Subjects characteristics
The T2D were older (p < 0.0001; average age 58 years) and had higher BMI (p < 0.0001; 29 kg/m2) compared to the healthy subjects (average age 42 years and BMI 23 kg/m2) (Table 1). Fasting glucose levels were higher (p < 0.0001) in T2D (average of 10.2 mmol/L) compared to the healthy subjects (average of 5.4 mmol/L).
Table 1 Demographic characteristics and fasting laboratory values of subjects at inclusion (mean (SD))
PhenFlex test response characterization in healthy subjects
Statistical analysis revealed that 110 of 132 parameters responded significantly to PFT drink in healthy subjects (Additional file 1: Table S1, Fig. 2). These 110 significantly modulated plasma parameters were grouped into 5 discrete response time course clusters (Fig. 3). Cluster 1 was the biggest cluster represented by 37 out of 110 plasma parameters. These parameters increased during most of the 8 h time course. Cluster 2 represents 8 plasma parameters that mainly decreased during most of the time course of 8 h. Time profile cluster 3 represented the 31 plasma parameters that decreased upon PFT, with a subsequent recovery phase. Cluster 4 represented 8 plasma parameters with a classical absorption profile, reaching maximum concentrations around 4 h after PFT challenge, followed by a continued reduction towards baseline values at the final (8 h) time point. Cluster 5, represented by 26 plasma parameters has a similar profile to cluster 4, with the main difference that parameters in cluster 5 reached maximum values around 1 h and that minimum concentrations were reached at the final time point (8 h), which were below baseline values. The healthy physiological responses of main metabolic processes modulated by PFT are described in more detail below.
Gut
Gut hormone production
In response to the lipid load, the gut secretes the incretin GIP. GIP shows a temporary increase in response to PFT drink, with maximum concentrations 4 h postprandially (cluster 4), similar as to TG response profile. Also, GLP-1 showed a similar time profile as compared to GIP (cluster 4).
Adipose tissue
Lipolysis
Plasma concentrations of both NEFA and glycerol increased after a lag time (cluster 1). Insulin, immediately released after PFT intake, suppresses fat mobilization for energy production. In the late (catabolic) phase of the 8-h time course, adipose tissue triglycerides are hydrolyzed by hormone-sensitive lipase for beta-oxidation, evidenced by the observed increased plasma levels of NEFA and glycerol. The various plasma free fatty acids either showed the same time cluster 1 response as NEFA or decreased plasma concentrations with subsequent recovery (cluster 3 response). Subsequently, also the plasma monoglycerides and diglycerides showed increased concentrations after a lag time (cluster 1).
Adipokine production
Leptin, the hormone that regulates the amount of fat storage, is secreted when the amount of fat storage has reached a certain threshold. This adipokine showed increased concentrations in the late time frame of PFT response (cluster 1).
Kidney
(Re)absorption
Measuring serum creatinine is the most commonly used indicator of renal function. Creatinine showed decreased concentrations in response to PFT (cluster 2).
Urea cycle
Urea, which is formed in the urea cycle by deamination of amino acids in the liver, is a waste product excreted by the kidney in the urine. Also, urea showed decreased concentrations in response to PFT (cluster 2). Together, these observations suggest that glomerular filtration rate of kidneys increased in response to PFT.
Vasculature
Endothelial integrity
The plasma total, HDL, and LDL cholesterol concentrations decreased in response to PFT with subsequent recovery (cluster 3). Also, the plasma adhesion markers sICAM and sVCAM showed the same response to PFT as compared to cholesterol parameters. Finally, SAA showed decreased concentrations in response to PFT (cluster 2). Together, this suggests that there was a temporarily reduced vascular response after PFT.
Muscle
Protein metabolism
Most plasma amino acids showed a classic absorption profile in response to PFT (cluster 5). This is a rapid increase which returns to baseline within 4–5 h and with concentrations below fasting concentration at the final time point (8 h). Some of the amino acids, however, showed decreased concentrations in response to PFT (glycine and tryptophan, cluster 2).
Muscle tissue injury control
The amino acid derivatives 3-methylhistidine and 1-methylhistidine together with creatinine that originate from muscle showed linear decreasing concentrations in response to PFT (cluster 2). Together, this suggests that muscle turns into an anabolic state after consumption of PFT.
Liver
Core metabolism
The glycolysis intermediate pyruvate showed a classic absorption profile (cluster 5), similar to glucose and most amino acids. Glycerol-3-phosphate, an intermediate metabolite derived from glycolysis, accumulated in plasma (cluster 1 profile). Plasma lactate showed linear decreased concentrations (cluster 2). This suggests that ATP was mainly aerobically produced and that the process of oxidative phosphorylation may have reached its maximum capacity. The TCA cycle intermediates succinate, malate, and citrate showed temporarily decreased plasma concentrations in response to PFT (cluster 3), whereas alpha-ketoglutarate showed increased concentrations after a lag phase (cluster 1) in response to PFT.
Lipoprotein production
Hepatic very low-density lipoprotein (VLDL) production (represented by free cholesterol and sphingomyelins) showed continuously increasing concentrations in response to PFT after a lag phase of about 2 h (cluster 1). Highest levels were reached at the final time point (8 h). After about 4 h maximum TG, plasma concentrations were reached (cluster 4), which were normalized at the final time point (8 h).
Hepatic tissue injury control
Similar cluster 1 responses were observed for liver integrity enzymes (ALAT, ASAT, GGT, ALP) and the process of ketogenesis (3-hydroxybutanoic acid, acetoacetate, 2-hydroxybutanoic acid).
Pancreas
β cell function
PFT caused a temporary release of both C-peptide and insulin from the pancreatic beta cells into the circulation with maximum concentrations around 1 h (cluster 5).
α cell function
Glucagon also showed a temporary release during PFT but at a later stage with maximum concentrations around 4 h (cluster 4).
Systemic stress
Systemic insulin sensitivity
PFT drink that contained 75 g of glucose (33 E%) caused a temporary increase in plasma concentrations of both glucose and fructose (cluster 5).
Oxidative stress
Several markers related to oxidative stress responded to PFT. The antioxidant uric acid showed temporarily increased concentrations (cluster 5), whereas the antioxidant vitamin E and mannose (ER stress) showed temporary reduced concentrations (cluster 3 response). Finally, erythronic acid, a molecule formed when N-acetyl-d-glucosamine is oxidized, showed temporarily increased concentrations in the late phase of the time course (cluster 4 response).
Adaptation carbohydrate and lipid switch
The RQ showed a significant increase in response to PFT drink with maximum reached at 2 h. RQ returned to fasting values at 150 min.
Comparison of healthy vs. T2D subjects at fasting
Levels of several biomarkers were significantly different in T2D as compared to healthy subjects at fasting (Table 2). A total of 18 out of 132 parameters were statistically different in T2D as compared to healthy subjects.
Table 2 Fasting levels of markers that were significantly different between 20 healthy male subjects and 20 type 2 diabetic male subjects, related to adipose tissue, vasculature, kidney, muscle, liver, pancreas, and systemic stress
Glucose metabolism
Markers related to insulin and glucose metabolism were significantly different between the T2D and healthy subjects at fasting such as increased glucose, insulin, and HbA1C. All fasting indexes (lower HOMA-B; higher HOMA-IR, HIRI, and AIR) were also different between the two subject groups. Other plasma metabolites related to glucose metabolism showed lower (1,5-anhydroglucitol, glycine) or higher (mannose, fructose, valine, leucine, 2-hydroxybutanoic acid) plasma concentrations in T2D.
Lipokine and adipokine production
Leptin concentrations were significantly higher in the T2D patients, suggesting higher adiposity, which was confirmed by a higher BMI in T2D.
Liver integrity
Plasma concentrations of ALAT and GGT were found to be significantly elevated as compared to the healthy volunteers at fasting, suggesting reduced liver integrity in T2D.
Inflammatory state
Higher levels of CRP and sICAM were found, suggesting an inflammatory state in T2D.
Comparison of healthy vs. T2D subjects in response to PFT
A total of 58 out of 132 parameters showed a statistically different challenge effect in T2D as compared to healthy subjects (Fig. 4). Furthermore, four PFT-based indexes related to insulin sensitivity and β cell functioning (IGI, DI, ISI, and MISI) were also statistically different (Additional file 1: Table S1; Additional file 2: Figures S1 A–F). The responses of the phenotypic processes that were differentially modulated by PFT in T2D as compared to healthy controls are described in more detail below.
Glucose metabolism
The insulin and C-peptide PFT response was significantly different in T2D as compared to healthy subjects. A delayed insulin response was found with maximum concentrations at the 2-h time point in T2D, as compared to the 1-h time point in healthy subjects. For C-peptide, maximum concentrations were reached at the 4-h time point in T2D, as compared to the 2-h time point in healthy subjects. DI and IGI indicated a decreased β cell function, whereas the α cell of the pancreas secreted higher levels of the hormone glucagon in T2D. Subsequently, T2D had a higher glucose PFT response with higher maximum concentrations as well as decreased ISI, indicating reduced systemic insulin sensitivity in T2D. The gut hormone GLP-1, closely connected to glucose metabolism, showed higher maximum concentrations as well as a faster return to homeostatic levels in T2D as compared to healthy subjects.
Adipose tissue
The response to PFT revealed a diminished lipolysis rate and leptin response in T2D as compared to healthy subjects. A blunted NEFA and glycerol response was observed in T2D. The individual non-essential FFAs showed the same blunted response profiles (C12:0, C14:0, C16:0, C16:1, C17:0, C18:0, C18:1 and C18:2) in T2D, whereas this was not the case for the essential FFA (C20:4 and C22:6). No difference in response was observed for the essential FFA between T2D and healthy male volunteers.
Muscle
A distinctly different response to PFT between T2D and healthy subjects was observed for most proteinogenic amino acids. This suggests a different protein metabolism between the two groups. Fourteen out of 19 proteinogenic amino acids showed a differential PFT response. T2D showed for all plasma BCAAs and derivatives a higher amplitude in response to PFT as compared to healthy subjects. Similar differences in PFT response profiles were observed for serine, lysine, threonine, glutamate, and tyrosine. Finally, alanine, asparagine, glutamine, cysteine, and phenylalanine also showed a differential PFT response in T2D as compared to healthy that differed from the above described amino acids.
Three metabolites that are intermediates of energy metabolism responded differently to PFT between T2D and healthy subjects. These were alpha-ketoglutarate, succinate, and pyruvate. Together, PFT response profiles from these metabolites suggest that T2D have a diminished carbohydrate metabolism shown by higher amplitudes for glycolysis and TCA cycle as compared to healthy subjects.
Liver
The ketone bodies 3-hydroxybutanoic acid and acetoacetate both involved in ketogenesis showed a reduced response in T2D as compared to healthy subjects. The minimum concentrations were reached at a later time point (4 vs. 2 h in healthy subjects) and the plasma increase at late time points is reduced, suggesting a diminished β-oxidation. Substrate oxidation measures showed that T2D have a significant lower RQ, indicating that T2D use a higher fat% and a lower carbohydrate% as an energy source compared to healthy subjects. The response to PFT revealed that ALAT and ASAT had a higher amplitude to PFT in T2D as compared to healthy subjects. Together these data further confirmed a reduced hepatic tissue injury control in T2D.
Systemic stress
Finally, in response to PFT, several markers related to oxidative stress showed a differential response in T2D as compared to healthy subjects. Glutathione ratio and levels of ribose were significantly lower in T2D as compared to healthy subjects. The response of erythronic acid was found to be strikingly different between the two groups. Healthy subjects showed a clear response (cluster 4) to PFT, whereas this response was lacking in T2D. Finally, CRP levels, a biomarker representing the process of inflammation, were significantly higher upon PFT in T2D as compared to healthy subjects.
Comparison of glucose and insulin PFT responses to OGTT
Similar glucose and insulin patterns were observed between the two subjects groups (Fig. 5a, b). Although, plasma glucose and insulin concentrations in T2D and plasma glucose in healthy subjects were higher in response to OGTT than in PFT. Indexes related to glucose metabolism that can be calculated from an OGTT were also calculated for PFT and compared (Table 3). DI, ISI, IGI, and MISI were all significantly different between healthy and T2D after both OGTT and PFT. Although the direction of change was the same for all indexes after OGTT and PFT, the absolute values for the indexes were different after the two challenges, especially for DI (6.31 for OGTT vs. 24.63 for PFT in healthy subjects). Furthermore, it was observed that healthy subjects had a remarkable higher standard deviation for the different indexes as compared to T2D.
Table 3 Overview of indexes related to insulin resistance, insulin secretion, and β cell function and their values in response to OGTT as compared to PFT that both contain 75 g of glucose in 20 healthy male subjects and 20 type 2 diabetic male subjects