The glucose clamp
Rather than using exogenous agents that suppress β-cell insulin secretion, the glucose clamp uses external feedback control to “open the loop” between insulin secretion and sensitivity (Figure 15.2). The glucose clamp is a powerful and widely used method to attain a quantitative measure of insulin sensitivity, and has been applied in many hundreds of experimental studies.
DeFronzo and colleagues published the most widely applied clamp methodology [26,27]. In the simplest “euglycemic” manifestation, catheters are inserted in the right and left arms of the subject, and basal samples are collected, after which insulin is infused intravenously at a constant rate. The dose of insulin has varied widely [26–34]; however, the usual dose is 40 mU min−1 m−2 . Infusing insulin would normally lower the blood glucose; however, the lowering is prevented by infusing glucose concomitant with the insulin infusion. It is not possible a priori to know the rate of glucose infusion to prevent insulin from changing; therefore, a separate external feedback loop is established wherein glucose is measured frequently (usually every 5 min) and infusion rate is chosen to attempt to keep the plasma glucose concentration from changing.
In normal subjects plasma glucose concentration is usually “clamped” at or near 5 mM. Clearly, if a subject is extremely insulin resistant (sensitivity ∼ 0), very little exogenous glucose would be needed to be infused despite increased insulin; if a subject is very sensitive to insulin, the needed exogenous glucose infusion rate will be substantial. The rate of glucose infusion is considered a reflection of insulin sensitivity. Insulin sensitivity is normalized to body size in some manner—either per kg body weight , per surface area [36,37], or per unit fat-free mass [38,39].
It is not a simple matter to determine in real time the exact glucose infusion rate needed to keep glucose at a predetermined “target” value. There have been numerous attempts to automate the external feedback loop, which includes the glucose measuring device as well as the glucose infusion device. Algorithms have been made available to calculate glucose infusion rate independently [26,40–42]. These algorithms have the advantage of objectivity; however, in most laboratories even today exogenous glucose infusion rate is determined at the bedside by a trained individual. The need for frequent monitoring of the blood glucose, and the need for a practiced individual to control the glucose infusion rate as a function of time, renders the glucose clamp method labor intensive and costly. Although providing an accurate measure of insulin sensitivity, under most circumstances three trained individuals are needed to carry on a safe and effective glucose clamp experiment on a human subject.
Components of insulin action calculated from the euglycemic clamp
Under fasting conditions in normal individuals, the blood glucose is determined by a balance between endogenous glucose production by the liver (and kidneys) and glucose uptake by insulin-independent (brain, red blood cells, gut) and insulin-dependent tissues (skeletal muscle, heart, adipose). Infusing insulin lowers the blood glucose both by inhibiting endogenous glucose production (EGP) and by stimulating glucose uptake (Rd). One of the great assets of the glucose clamp is the potential for teasing apart these two effects of insulin on glucose handling. Over three decades accurate methods were established to measure rates of endogenous glucose production — even under conditions of substantial glucose turnover that is observed during clamps [26,43–46]. These methods involve the infusion of glucose either labeled with tracer (usually 3-3H-glucose), or stable isotopes such as 6,6 dideuterated glucose. Glucose output is calculated based upon the dilution by unlabeled endogenously produced glucose of the labeled glucose pool in extracellular fluid (Figure 15.3).
Great care is necessary to make accurate calculations of endogenous glucose production and glucose uptake during glucose clamps [26,45–50]. The large potential error is due to the considerable glucose turnover stimulated by hyperinsulinemia. Endogenous production is the difference between two large numbers: total rate of glucose disposal minus the rate of exogenous glucose infusion. Small errors in estimating either of these rates will cause a very large error in calculation of glucose output. Finegood and colleagues proved that to make an accurate calculation, exogenous glucose must be labeled at a specific activity or enrichment similar to that attained when labeled moiety is infused into the fasting patient . Under such conditions plasma specific activity remains near steady-state and accurate EGP is calculated. Impurities in commercial labeled glucose preparations can lead to spurious results ; thus tracer should be checked and purified if necessary. Also, even being unaware that so-called 50% D/W dextrose solution is only 45.4% glucose can lead to large errors. Thus, it is important to be vigilant in these calculations and to be familiar with the literature on these subjects.
Based upon accurate methods applied to normal individuals, suppression of glucose output by liver is much more sensitive to insulin (ED50 ∼ 25 mU L−1 ) than stimulation of glucose uptake (ED50 ∼ 60 mU L−1 ) [48,50]. Reduced insulin sensitivity as assessed by the clamp method has been demonstrated in an enormous variety of physiologic and pathophysiologic situations including fasting, impaired glucose tolerance (IGT), polycystic ovarian syndrome, pregnancy, children of low birth weight, sedentary lifestyle, obesity, and acromegaly, as well as many ethnic groups from around the globe. Comparing insulin resistance in these different conditions and populations is important because insulin resistance is a primary risk factor for diabetes, but also for many other chronic diseases, including cardiovascular disease, hypertension, and cancer.
A quantitative index from the glucose clamp
As stated, glucose infusion rate during the clamp is an index of insulin sensitivity. Because insulin suppresses endogenous glucose output and increases glucose disposal, infusion of glucose must compensate for both changes under hyperinsulinemic but euglycemic conditions; the infusion rate is thus the sum of these two effects:
Glucose infusion rate = Increase in glucose disposal + Suppression of endogenous glucose production
Changes in either of these components on the right-hand side of this equation may be regarded as contributing to insulin sensitivity; percent suppression of EGP is often considered hepatic insulin sensitivity, whereas increase in glucose disposal (Rd: expressed as mass per unit time per unit body size) is reflective of “peripheral” insulin sensitivity (usually considered the effect of hyperinsulinemia on glucose uptake by skeletal muscle). In populations of normal subjects knowing EGP and Rd changes are often sufficient; however, in pathologic conditions they may be misleading. Glucose uptake is a nonlinear function of glucose and insulin [43,51,52]. It therefore may not be correct to compare Rd values in individuals assessed at different glucose levels or at different insulin levels [43,51]. Generally subjects with fasting hyperglycemia may have their fasting glucose values normalized by infusing exogenous insulin acutely, or overnight [53–55].
Insulin is degraded very rapidly under normal conditions. Because the liver degrades 50% or more of the insulin presented to it during a single passage of blood from the abdominal portal vein to the hepatic veins, the plasma level of insulin achieved during a euglycemic clamp can vary substantially. Generally insulin clearance is reduced in insulin-resistant situations, resulting in proportionately greater plasma insulin levels during insulin infusion during the clamp [56 – 58]. For example, in the canine model, simple fat feeding for 6 weeks decreased fractional insulin clearance by the liver from 60 to 40% for a single passage . To compensate for differences in insulin clearance, many investigators divide glucose disposal (Rd) by the increment in insulin under steady-state conditions (M/I).
We proposed a single variable, the “insulin sensitivity index,” which attempts to correct or normalize for differences in plasma glucose and insulin concentration at which clamps may be performed [60 – 63]. The clamp-based index is defined as the following:
SiP = ΔI ∗ G
in which SiP represents peripheral insulin sensitivity, the numerator is the increase in glucose disposal during a clamp, and the denominator is the product of the insulin increment during the clamp, and the ambient glucose at which the clamp is performed. Thus, this equation calculates increase in glucose clearance per unit increase in the plasma insulin value (Figure 15.4).