The association of plasma pancreastatin levels with insulin resistance in patients with gestational diabetes mellitus

Abstract

Introduction

Gestational diabetes mellitus (GDM) occurs on the background of increased insulin resistance. We aimed to investigate the levels of plasma pancreastatin (PST) levels and its association with metabolic, demographic, and anthropometric parameters in gestational diabetic and normal glucose-tolerant pregnant women.

Materials and methods

A total of 165 pregnant women in the 24th–28th week of pregnancy were enrolled in this cross-sectional study. PST levels were measured using ELISA method.

Results

Median PST levels were similar between GDM (n = 62, 37.6%) and normal glucose-tolerant control group (n = 103, 62.4%). In the GDM group, PST level showed a significant positive correlation with fasting insulin and the homeostasis model assessment of insulin resistance (HOMA-IR). In binary logistic regression analysis, PST levels did not predict the presence of GDM significantly.

Conclusion

Positive correlation of PST with fasting insulin and HOMA-IR suggests that PST could be associated with insulin resistance. Further studies are needed as regards to the role of PST in GDM pathogenesis.

Introduction

Gestational diabetes mellitus (GDM) is traditionally defined as any glucose intolerance first diagnosed in the second or third trimester of pregnancy. ¹ Prevalence of GDM is increasing throughout the world, parallel to the increasing prevalence of obesity and related conditions. Therefore it is considered as a worldwide public health concern. Although the prevalence varies on which diagnostic test is used, Guariguata et al. ² reported that prevalence of hyperglycemia in pregnancy worldwide in 2013 was 16.9%. In our country, the prevalence of GDM was found to be 16.2% ³ using the International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria.

GDM is associated with many short-term or long-term adverse outcomes. The short-term adverse outcomes are increased risk of hypertensive disorders of pregnancy (preeclampsia, gestational hypertension), large for gestational age (LGA) or macrosomia, birth trauma, polyhydramnios, neonatal respiratory problems, neonatal hypoglycemia, hypocalcemia, and perinatal mortality. Long-term outcomes are increased cardiovascular disease and type 2 DM risk in mother, and obesity, abnormal glucose tolerance, hypertension, and metabolic syndrome in the offspring.

Universal screening is proposed by many international associations and societies. Both American Diabetes Association (ADA) and American College of Obstetrics and Gynecology (ACOG) recommend screening for risk factors for the first visit and universal screening with oral glucose tolerance test (OGTT) between 24th and 28th weeks of gestation.^1,4

Pregnancy is mainly accompanied by insulin resistance mediated by increased levels of diabetogenic hormones including growth hormone, corticotrophin-releasing hormone, human placental lactogen (chorionic somatomammotropin), prolactin, and progesterone. Leptin resistance and decreased levels of adiponectin also contribute to this situation. This increased insulin resistance overwhelms the woman's pancreatic insulin secretion capacity and hyperglycemia ensues.^5,6

Pancreastatin (PST) is a peptide derived from proteolysis of chromogranin-A and is shown to play a role in energy modulation and counteract insulin action.^7,8 PST activates the receptor signaling system of Gq-PLCβ-calcium-PKC signaling pathway. ⁷ This pathway induces hepatic glycogenolysis and lipolysis in adipocytes.^7,9

In a study, prediabetic or type 2 diabetic patients were found to have higher postprandial PST levels than healthy controls.^10,11 Fasting levels did not differ between patients and healthy controls. This finding was proposed to be related to the effect of PST on glucose-stimulated insulin secretion.^11,12 Also obese type 2 diabetic patients are found to have higher PST levels than non-obese healthy and non-diabetic obese patients. ¹²

PST was also studied in GDM. Sanchez-Margalet et al. ¹³ found increased levels of PST in a small number of gestational diabetic patients. In their study, PST levels were positively correlated with epinephrine and norepinephrine.

In our study, we aimed to investigate the levels of plasma PST levels and its association with metabolic, demographic, and anthropometric parameters in gestational diabetic and normal glucose-tolerant pregnant women.

Material and methods

Ethics statement

This study was conducted after local ethics committee approval from Ege University School of Medicine’s ethical committee. The patients gave oral and written informed consent before recruitment (Ethics approval number 22–3.1 T/68).

Subjects and study design

We designed a cross-sectional study and conducted between January 2021 and January 2022 in the Ege University Faculty of Medicine Department of Endocrinology and Metabolism Disorders and Department of Obstetrics and Gynecology. A total of 169 consecutive pregnant women between 24th and 28th weeks of pregnancy referred for GDM screening were included. After applying exclusion criteria a total of 165 women were analyzed.

GDM was diagnosed according to IADPSG criteria ¹⁴ ; at least one of the glucose measurements during the test is above the predefined thresholds as fasting plasma glucose ≥92 mg/dL, 1st hour ≥180 mg/dL, and 2nd hour ≥153 mg/dL. The control group was recruited from screened women whose OGTT was normal.

The patients excluded were those with overt pregestational diabetes, overt hypothyroidism or hyperthyroidism, ALT/AST levels higher than three times the upper limit of normal, estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m², acute infection in the previous 14 days, any known chronic inflammatory and autoimmune disease, and women with multiple pregnancies. Women on any medications with possible effects on plasma glucose levels (e.g., antiepileptics, corticosteroids) were also excluded.

Statistical analysis

Descriptive statistics for continuous variables were calculated with median and inter-quartile range; frequencies and percentages were given for categorical variables. The Shapiro–Wilk test was used to check the normality assumption of the continuous variables. Because the data were not normally distributed, The Wilcoxon rank-sum (Mann–Whitney U) test was performed to compare continuous variables between groups. The Pearson Chi-square test was used for the analysis of categorical variables in two groups. The correlations between continuous variables were examined with Spearman's rank correlation coefficient. Predictors of GDM were evaluated with binary logistic regression analysis of univariate models on single variables, then a final model was constructed on all variables, and selected with backward stepwise method. A p-value of less than 0.05 was considered statistically significant. All statistical analyses were performed by using IBM SPSS version 25.0 (Chicago, IL, USA).

Anthropometric evaluation

A detailed history was taken and comprehensive physical examination was performed on all subjects. Height (centimeter) and weight (kilogram) were measured with the subject barefoot, in light daily clothes, and fasting state in the morning with a standardized scale. Body mass index (BMI) was calculated as weight in kg divided by height in square meters. Mid-upper arm circumference was measured from the right arm, from the middle of the tip of the acromion and olecranon process. Neck circumference was measured just below the larynx, in the standing position, with shoulders and head straight. Blood pressure was measured after 15 min of rest, in the sitting position.

Biochemical evaluation

Patients were instructed to consume large amounts of carbohydrate in the preceding 3 days. After an overnight fast, fasting blood samples were taken. Patients were ordered to drink the 250 mL of standardized commercial glucose solution containing 75 g of glucose in 10 min. Then blood was drawn in 60th and 120th min. Blood samples for PST levels were drawn in the fasting state from the antecubital veins.

Insulin resistance was calculated for each participant using the homeostasis model assessment of insulin resistance (HOMA-IR = fasting insulin (μU/mL) × fasting glucose (mg/dL)/405). ¹⁵

Plasma PST levels were measured by ELISA method using human pancreastatin (CAT: E0983Hu). Plasma measurements were detected using a BT-LAB (Bioassay Technology Laboratory) ELISA KIT (96T) method according to the manufacturer's instructions. In the ELISA method, two replications were performed using the standard determined in the kit as a standard. 10 µl of biotin was added to all samples and then 50 µl of streptavidin was seeded on all samples and standards except the blank. Afterward, it was incubated at 37° for 60 min. After this process, washing was performed 5 times. Then, 50 μl of chromogen A and B were added and incubated for another 10 min. The reaction was stopped with 50 µl of stop reagent and the measurement was performed spectrophotometrically at OD 450 nm.

Results

Data from a total of 165 pregnant women were analyzed. Sixty-two (37.6%) women were diagnosed with GDM, and 103 women with normal OGTT were considered as controls (62.4%). Table 1 shows the demographic data of the participants. The GDM group was older (median 28 [IQR 6] vs 32 [7] years, p < 0.001) and more obese (BMI 24 [6.83] vs 26.9 [7.45] kg/m², p = 0.003) than controls. While mid-upper arm circumference was not different between groups (27.75 [4] vs 29 [5] cm, p = 0.057) neck circumference was significantly higher in GDM group (33 [3] vs 34 [3] cm, p = 0.006).

Table 1.

Demographics of patients, median (IQR).

Variables	Non-GDM (n = 103, 62.4%)	GDM (n = 62, 37.6%)	p-value*
Age (years)	28 (6)	32 (7)	<0 . 001*
Pancreastatin (ng/L)	1136.82 (1012.5)	1232.72 (2055)	0.346
Pregestational weight (kg)	62.50 (20)	70.00 (21)	0 . 002*
BMI (kg/m2)	24 (6.83)	26.90 (7.45)	0.003*
Mid-upper arm circumference (mm)	27.75 (4)	29.00 (5)	0.057
Neck circumference (mm)	33 (3)	34.00 (3)	0.006*
Fasting insulin (mU/L)	10.90 (5.98)	14.7 (10)	0 . 008*
Glucose AUC	258.25 (63.88)	365 (101.25)	0 . 00*
HOMA-IR	2.12 (1.23)	3.34 (2.17)	<0 . 001*
HbA1c (%)	4.9 (0.3)	5.1 (0.6)	0 . 006*
1st-hour glucose in OGTT (mg/mL)	122.5 (44.75)	184.0 (49.75)	<0 . 001*
Birth weight (grams)	3177.5 (761.25)	3317 (675)	0.132

BMI: body mass index; HbA1c: hemoglobin A1c; HOMA-IR: homeostasis model assessment of insulin resistance; OGTT: oral glucose tolerance test.

*p < 0.05.

There was no significant difference in weight gain until OGTT screening, smoking status, family history of diabetes, birth weight, and pregnancy complications between groups.

We did not find any significant difference regarding PST levels between groups (1136.82 [1012.5] vs 1232.72 [2055] ng/L, p = 0.346).

In the GDM group, PST levels were negatively correlated with age (rho: −463, p = 0.001). Also in GDM group, significant positive correlation was shown with fasting insulin (rho: 0.392 p = 0.024). First-hour postload, glucose levels were negatively correlated with PST levels (rho: −0.329, p = 0.027) and glucose area under the curve (AUC) levels showed no correlation with PST levels in neither of the groups. In control group, PST was not correlated with any of the variables (Table 2).

Table 2.

Correlation of variables in GDM and non-GDM groups.

	Non-GDM		GDM
	ng/L		ng/L
	rho	p	rho	p
Age (years)	−0.073	0.479	−0.448	0.001*
Pregestational weight (kg)	0.033	0.753	0.036	0.813
BMI (kg/m2)	−0.015	0.885	−0.118	0.430
Mid-upper arm circumference (mm)	−0.101	0.338	−0.006	0.967
Neck circumference (mm)	−0.146	0.165	0.016	0.916
Fasting plasma glucose (mg/dL))	−0.62	0.549	−0.191	0.189
Fasting insulin (mU/L)	0.094	0.440	0.456	0.008*
HOMA-IR	0.094	0.438	0.378	0.030*
HbA1c (%)	0.121	0.254	−0.269	0.074
1st-hour glucose in OGTT (mg/mL)	−0.030	0.776	−0.329	0.027*
Glucose AUC	−0.35	0.739	−0.257	0.075
Birth weight (grams)	0.019	0.855	0.094	0.536

BMI: body mass index; HbA1c: hemoglobin A1c; HOMA-IR: homeostasis model assessment of insulin resistance; OGTT: oral glucose tolerance test.

*p < 0.05.

The median birth weight of fetuses was not different between GDM and control groups (3317 [675] vs 3036 [761.25] gr, p = 0.132). PST levels did not show any correlation with birth weight in neither of the groups (rho = 0.019, p = 0.855 for control group and r = 0.095, p = 0.536 for GDM group). In univariate logistic regression analysis; age, pregestational weight, BMI, mid-upper arm and neck circumference, fasting insulin, HOMA-IR, and HbA1c were found statistically significant to predict GDM (p < 0.05; Table 3). However, a final model result showed that only the 1st-hour glucose in OGTT (mg/dL) was found significant to predict GDM (p < 0.001).

Table 3.

Predictors of GDM with logistic regression analysis.

Variables	Unadjusted model on single variables		Final adjusted model a
	OR (95% CI)	p-value	OR (95% CI)	p-value
Age	1.152 (1.074–1.237)	<0.001*	–	–
Pancreastatin (ng/L)	1.000 (1.000–1.001)	0.152	–	–
Pregestational weight (kg)	1.031 (1.008–1.055)	0.008*	–	–
BMI (kg/m2)	1.096 (1.027–1.170)	0.006*	–	–
Mid-upper arm circumference (mm)	1.100 (1.006–1.202)	0.036*	–	–
Neck circumference (mm)	1.205 (1.056–1.374)	0.006*	–	–
Fasting insulin (mU/L)	1.087 (1.020–1.157)	0.010*	–	–
HOMA-IR	1.692 (1.238–2.311)	0.001*	–	–
HbA1c (%)	3.201 (1.367–7.497)	0.007*	–	–
1st-hour glucose in OGTT (mg/dL)	1.063 (1.043–1.083)	<0.001*	1.073 (1.042–1.106)	<0.001*

BMI: body mass index; CI: confidence interval; HbA1c: hemoglobin A1c; HOMA-IR: homeostasis model assessment of insulin resistance; OGTT: oral glucose tolerance test; OR: odds ratio.

^a Multiple regression final model was executed on all variables, included together in the model, and selected with stepwise method, *p < 0.05.

Discussion

In our study, we revealed that PST did not show a significant difference regarding GDM status. However in gestational diabetic population, PST levels were positively correlated with fasting insulin and HOMA-IR while no correlations were detected in non GDM group.

In a study, prediabetic or type 2 diabetic patients were found to have higher postprandial PST levels than healthy controls.^10,11 Fasting PST levels did not differ between patients and healthy controls. This finding was proposed to be related to the effect of PST on glucose-stimulated insulin secretion.^11,12

In the study of O’Connor et al. ¹² PST levels were approximately 3.7-fold elevated in obese patients with type 2 DM, compared to controls or non-diabetic obese subjects. A relatively modest state of insulin resistance in obesity was not related to elevated PST levels. This suggests that only extreme levels of insulin resistance might be associated with elevated PST levels. Interestingly, the same study did not show any decline in PST levels with substantial weight loss. This suggests that the mechanism beyond elevated PST in type 2 diabetes is more complicated than simply insulin resistance. ¹²

Although Sanchez-Margalet et al. ¹³ found increased levels of PST in gestational diabetics, our study failed to retrieve significant differences in terms of PST between GDM and non-GDM pregnancies. However the glucose criteria for diagnosing GDM in that study ¹³ were higher than we used in our study. Besides, median gestational week of the patients was higher than in our study. As insulin resistance increases with the progression of pregnancy, PST may not show significant difference in our study due to the relatively earlier gestational weeks and relatively lower glycemic thresholds for GDM diagnosis of our patients.

In healthy humans, PST has been shown to increase in response to a standard meal. ¹⁶ Siegel et al. ¹⁷ have demonstrated that infusion of human pancreastatin-16 mitigates the elevation in serum insulin and glucose levels in response to oral glucose load, in healthy humans. In our study, we did not apply dynamic studies. If we would have the opportunity to study PST levels after glucose load, we might have a better understanding of dynamic changes in PST.

O’Connor et al. ¹² also studied PST variants and found out Gly297Ser variant to be more potent than wild type in terms of inhibition of insulin-stimulated glucose uptake. This suggests that some genetic alterations in PST's structure may result in interindividual variations in action.

No correlations between PST and any of the studied parameters were detected in non-GDM group, whereas in GDM group, PST was positively correlated with fasting glucose, fasting insulin, and HOMA-IR. PST reduces insulin sensitivity and inhibits insulin secretion thus leading to hyperglycemia.^17,18 Additionally, PST has regulatory effects on hepatic glycogenesis, lipid metabolism, and adipocytes. PST stimulates hepatic gluconeogenesis, while inhibiting glucose uptake and glycogen synthesis in adipocytes and hepatocytes. Our finding of positive correlation of PST with insulin resistance parameters is consistent with previous studies relating PST with insulin resistance. PST was negatively correlated with 1st-hour postload glucose in GDM group although the correlation was weak. When analyzed for AUC, no correlation was found with PST levels in either group.

Age, pregestational weight, BMI, arm and neck circumference, fasting insulin, HOMA-IR, HbA1c, 1st-hour glucose, and PST were included in the multiple regression model. PST levels did not significantly predict GDM. In the final model, none of the variables other than 1st-hour glucose which is also a diagnostic criterion of GDM was predictive of GDM.

To the best of our knowledge, this is the first study in the literature demonstrating lack of significant difference between GDM and non-GDM in terms of PST. Although our study group was larger than the previous study ¹³ still further studies with larger populations are needed. Relatively small population is one of our study limitations. The other limitation is that we only studied fasting PST levels in the 24th–28th gestational week. As a recommendation further studies can be performed analyzing PST levels in different gestational weeks, and different time points after glucose load to understand the secretory dynamics of PST.

In conclusion, our study did not reveal any significant difference of PST between GDM and non-GDM groups. Correlation between insulin resistance parameters and PST in GDM patients suggests some relationship of PST with insulin resistance of GDM. But further studies are needed to enlighten any possible relationship.