Incretin Hormones and Insulin Responses During OGTT in Newly Diagnosed T2DM Patients

Several research groups have reported variable results about incretin effects of glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1(GLP-1) particularly, as well as cytokines balance in type 2 diabetes mellitus (T2DM). The present case-control prospective interventional study was conducted in 2014-2015 at Medical College for Women & Hospital, Dhaka, investigating responses of incretin hormones and insulin to oral glucose tolerance test (OGTT) in Bangladeshi subjects. Blood samples were collected from 36 OGTT positive adult T2DM patients (Pt) and 30 normal adults (NC) at ‘0’ minute (fasting) and at 2 hours after OGTT. Routine laboratory investigations in blood were done and special parameters in serum, i.e. insulin, HGH, TSH, GIP and GLP-1 were analyzed using enzyme immunoassay kits. Pt had FBG and BG2Hr levels much higher than NC (p<0.001). In Pt, F-Insulin and Insulin2Hr were much lower than NC subjects (p<0.001), although Insulin2Hr level was higher than F-Insulin level in Pt. FGIP (p=0.309) and F-GLP-1(p=0.984) levels were similar between Pt and NC. Interestingly, NC responded to OGTT by increasing GIP2Hr and GLP-1,2Hr levels about 3 times, whereas Pt responded by raising about 1.5 times only compared to F-GIP and F-GLP-1 (p< 0.001). Reduced insulin levels, both fasting and postprandial, were possibly due to decreased responses of GIP and GLP-1 to glucose load in T2DM patients. Further studies with a larger sample size including cytokines are warranted.


Introduction
Diabetes mellitus (DM) is a serious global health issue with type 2 diabetes mellitus (T2DM) accounting for about 90-95% of all cases. It is estimated that about 7.8% of the world population aged between 20 and 79 years will have T2DM by 2030. 1,2 Although glucose-tolerant individuals are capable of adjusting their insulin secretion to their actual insulin sensitivity, people with T2DM are incapable of doing so. Beta-cell failure is the hallmark of this disease, although failure may be precipitated by the development of insulin resistance. In healthy subjects, a considerable part of the postprandial insulin response is due to the actions of the incretin hormones, particularly glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). Together, these two hormones are mainly responsible for the socalled incretin effect. 3 Although frequently ignored, the effect strongly depends on the dose of glucose. A convenient way of describing the effect is to calculate the gastro-intestinally mediated glucose disposal (GIGD). 4 A convenient way of describing the effect, which depends on the dose of glucose, is to calculate the gastro-intestinally mediated glucose disposal (GIGD). 4,5 Here, the amount of glucose required by intravenous infusion to copy the glucose excursions after the oral load is related to the oral load. Thus, if 25 g is required to copy a 75g oral glucose load, the GIGD amounts to 100 × (75-25)/75=66%. In other words, mechanisms associated with and activated by the oral ingestion resulted in a disposal of 75-25=50 g of the ingested glucose. 5 Most of the GIGD in healthy subjects is accounted for by the actions of the incretin hormones, but inhibition of hepatic glucose production by suppression of glucagon secretion, hepatic uptake of glucose from the portal vein, and gut-brain or liver-brain reflex activity may also play a role. The healthy human body has a remarkable capacity to handle the intake of increasing amounts of glucose and is therefore capable of maintaining almost unchanged postprandial glucose excursions, regardless of the oral load. In people with T2DM, this ability is dramatically reduced as illustrated by calculation of the GIGD, which may be close to zero. 5 The almost complete loss of GIGD is typically accompanied by a greatly reduced difference between the insulin responses to the oral and the intravenous glucose load, i.e. the incretin effect. 5, 6 As indicated by the almost complete loss of GIGD, the incretin effect remaining in the patients with T2DM has little effect on glucose disposal, probably as a result of the simultaneously occurring insulin resistance. The loss of incretin effect is therefore likely to contribute importantly to the postprandial hyperglycemia in T2DM. Research carried out by several groups during the last decades has indicated that the incretin effect is mediated mainly by GIP and GLP-1 and no other gut hormones fulfill all criteria to act as incretin hormones, i.e. being secreted during glucose ingestion and being capable of stimulating insulin secretion during similar glycemic levels and in those concentrations that are reached during glucose ingestion. [5][6][7][8][9][10] The concentrations of GIP have been reported to be elevated, decreased and unchanged in patients with T2DM. Toft-Nielsen et al found slightly decreased postprandial GIP concentrations in a large group of T2DM patients compared with a matched control group of nondiabetic subjects. 11 A major secretary defect regarding GIP secretion did not seem to exist in T2DM. When GLP-1 was identified as the other important incretin hormone, it was relevant to evaluate GLP-1 secretion in T2DM also. 12 Toft-Nielsen et al found a pronounced impairment of the postprandial GLP-1 response, particularly during the later postprandial phase (after the first 60 min). 11 The decreased secretion was thought to be secondary to the disease, since it was noted only in the diabetic twins or identical twins discordant for diabetes . 13,14 In addition, first-degree relatives of T2DM patients exhibited normal 24-h GLP-1 profiles. 15 Some other studies could not confirm decreased GLP-1 responses in T2DM patients and reported contradictory results. 16,17 So, it needs to be settled through further research in human. Literature survey has indicated that so far no study has been done or reported on incretin hormones and their responses to glucose load in Bangladeshi patients with T2DM. Thus, it was intended to investigate serum incretin hormones (GIP, GLP-1), insulin, thyroid stimulating hormone (TSH) and human growth hormone (HGH) status and their responses to oral glucose load (OGTT) in Bangladeshi T2DM patients and healthy subjects.

Materials and Methods
This case-control prospective interventional study conducted during the period from Hrs (120 min) after OGTT. Samples were aliquoted for routine analyses and special research investigations. Among the routine analysis, glucose level and other usual routine tests in blood, serum and urine were done. Serum aliquots were preserved for a short time at -20 o C to -30 0 C for incretin hormones, and other special investigations. A total of 36 OGTT positive adult patients (male: 15, female: 21) were included in this casecontrol prospective interventional study. Only newly diagnosed DM patients were included in this study. Those who had other diseases such as thyroid diseases, any endocrine diseases, renal diseases, hypertension, etc were excluded. And 30 normal healthy adults (male: 12, female: 18) were investigated in parallel as normal control subjects. Routine laboratory investigations such as blood glucose, CBP, HbA1C, LFTs, TFTs, RFTs, etc were made according to usual clinical laboratory methods as practiced in the hospital laboratory. The special investigations, i.e. serum Insulin, TSH, HGH, GIP and GLP-1, were made by adopting enzyme immunoassay (EIA) methods using kits obtained from reputed commercial companies such as R&D Systems (USA), Calbiotech (USA), Novatech (Germany) etc. Statistical analyses were perfomed by Paired t-test and Student's t-test using SPSS programme.

Results
The results of the laboratory investigations and their statistical analysis by Student's t-test for T2DM patients (Pt) group and normal control (NC) subjects are stated in glucose stimulated values were also less than the healthy subjects. Therefore, the data support the concept that in T2DM, like that of insulin, incretins abnormality is also an early component in pathophysiology of diabetes.

Discussion
In this study, T2DM patients (Pt) had FBG (p< 0.001) and BG2Hr (p< 0.001) levels much higher than NC subjects (p< 0.001) which were expected (table-I). No significant differences were observed between Pt and NC for F-TSH (p=0.927), TSH2Hr (p=0.413), F-HGH (p=0.532) and HGH2Hr (p=0.773) levels. In Pt, F-Insulin (p< 0.001) and Insulin2Hr (p< 0.001) were much lower than NC subjects, although Insulin2Hr level was higher than F-Insulin level in Pt. It was observed that F-GIP (p=0.309) and F-GLP-1 (p=0.984) levels were similar between Pt and NC subjects. Interestingly, NC subjects responded to OGTT by increasing GIP2Hr and GLP-1,2Hr levels about 3 times compared to F-GIP and F-GLP-1. In Pt, F-GIP and F-GLP-1 levels were also raised responding to OGTT but by about 1.5 times only (table-I).
Most of the GIGD in healthy subjects is accounted for by the actions of the incretin hormones, but inhibition of hepatic glucose production by suppression of glucagon secretion, hepatic uptake of glucose from the portal vein and gut-brain or liver-brain reflex activity may also play a role. There is no doubt that the incretin hormones play a major role in GIGD in healthy subjects and it can be concluded that the incretin effect plays a major role for normal glucose tolerance. In patient with T2DM, this ability is dramatically reduced as shown in our result similar to some other reports. 5 The loss of incretin effect is therefore likely to contribute importantly to the postprandial hyperglycemia in T2DM.
Results of this study were the first to be reported from this geographical region. However, results were similar to those of Toft-Nielsen et al, who suggested that a major secretary defect regarding GIP secretion did not seem to exist in T2DM, but they reported pronounced impairment of the postprandial GLP-1 response in T2DM subjects, particularly during the later postprandial phase (after the first 60 min). 9 However, presence of some other unidentified incretin hormones not yet discovered cannot be ruled out. Holst et al concluded that the dramatic loss in T2DM patients of the ability to dispose of orally ingested glucose (GIGD) is related to the inability of the incretin hormones to increase insulin secretion after meal or glucose load. 18,19 Several lines of evidence support that the loss of incretin effect is secondary to development of diabetes. [19][20][21][22] However, more recent findings suggest that the loss of incretin effects in T2DM patients can only be explained by a specific loss of insulinotropic activity of the incretin hormones at physiological level. 19,2325 In overt T2DM, the consequence of the impaired incretin effect is that the ability of the patients to efficiently dispose of orally as opposed to intravenously administered glucose is completely lost. 19 Findings of this study, relatively reduced capacity of T2DM Patients to produce GIP and GLP-1 seemed to be relevant and interesting. These incretin responses may be linked to proinflammatory cytokines such as interleukin (IL)-1, IL-6, tumor neurosis factor- (TNF-), etc. These cytokines are thought to impair insulin signalling and abnormally high levels of them are associated with insulin resistance and T2DM. [26][27][28][29][30] However, conflicting and contrasting results of increase in postprandial plasma IL-6 and TNF- levels were reported in T2DM patients. 24,31 The involvement of proinflammatory cytokines was substantiated by the observation that IL-1 blockade attenuates -cell dysfunction by islet amyloid-induced inflammation in T2DM. 32 It has been reported that TNF- induced insulin resistance is associated with an elevated expression of IL-18 in human skeletal muscle tissue, which suggest, a possible role for IL-18 in the pathogenesis of TNF- induced insulin resistance in humans. 33,34 Also, it has been reported that antiinflammatory cytokines (IL-4, IL-10, IL-13, etc) counteracts the cytotoxic effects of proinflammatory cytokines, i.e. IL-1, TNF-, IL-17, etc in insulin-producing cells through the reduction of nitrosative stress. 35 Thus, a balance between the anti-inflammatory and the proinflammatory cytokines is of crucial importance for the prevention of pancreatic -cell destruction. It is therefore evident that perturbation of this delicate balance in favour of pro-inflammatory cytokines is a strong possibility as the pathogenetic mechanism towards development of T2DM.

Conclusion
In conclusion, data generated through this study that can be used as normogram of incretin hormones (GIP, GLP-1) in Bangladeshi population. The findings have supported the concept of early involvement of incretin hormones in T2DM patients and thereby rationality of selecting incretin-based therapy as a first line option for T2DM individuals. However, further studies on incretin hormones and their responses to OGTT including serum pro-inflammatory and anti-inflammatory cytokines in Bangladeshi T2DM patients are warranted.