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