Assessing Insulin Sensitivity and Postprandial Triglyceridemic Response Phenotypes With a Mixed Macronutrient Tolerance Test

Abstract

The use of meal challenge tests to assess postprandial responses in carbohydrate and fat metabolism is well established in clinical nutrition research. However, challenge meal compositions and protocols remain a variable. Here, we validated a mixed macronutrient tolerance test (MMTT), containing 56-g palm oil, 59-g sucrose, and 26-g egg white protein for the parallel determination of insulin sensitivity and postprandial triglyceridemia in clinically healthy subjects. The MMTT was administered in two study populations. In one, women with overweight/obese BMIs (n = 43) involved in an 8-week dietary intervention were administered oral glucose tolerance tests (OGTTs) and MMTTs within 2 days of each other after 0, 2, and 8 weeks of the dietary intervention. In the other, 340 men and women between 18 and 64 years of age, with BMI from 18-40 kg/m2, completed the MMTT as part of a broad nutritional phenotyping effort. Postprandial blood collected at 0, 0.5, 3, and 6 h was used to measure glucose, insulin, and clinical lipid panels. The MMTT postprandial insulin-dependent glucose disposal was evaluated by using the Matsuda Index algorithm and the 0- and 3 h blood insulin and glucose measures. The resulting MMTT insulin sensitivity index (ISIMMTT) was strongly correlated (r = 0.77, p < 0.001) with the OGTT-dependent 2 h composite Matsuda index (ISIComposite), being related by the following equation: Log (ISIComposite) = [0.8751 x Log(ISIMMTT)] -0.2115. An area under the triglyceride excursion curve >11.15 mg/mL h-1 calculated from the 0, 3, and 6 h blood draws established mild-to-moderate triglyceridemia in agreement with ∼20% greater prevalence of hypertriglyceridemia than fasting indications. We also demonstrated that the product of the 0 to 3 h and 3 to 6 h triglyceride rate of change as a function of the triglyceride incremental area under the curve optimally stratified subjects by postprandial response patterns. Notably, ∼2% of the population showed minimal triglyceride appearance by 6 h, while ∼25% had increasing triglycerides through 6 h. Ultimately, using three blood draws, the MMTT allowed for the simultaneous determination of insulin sensitivity and postprandial triglyceridemia in individuals without clinically diagnosed disease.

Clinical trial registration: [https://ichgcp.net/clinical-trials-registry/NCT02298725" title="See in ClinicalTrials.gov">NCT02298725; NCT02367287].

Keywords: fat tolerance test; insulin patterns; insulin sensitivity; meal challenge test; phenotyping; postprandial triglyceridemia; triglyceridemia.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2022 Newman, Krishnan, Borkowski, Adams, Stephensen and Keim.

Figures

FIGURE 1

Study recruitment and data restrictions for the mixed macronutrient tolerance test (MMTT) analysis. (A) The individual Metabolism and Physiological Signatures Study (iMAPS) recruited 52 female (F) individuals with BMIs in the overweight to the obese range. Of these, 44 completed the entire 8-week intervention. A single individual was lost to the triglyceride analysis due to difficulty in blood collection (B) The WHNRC nutritional phenotyping study recruited 393 male (M) and F participants with attempts to balance into 3 age and BMI ranges. Of these, 340 were retained for analyses.

FIGURE 2

Glucose and insulin responses of insulin-sensitive and insulin-resistant iMAPS participants to a 75 g glucose oral glucose tolerance test (OGTT) and a 50 g sucrose containing mixed macronutrient meal challenge test (MMTT). The OGTT (A) glucose and (B) insulin responses of participants identified as insulin sensitive (n = 8) or insulin sensitive (n = 35) by the 2 h ISIComposite. The MMTT (C) glucose and (D) insulin responses of the participants identified as insulin sensitive (n = 10) or insulin sensitive (n = 33), the 3 h ISIMMTT. In panels (A–D): • = insulin sensitive; ○ = insulin resistance. (E) iMAPS participant MMTT-dependent postprandial insulin response patterns. (F) Phenotyping participant MMTT-dependent postprandial insulin response patterns. Postprandial response patterns were adapted from previously reported 4 h OGTT-dependent insulin response patterns (–31). Pattern I – normal = peak insulin at 0.5 h, 3 h insulin <20% of 0.5 h insulin; Pattern II – delayed insulin decline = peak insulin at 0.5 h, 3 h insulin <between 20 and 65% of 0.5 h insulin; Pattern III – delayed peak insulin = 3 h insulin >65% of 0.5 h insulin; Pattern IV – high-fasting insulin – 0 h insulin >50-μ units/ml; Pattern V- low insulin = no insulin >15-μ units/ml. iMAPS results are the mean ± standard error of the means of triplicate measurements at 0, 2, and 8 weeks of intervention. Phenotyping results are means ± standard errors of the participants within each postprandial insulin pattern.

FIGURE 3

Postprandial triglyceride response in phenotyping cohort individuals characterized with normal triglyceridemia, normal triglyceridemia to mild hypertriglyceridemia, mild-to-moderate hypertriglyceridemia. Cutoffs established for the normal, normal-mild, and mild-moderate postprandial triglyceridemia were 11.15 and 14.47 mg/ml h–1 for the 6 h area under the triglyceride curve calculated from the 0, 3, and 6 h plasma measurements (i.e., 6h AUCTG). Measurements that do not share annotations differ at p < 0.05 by Tukey’s HSD post hoc analysis. The normal group 0 to 3 h slopes differ from other groups (p < 0.0001). The normal-mild group 3 to 6 h slopes differ from other groups (p < 0.0016). Results represent means ± standard deviations.

FIGURE 4

The postprandial triglyceride area under the curve x kinetic response groups among 340 clinically healthy free-living individuals. Data shown are the mean ± SD for the concentrations of the participants identified within five equal intensity groups of the population-wide Log [AUCTG + 1] and one of 4 postprandial kinetic behaviors (Groups I–IV). (A) Group I plasma triglycerides appeared quickly and disappeared rapidly (n = 54; 16%); (B) Group II plasma triglycerides appeared moderately and disappeared slowly (n = 231; 68%); (C) Group III plasma triglycerides appeared continuously through 6 h (n = 49, 14%); (D) Group IV plasma triglycerides showed low or delayed postprandial appearance (n = 6, 1.8%). Postprandial responses were assigned using the following rules: Group I – [(ka*ke)/AUCTG] < 0.033 and [ka/AUCTG] >0.021; Group II –0.33 ≤[(ka*ke)/AUCTG] < 0.0056 and [ka/AUCTG] >0.01; Group III – [(ka*ke)/AUCTG] ≥0.0056 and (ka/AUCTG) >0.01; Group IV – (ka/AUCTG) ≤0.01]. Time-dependent changes in triglyceride levels within identified kinetic groups were evaluated using least squares regression mixed models with plasma triglyceride levels as the outcome variables with time, the AUCTG kinetic pattern group and the AUCTG intensity group as fixed effects, with the participant as a random effect, followed by Tukey’s HSD post hoc testing. Time points annotated with different letters within each TG kinetic group are different at p < 0.05.

FIGURE 5

Phenotyping cohort postprandial triglyceride kinetic analysis demonstrated significant phenotypic variance. Panels show: (A) the triglyceride rate of change in the 0 to 3 h early phase (kEP) as a function of the incremental area under the triglyceride curve (incAUCTG); (B) the triglyceride rate of change in the 3 to 6 h late phase (kLP) as a function of the incAUCTG; (C) A Johnson-normalized kEP x kLP product as a function of the Johnson-normalized incAUCTG. The quartiles of the Johnson Su [(kEP x kLP)/incAUCTG] defined four kinetic response groups (A-D): Group A – early-phase increase/substantial late-phase decrease (orange; n = 86); Group B – early-phase increase/minimal late-phase decrease (blue; n = 83); Group C – early-phase increase/no late-phase decrease (green; n = 85); Group D – early-phase increase/late-phase increase (purple; n = 84). Note, quartiles do not have the same number of participants due to a small percentage of individuals with identical values. The Johnson Su [(kEP x kLP)/incAUCTG] and kLP/incAUCTG differ between each kinetic group by one-way ANOVA with a Tukey post hoc analysis (p < 0.05). Symbols indicate the estimated postprandial triglyceridemia: • = Normal (6h AUCTG <11.15 mg/ml h–1); ○ = mild-moderate (6h AUCTG >11.15 mg/ml h– 1).

FIGURE 6

The postprandial triglyceride incremental area under the curve x kinetic response patterns of 340 clinically healthy free-living individuals. Triglyceride kinetic response types A, B, C, and D are defined by the quartiles of the Johnson Su [(kEP x kLP)/incAUCTG], which describes the product of the 0 to 3 h and 3 to 6 h triglyceride rates of change in relation to the incremental area under the triglyceride curve. The range of the population-wide Log [incAUCTG + 1] was further subdivided into five equal TG concentration intensity groups. (A) Plasma triglyceride kinetic response Group A showed a rapid early period increase and late-period decrease. (B) Plasma triglyceride kinetic response Group B showed modest early-period increase with minimal but significant late-period decrease. (C) Plasma triglyceride kinetic response Group C showed apparent modest early-period increase but insignificant TG change in the late period. (D) Plasma triglyceride kinetic response Group D showed elevating TG levels in both the early and late periods. The occurrence of mild-to-moderate triglyceridemia by the kinetic group was A (n = 38; 44%), B (n = 19; 22%), C (n = 20; 23%) and D (n = 30; 36%). Results are means ± stdev of each intensity group. Time-dependent changes in triglyceride levels within identified kinetic groups were evaluated using least squares regression mixed models with fasting-corrected plasma triglyceride levels as the outcome variables, time, the incAUCTG kinetic pattern group, and the incAUCTG intensity group as fixed effects, and the participant as a random effect, followed by Tukey’s HSD post hoc testing. Time points annotated with different letters within each TG kinetic group are different at p < 0.05.

FIGURE 7

The intra-individual variance in (A) early-phase postprandial triglyceride change (kEP) and (B) late-phase postprandial triglyceride change (kLP) from the plasma measured at 0, 2, and 8 weeks of the iMAPS dietary intervention. The 43 participants are ordered by decreasing kEP x kLP and colored by the median kEP x kLP kinetic quartile group (A, B, C, or D) of the three measurements. Considering all participants, and controlling for the participant as a random effect, EP increased (p = 0.012) and kLP tended to decrease (p = 0.06) over the 8-week intervention, but was not affected by diet type, age, or BMI.

FIGURE 8

Resting and postprandial energy metabolism shows subtle differences among MMTT triglyceride kinetic response groups. Mixed models of (A) resting and postprandial energy expenditure (EE) and (B) the respiratory exchange ratio (RER) were constructed using lean body mass, BMI, time, and the Johnson (kEP x kLP)/incAUCTG kinetic quartile group as fixed effects and the participant as random effect. Results are adjusted least square means ± standard errors. Results of contrast post-tests of Group A vs. other kinetic groups are shown. Constructing the same models with Johnson (kEP x kLP)/incAUCTG as a continuous variable indicated a negative correlation between this factor and EE (p = 0.0056) but not RER (p = 0.1).