Diabetes-Associated Variation in TCF7L2 Is Not Associated With Hepatic or Extrahepatic Insulin Resistance

The TCF7L2 locus encodes a transcription factor that is involved in various pathways, most notably the Wnt signaling pathway, and predisposes individuals to type 2 diabetes. Although the diabetes-associated allele in TCF7L2 affects insulin secretion and β-cell function (1), there have been reports that it may also impair insulin action and hepatic glucose metabolism (2–5), thereby potentially leading to increases in both fasting glucose and endogenous glucose production (EGP). Both of these changes occur early in the evolution of type 2 diabetes (6,7).

Previous investigators (8) have reported that variation in TCF7L2 does not alter insulin action. However, the experiment used insulin concentrations (1 mU/kg/min) that result in the maximum suppression of EGP, thereby preventing the assessment of hepatic insulin action. On the other hand, Pilgaard et al. (5) reported increased fasting EGP in a cohort of 37 males heterozygous or homozygous for the diabetes-associated allele at TCF7L2. In this experiment, low-dose insulin infusion (∼0.3 mU/kg/min) suppressed EGP equally regardless of the presence or absence of the diabetes-associated allele at TCF7L2, whereas the effects of high-dose insulin (∼1 mU/kg/min) were impaired in people with diabetes-associated variation in TCF7L2 (5). In contrast, Rasmussen-Torvik et al. (8) used an insulin infusion (∼1 mU/kg/min) during a euglycemic clamp in 40 subjects homozygous for the T allele at rs7903146 and did not detect differences in insulin sensitivity as measured by glucose infusion rate. Lyssenko et al. (9) studied a larger cohort composed of subjects with diabetes, impaired glucose tolerance, or normal glucose tolerance and reported higher basal EGP in individuals with one or two copies of the (T) allele of rs7903146.

Even if TCF7L2 does not alter insulin action, it may alter the relative contributions of gluconeogenesis and glycogenolysis to EGP (10–12) because it regulates multiple genes associated with hepatic glucose metabolism. In people with impaired fasting glucose production, the ability of insulin to suppress gluconeogenesis is impaired, resulting in higher rates of EGP compared with people with normal fasting glucose production (13). It is unknown whether diabetes-associated genetic variation in TCF7L2 also alters insulin-induced regulation of gluconeogenesis and glycogenolysis.

We therefore sought to determine the association of genetic variation in TCF7L2 with insulin-induced suppression of EGP and insulin-induced stimulation of glucose uptake in humans without diabetes. We hypothesized that individuals who are homozygous for the diabetes-associated allele (TT) at rs7903146 have impaired insulin-induced suppression of EGP and of gluconeogenesis compared with individuals with the diabetes-protective (CC) genotype. To do so, we studied subjects without diabetes who had otherwise been matched for anthropometric characteristics using a euglycemic clamp together with a variation of the deuterated water method to accurately measure gluconeogenesis. We report that diabetes-associated variation in TCF7L2 does not alter either hepatic or extrahepatic insulin action or the suppression of gluconeogenesis by insulin.

After approval from the Mayo Clinic Institutional Review Board, we used the Mayo Clinic Biobank, a repository of 20,000 DNA samples collected from volunteers, to perform genotyping of 4,000 individuals at rs7903146. Genotyping was performed using TaqMan (Applied Biosystems, Foster City, CA). The individuals genotyped were randomly selected from the Biobank cohort, as previously described (14). Subjects who were homozygous for the disease-causing allele (TT) were matched for age, gender, fasting glucose level, and body weight with subjects who were homozygous for the disease-protective allele (CC), and were invited in writing to participate in the study. After informed written consent was obtained, subjects underwent a screening examination to ensure that they were good health and were not receiving medications that could influence glucose metabolism.

Participants were admitted to the Clinical Research Unit at 1700 h on the day before the study. After a standard 10 kcal/kg caffeine-free meal, blood was sampled for baseline enrichment, and the subjects fasted overnight. A total 1.67 g/kg deuterated water (²H₂O) was then given in three divided doses at 2200, 2400, and 0200 h.

The following morning at 0600 h, a dorsal hand vein was cannulated and placed in a heated Plexiglas box and maintained at 55°C to allow sampling of arterialized venous blood. The contralateral forearm vein was cannulated for tracer, glucose, and hormone infusions. At 0630 h (−180 min), a primed, continuous infusion of [3-³H] glucose (12 μCi prime, 0.12 μCi/min continuous) and [1-¹³C] acetate (2.5 μmol/kg/min) was started and continued for the duration of the experiment. At 0930 (0 min), an infusion of somatostatin (60 ng/kg/min), glucagon (0.65 ng/kg/min), and growth hormone (0.25 ng/kg/min) was started and maintained for the duration of study. Insulin was also infused at 0.30 mU/kg/min. At this time, a variable infusion of 50% dextrose containing [3-³H] glucose commenced, with the infusion rate varied to maintain glucose at ∼5.5 mmol/L over the period of study. Arterialized venous blood samples were collected to allow the measurement of hormone, tracer, and substrate concentrations.

All blood was immediately placed on ice, centrifuged at 4°C, separated, and stored at −80°C until assay. Glucose concentrations were measured using a glucose oxidase method (Yellow Springs Instruments, Yellow Springs, OH). Plasma insulin was measured using a chemiluminescence assay (Access Assay; Beckman, Chaska, MN). Plasma glucagon and C-peptide were measured by radioimmunoassay (Linco Research, St. Louis, MO). Plasma [3-³H] glucose-specific activity was measured by liquid scintillation counting. The method of Jones et al. (15) was used to measure and analyze deuterium enrichment on the second and fifth carbon of plasma glucose. To correct for errors introduced by transaldolase exchange (16), the estimation of transaldolase exchange was accomplished by the measurement of [3-¹³C] glucose and [4-¹³C] glucose enrichment by nuclear magnetic resonance spectroscopy, as previously described (17).

Glucose appearance and disappearance were calculated using the steady-state equations of Steele et al. (18), where the actual tracer infusion rate was used. The volume of distribution of glucose was assumed to be 200 mL/kg with a pool correction factor equal to 0.65. EGP was calculated by subtracting the glucose infusion rate from the tracer-determined rate of glucose appearance. Fasting and clamp rates represent the mean of the −30- to 0-min and 210- to 240-min values, respectively. All rates of infusion and turnover were expressed per kilogram of lean body mass.

The rate of gluconeogenesis was calculated by multiplying the C5/C2 ratio by the respective EGP. The C5 enrichment was corrected for transaldolase exchange using the following equation: C5_corrected = C5_observed − {([3-¹³C]glucose/[4-¹³C]glucose) × C5_observed}, as previously described (17). Glycogenolysis was then calculated by subtracting the rate of gluconeogenesis from EGP.

Data in the text and figures are expressed as the mean ± SEM. All rates are expressed per kilogram of lean body mass. An unpaired, two-tailed Student t test (or a Mann-Whitney U test for values that were not normally distributed) was used to test differences between the genotype groups. A P value of <0.05 was considered to be statistically significant. The area under the curve was calculated using the trapezoidal rule.

We studied a total of 58 subjects without diabetes. Of these, 28 were homozygous for the diabetes-associated allele (TT) at rs7903146 of TCF7L2 and 30 were homozygous for the diabetes-protective allele (CC). Subjects were matched for age, BMI, and lean body mass (Table 1).

Plasma glucose concentrations (Fig. 1A) did not differ between the CC and TT groups either before (5.0 ± 0.1 vs. 5.0 ± 0.1 mmol/L, P = 0.90) or during the clamp (5.5 ± 0.1 vs. 5.5 ± 0.1 mmol/L, P = 0.89).

Insulin concentrations (Fig. 1B) did not differ before (33.1 ± 4.8 vs. 30.1 ± 2.2 pmol/L, P = 0.59) and during the clamp (90.1 ± 5.5 vs. 92.5 ± 4.6 pmol/L, P = 0.88). Similarly, C-peptide concentrations (Fig. 1C) did not differ before (0.66 ± 0.04 vs. 0.67 ± 0.04 pmol/L, P = 0.84) and during the clamp (0.10 ± 0.02 vs. 0.09 ± 0.01 pmol/L, P = 0.45).

Fasting glucagon concentrations (Fig. 1D) (−81.0 ± 4.4 vs. 94.1 ± 10.4 ng/L, P = 0.24) were slightly but not significantly higher in the TT group compared with the CC group. During the clamp, glucagon concentrations did not differ between the groups.

The glucose infusion rate (Fig. 2A) during the final 30 min of the clamp (3.3 ± 0.4 vs. 3.5 ± 0.5 mg/kg/min, P = 0.79) did not differ between groups. Similarly, specific activity (Fig. 2B) did not differ between the groups and was constant during the fasting and clamp period.

Fasting EGP (13.9 ± 0.5 vs. 13.8 ± 0.4 μmol/kg/min, P = 0.83) and glucose disappearance did not differ between the groups. Insulin-stimulated glucose disappearance (Fig. 2C) (−28.7 ± 2.3 vs. 29.6 ± 3.2 μmol/kg/min, P = 0.82) and insulin-induced suppression of EGP (Fig. 2D) (−6.7 ± 0.9 vs. 6.4 ± 1.0 μmol/kg/min, P = 0.80) also did not differ between the groups (Supplementary Table 1).

After correction for the transaldolase reaction, the rates of fasting gluconeogenesis (Fig. 3A) (−7.2 ± 0.4 vs. 7.4 ± 0.5 μmol/kg/min, P = 0.78) and glycogenolysis (Fig. 3C) (−6.3 ± 0.3 vs. 6.2 ± 0.4 μmol/kg/min, P = 0.55) did not differ between the genotype groups. Similarly, insulin-induced suppression of gluconeogenesis (Fig. 3B) (−2.7 ± 0.3 vs. 2.9 ± 0.5 μmol/kg/min, P = 0.99) and glycogenolysis (Fig. 3D) (−3.3 ± 0.5 vs. 3.3 ± 0.7 μmol/kg/min, P = 0.85) did not differ between the genotype groups (Supplementary Figs. 1–4).

Elucidating the phenotype conferred by the diabetes-associated allele in the TCF7L2 locus has been limited by the relative complexity of the in vivo experiments and the small effect size subject to multiple confounding factors. We sought to ascertain an effect of diabetes-associated genetic variation in TCF7L2 on hepatic glucose metabolism and insulin sensitivity. To do so, we used a cohort of subjects without diabetes who were homozygous for either the diabetes-associated or diabetes-protective allele at rs7903146. The insulin infusion rate was chosen to avoid complete suppression of EGP, and gluconeogenesis was measured using a methodology that avoids a potential source of error in the deuterated water method (16). In these experimental conditions, no effect of variation at TCF7L2 was discerned.

Although the study was powered (80% power [two-sample t test, two-sided α = 0.05]) to detect a relatively small (∼15%) change in EGP or glucose disappearance, it is possible that we missed a smaller effect of the locus on these parameters. However, the similar mean values for the rates of EGP, glucose disappearance, gluconeogenesis, and glycogenolysis suggest that differences between genotype groups, if any, are small and would require unfeasibly large numbers of subjects to study. For example, given the variation in EGP observed, to detect a 5% difference would require more than 200 subjects per genotype group. A limitation of the current study is that we did not attempt to suppress EGP completely using higher doses of insulin (5). This raises the possibility that we missed an effect of TCF7L2 on EGP suppression by high-dose insulin (∼1 mU/kg/min).

It is likely that the diabetes-associated variant in TCF7L2 affects hepatic glucose metabolism indirectly via effects on β-cell and α-cell function. These effects were not apparent in the fasting state during this experiment in which subjects were matched for fasting glucose concentrations at the time of screening. By design, endogenous glucagon (and insulin) secretion during the clamp were inhibited by somatostatin to ensure comparable portal insulin and glucagon concentrations, thereby permitting direct assessment of insulin action. It remains possible that, under conditions of daily living, portal concentrations of glucagon differ between groups (19). Intriguingly, Daniele et al. (20) recently reported that the T allele at rs7903146 was associated with lower glucagon concentrations and decreased systemic meal appearance. This contrasts with our recent findings (14) and suggests the need for further study of hepatic extraction of ingested glucose using techniques designed to minimize measurement error of meal appearance (21).

In conclusion, diabetes-associated genetic variation in TCF7L2 does not impair insulin-induced suppression of EGP, gluconeogenesis, and glycogenolysis. Similarly, it does not impair insulin-induced stimulation of glucose uptake. The data from this experiment suggest that the diabetes-associated variant at TCF7L2 predisposes individuals to diabetes through mechanisms that do not alter insulin action.

Funding. This study was supported by funds from the Mayo Clinic General Clinical Research Center (UL1-TR-000135) and by the National Institutes of Health (DK-78646 and DK-82396). R.T.V. is supported by training grant 5T32-DK-007352-37. Structural funding for the Center for Neurosciences and Cell Biology and the University of Coimbra Nuclear Magnetic Resonance facility is supported in part by FEDER – European Regional Development Fund through the COMPETE Programme and the Portuguese Foundation for Science and Technology through grants EXCL/DTP-PIC/0069/2012, PEst-C/SAU/LA0001/2011, REEQ/481/QUI/2006, RECI/QEQ-QFI/0168/2012, CENTRO-07-CT62-FEDER-002012, and Rede Nacional de Ressonância Magnética Nuclear.

Duality of Interest. A.V. is an investigator in multicenter studies sponsored by Novartis and GI Dynamics and has consulted for XOMA, Sanofi, Novartis, and Bristol-Myers Squibb during the past 5 years. No other potential conflicts of interest relevant to this article were reported.

Author Contributions. R.T.V. and M.S. researched the data and ran the studies. I.V., C.B., and C.M. measured the rates of gluconeogenesis. R.A.R. contributed to the discussion and reviewed and edited the manuscript. J.G.J. oversaw the measurement of gluconeogenesis, contributed to the discussion, and reviewed and edited the manuscript. A.V. designed the study, oversaw its conduct, researched the data, and wrote the manuscript. A.V. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.