Investigación sobre factores de calidad en centros EAPI

This technique is recognised as being the ‘gold standard’ for determining whole body glucose sensitivity to insulin in vivo (Muniyappa, Lee et al. 2008). It differs from the HOMA assessment in that it mimics the postprandial, as opposed to fasted, state and also it predominantly assesses tissue sensitivity to insulin-mediated glucose uptake as opposed to hepatic sensitivity to insulin-insulin-mediated regulation of gluconeogenesis.

The delivery of exogenous insulin, as opposed to relying on endogenous insulin production, increases its reproducibility over homeostasis models and glucose tolerance tests. The clamp procedure can be used to quantify sensitivity to several of insulin’s actions beyond that of whole body glucose disposal. Adipose tissue sensitivity to insulin-mediated lipolysis can be assessed by free fatty acid concentrations during the procedure. Hepatic insulin sensitivity and glucose output can be determined with the addition of a glucose tracer, as will be discussed further. Finally, the combination with indirect calorimetry facilitates the quantification of glycogen synthesis (i.e. non-oxidative glucose disposal) as previously described.

Determining systemic insulin sensitivity by use of a hyperinsulinaemic euglycaemic clamp

Using the protocol described by DeFronzo et al. (DeFronzo, Tobin et al.

1979), exogenous insulin is delivered as a loading bolus over 10 minutes followed by a constant infusion at a pre-determined dose. A stable plasma glucose concentration is maintained by the simultaneous infusion of glucose. The delivery of a high insulin concentration mimics the postprandial state and is assumed to fully suppress endogenous glucose production. As a result of the suppression of endogenous production, the rate of exogenous glucose supplied equals the rate of whole body glucose disposal at steady state plasma glucose (M). M is typically

normalised to fat free mass as glucose uptake can only occur in these tissues (Ferrannini and Mari 1998).

From the above it is clear that the determination of the timing of ‘steady state’ is critical to the analysis of the data generated. Two approaches can be taken, either defining an arbitrary time point towards the end of the clamp study, or by taking data from a period of objective stability more than an hour into the clamp (Muniyappa, Lee et al. 2008). The objective criteria proposed include a period greater than 30 minutes during which the coefficient of variation of plasma glucose, plasma insulin, and glucose infusion rate (GIR) is less than 5%. The latter approach is more rigorous, though may not be achieved during every clamp.

Determining adipocyte insulin sensitivity

Insulin suppresses lipolysis in adipocytes via inhibition of hormone-sensitive lipase and thus reduces circulating plasma NEFA concentrations. The relative reduction in plasma NEFA concentrations following insulin delivery during a clamp can thus be used to assess adipocyte insulin sensitivity.

Determining hepatic insulin sensitivity

This was performed by using a stable glucose isotope dilution technique during basal and insulin stimulated periods. The addition of a label to exogenously delivered glucose enables differentiation of the origin of circulating glucose from exogenous and endogenous sources.

Deuterated glucose is a stable isotope of glucose containing ²H as opposed to the naturally occurring ¹H. The differing atomic masses of ²H and ¹H glucose facilitate their individual quantification by mass spectrometry. The location of the isotopic label in the glucose molecule determines which metabolic pathways the tracer enters post glucose catabolism. Dideuterated glucose with deuterium in the 6^th position ([6,6-²H₂]) was selected as it is less susceptible to intermediary step analyses than the 2^nd or 3^rd position, and hence estimates total glucose turnover with greater accuracy (Choukem and Gautier 2008). The rate of appearance (Ra) of [6,6-²H2] is its rate of infusion, and hence at steady glucose state the proportion of [6,6-²H2] dideuterated glucose amongst total glucose facilitates estimations of endogenous glucose production.

There are 3 main assumptions made about the behaviour of tracer glucose:

1. The tracer is treated and distributed the same as unlabelled glucose.

2. At steady state the rate of tracer infusion is proportional to its disposal.

3. The tracer is not recycled

The first two assumptions have been shown to be essentially valid (Wolfe RR 2004). However, recycling has been shown to occur. Labeled glucose can be taken up peripherally and metabolised to pyruvate and then lactate. The labeled lactate can then return back to the liver and re-form glucose. The resultant endogenous, hepatically synthesised, glucose will be labeled and circulate in the plasma as either ([6,6-²H2]) or ([3,3-²H2]) glucose. As a result some of the products of this recycling and endogenous re-synthesis are indistinguishable from exogenous labeled glucose. The magnitude of this effect is not believed to be great (Finegood, Bergman et al. 1987), though it results in an underestimate of the rate of hepatic glucose production.

Equations of Steele:

The most commonly used equations to determine hepatic insulin sensitivity are the equations of Steele (Steele 1959). These equations rely on the assumptions that there is only a single compartment of distribution and that it has a constant volume. Several authors have questioned these assumptions (Cobelli, Mari et al. 1987; Finegood, Bergman et al. 1987), though these original equations remain the most widely used (Choukem and Gautier 2008).

C^*+ C Ra^*

Rd^*

Ra Rd

Figure 2.1. Depiction of a one compartment model (adapted from (Choukem and Gautier 2008)). Ra: rate of appearance of unlabelled (hepatic + exogenous sources) glucose. R_a^*: rate of appearance of tracer glucose. C: unlabelled plasma glucose concentration. C^*: labeled plasma glucose concentration. R_d: rate of disappearance of unlabelled glucose. Rd*

: rate of disappearance of tracer glucose.

As depicted above in figure 2.1., during steady-state conditions, the combined rate of glucose appearance (R_a + R_a^*) equals its combined disappearance rate (R_d + R_d^*). Furthermore, the ratio of labeled to unlabeled plasma glucose (C^* / C) also reflects the relative rate of the labeled tracer infusion to the rate of endogenous glucose production. So the equations of Steele for hepatic glucose production (HGP) are:

HGP = TIR / (C^* / C)

Where TIR is tracer infusion rate. The greater the HGP the more resistant the liver is to the effects of insulin. HGP can be further related to plasma insulin to form a hepatic insulin resistance index (Matsuda and DeFronzo 1999).

Hepatic insulin resistance index = 1 / (HGP x plasma insulin) x 100

It bears remembering that the liver is not the only glucose-producing organ during fasting conditions. The renal contribution is at least 5% (Choukem and Gautier 2008).

Modifications to the equations of Steele:

In the postabsorptive (fasted) state there is essentially no glycolysis of glucose within the liver, and therefore all hepatically produced glucose enters the circulation. A one-compartmental model can therefore be used in this setting.

However as previously discussed, in the presence of glucose and insulin administration glucose is disposed of in muscle and liver either by oxidation or by non-oxidation (glycogen formation). This results in a non-static volume of distribution during a clamp.

If ‘A’ is the amount of tracer (C^* in figure 2.1.) and ‘B’ is the amount of tracee (C in figure 2.1.), then the enrichment ‘E’ is:

E = A/B

If we determine values at a specific time (t) and changes as then:

At/ t = Et ( Bt/ t)+ Bt( Et/ t) (equation 1)

Changes in the total amount of tracee within a pool ( Bt) over a time period ( t) are equal to the differences between its rate of entry (Ra) and disappearance (Rd):

Bt/ t = Ra – Rd (equation 2)

Changes in the total amount of tracer within a pool ( At) over a time period ( t) are equal to the rate that it is infused (F) minus the rate that it leaves the pool (RdE):

At/ t = F – RdE (equation 3) combining equations 3 and 1:

Et ( Bt/ t)+ Bt( Et/ t) = F – RdE Combining this with equation 2:

Et (Ra – Rd) + Bt( Et/ t) = F – RdE Hence

Ra = [F- Bt( Et/ t)] / Et

As glucose tracee and tracer do not mix instantaneously additional factors need to be added, where ‘p’ is the fraction of the total pool that does not rapidly mix, and the total extracellular glucose space is ‘V’. As a result the amended formula is:

Ra = [F- pV ((C2 + C1)/ 2) * ((E2 – E1) / (t2 – t1))] / ((E2 + E1) / 2)

Where C is concentration, E enrichment and t is time at two time points (1 and 2).

The commonly used value for p is 0.65 (Cowan and Hetenyi 1971), and 25% of body weight for V (Rebrin, Steil et al. 1999).

A large volume of exogenous glucose is typically infused during a glucose clamp which results in a fall in glucose enrichment. The problem of negative values for glucose production can be avoided by the addition of tracer to the exogenous glucose infused, a so-called ‘hot infusion’ protocol (Finegood, Bergman et al.

1987) (Levy, Brown et al. 1989). Such an approach was used by Powrie et al. who developed a formula for Ra (hepatic glucose production) (Powrie, Smith et al.

1992):

Ra = (F / Ep(t)) + (Evar * Ivar(t)) / Ep(t)) – ((p*V*G(t)* ( Ep(t) / t) / Ep(t))

Where F is the glucose tracer constant infusion rate, Ep(t) is the plasma enrichment at time t, Evar is the enrichment of the variable glucose solution used during the

clamp, Ivar_(t) is the infusion rate of that solution, and G is the prevailing glucose concentration.

It will be noted that at steady state the value for Ep(t) is zero and hence Ra is the sum of infusion divided by enrichment of the constant and variable solutions.

From this, endogenous (hepatic) glucose production = Ra – exogenous glucose infusion rate