Whole-Body Glycolysis Measured by the Deuterated-Glucose Disposal Test Correlates Highly With Insulin Resistance In Vivo

The ²H-GDT consists of a 75- or 30-g glucose load containing 15 g of [6,6-²H₂]glucose. For C-6–labeled glucose, >90% of C–H hydrogen atoms are lost to tissue water after glycolytic metabolism to pyruvate and oxaloacetate, but C–H hydrogen atoms are otherwise nonlabile and are retained in the glucose molecule (27). Complete glycolytic disposal of 15 g of [6,6-²H₂]glucose results in the release of 0.0824 mol of ²H₂O. Dispersion in the body water pool (∼2,200 mol/70-kg human) results in body water enrichment of about 0.0037% (250 δ units) in humans, well above the detection limit (∼1 δ) for ²H₂O by isotope ratio-mass spectrometry (IR-MS). The ²H-GDT was designed to adhere to the following principles: 1) ambient glucose and insulin concentrations should reflect metabolic conditions present physiologically in daily life; 2) the test should primarily quantify insulin-mediated glucose utilization by tissues, rather than other aspects of insulin action, such as suppression of hepatic glucose production; and 3) the metabolic conditions should be comparable to those present in tests proven to be predictive for cardiovascular outcomes and type 2 diabetes risk (i.e., euglycemic-hyperinsulinemic clamp and SSPG test).

Some features of glucose homeostasis are worth noting. First, suppression of endogenous glucose production (EGP) is essentially complete during a clamp or SSPG test (28). Second, insulin concentrations influence the relative contributions from glycolytic and glycogenic routes of glucose disposal. At supraphysiologic concentrations (e.g., >1,000 pmol/l), glucose disposal is dominated by the pathway in the body with the greatest glucose utilization capacity (muscle glycogen storage), whereas glycolysis reaches a maximum and becomes less informative. At low insulin concentrations (e.g., <120–180 pmol/l), hepatic glucose production is not fully suppressed (29), and insulin-independent glucose utilization (e.g., by the brain) makes a proportionately greater contribution to glucose utilization. Accordingly, our criteria for a glycolysis-based test of insulin resistance are best fulfilled at serum insulin concentrations in the dynamic range, as achieved by a 75-g oral glucose load (15).

Operationally, any measure of tissue insulin sensitivity must either control insulin concentrations (e.g., the clamp or SSPG test) or correct for them (e.g., HOMA or frequently sampled intravenous glucose tolerance test). Glucose utilization rates cannot be compared directly as a metric of tissue insulin sensitivity if insulin levels are different. Accordingly, the observed glycolytic rate was corrected here for ambient insulin concentrations to calculate insulin sensitivity of tissues. If glucose concentrations are variable or out of the normal range, correction for glucose concentrations can also be performed to account for glucose effectiveness (30).

All subjects gave written, informed consent. Protocols were approved by the appropriate institutional review boards. Two studies were performed.

Ten healthy nonobese subjects and 10 subjects with the metabolic syndrome were recruited and studied at the Diabetes and Glandular Disease Center in San Antonio, Texas (a fee for service clinical trial site). Metabolic syndrome was defined by the presence of three of five modified Adult Treatment Panel III criteria (20): blood pressure ≥130/85 mmHg or taking antihypertensive medication, fasting triacylglycerol concentrations ≥1.7 mmol/l (150 mg/dl) or treated with gemfibrozil or fenofibrate, fasting glucose concentrations ≥5.5 mmol/l (100 mg/dl), fasting HDL cholesterol ≤1.0 mmol/l (40 mg/dl) for men or ≤1.3 mmol/l (50 mg/dl) for women, and waist circumference ≥102 cm for men or ≥88 cm for women. Subjects with known type 2 diabetes, subjects with fasting glucose ≥7.0 mmol/l (126 mg/dl), or subjects being treated with medications known to alter insulin sensitivity were excluded. Subjects underwent a 75-g ²H-GDT followed within 2 weeks by a clamp study. Subjects then returned 6–8 months later for a 30-g ²H-GDT.

Overweight (n = 12) and obese subjects (n = 6) screened for a weight loss study at Stanford University underwent both an SSPG test and a 75-g ²H-GDT. Subjects with BMI >35 kg/m², fasting plasma glucose ≥7.0 mmol/l, variable weight, or a history of major organ disease and subjects taking drugs known to alter insulin sensitivity were excluded.

After a 10- to 12-h overnight fast, subjects drank 75 or 30 g of glucose, of which 15 g was [6,6-²H₂]glucose, dissolved in 300 ml of water. Plasma samples for glucose, insulin, and ²H₂O content were obtained at baseline and hourly for up to 4 h.

The clamps were performed as described by DeFronzo et al. (7). Briefly, after a 10-h overnight fast subjects received a primed, continuous insulin infusion (40 mU/m² per min) for 110 min. Blood glucose concentrations were clamped at 5 mmol/l with an exogenous glucose infusion (20% wt/vol).

Insulin-mediated glucose disposal was quantified as described previously (11). Briefly, subjects were admitted to the Stanford General Clinical Research Center after a 12-h overnight fast for 180-min infusions of octreotide (0.27μg/m² per min), insulin (25 mU/m² per min), and glucose (240 mg/m² per min).

Total body water (TBW) in the clamp study was measured by bioelectrical impedance analysis (Tanita model TBF 300A). As a comparison, TBW was also calculated from weight and height using the Hume formula (31). TBW for the SSPG study was calculated using the Hume formula.

For ²H₂O content, 100-μl aliquots of plasma in the cap of an inverted vial were placed in a 70°C glass bead–filled heating block overnight. Water distillate inside the vial was then collected. Samples were run in triplicate.

Plasma glucose was measured by the glucose-oxidase method. Plasma insulin concentrations were measured by radioimmunoassay (Linco, St. Charles, MO).

The deuterium content of plasma samples was determined using a ThermoFinnigan High Temperature Conversion/Elemental Analyzer coupled with a ThermoFinnigan MAT 253 isotope ratio-mass spectrometer via a Conflo-III Interface. The deuterium isotope abundance is first calculated in δ ²H values relative to the international Vienna Standard Mean Ocean Water standard and then transformed to atom percent excess by using a calibration curve of standards.

²H₂O enrichment from IR-MS was converted to millimoles by multiplying enrichment by the TBW pool size and dividing by 20 (molecular weight ²H₂O). Plasma insulin and glucose areas under the curves (AUCs) were calculated using the trapezoidal method.

The ²H-GDT parameter of insulin sensitivity was calculated as ²H₂O production (millimoles) per unit of insulin exposure (insulin AUC). M values (milligrams per kilogram per minute), SSPG results, and steady-state plasma insulin concentrations were determined during the last 30 min of the clamp. HOMA (12), QUICKI (14), and insulin sensitivity indices (Matsuda) (15) were calculated as referenced. Plasma glucose was determined during the last 30 min of the SSPG test; the higher the SSPG concentration, the more insulin resistant an individual is.

Differences between independent groups were determined by the Mann-Whitney U test. Correlations were calculated with the Pearson's correlation test. P ≤ 0.05 was considered statistically significant.

Healthy lean subjects and subjects with the metabolic syndrome were recruited to compare the 75-g ²H-GDT with the clamp across a range of insulin sensitivities. Technical problems occurred with the clamp in three subjects, preventing completion of the study, leaving 17 subjects. Fourteen subjects also had a follow-up 30-g ²H-GDT to determine whether a lower glucose load allowed a shorter ²H-GDT. Clinical characteristics of subjects are shown in Table 1.

During the clamp, SSPG concentrations were 5.1 ± 0.3 mmol/l for control subjects vs. 4.9 ± 0.3 mmol/l for subjects with metabolic syndrome and steady-state plasma insulin concentrations were 382 ± 54 vs. 502 ± 116 pmol/l, respectively (P < 0.05). Peak plasma insulin concentrations after the 75-g oral glucose challenge were 309 ± 123 pmol/l for control subjects and 837 ± 352 pmol/l for subjects with metabolic syndrome. After the 30-g oral glucose challenge, peak insulin concentrations were 155 ± 73 pmol/l for control subjects and 371 ± 191 pmol/l for subjects with metabolic syndrome. No significant difference between groups was seen in glucose AUC for the 75-g ²H-GDT, although a significant difference between groups was seen after a 30-g glucose load (2 h, P < 0.01).

The M value from the clamp was significantly lower in subjects with metabolic syndrome (Fig. 1A). The 75-g (Fig. 1B) and the 30-g (data not shown) ²H-GDT results revealed a proportional decrease in insulin sensitivity almost identical to that with the clamp in subjects with metabolic syndrome. The 75-g ²H-GDT correlated extremely closely with the M value across all subjects (r = 0.93 at 3 h and r = 0.95 at 4 h) (Figs. 1C and D). Use of the Hume formula instead of bioelectrical impedance analysis to determine TBW did not change correlations. A significant correlation with the M value was seen for the 30-g ²H-GDT as early as 1–2 h after the glucose load (r = 0.88) (Figs. 1E and Table 2), better than the 2-h correlation seen with the 75-g test (r = 0.75). Adjusting the 75-g ²H-GDT values for differences in glucose concentrations (glucose AUC) did not improve correlations, although a slight improvement was seen for 30-g ²H-GDT values (r = 0.92).

Correlations between the clamp, ²H-GDT, and other indexes of insulin resistance are shown in Table 2. When data from all subjects were included, the best correlation was with the 75-g ²H-GDT, whereas only modest correlations were seen for fasting indexes of insulin resistance and Matsuda insulin sensitivity indices. Considering subjects in the metabolic syndrome group alone, only the 75-g ²H-GDT correlated significantly with the M value. Correlation coefficients improved slightly when glucose AUC was included. For subjects in the control group alone, both the 75- and the 30-g ²H-GDT correlated significantly with the clamp. Insulin AUC had a weaker but significant correlation at 4 h after the 75-g glucose load. When differences in glucose AUC were taken into account in the control group, the correlation coefficients improved for the 30-g but not for the 75-g ²H-GDT.

Clinical characteristics of subjects who participated in the SSPG study are shown in Table 1. SSPG values in these obese and overweight subjects ranged from 2.7 to 14.9 mmol/l, indicating a broad range of insulin sensitivities. Previous studies have shown that SSPG values >10 mmol/l represent the top tertile denoting insulin resistance (32). Correlation between the SSPG value and the ²H-GDT (Fig. 1F) was excellent (r = −0.874).

We demonstrate here that, in humans, insulin-mediated whole-body glycolysis correlates closely with insulin sensitivity in states of normal and reduced insulin sensitivity and may be useful as a potential metric of insulin resistance.

Insulin-mediated whole-body glycolytic disposal of a glucose load was measured by the ²H-GDT in insulin-resistant and insulin-sensitive individuals and correlated extremely well with M values during the clamp. Correlations between the clamp and ²H-GDT were stronger than correlations with indirect markers of insulin resistance and remained strong even in lean control subjects alone, for whom correlations with other markers have historically been poor (19). The SSPG test also correlated well with the ²H-GDT, whereas other markers of insulin resistance have traditionally correlated poorly (33).

Metabolic influences on glycolysis are worth considering. Factors that reduce glucose transport and/or phosphorylation should impair glycolysis and glycogen synthesis in parallel. In contrast, high-fat diet feeding to rats was reported (34) to reduce glycolytic disposal before reducing glycogen synthesis. It is possible that fatty acid oxidation products may inhibit glycolytic enzymes, such as phosphofructokinase, more than glucose phosphorylation or transport. We did not see dissociation between insulin-mediated glycolysis and insulin-mediated total glucose disposal in humans with insulin resistance, however, based on comparisons of the ²H-GDT to clamps and the SSPG test.

The ²H-GDT was designed to measure glycolysis, not to complete oxidation of a glucose load. Another stable isotope method, based on measurement of whole-body oxidation of [U-¹³C₆] glucose to ¹³CO₂, has recently been described for assessing insulin resistance. This approach gave a lower correlation with clamps (r = 0.69) (35) than the ²H-GDT. Several factors may account for the lower correlation. A ¹³CO₂ collection represents oxidation at a single time point, whereas measurement of ²H₂O production reveals integrated glycolytic flux over the preceding 3- to 4-h period. In addition, extensive exchange of the ¹³C label from ¹³CO₂ into cellular metabolites reduces recovery of ¹³CO₂ in breath in an unpredictable manner (36), whereas ²H₂O distributes predictably into body water. Dietary and endogenous fatty acids may also affect pyruvate dehydrogenase activity, independent of insulin sensitivity (37), so that complete oxidation of glucose may be dissociated from glycolysis or glycogen synthesis. Finally, the low total glucose load given (15 g) for the breath ¹³CO₂ test (35) interrogates a different physiological state and insulinemic level than is present in clamps (e.g., EGP or insulin-independent glucose utilization may confound results).

Hyperglycemia per se can stimulate uptake and glycolytic disposal of glucose (“glucose effectiveness”) (30), potentially increasing ²H₂O production. Accordingly, we corrected for the glucose AUC, although this correction had, at most, a minor impact on calculated insulin sensitivity in the euglycemic subjects studied here. Conversely, hyperglycemia can also reduce ²H₂O production. A glucose load mixes into the whole-body pool of free glucose of 15–20 g, so that the throughput of glucose after a 75-g glucose challenge is much higher than the pool size present before the load. If the fasting blood glucose concentration is 11 mmol/l, the pool size increases to 30–40 g; at >17 mmol/l, the fasting pool size of glucose becomes quantitatively significant in comparison with the glucose load. Thus, the presence of fasting hyperglycemia may dilute exogenous-labeled glucose and reduce recovery of ²H₂O from an exogenous load, but this effect is modest unless hyperglycemia is severe.

The effects of EGP on the ²H-GDT are also worth considering. One of the advantages of the ²H-GDT (compared with glucose concentrations, for example) is that it primarily reflects glucose utilization, not hepatic insulin resistance. EGP might dilute exogenous [²H]glucose, however, and reduce recovery of ²H₂O. An oral glucose load normally suppresses fasting EGP by ∼60%, from ∼2 to ∼0.8 mg · kg⁻¹ · min⁻¹ during the next 3–4 h (38). Endogenously produced glucose (roughly 14 g over 4 h) mixes with exogenous glucose (75 g), for a total flux rate of ∼90 g of glucose through the bloodstream. This, in turn, mixes with the ∼20 g of free glucose in the body before the glucose load. Total “exposure” to glucose over the 3–4 h after a 75-g glucose load is therefore normally about 110 g, with EGP providing about 11–14 g, so that dilution of labeled glucose by EGP normally affects ²H₂O recovery only modestly. If a euglycemic, insulin-resistant subject starts with a higher EGP (2.5–3.0 mg · kg⁻¹ · min⁻¹, for example) and EGP is only suppressed by 40% (to 1.5–1.8 mg · kg⁻¹ · min⁻¹), EGP will be about twice normal (∼25–30 g over 4 h). Total flux of glucose will increase to ∼100–105 g/4 h, and the total glucose exposure will increase to 120–125 g. Thus, if EGP after a glucose load is twice normal, the net effect is to dilute exogenous label by about 15–20% and potentially reduce ²H₂O production proportionately. This result is less than the differences observed between lean control subjects and subjects with metabolic syndrome (Fig. 1B). In diabetic patients, EGP may be even higher and less suppressible. Although we did not measure EGP here, it could be measured after a ²H-GDT based on the die-away curve of plasma [²H]glucose. Given these factors, the ²H-GDT is most easily interpreted in the presence of normal or near-normal glucose concentrations, which is the setting in which metrics of insulin resistance are most lacking. Independent validation of the ²H-GDT in diabetes will be required.

On the basis of these findings, the ²H-GDT is of interest as an index of insulin resistance. The ²H-GDT correlates closely with the euglycemic-hyperinsulinemic glucose clamp and the SSPG test. In addition, this measurement approach has a fundamental advantage over measures of insulin resistance that are based on serum insulin concentrations, including derived parameters such as HOMA or QUICKI. The loss of β-cell function that develops in the progression to type 2 diabetes (39,40) represents a fundamental problem for use of insulin concentrations alone as markers of insulin resistance. An individual who exhibits progressively lower insulin levels over time might either have improving insulin sensitivity or be progressing to β-cell failure. Indeed, a low insulin sensitivity index is better at predicting cardiovascular disease (41) than high postchallenge insulin concentrations. In contrast, the ²H-GDT overcomes the problem of pancreatic response, because the ratio of ²H₂O production to insulin AUC remains low even if insulin secretion is failing.

The ²H-GDT does have some limitations. IR-MS is not a routine technique in clinical laboratories and requires special expertise. Future analytic advances (e.g., laser-based spectroscopic instruments) may overcome this limitation. Other limitations include the 2–3 h required to complete the test, with blood samples being drawn during that time. Although the ²H-GDT is considerably easier than the clamp, it does not have the ease of a single blood sample.

In summary, the ²H-GDT, a measure of whole-body glycolysis in response to a physiological glucose load, has excellent correlation with the euglycemic-hyperinsulinemic glucose clamp and SSPG test in humans across a wide range of insulin sensitivities. Accordingly, impaired insulin-mediated whole-body glycolytic disposal of a glucose load is a feature of insulin resistance in humans and provides a quantitative metric of insulin sensitivity.

We thank Mark Kipnes and the Diabetes and Glandular Disease Center in San Antonio, Texas, for performing the clamps and Alex Fong for his assistance.