Variaciones en el tipo de cambio real

The IL-6 and TNF-α responses are presented in Figure 3.5. Resting concentrations of IL-6 and TNF-α were positively related to length of diabetes (IL-6: r = 0.701, p = 0.033; TNF-α: r = 0.632, p = 0.042) and inversely related to HbA1c (IL-6: r = -0.699, p = 0.020; TNF-α: r = -

0.698, p = 0.039), but not V̇O2peak (IL-6: r = -0.463, p = 0.528; TNF-α: r = -0.327, p = 0.356).

Both plasma IL-6 and TNF-α concentrations were significantly raised from rest at 15 minutes post-exercise (IL-6: Full Δ+2.02  1.01 (125 %) pg.ml-1; p = 0.032, 50% Δ+1.34  0.98 (116 %) pg.ml-1; p = 0.030; TNF-α: Full Δ+2.83  0.86 (147 %) pg.ml-1; p = 0.025, 50% Δ+2.99  0.74 (144 %) pg.ml-1; p = 0.021). Following the post-exercise meal, IL-6 concentrations were significantly greater under 50% (Figure 3.5), although TNF-α was similar between conditions. Despite this, both cytokines under 50% remained similar to pre-meal and resting measures (p > 0.05; Figure 3.5). Under 50% mean IL-6 concentrations over the post-exercise period were positively related to mean TNF-α concentrations (r = 0.676, p < 0.001) and serum insulin

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concentrations were inversely related to IL-6 (r = -0.484, p = 0.017), but not TNF-α (r = - 0.169, p = 0.430). No significant relationships existed between mean blood glucose and IL-6 (r = 0.299, p = 0.155) or TNF-α (r = 0.005, p = 0.980) over the post-meal period under 50%. No relationships were found between any other measures under Full.

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Table 3.2. Metabolic and counter-regulatory hormone responses to reductions in pre- and post-exercise rapid-acting insulin dose

ANOVA p Rest 60 E 0 15 30 Pre-Meal 30 60 90 120 150 180 T T*C Plasma Glucagon (pg.ml-1) Full 76070 69781 115299† 83283† 69861 71069 101579†‡ 1597116†‡ 1590102†‡ 140294†‡ 1332137†‡ 1099124†‡ <0.001 =0.006 50% 78985 62885†* 1067128† 854127 81589 60998† 97582†‡ 135096†*‡ 125197†*‡ 117790†*‡ 108586†*‡ 88982*‡ Plasma Adrenaline (nmol.l-1₎ Full 0.29±0.05 0.12±0.03† 0.56±0.09† 0.40±0.10 0.31±0.07 0.21±0.05 0.20±0.05 0.18±0.07 0.12±0.04† 0.15±0.05 0.18±0.06 0.13±0.04†‡ =0.029 =0.577 50% 0.27±0.05 0.19±0.04 0.59±0.07† 0.29±0.04 0.20±0.04 0.15±0.03 0.15±0.04 0.14±0.03 0.15±0.04 0.15±0.04 0.15±0.02 0.12±0.03 Plasma Noradrenaline (nmol.l-1₎ Full 2.13±0.24 1.76±0.20 11.75±1.37† 4.30±0.56† 2.87±0.30† 2.49±0.27 2.51±0.25 2.86±0.33 2.53±0.29 2.61±0.40 2.37±0.31 2.36±0.32 <0.001 =0.537 50% 2.51±0.28 2.42±0.29 12.68±1.38† 4.09±0.27† 3.35±0.21† 2.74±0.27 2.79±0.39 2.97±0.44 2.70±0.37 2.90±0.52 2.82±0.49 2.80±0.44 Serum Cortisol (µmol.l-1) Full 0.22±0.03 0.21±0.03 0.19±0.02 0.23±0.03 0.20±0.03 0.17±0.01† 0.16±0.02† 0.15±0.03† 0.12±0.02†‡ 0.11±0.02†‡ 0.10±0.01†‡ 0.10±0.01†‡ <0.001 =0.256 50% 0.23±0.03 0.19±0.03 0.20±0.03 0.23±0.04 0.22±0.03 0.17±0.02†‡ 0.14±0.02†‡ 0.12±0.02†‡ 0.11±0.02†‡ 0.10±0.02†‡ 0.08±0.01†‡ 0.07±0.01†‡ Blood Lactate (mmol.l-1₎ Full 0.49±0.12 0.82±0.13 3.63±0.61† 1.49±0.25† 1.07±0.16† 0.79±0.10 0.62±0.10 0.74±0.17 0.66±0.19 0.56±0.16 0.51±0.13‡ 0.50±0.13‡ <0.001 =0.789 50% 0.53±0.17 0.80±0.10 3.68±0.48† 1.64±0.29† 0.96±0.19† 0.74±0.15 0.68±0.19 0.69±0.11 0.59±0.13 0.54±0.12‡ 0.55±0.1‡2 0.50±0.12‡ Serum NEFA (mmol.l-1₎ Full 0.48±0.08 0.23±0.04† 0.24±0.04 0.42±0.11 0.34±0.06 0.35±0.07 0.30±0.06† 0.18±0.04†‡ 0.17±0.04†‡ 0.27±0.04† 0.25±0.03† 0.25±0.02† =0.012 =0.448 50% 0.40±0.06 0.24±0.07† 0.31±0.11 0.46±0.16 0.32±0.14 0.38±0.14 0.33±0.08 0.21±0.06† 0.24±0.05† 0.37±0.05 0.44±0.07 0.51±0.07

Note: Data presented as mean  SEM. 75% trial was omitted from analysis. Test meal and insulin were administered immediately following rest and pre-meal sample points. * indicates significantly different from Full (p ≤ 0.05). † indicates significantly different from rest. ‡ indicates significantly different from pre-meal. Exercise commenced 60 minutes after rest. T = Time, C = Condition, E = Exercise.

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Figure 3.5 A-C. Time-course changes in (A) plasma IL-6, (B) plasma TNF-α, and (C) serum β-

hydroxybutyrate from rest. Data presented as mean ± SEM. IL-6 and TNF-α (n = 8). Black squares =

Full, red diamonds = 50%. Transparent sample point within a condition indicates a significant

difference from pre-meal concentrations (p ≤ 0.05). * indicates significantly different from Full (p ≤ 0.05). Thatched area indicates exercise. Vertical dashed line break indicates carbohydrate meal and insulin administration. Note: Test meal and insulin were administered immediately following 60 minutes post-exercise sample point.

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3.7 Discussion

This study demonstrates that reducing pre- and also post-exercise rapid-acting insulin dose, as a strategy for preventing early-onset post-exercise hypoglycaemia, does not cause adverse metabolic, counter-regulatory-hormonal or inflammatory disturbances. Specifically, the data indicates that large reductions in rapid-acting insulin dose administered before and also after intensive running does not augment ketonaemia, nor cause significant elevations in inflammatory cytokines IL-6 and TNF-α above fasting concentrations, despite periods of postprandial hyperglycaemia following the post-exercise meal, in patients with type 1 diabetes.

Completing the exercise protocol caused a significant metabolic stress to patients, inducing large increases in blood lactate (~392 %) and catecholamines (adrenaline ~287 %, noradrenaline ~591 %), and large decreases in blood glucose (Δ~7.6 mmol.l-1; chapter 3A). As described in chapter 3A, all patients under 50% were protected from hypoglycaemia throughout their laboratory stay despite reductions in glycaemia following exercise and the administration of a second dose of rapid-acting insulin 60 minutes later. However, as a consequence of preventing hypoglycaemia, the majority of patients following the 50% reduction (82 %) were exposed to periods of hyperglycaemia following the post-exercise meal.

Hyperglycaemia plays a central pathophysiological role in the development of long-term diabetes related complications (Nathan et al. 2005, Ceriello et al. 2013), but is also of immediate concern because hypoinsulinaemic hyperglycaemia is associated with an acute increase in lipolysis and ketogenesis (Laffel 2000, Wallace and Matthews 2004). Indeed, the present study revealed a positive association between increased blood glucose and NEFA, and increased blood glucose and β-hydroxybutyrate appearance. Although temporal changes in both NEFA and β-hydroxybutyrate concentrations were evident following the post-exercise meal under 50%, concentrations remained similar between both conditions, and by 180 minutes both metabolites were similar to fasting rested concentrations. From a clinical

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perspective, concentrations in these ranges are not deemed significant (< 1.0 mmol.l-1) (Laffel 2000).

Although a large reduction in rapid-acting insulin dose was applied, serum insulin concentrations were elevated above resting and pre-meal measures under 50% (chapter 3A). Despite unexplained differences in glucagon concentrations, the administration of even small amounts of rapid-acting insulin, under conditions of unchanged basal insulin dose, is likely to have raised circulating insulin concentrations whereby lipolysis is inhibited (through dephosphorlyation of hormone-sensitive lipase) and lipogenesis is increased (via activation of acetyl CoA carboxylase). Thus, reducing the capacity for β-oxidation of NEFA and ultimately limiting substrate availability for ketogenesis (Mcgarry 1996), and potentially promoting peripheral ketone body disposal (Balasse and Féry 1989). Moreover, temporal changes in catecholamine concentrations, the main lipolytic stimulus (Kalra and Tigas 2002), and cortisol (Fowler 2008) were not statistically significant between conditions. The consumption of a large carbohydrate based meal would have helped supplement muscle and liver glycogen, reducing the energy deficit created by exercise, and limiting the appearance of catecholamines and cortisol.

A logical extension of the results from chapter 3A was to question whether post-exercise hyperglycaemia would exacerbate the appearance of inflammatory cytokines in the patients in this investigation, as this would likely be further increased if patients experienced hyperketonaemia. Increased markers of inflammation are strongly related to glycaemic management and the pathogenesis of diabetes related complications (Targher et al. 2001, Fowler 2008). Patients with type 1 diabetes exhibit chronically elevated levels of inflammatory markers at rest (Targher et al. 2001, Esposito et al. 2002, De Rekeneire et al. 2006, Galassetti et al. 2006, Rosa et al. 2008). Indeed, a positive relationship was observed between resting inflammatory cytokine concentrations, and diabetes duration, which was inversely related to HbA1c. It is worthy to note however, that baseline measures in this study were elevated above

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2008, 2010, Rosa et al. 2011). However it would be naïve to think that this was not influenced by the overnight fast or low circulating concentrations of insulin. Furthermore, most studies implement glucose and/or insulin clamp procedures and recruit children or adolescents who are usually recently diagnosed (Galassetti et al. 2006, Galassetti et al. 2006, Rosa et al. 2008, 2010, Rosa et al. 2011). This study population consisted of a relatively young (~24 years) group of individuals all in good glycaemic control (~7.7 % / 61 mmol/mol); exposure to inflammatory stimuli is likely to be much greater in the general diabetes population who are older, have a longer duration of diabetes, and in those with excess adiposity (De Rekeneire et al. 2006).

Only modest increases in IL-6 (~22%) and TNF-α (~45%) were observed following exercise, which is likely due to the consumption of the pre-exercise meal and concomitant insulin administration. The large carbohydrate bolus (1.0 g.carbohydrate.kg-1 BM) would have helped supplement glycogen reserves (Jentjens and Jeukendrup 2003), which may have attenuated the exercise-induced increases in IL-6 (Stouthard et al. 1995, Pedersen and Febbraio 2008) and even completely inhibited IL-6 release from contracting skeletal muscle (Pedersen and Febbraio 2008). In addition, insulin carries anti-inflammatory properties (Viardot et al. 2007), of which its administration, even in small doses, may have partially combatted the pro- inflammatory effects of TNF-α. Indeed, IL-6 concentrations were inversely related to circulating insulin concentrations. IL-6 has anti- as well as pro-inflammatory properties (Ostrowski et al. 1999, Petersen and Pedersen 2006), with some studies demonstrating IL-6 to exert inhibitory effects on TNF-α (Petersen and Pedersen 2006). In the present study there was a positive correlation between IL-6 and TNF-α; although a relationship does not necessarily indicate cause-effect, speculatively, increases in IL-6 may indeed have been in direct response to reductions in TNF-α (see section 1.5). Regardless of the underpinning mechanisms, data presented herein indicates that reductions in rapid-acting insulin after exercise do not significantly elevate the pro-inflammatory cytokine TNF-α, and that both TNF-α and IL-6 are not elevated above fasting concentrations.

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IL-6 and TNF-α were selected, in part, because both display the greatest quantitative change in individuals with and without type 1 diabetes (Pedersen and Febbraio 2008), and therefore have a likelihood to yield distinct differences between study conditions (Ostrowski et al. 1999, Pedersen and Hoffman-Goetz 2000, Galassetti et al. 2006, Rosa et al. 2011). There is however, a known and marked inherent variability of many inflammatory markers (Rosa et al. 2008, Rosa et al. 2011), which reflects the remarkable metabolic complexity of the patient with type 1 diabetes, in which permutations in inflammation status are variable across patients and also within the same individuals over time (Rosa et al. 2008). Some of this variability can be attributed to antecedent hyperglycaemia (Sprenger et al. 1992, Drenth et al. 1995, Nehlsen- Cannarella et al. 1997, Ostrowski et al. 1999, Pedersen and Hoffman-Goetz 2000, Nemet et al. 2002, Galassetti et al. 2006, Rosa et al. 2008, 2010), however, it is important to note that patients in this study were kept under free-living conditions before experimentation and without correction using euglycaemic clamp procedures. Patients outside of this study are therefore likely to closely experience the responses in day-to-day life that were found in this study.

An interesting, if not surprising, finding was that plasma glucagon concentrations following the post-exercise meal were elevated under both Full and 50%, but were significantly greater under Full. Although the majority of patients under this condition experienced hypoglycaemia (blood glucose ≤ 3.9 mmol.l-1

; Full n = 5, 50% n = 0; chapter 3A), patients were treated with a corrective bolus of carbohydrate such that blood glucose levels (group mean blood glucose ~6.6 mmol.l-1; chapter 3A) remained above the glycaemic threshold for plasma glucagon release (blood glucose ~ < 3.0 mmol.l-1; Cryer 2008). Even if an appropriate glycaemic threshold was achieved to stimulate glucagon release, it would remain a surprise to find any increase in glucagon concentrations in these patients (length of diagnosis: range 4-31 years) as its secretion under hypoglycaemic conditions is largely attenuated in long-standing type 1 diabetes (Cryer 2008). One possible explanation for the increase in glucagon concentrations could be the consumption of a mixed-meal. Brown et al (2008) observed increased glucagon

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concentrations in response to a mixed-meal in type 1 diabetes patients, as have other studies (Müller et al. 1970, Gerich et al. 1975, Ternand et al. 1982, Porksen et al. 2007), suggestive that the α-cell secretory reserve may be unaffected by the progression of the autoimmune process (Brown et al. 2008). If this were true, increases in glucagon may be attributed to neural stimulation, increased α-cell stimuli such as gastric inhibitory polypeptide (GIP) or a lack of glucagon-like peptide (GLP) which would otherwise promote postprandial endogenous glucagon secretion, although this is purely speculative. Whereas GLP usually inhibits glucagon secretion in non-diabetes patients, GLP is largely deficient in those with type 1 diabetes (Aronoff et al. 2004). Casual factors underpinning this are yet to be elucidated, although some authors suggest that this is consequential of lower intra-islet insulin levels, rather than systemic insulin concentrations per se (Greenbaum et al. 2002). Of note, the meal administered in the study by Brown et al (2008), was similar in nutritional content to the post-exercise meal given to patients in this study (Carbohydrate: 56 vs. 53 %, Protein: 21 vs. 25 %, Fat: 21 vs. 22 %), although smaller (~1.7 vs. ~2.8 MJ). In the present study, glucagon increased under both conditions, but was significantly greater following Full; as meals were identical in composition and weight, this may suggest a conditional effect following changes in rapid- acting insulin dose, however glucagon failed to correlate with changes in insulin concentrations, similarly to Brown et al (2008) and Potter and colleagues (1989). Interestingly, there was a positive association between glucagon and noradrenaline concentrations over the post-exercise post-prandial period. In individuals without diabetes, a sustained rise (≤ 120 minutes) in plasma noradrenaline has previously been demonstrated following a carbohydrate- rich mixed meal (Potter et al. 1989) with some initial mechanistic data indicating a complex synergy between glucagon and norepinephrine in appetite hormone regulation (Gagnon and Anini 2013). However, this has never been demonstrated in type 1 diabetes patients. Temporal rises in mean noradrenaline were evident at 60 minutes following the post-exercise meal under both conditions in this study, although changes from pre-meal concentrations were very small (~0.4 nmol.l-1) and did not reach statistical significance.

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The aim of this study was to assess the acute metabolic, inflammatory, and counter-regulatory hormonal effects of reducing post-exercise rapid-acting insulin dose under conditions of reduced pre-exercise rapid-acting insulin dose. The results from this study indicate that heavily reducing the dose of pre- and post-exercise rapid-acting insulin, as a measure to combat post- exercise hypoglycaemia, does not induce hyperketonaemia, increase the inflammatory cytokines IL-6 or TNF-α above fasting concentrations, or cause other metabolic or hormonal disturbances in type 1 diabetes patients. With this, diabetes care staff can have confidence that the only adverse effect of this strategy is hyperglycaemia. There is now a need to normalise post-exercise post-prandial glycaemia through modifications to dietary intake whilst under conditions of reduced rapid-acting insulin dose.

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CHAPTER 4A

THE GLYCAEMIC RESPONSES TO MANIPULATING THE