Toffolo, Gianna

Insulin and nutrients have profound effects on proteome homeostasis. Currently no reliable methods are available to measure postprandial protein turnover. A triple-tracer method was developed using phenylalanine stable isotope tracers to estimate appearance rates of ingested (R a meal ) and endogenous phenylalanine and the rate of phenylalanine disposal (R d ). This was compared with the “traditional” dual-tracer method, using one (1-CM)- and two (2-CM)-compartment models. For both methods, [ 13 C 6 ]phenylalanine was given orally, and [ 15 N]phenylalanine was constantly infused; the triple-tracer method added [ 2 H 5 ]phenylalanine, infused at rates to mimic meal [ 13 C 6 ]phenylalanine appearance. Additionally, incorporation of meal-derived phenylalanine into specific proteins was measured after purification by two-dimensional electrophoresis. The triple-tracer approach reduced modeling errors, allowing improved reconstruction of R a meal with a tracer-to-tracee ratio that was more constant and better estimated R d . The 2-CM better described phenylalanine kinetics and R d than 1-CM. Thus, the triple-tracer approach using 2-CM is superior for measuring non-steady-state postprandial protein turnover. This novel approach also allows measurement of postprandial synthesis rates of specific plasma proteins. We offer a valid non-steady-state model to measure postprandial protein turnover and synthesis of plasma proteins that can safely be applied in adults, children, and pregnant women.

Fasting hyperglycemia occurs when an excessive rate of endogenous glucose production (EGP) is not accompanied by an adequate compensatory increase in the rate of glucose disappearance (Rd). The situation following food ingestion is more complex as the amount of glucose that reaches the circulation for disposal is a function of the systemic rate of appearance of the ingested glucose (referred to as the rate of meal appearance [Rameal]), the pattern and degree of suppression of EGP, and the rapidity of stimulation of the Rd. In an effort to measure these processes, Steele et al. proposed what has come to be referred to as the dual-tracer method in which the ingested glucose is labeled with one tracer while a second tracer is infused intravenously at a constant rate. Unfortunately, subsequent studies have shown that although this approach is technically simple, the marked changes in plasma specific activity or the tracer-to-tracee ratio, if stable tracers are used, introduce a substantial error in the calculation of Rameal, EGP, and Rd, thereby leading to incorrect and at times misleading results. This Perspective discusses the causes of these so-called “nonsteady-state” errors and how they can be avoided by the use of the triple-tracer approach.

Numerous studies have used the dual-tracer method to assess postprandial glucose metabolism. The present experiments were undertaken to determine whether the marked tracer nonsteady state that occurs with the dual-tracer approach after food ingestion introduces error when it is used to simultaneously measure both meal glucose appearance (Ra meal) and endogenous glucose production (EGP). To do so, a novel triple-tracer approach was designed: 12 subjects ingested a mixed meal containing [1-13C]glucose while [6-3H]glucose and [6,6-2H2]glucose were infused intravenously in patterns that minimized the change in the plasma ratios of [6-3H]glucose to [1-13C]glucose and of [6,6-2H2]glucose to endogenous glucose, respectively. Ra meal and EGP measured with this approach were essentially model independent, since non-steady-state error was minimized by the protocol. Initial splanchnic glucose extraction (ISE) was 12.9% ± 3.4%, and suppression of EGP (EGPS) was 40.3% ± 4.1%. In contrast, when calculated with the dual-tracer one-compartment model, ISE was higher ( P < 0.05) and EGPS was lower ( P < 0.005) than observed with the triple-tracer approach. These errors could only be prevented by using time-varying volumes different for Ra meal and EGP. Analysis of the dual-tracer data with a two-compartment model reduced but did not totally avoid the problems associated with marked postprandial changes in the tracer-to-tracee ratios. We conclude that results from previous studies that have used the dual-tracer one-compartment model to measure postprandial carbohydrate metabolism need to be reevaluated and that the triple-tracer technique may provide a useful approach for doing so.

The simultaneous assessment of insulin action, secretion, and hepatic extraction is key to understanding postprandial glucose metabolism in nondiabetic and diabetic humans. We review the oral minimal method (i.e., models that allow the estimation of insulin sensitivity, β-cell responsivity, and hepatic insulin extraction from a mixed-meal or an oral glucose tolerance test). Both of these oral tests are more physiologic and simpler to administer than those based on an intravenous test (e.g., a glucose clamp or an intravenous glucose tolerance test). The focus of this review is on indices provided by physiological-based models and their validation against the glucose clamp technique. We discuss first the oral minimal model method rationale, data, and protocols. Then we present the three minimal models and the indices they provide. The disposition index paradigm, a widely used β-cell function metric, is revisited in the context of individual versus population modeling. Adding a glucose tracer to the oral dose significantly enhances the assessment of insulin action by segregating insulin sensitivity into its glucose disposal and hepatic components. The oral minimal model method, by quantitatively portraying the complex relationships between the major players of glucose metabolism, is able to provide novel insights regarding the regulation of postprandial metabolism.

OBJECTIVE Glucagon-like peptide (GLP)-1 receptor is encoded by GLP1R. The effect of genetic variation at this locus on the response to GLP-1 is unknown. This study assessed the effect of GLP1R polymorphisms on insulin secretion in response to hyperglycemia and to infused GLP-1 in nondiabetic subjects. RESEARCH DESIGN AND METHODS Eighty-eight healthy individuals (aged 26.3 ± 0.6 years, fasting glucose 4.83 ± 0.04 mmol/l) were studied using a hyperglycemic clamp. GLP-1 was infused for the last 2 h of the study (0.75 pmol/kg/min over 121–180 min, 1.5 pmol/kg/min over 181–240 min). β-Cell responsivity (ΦTotal) was measured using a C-peptide minimal model. The effect of 21 tag single nucleotide polymorphisms (SNPs) in GLP1R on ΦTotal was examined. RESULTS Two SNPs (rs6923761 and rs3765467) were nominally associated with altered β-cell responsivity in response to GLP-1 infusion. CONCLUSIONS Variation in GLP1R may alter insulin secretion in response to exogenous GLP-1. Future studies will determine whether such variation accounts for interindividual differences in response to GLP-1–based therapy.

In this article, a first aim was to develop a minimal modeling approach to noninvasively assess hepatic insulin extraction in 204 healthy subjects studied with a standard meal by coupling the already available meal C-peptide minimal model with a new insulin model. The ingredients of this model are posthepatic IDR, which in turn is described in terms of pancreatic ISR and hepatic insulin extraction HE, and a linear monocompartmental model of insulin kinetics. Even if ISR is provided by the C-peptide minimal model, the simultaneous assessment of HE and insulin kinetics is critical, since compensations may arise between parameters describing these two processes. Therefore, as a second aim of this study, a method was developed to predict standard values of insulin kinetic parameters in an individual on the basis of the individual's anthropometric characteristics. The statistical analysis, based on linear regression of insulin kinetic parameters estimated from IM-IVGTT data performed on the same subjects, demonstrated that insulin kinetic parameters can be accurately predicted from age and body surface area. Once kinetic parameters of the new insulin model were fixed to these values, HE profile and indexes during a meal were reliably estimated in each individual, indicating a significant suppression during the meal since the overall index of HE, equal to 60 ± 1% in the basal state, is reduced to 40 ± 1% during a meal. However, standard parameters provide an approximation of the individual one; thus, the third aim was to define the impact on estimated indexes of using standard instead of individually estimated values. Our results showed that the 25% uncertainty affecting as an average insulin kinetic parameters of an individual, when they are predicted from age and body surface area, translates into a similar relative uncertainty in the individual's hepatic insulin extraction indexes.

OBJECTIVE In this study, we sought to determine whether postprandial insulin secretion, insulin action, glucose effectiveness, and glucose turnover were abnormal in type 2 diabetes. RESEARCH DESIGN AND METHODS Fourteen subjects with type 2 diabetes and 11 nondiabetic subjects matched for age, weight, and BMI underwent a mixed-meal test using the triple-tracer technique. Indexes of insulin secretion, insulin action, and glucose effectiveness were assessed using the oral “minimal” and C-peptide models. RESULTS Fasting and postprandial glucose concentrations were higher in the diabetic than nondiabetic subjects. Although peak insulin secretion was delayed (P &lt; 0.001) and lower (P &lt; 0.05) in type 2 diabetes, the integrated total postprandial insulin response did not differ between groups. Insulin action, insulin secretion, disposition indexes, and glucose effectiveness all were lower (P &lt; 0.05) in diabetic than in nondiabetic subjects. Whereas the rate of meal glucose appearance did not differ between groups, the percent suppression of endogenous glucose production (EGP) was slightly delayed and the increment in glucose disappearance was substantially lower (P &lt; 0.01) in diabetic subjects during the first 3 h after meal ingestion. Together, these defects resulted in an excessive rise in postprandial glucose concentrations in the diabetic subjects. CONCLUSIONS When measured using methods that avoid non–steady-state error, the rate of appearance of ingested glucose was normal and suppression of EGP was only minimally impaired. However, when considered in light of the prevailing glucose concentration, both were abnormal. In contrast, rates of postprandial glucose disappearance were substantially decreased due to defects in insulin secretion, insulin action, and glucose effectiveness.

OBJECTIVE—The purpose of this study was to determine the mechanism by which dipeptidyl peptidase-4 inhibitors lower postprandial glucose concentrations. RESEARCH DESIGN AND METHODS—We measured insulin secretion and action as well as glucose effectiveness in 14 subjects with type 2 diabetes who received vildagliptin (50 mg b.i.d.) or placebo for 10 days in random order separated by a 3-week washout. On day 9 of each period, subjects ate a mixed meal. Insulin sensitivity (SI), glucose effectiveness, and β-cell responsivity indexes were estimated using the oral glucose and C-peptide minimal models. At 300 min 0.02 unit/kg insulin was administered intravenously. RESULTS—Vildagliptin reduced postprandial glucose concentrations (905 ± 94 vs. 1,008 ± 104 mmol/6 h, P = 0.02). Vildagliptin did not alter net SI (7.71 ± 1.28 vs. 6.41 ± 0.84 10−4 dl · kg−1 · min−1 · μU−1 · ml−1, P = 0.13) or glucose effectiveness (0.019 ± 0.002 vs. 0.018 ± 0.002 dl · kg−1 · min−1, P = 0.65). However, the net β-cell responsivity index was increased (35.7 ± 5.2 vs. 28.9 ± 5.2 10−9 min−1, P = 0.03) as was total disposition index (381 ± 48 vs. 261 ± 35 10−14 dl · kg−1 · min−2 · pmol−1 · l−1, P = 0.006). Vildagliptin lowered postprandial glucagon concentrations (27.0 ± 1.1 vs. 29.7 ± 1.5 μg · l−1 · 6 h−1, P = 0.03), especially after administration of exogenous insulin (81.5 ± 6.4 vs. 99.3 ± 5.6 ng/l, P = 0.02). CONCLUSIONS—Vildagliptin lowers postprandial glucose concentrations by stimulating insulin secretion and suppressing glucagon secretion but not by altered insulin action or glucose effectiveness. A novel observation is that vildagliptin alters α-cell responsiveness to insulin administration, but the significance of this action is as yet unclear.

OBJECTIVE—We sought to determine whether, and if so the mechanism by which, testosterone replacement improves carbohydrate tolerance. RESEARCH DESIGN AND METHODS—Fifty-five elderly men with relative testosterone deficiency ingested a labeled mixed meal and underwent a frequently sampled labeled intravenous glucose tolerance test before and after either placebo or treatment with testosterone patch (5 mg/day) for 2 years. RESULTS—Despite restoring bioavailable testosterone to values observed in young men, the change (24 months minus baseline values) in fasting and postprandial glucose, insulin, and C-peptide concentrations and meal appearance, glucose disposal, and endogenous glucose production were virtually identical to those observed after 2 years of placebo. The change over time in insulin and C-peptide concentrations post–intravenous glucose injection also did not differ. Furthermore, the change over time in insulin action and glucose effectiveness (measured with the unlabeled and labeled “oral” and “intravenous” minimal models), as well as insulin secretion and hepatic insulin clearance (measured with the C-peptide model), did not differ in the testosterone and placebo groups. CONCLUSIONS—We conclude that 2 years of treatment with testosterone in elderly men does not improve carbohydrate tolerance or alter insulin secretion, insulin action, glucose effectiveness, hepatic insulin clearance, or the pattern of postprandial glucose metabolism. Thus, testosterone deficiency is unlikely the cause of the age-associated deterioration in glucose tolerance commonly observed in elderly men.

OBJECTIVE—To determine the contribution of hepatic insulin resistance to the pathogenesis of impaired fasting glucose (IFG). RESEARCH DESIGN AND METHODS—Endogenous glucose production (EGP) and glucose disposal were measured in 31 subjects with IFG and 28 subjects with normal fasting glucose (NFG) after an overnight fast and during a clamp when endogenous secretion was inhibited with somatostatin and insulin infused at rates that approximated portal insulin concentrations present in IFG subjects after an overnight fast (∼80 pmol/l, “preprandial”) or within 30 min of eating (∼300 pmol/l, “prandial”). RESULTS—Despite higher (P &lt; 0.001) insulin and C-peptide concentrations and visceral fat (P &lt; 0.05), fasting EGP and glucose disposal did not differ between IFG and NFG subjects, implying hepatic and extrahepatic insulin resistance. This was confirmed during preprandial insulin infusion when glucose disposal was lower (P &lt; 0.05) and EGP higher (P &lt; 0.05) in IFG than in NFG subjects. Higher EGP was due to increased (P &lt; 0.05) rates of gluconeogenesis in IFG. EGP was comparably suppressed in IFG and NFG groups during prandial insulin infusion, indicating that hepatic insulin resistance was mild. Glucose disposal remained lower (P &lt; 0.01) in IFG than in NFG subjects. CONCLUSIONS—Hepatic and extrahepatic insulin resistance contribute to fasting hyperglycemia in IFG with the former being due at least in part to impaired insulin-induced suppression of gluconeogenesis. However, since hepatic insulin resistance is mild and near-maximal suppression of EGP occurs at portal insulin concentrations typically present in IFG subjects within 30 min of eating, extrahepatic (but not hepatic) insulin resistance coupled with accompanying defects in insulin secretion is the primary cause of postprandial hyperglycemia.