Consideraciones finales y manera de salir adelante

Evidence from laboratory studies in humans examining daily rhythms of the hormones measured in this analysis are limited, and conflicting. GI hormones are primarily influenced by food quantity or quality, or indeed the lack of it, and differences in study design, such as meal timing, may explain these discrepancies. However, it is generally accepted insulin, C-peptide and glucagon levels peak in the morning, visfatin levels peak in the afternoon and ghrelin and leptin levels peak in the middle of the night. GIP, GLP-1 and resistin do not display significant diurnal rhythmicity. PAI-1 levels peak in the early morning. However, PAI-1 is not

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a GI hormone, it influences the endothelium and levels are not affected by nutritional status.

Many endogenous and exogenous factors affect the synthesis, secretion and elimination of circulating hormones. There is also a large inter-individual variation in hormone concentrations reported in the literature. Homeostatic mechanisms keep some hormones, such as circulating insulin and glucose concentrations, within safe limits whereas other analytes fluctuate over a large concentration range resulting in a wide inter-individual variation. The manufacturers of the multiplex assay used in this study demonstrates the large inter-individual variation within normal and T2DM samples. This inter-individual concentration variation was removed when examining the diurnal rhythms by performing the cosinor analysis using the relative concentration (% of the mean). The results were more powerful when the group mean data were analysed than when a cosine curve was fitted to each individual within the group. This was most likely because the daily rhythms of each individual within each study group peaked at a similar time of day, strengthening the rhythm. Previous studies have shown that when the daily rhythms of individuals within a group do not peak at the same of day they essentially cancel each other out, dampening the observed rhythm (Chua et al., 2013).

It was notable that the glucose rhythm peaked, in the afternoon, at a similar time of day as C-peptide, insulin and GIP. These hormones are all closely associated with glucose metabolism. GIP is an incretin hormone and is secreted in response to nutrients in the gut to stimulate the release of insulin (Section 3.4.3.1). C-peptide and insulin, ex-partners of the same molecule, proinsulin, are released from the pancreas at the same time (Section 1.4.1). In the absence of food (individuals fasted over a 72-h period) plasma insulin and C-peptide rhythms peaked at 08:00 h (Merl et al., 2004). A forced desynchrony protocol showed that insulin peaked during the late biological night/early morning (Morris et al., 2012). Elevated insulin, C-peptide and GIP, seen in the wake period in the OW/OB and T2DM groups of the present study, have been observed elsewhere, albeit in a different protocol (Nyholm et al., 1999). The elevated postprandial hormone levels

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in the obese first degree relatives of T2DM patients were attributed to impaired pancreatic β cell function in response to food, which was given to the participants 3 times a day (Nyholm et al., 1999).

In the present study, significant daily rhythms GIP displayed in all participant groups was most likely due to decreased concentrations during the dark period because no meals were served after 21:30 h. A similar observation was seen in rats where GIP concentrations correlated with the feeding schedule; GIP rhythms observed with feeding schedules were absent in fasted rats (Rubin et al., 1988). A study reporting daily GIP and GLP-1 rhythms in morbidly obese women with and without T2DM (Mingrone et al., 2009) conflicts with a well-controlled study using a forced desynchrony protocol that examined GIP and GLP-1 did not observe a circadian rhythm in either hormone (Morgan et al., 1998). These findings suggest that GIP and GLP-1 are food driven rather than circadian.

Glucagon has opposing actions to insulin so it may be expected to have an opposing daily rhythm. In the present study, glucagon displayed a significant daily rhythm in the lean group only, peaking at the start of active/feeding period in the early morning, 3.5 h earlier than the peak time of insulin. The peak time of the glucagon rhythm in the present study has also been observed in SCN-derived daily rhythms in rats which peaked at the start of the active period with acute postprandial peaks (Ruiter et al., 2003).

The correlation with leptin and adiposity (Sharifi et al., 2013) and the peak time of daily rhythms (Benedict et al., 2012, Mantele et al., 2012b, Saad et al., 1998, Shea et al., 2005, Maentele et al., 2012) is well established in the literature. In the current study leptin displayed significant cosine rhythms that peaked around midnight in all the study groups. These results confirmed the results from a previous study at the University of Surrey, which measured leptin in the same samples by RIA. The results of which showed that leptin had a clear diurnal rhythm in all groups, peaking on average at 00:31 h (Maentele et al., 2012). The study by Maentele et al. served to validate the multiplex assay used in the present analysis. The night-time leptin peak (02:04 h) has been reported elsewhere, in a study with a similar sleep wake-pattern (23:00–07:00 h) (Benedict et al., 2012). Furthermore,

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a study observing this nocturnal rise in leptin levels associated wakefulness and food intake with lower levels of leptin and lower glucose and insulin levels were attributed to the effects of sleep-fasting (Shea et al., 2005).

Similar to glucagon and leptin, ghrelin also seems to be regulated by more than one mechanism. It is intriguing that the diurnal rhythms of leptin and ghrelin both peak in the middle of the night (Bodosi et al., 2004, Cummings et al., 2001) although they have opposing roles on appetite; leptin is a satiety hormone and ghrelin acting as an appetite stimulator. Under free-living conditions, the diurnal rhythms of leptin and ghrelin are entrained to meal timing (Schoeller et al., 1997), seen as increased postprandial leptin levels and decreased postprandial ghrelin levels, but in the present study the masking effect of the meals was minimised with the inclusion of hourly isocaloric meals in the wake period. The night time peak (02.00 h) concords with Cummings et al. who observed a peak in ghrelin levels at 01:00 h (Cummings et al., 2001). There is evidence to suggest that the circadian clock also plays a role in ghrelin regulation within the MBH/SPV in the hypothalamus (Turek et al., 2005). In this study, reduced ghrelin mRNA expression was observed in Clock mutant mice at virtually all time points of the 12L:12D cycle.