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Environmental Science and Pollution Research

, Volume 26, Issue 3, pp 2864–2872 | Cite as

A low GLP-1 response among patients treated for acute organophosphate and carbamate poisoning: a comparative cross-sectional study from an agrarian region of Sri Lanka

  • Devarajan RathishEmail author
  • Indika Senavirathna
  • Channa Jayasumana
  • Suneth Agampodi
  • Sisira Siribaddana
Research Article
  • 67 Downloads

Abstract

Higher incidence of diabetes along with increased use of pesticides is seen in Southeast Asia. Recent hypothesis postulated a link between acetylcholinesterase inhibitor insecticides and type 2 diabetes through the GLP-1 pathway. This study compares the GLP-1 response between groups with low and high red blood cell acetylcholinesterase (RBC-AChE) activity. A comparative cross-sectional study was conducted amongst patients who were within 3 months after an acute organophosphate or carbamate poisoning (acute group) and amongst vegetable farmers with low (chronic group) and high (control group) RBC-AChE activity. Acute (366 mU/μM Hb) and chronic (361 mU/μM Hb) groups had significantly lower RBC-AChE activity in comparison to the control (471 mU/μM Hb) group (P < 0.0001). Only the acute group, which has had atropine therapy, showed a significantly lower 120 min value in comparison to the control group (P = 0.0028). Also, the acute group had significantly low late (P = 0.0287) and total (P = 0.0358) responses of GLP-1 in comparison to the control group. The findings of the study allude towards attenuation of GLP-1 response amongst patients after acute organophosphate and carbamate poisoning. The possibility of an atropine-mediated attenuation of GLP-1 response was discussed.

Keywords

Atropine Organophosphate Carbamate Diabetes mellitus Glucagon-like peptide-1 Incretin effect Acetylcholinesterase activity 

List of abbreviations

ACh

acetylcholine

AChE

acetylcholinesterase

AUC

area under the curve

BMI

body mass index

CKD-EPI

Chronic Kidney Disease Epidemiology Collaboration

eGFR

estimated glomerular filtration rate

ELISA

enzyme-linked immunosorbent assay

GIP

glucose-dependent insulinotropic polypeptide

GLP-1

glucagon-like peptide-1

IQR

interquartile range

NA

not applicable

OGTT

oral glucose tolerance test

OPI

organophosphate insecticide

RBC

red blood cell

T2DM

type 2 diabetes mellitus

tAUC

area under the curve for total response

Background

Type 2 diabetes mellitus in Southeast Asia

Type 2 diabetes mellitus (T2DM) is a chronic disease influenced by genetic and environmental factors. Dysfunction of beta cells of the pancreas and insulin resistance is implicated in its pathogenesis (Scheen 2003). The global, Southeast Asian, Sri Lankan and North Central Provincial prevalence of diabetes was 8.8%, 8.5%, 8.6% and 9.6% respectively (Katulanda et al. 2012; Karuranga et al. 2017). Majority of the Southeast Asian adults with diabetes are living outside the cities. This region had 1.1 million deaths due to diabetes in 2017, comprising 14% of global mortality, and that is second only to the Western Pacific region. More than half of these deaths occurred amongst people less than 60 years old (Karuranga et al. 2017). Diabetes is observed amongst young Asians and in people with low body mass index (Chan et al. 2009). Also, an increase in the incidence of diabetes with the increased use of pesticides is observed in Southeast Asia (Gifford et al. 2015). Patients with T2DM have shown attenuation of the incretin effect (Nauck et al. 1986; Holst 2007), and incretin modulators are increasingly used in their management. Asians have shown higher glucose-lowering effect with incretin modulators compared to non-Asians (Kim et al. 2013; Singh 2015).

The incretin effect and the GLP-1 response

The incretin effect is known as a 40–60% increase in insulin secretion with oral when compared to intravenous glucose (Elrick et al. 1964; Perley and Kipnis 1967). Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are the two main incretin hormones with glucose-dependent insulinotropic activity (Prins 2008; Seino et al. 2010). Thus, attenuation of GLP-1 secretion can result in hyperglycaemia. K cells of the duodenum and proximal jejunum secrete GIP and L cells of the distal ileum and colon secrete GLP-1 (Prins 2008; Seino et al. 2010). Glucose transporter-2 and calcium-sensing receptors regulate the release of both GIP and GLP-1 (Mace et al. 2012).

GLP-1 secretion occurs in two phases. In the early secretory phase, GIP indirectly stimulates the secretion of GLP-1 via the vagus nerves. ACh is involved in this pathway and atropine inhibits the secretion of GLP-1 (Herrmann-Rinke et al. 1995; Balks et al. 1997; Ahrén and Holst 2001; Anini et al. 2002). The late secretory phase is directly stimulated by the nutrients (Anini et al. 2002). Dipeptidyl peptidase-4 enzyme rapidly inactivates incretin hormones. Hence, measurement of total GLP-1 is needed to estimate the secretion of GLP-1 (Kuhre et al. 2015).

The fasting GLP-1 levels were found to be high in men compared to women. However, the 30- and 120-min GLP-1 concentrations after an oral glucose tolerance test (OGTT) were higher in women (Faerch et al. 2015). Older age, low body mass index (BMI) and low waist circumference are associated with the higher GLP-1 response. Increased GLP-1 response with increased age is suspected due to reduced renal clearance of GLP-1 (Idorn et al. 2014; Faerch et al. 2015). Following an OGTT, mean area under the curve (AUC) for total GLP-1 amongst T2DM patients and controls was 3113 pmol min L−1 and 3599 pmol min L−1 respectively (Vilsbøll et al. 2001).

Acetylcholinesterase inhibitor insecticides induced T2DM

Evidence suggests organophosphate insecticide (OPI) and carbamate induced disruption of glucose homeostasis (Montgomery et al. 2008; Hectors et al. 2011; Karami-Mohajeri and Abdollahi 2011; Joshi and Rajini 2012; Thayer et al. 2012; Lasram et al. 2014). Formation of advanced glycosylation end products, accumulation of lipid metabolites and activation of inflammatory pathways and oxidative stress were proposed as possible mechanisms (Lasram et al. 2014). OPI is thought to cause a prolonged hyperglycaemia due to glycogenolysis and gluconeogenesis. OPI can also cause cell damage which can induce inflammation and oxidative stress (Lasram et al. 2014).

Acetylcholinesterase inhibitor insecticide poisoning

Acute poisoning from OPI and carbamate is mainly due to self-harm and accidental exposure. Chronic poisoning occurs from the occupational exposure amongst farmers. Globally, almost 250,000 per year die due to pesticide poisoning (McNab 2006). Case fatality rates of 5–20% and 5.8% were found in Asia and North Central Province of Sri Lanka for OPI (Senarathna et al. 2012; Thomas and White 2014).

Absorption of these insecticides is rapid as most are lipophilic and unionised (Vale 1998). The majority is stored in fat leading to prolonged intoxication and clinical relapse. The above insecticides inactivate the enzyme acetylcholinesterases (AChEs) which degrade acetylcholine (ACh) (Fukuto 1990) leading to accumulation of ACh causing overstimulation and subsequent downregulation of ACh receptors (Organophosphates 2018). Although acute OPI toxicity is a clinical diagnosis, confirmation is based on the measurement of red blood cell acetylcholinesterase (RBC-AChE) activity (Katz 2015). Atropine (a non-selective anti-muscarinic drug) and pralidoxime (an AChE reactivator) are the antidotes used (Thomas and White 2014). Chronic poisoning involves disturbances to the endocrine and nervous systems (Organophosphates 2018).

The hypothesis

We recently hypothesised that attenuation of GLP-1 response would occur amongst patients treated for OPI and carbamate poisoning due to the following two mechanisms (Rathish et al. 2016):
  1. 1.

    Excess ACh-mediated attenuation of GLP-1 secretion due to the overstimulation and subsequent downregulation of the muscarinic receptors

     
  2. 2.

    Atropine-mediated inhibition of GLP-1 secretion due to competitive antagonism of ACh action at the muscarinic receptors

     

This study aims to assess total GLP-1 response against RBC-AChE activity in acute and chronic OPI or carbamate poisoning. Hence, it intends to compare total GLP-1 values between groups with low and high RBC-AChE activity. A possible association would lead towards early preventive measures and novel treatment options.

Materials and methods

Study setting

Anuradhapura had a population of nearly 856,500 in 2012 (Census of Population and Housing, Department of Census and Statistics, Ministry of Finance and Planning, Sri Lanka 2012). Majority of its population (94.6%) are rural (Census of Population and Housing, Department of Census and Statistics, Ministry of Finance and Planning, Sri Lanka 2012), and agriculture is their main (55%) employment (Annual Bulletin 2014). Being an agricultural area, Anuradhapura has been heavily exposed to OPI and carbamates during the past few decades. Case fatality rate after acute OPI poisoning was 5.8% in Anuradhapura (Senarathna et al. 2012).

A comparative cross-sectional study was conducted amongst poisoned patients and vegetable farmers. Patients discharged after treatment for acute OPI and carbamate poisoning at Teaching Hospital Anuradhapura (THA) and Base Hospital Thambuttegama (BHT) were selected. These hospitals provide universal-free health care. THA is the only tertiary care hospital available for the entire North Central Province of Sri Lanka. BHT is the highest grade secondary care institution of the Anuradhapura district. Although atropine is available in primary care institutions (Rathish et al. 2017), pralidoxime is considered as an essential medicine only for secondary and tertiary care institutions (National list of essential medicines 6th edn 2013-2014). Also, ventilator facilities are scarce at peripheral hospitals. Therefore, the majority of patients with acute OPI and carbamate poisoning in Anuradhapura district are treated at THA and BHT.

Sample size

Minimum sample size was calculated, as 23 each for farmers with high RBC-AChE activity (control group) and farmers with low RBC-AChE activity (chronic group) using data from previous literature (Vilsbøll et al. 2001) and the formula nB = (1 + 1/k) [σ × (Z1−α/2 + Z1−β)/(μAμB)]2. Where, nB is the calculated sample size, k is nA/nB (matching ratio) (= 1), σ is the standard deviation of AUC for total GLP-1 (= 579), Z1−α/2 is type I error (= 1.96), Z1−β is power (= 0.84), μA is mean AUC for total GLP-1 related to chronic group (= 3113 pmol min L−1) and μB is mean AUC for total GLP-1 related to control group (= 3599 pmol min L−1). Similarly, the sample size for the patients treated for acute OPI and carbamate poisoning (acute group) was calculated as 23 against the control group.

Selection of participants

Vegetable farmers are known to be occupationally exposed to OPI and carbamates. Purposive sampling was done to recruit 46 vegetable farmers at Anuradhapura. All 46 were measured for their RBC-AChE activity, and the farmers with the highest 23 values were considered as the control group, the lowest 23 the chronic group. Only Sinhalese males were selected in view of having homogenous groups concerning ethnicity and gender. The above would minimise confounding on plasma glucose and total GLP-1 levels.

Patients who were within 3 months after an acute OPI or carbamate poisoning were recruited. They were informed of the study via a telegram to the addresses retrieved from the record rooms of THA and BHT. All who responded were sampled. The criterion of “within three months after ingestion of the insecticides” was strictly enforced. Only Sinhalese males were selected in view of having homogenous groups concerning ethnicity and gender. The above would minimise confounding on plasma glucose and total GLP-1 levels.

Selection criteria

Inclusion criteria for all three groups were the following: Sinhalese males between 18 and 65 years old, permanent resident of Anuradhapura for ≥ 5 years, body mass index ≤ 24.9 kgm−2, waist circumference < 90 cm, AST < 120 U/L, ALT < 120 U/L and estimated GFR ≥ 60 mL/min/1.73 m2 according to the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation (Levey et al. 2009). Exclusion criteria were the following: any acute illness, fasting plasma glucose of ≥ 126 mg/dL OR 120-min plasma glucose of ≥ 200 mg/dL during the OGTT (“Classification and diagnosis of diabetes: standards of medical care in diabetes—2018” 2018), history of diabetes mellitus, pancreatic injury, renal failure, malignancy, chronic gastrointestinal disorders, liver disease, immunosuppression, use of AChE inhibitor medications, haemoglobinopathies or anaemia, neuromuscular disorders, everyday smokers (Adult Tobacco Use Information 2015), heavy alcohol users (Drinking Levels Defined 2016) and cognitive impairment. People with a history of acute OPI or carbamate poisoning were excluded from the farmer (control and chronic) group.

Data collection, instruments and investigations

Data collection was done from September 2017 to December 2017, according to the convenience of the participants, either at the nearest collection centre of Durdans Hospital laboratory or their residence. The study was carried out to obtain demographic data, details on acute OPI and carbamate poisoning, anthropometric measurements and blood samples for eGFR, AST, ALT, RBC-AChE activity, OGTT (fasting, 30 min, 120 min) and total GLP-1 (fasting, 30 min, 120 min). OGTT was performed following administration of oral 75-g anhydrous glucose in 300 mL drinking water after a 10-hour-overnight-fasting (Martin 2015). Explaining the study, obtaining consent, collecting data and measuring anthropometry were done by the first author. All necessary measures were taken to preserve participant’s privacy and confidentiality. The atlas of commonly used pesticides in Sri Lanka was used as a guide in identifying the OPI or carbamate implicated in farming and self-poisoning (Rathish and Jayasumana 2017).

Blood samples for eGFR, AST and ALT were analysed at the Durdans Hospital laboratory, Anuradhapura (Durdans hospital laboratory network 2018). It is a Joint Commission International accredited hospital in Sri Lanka. Procedures for measurement of the above investigations were well established and routinely done at the above laboratory. The methods used for the analysis of serum creatinine and AST/ALT were enzymatic colorimetric assay and photometric rate (l-aspartate/l-alanine with 2-oxoglutarate) respectively. Quality control for serum creatinine, AST and ALT, was maintained using ROCHE (Precinorm U and Precipath U) (Precinorm and Precipath 2018).

Duplicate measurements of total GLP-1 and RBC-AChE activity in whole blood per haemoglobin concentrate were performed at the Department of Biochemistry, Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka using ELISA and modified Ellman procedure (Worek et al. 1999; Worek 2013) respectively. Total GLP-1 includes both the intact GLP-1 and the primary GLP-1 metabolite (Kuhre et al. 2015). For total GLP-1, Merck KGaA, USA GLP-1 Total ELISA kit and quality controls were used (EZGLP1T-36K) (Glucagon-like peptide-1 total ELISA 96-well plate assay 2013). ELISA reading was done using the microplate photometer of Thermo Scientific Multiskan FC, Finland (MultiskanTM FC Microplate Photometer 2018). For RBC-AChE activity, Sigma-Aldrich reagents, USA (Sigma-Aldrich, analytical reagents and solvents 2018), were used, and the quality control was done using the AChE-check-control (high/low) from Securetec Detektions-Systeme AG, Germany (AChE check Control high/low 2018). Spectrophotometric reading was done using Spectro 2000, Labomed, Inc, USA (Spectro 2000 2001). Wavelengths of 546 nm and 436 nm were used for the measurement of haemoglobin content and RBC-AChE activity respectively. The measurement of RBC-AChE activity was done at a pH of 7.4 and a temperature of 37 °C. The concentration in solution of Ellman’s reagent, ethopropazine hydrochloride and acetylthiocholine iodide was 10 mM, 6 mM and 28.4 mM respectively (Worek et al. 1999; Worek 2013).

Data analysis and description

Data were entered into a Microsoft Excel sheet (Online Resource 1). Descriptive statistics were used to describe data. Medians and interquartile range (IQR) were reported. Spearman rho (95% confidence interval) and regression analysis were performed for RBC-AChE activity against the ingested volume of insecticide. Using the trapezoid rule, total response for plasma glucose and GLP-1 of each participant was calculated from the area under the curve for 0 to 120 min (tAUC0-120). Similarly, early and late responses were calculated from the curves tAUC0-30 and tAUC30-120 respectively.

The outcome variable GLP-1 tAUC0-120 showed a non-normal distribution (Online Resource 1); therefore, Kruskal-Wallis test (and when significant post hoc Dunn’s multiple comparisons test and Cohen d effect size calculation) was performed to determine significant differences between the groups in relation to the medians of the following (P < 0.05): age, years residing at Anuradhapura, body mass index, waist circumference, eGFR, AST, ALT, RBC-AChE activity, fasting plasma glucose (0 min), 30-min plasma glucose, 120-min plasma glucose, OGTT tAUC0-30, OGTT tAUC30-120, OGTT tAUC0-120, fasting total GLP-1 (0 min), 30 min total GLP-1, 120 min GLP-1, GLP-1 tAUC0-30, GLP-1 tAUC30-120 and GLP-1 tAUC0-120. Spearman rho (95% confidence interval) correlation and regression analysis were performed for plasma glucose and total GLP-1 parameters against the RBC-AChE activity.

Spearman rho, regression analysis, Kruskal-Wallis test and its post hoc Dunn’s multiple comparisons test were performed using GraphPad Prism 7 software (GraphPad Prism 7 2018). Kruskal-Wallis H statistic and the subsequent Cohen d effect size calculation were calculated using social sciences statistics (Stangroom 2018) and psychometrica software (Lenhard and Lenhard 2017) respectively.

Data availability

All data generated or analysed during this study are included in this published article (and its Online Resource).

Results

Characteristics of the study groups

Details of four farmers were excluded during the selection process because both their fasting and 120-min plasma glucose were high (“Classification and diagnosis of diabetes: standards of medical care in diabetes—2018” 2018). Carbosulfan was the main acetylcholinesterase inhibitor insecticide used for farming by both the control (91%) and the chronic (78%) groups (Fig. 1). All farmers sprayed insecticides by hand.
Fig. 1

Number of participants of the three study groups for organophosphate and carbamate usage, AChE and GLP-1 study, Anuradhapura, 2017

Two patients were excluded during the selection process because both fasting and 120-min plasma glucose were high (“Classification and diagnosis of diabetes: standards of medical care in diabetes—2018” 2018). Most of the patients were “skilled agricultural, forestry and fishery workers” (39%). Carbosulfan (39%) was the most common acetylcholinesterase inhibitor insecticide implicated in self-poisoning followed by profenofos (35%) and quinalphos (4%). Also, 22% were unable to identify the toxic agent but were clinically managed as for OPI or carbamate poisoning. Only one had inhaled the insecticide accidentally and the rest ingested it for deliberate self-harm (96%). All were treated with atropine, and 13% (3/23) had pralidoxime. Characteristics of the acute poisoning were summarised in Table 1. Spearman rho (95% confidence interval) for RBC-AChE activity against the ingested volume of insecticide was − 0.5637 (− 0.7967 to − 0.1849) (Fig. 2). Regression of RBC-AChE activity against the ingested volume of insecticide was significant (P = 0.0002).
Table 1

Characteristics of acute organophosphate and carbamate poisoning, AChE and GLP-1 study, Anuradhapura, 2017 (n = 23)

Items

Median (IQR)

Amount ingested (mL)

60 (5–90)

Time taken to reach medical care (min)

30 (30–45)

Days of hospital stay

5 (4–9)

Days from discharge

21 (15–45)

Days from poisoning

27 (20–51)

AChE, acetylcholinesterase; GLP-1, glucagon-like peptide-1; IQR, interquartile

Fig. 2

Scatter plot for acetylcholinesterase activity against the ingested volume of insecticide, AChE and GLP-1 study, Anuradhapura, 2017

Comparison of the three study groups

The acute group had a significantly shorter residence at Anuradhapura district in comparison to the chronic group (P = 0.0257). The acute group had significantly lower BMI and waist circumference compared to both control (P = 0.0008 and P = 0.0007 respectively) and chronic (P = 0.0007 and P = 0.0002 respectively) groups. The acute group had significantly higher eGFR in comparison to the chronic group (P = 0.0033). Both chronic (P < 0.0001) and acute (P < 0.0001) groups had significantly lower RBC-AChE activity in comparison to the control group (Table 2).
Table 2

Characteristics of the study participants, AChE and GLP-1 study, Anuradhapura, 2017

Items

Farmers

Patients

Kruskal-Wallis P value (Cohen d effect size)

Significant pairs found from Dunn’s multiple comparisons test

Control (n = 23) median (IQR)

Chronic (n = 23) median (IQR)

Acute (n = 23) median (IQR)

Age (years)

40 (28–48)

47 (41–55)

40 (31–49)

0.0943

NS

Years at Anuradhapura

39 (24–44)

48 (37–51)

36 (23–45)

0.0239 (0.6)

Acute vs chronic

Body mass index (kgm−2)

22 (20–23)

21 (20–23)

18 (17–20)

0.0001 (1.127)

Acute vs control

Acute vs chronic

Waist circumference (cm)

81 (75–88)

82 (77–86)

70 (67–75)

<0.0001(1.198)

Acute vs control

Acute vs chronic

eGFR (mL/min)

101 (94–109)

98 (87–105)

113 (103–121)

0.0034 (0.814)

Acute vs chronic

AST (U/L)

22 (19–33)

20 (18–24)

19 (17–27)

0.2877

NS

ALT (U/L)

25 (16–35)

26 (19–36)

20 (16–46)

0.8145

NS

RBC-AChE activity (mU/μM Hb)

471 (431–515)

361 (323–392)

366 (264–435)

< 0.0001(1.987)

Acute vs control

Chronic vs control

Italic values indicate significance with a p-value of < 0.05

AChE, acetylcholinesterase; GLP-1, glucagon-like peptide-1; IQR, interquartile range; NS, not significant; RBC-AChE, red blood cell acetylcholinesterase

Plasma glucose and total GLP-1 response

The acute group had significantly lower 120 min total GLP-1 (P = 0.0028) (Fig. 3), GLP-1 tAUC30-120 (P = 0.0287) and GLP-1 tAUC0-120 (P = 0.0358) in comparison to the control group (Table 3). Spearman rho (95% confidence interval) correlation for plasma glucose and total GLP-1 parameters against the RBC-AChE activity was summarised in Online Resource 1. Regression of plasma glucose and total GLP-1 parameters against the RBC-AChE activity had no significance (Online Resource 1).
Fig. 3

Mean ± standard error of plasma glucose and total GLP-1 for the three study groups against time, AChE and GLP-1 study, Anuradhapura, 2017

Table 3

Plasma glucose and total GLP-1 response of the study participants, AChE and GLP-1 study, Anuradhapura, 2017

Items

Farmers

Patients

Kruskal-Wallis P value (Cohen d effect size*)

Significant pairs found from Dunn’s multiple comparisons test

Control (n = 23) median

(IQR)

Chronic (n = 23) median

(IQR)

Acute (n = 23) median

(IQR)

Fasting plasma glucose (mg/dL)

89 (82–100)

91 (83–97)

90 (82–95)

0.8948

NS

30 min plasma glucose (mg/dL)

128 (116–176)

153 (113–191)

146 (124–162)

0.7581

NS

120 min plasma glucose (mg/dL)

88 (66–98)

90 (68–115)

98 (77–109)

0.2623

NS

OGTT tAUC0-30 (mg min dL−1)

3315 (2985–4155)

3660 (2880–4275)

3495 (3210–3840)

0.8235

NS

OGTT tAUC30-120 (mg min dL−1)

10,125 (8775–11,880)

11,115 (8775–12,420)

10,485 (9540–12,300)

0.4672

NS

OGTT tAUC0-120 (mg min dL−1)

13,410 (11880–16,110)

14,580 (11775–16,170)

13,890 (12765–16,185)

0.5526

NS

Fasting total GLP-1 (pM)

55 (48–76)

53 (43–74)

47 (37–60)

0.1301

NS

30 min total GLP-1 (pM)

64 (47–86)

64 (49–81)

56 (44–70)

0.4917

NS

120 min total GLP-1 (pM)

59 (41–71)

49 (40–63)

40 (32–44)

0.0029 (0.828)

Acute vs control

GLP-1 tAUC0-30 (pM min)

1775 (1587–2245)

1904 (1525–2218)

1673 (1342–1925)

0.1557

NS

GLP-1 tAUC30-120 (pM min)

5672 (4343–6930)

5069 (4527–6085)

4261 (3258–5529)

0.0309 (0.57)

Acute vs control

GLP-1 tAUC0-120 (pM min)

7931 (5943–9216)

7362 (6252–8391)

5974 (4614–7229)

0.0335 (0.559)

Acute vs control

AChE, acetylcholinesterase; GLP-1, glucagon-like peptide-1; IQR, interquartile range; NS, not significant; tAUC, area under the curve for total response

Discussion

Patients treated for acute OPI or carbamate poisoning and farmers involved in vegetable cultivation were studied to determine the GLP-1 response following 75 g OGTT. Patients treated for acute poisoning had significantly lower GLP-1 response compared to farmers with higher RBC-AChE activity. This difference was mainly due to the GLP-1 level at 120 min. Farmers with low RBC-AChE activity were considered to have chronic exposure to AChE inhibitors (OPI and carbamates) (chronic group). However, the chronic group failed to show a significant attenuation of GLP-1 response in comparison to farmers with higher RBC-AChE activity despite having low GLP-1 values. Nevertheless, patients treated for acute poisoning (acute group) had significantly lower BMI, lower waist circumference and higher eGFR compared to the chronic group. Thus, the acute group had the minimum probability of having a lower GLP-1 response (Faerch et al. 2015).

Overstimulation and subsequent downregulation of the muscarinic receptors due to high ACh levels are possible explanations for the GLP-1 attenuation. Farmers with low RBC-AChE were also expected to have high ACh levels. However, only the patients treated for acute poisoning had significantly low GLP-1. The patients differed from farmers by being exposed to larger doses of AChE inhibitor insecticides and by receiving atropine (100%) and pralidoxime (13%). Except for the large dose of AChE inhibitors, the main difference was atropine which is a competitive antagonist of the muscarinic receptor. Atropine binds to the muscarinic receptors of the L cells and blocks the action of ACh. Further, muscarinic receptor downregulation decreases sensitivity to ACh and increases sensitivity to atropine (Li et al. 2003). This would lead to the attenuation of GLP-1 release from the L cells of the ileum and colon.

Previous literature has also shown atropine to inhibit a GLP-1 response (Herrmann-Rinke et al. 1995; Balks et al. 1997; Ahrén and Holst 2001; Anini et al. 2002). Enhancement of GLP-1 secretion in rats by arterial infusion of cholinergic agonists was impeded by atropine (Herrmann-Rinke et al. 1995). Atropine infusion to the anaesthetised rats 20 min before an intra-duodenal administration of corn oil completely blocked nutrient-induced GLP-1 secretion (Anini et al. 2002). Atropine did not affect baseline GLP-1 of healthy men (Balks et al. 1997) and women (Ahrén and Holst 2001). However, it delayed GLP-1 response significantly amongst healthy men (Balks et al. 1997). Also, atropine significantly reduced the post-absorptive GLP-1 response of women (Ahrén and Holst 2001).

In the present study, GLP-1 value at 120 min was low, but the fasting and 30 min values were normal. Low reserves of GLP-1 in the L cells of ileum and colon would have contributed to the above finding. Also, the ACh level is expected to be at its lowest by 120 min when compared to fasting and 30 min. Thus, the action of atropine would be predominant at 120 min.

A mechanistic theory was proposed to explain the effect of atropine on the GLP-1 response of the patients treated for acute OPI or carbamate poisoning. However, the half-life of intravenous atropine is 3.0 ± 0.9 h amongst adults (Drugbank 2018). On average, the samples of the patients were collected after 29 days from discharge. Thus, the proposed action of atropine has occurred beyond the biological duration of its action. Nevertheless, a cross-sectional study cannot be expected to reveal a definitive causal relationship.

The arbitrary division of the control and chronic groups amongst farmers was a drawback of the study. It would have been ideal to recruit at least three times the minimum sample size and select the farmers with highest and lowest one third of RBC-AChE activities as the control and the chronic groups respectively. However, it was not feasible. Nevertheless, the chronic group had significantly lower RBC-AChE activity when compared to the control group.

This study at a rural, agrarian region of Sri Lanka is a vital lead for future receptor level analysis on atropine-mediated attenuation of GLP-1 response. Further, studies on GLP-1 response amongst patients treated with atropine for indications other than OPI and carbamate poisoning can anticipate this finding. Prospective cohort studies are needed to find the long-term effects on glucose homeostasis and GLP-1 response amongst patients treated for poisoning.

Conclusion

Patients treated with atropine for acute OPI and carbamate poisoning had an attenuated GLP-1 response. Farmers with low RBC-AChE activity did not show such response. This study provides evidence that atropine may be the cause for an attenuated GLP-1 response.

Notes

Authors’ contributions

DR conceived the idea of the study and all authors participated in designing the study. DR was involved in data collection and analysis. DR and IS were involved in biochemical analysis. All authors were involved in the interpretation of data. DR drafted the manuscript and IS, CJ, SA and SS critically revised it. All authors read and approved the final manuscript.

Funding information

The study was partially funded by the grant awarded by the Research, Publication & Higher Degrees Committee, Rajarata University of Sri Lanka to DR (grant number RJT/RP&HDC/2017/FMAS/R/01) and the sponsorship awarded to DR by the State Pharmaceutical Corporation, Sri Lanka. The above agents did not influence the design of the study, collection, analysis and interpretation of data or the writing of the manuscript.

Compliance with ethical standards

Competing interests

The authors declare that they have no competing interests.

Ethics approval and consent to participate

Ethical clearance was obtained from the Ethics Review Committee of Faculty of Medicine and Allied Sciences, Rajarata University of Sri Lanka (ERC/2016/80). Institutional permission was obtained from the institutional heads of Teaching Hospital Anuradhapura and Base Hospital Thambuttegama. Informed written consent to participate was obtained from all participants. All necessary measures were taken to preserve participant’s privacy and confidentiality.

Consent for publication

Consent to publish the information provided by the participants was obtained, provided that it will not be possible to identify individual participants in any way.

Supplementary material

11356_2018_3818_MOESM1_ESM.xls (84 kb)
Online Resource 1 (XLS 83 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Devarajan Rathish
    • 1
    Email author
  • Indika Senavirathna
    • 2
  • Channa Jayasumana
    • 1
  • Suneth Agampodi
    • 3
  • Sisira Siribaddana
    • 4
  1. 1.Department of Pharmacology, Faculty of Medicine and Allied SciencesRajarata University of Sri LankaSaliyapuraSri Lanka
  2. 2.Department of Biochemistry, Faculty of Medicine and Allied SciencesRajarata University of Sri LankaSaliyapuraSri Lanka
  3. 3.Department of Community Medicine, Faculty of Medicine and Allied SciencesRajarata University of Sri LankaSaliyapuraSri Lanka
  4. 4.Department of Medicine, Faculty of Medicine and Allied SciencesRajarata University of Sri LankaSaliyapuraSri Lanka

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