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J. Endocrinol. Invest. 36: 734-738, 2013DOI: 10.3275/8934

ABSTRACT. Background: Short-term intensive insulin therapy(IIT) in patients with Type 2 diabetes mellitus (T2DM) has ben-eficial effects on insulin secretion. However, IIT effect onglucagon and glucagon-like peptide-1 (GLP-1) secretion is un-known. Aim: We evaluated short-term intensive glycemic con-trol effects on insulin, glucagon, and GLP-1 secretory dynam-ics in T2DM. Materials and methods: Twenty-six patients withT2DM were hospitalized and treated with IIT for 10-14 days. Ameal tolerance test was performed before and after IIT andthe differences in serum immunoreactive insulin (IRI) and C-peptide immunoreactivity (CPR) as well as plasma glucagonand active GLP-1 levels were evaluated. Results: Glycoalbu-min levels decreased significantly from 23.0% before to 19.6%

after IIT (p<0.001). However, pre- and post-IIT, IRI and CPRlevels were not significantly different; post-IIT glucose levelswere significantly decreased. The post-IIT glucagon levels at0 and 60 min were lower than pre-IIT levels. Moreover, post-IIT area under the curve (AUC) of glucagon significantly re-duced from 6755±996 pg/dl·60 min to 5796±1074 pg/dl·60min (p<0.001). Furthermore, post-IIT GLP-1 levels and AUCwere significantly higher than pre-IIT values. Conclusions: Ourresults suggest that patients with T2DM who received short-term IIT demonstrated decreased postprandial glucagon lev-els and increased GLP-1 levels following a meal tolerance test.(J. Endocrinol. Invest. 36: 734-738, 2013)©2013, Editrice Kurtis

INTRODUCTION

The pathogenesis of Type 2 diabetes mellitus (T2DM) in-volves multiple metabolic defects, of which β-cell dys-function and insulin resistance are possibly the most im-portant. The deterioration of β-cell function and survivaldue to chronic exposure to supraphysiological glucoselevels is termed glucotoxicity. The elimination of gluco-toxicity and improved insulin secretion were achieved notonly through intensive insulin therapy (IIT) but also withoral antidiabetic therapy (1, 2). Although many studieshave reported that rapid correction of hyperglycemia im-proves β-cell function and insulin resistance, the mecha-nisms by which it improves β-cell function remain unclear.Besides β-cell dysfunction and insulin resistance, abnor-mal regulation of glucagon (3, 4) and reduced glucagon-like peptide 1 (GLP-1) secretion are common features indiabetic patients (5, 6). Glucagon is the main hormonethat counter-regulates insulin for maintaining glucosehomeostasis in humans. In the fasting state, glucagonstimulates glycogenolysis and gluconeogenesis therebyincreasing hepatic glucose output to maintain eug-lycemia. Another important regulator of glucagon secre-tion is GLP-1, which inhibits glucagon secretion from pan-creatic α-cells. This effect results in lower plasma glucoselevels due to decreased hepatic glucose output.

Recent studies have demonstrated that short-term IIT inpatients with newly diagnosed T2DM has beneficial ef-fects on β-cell function, glycemic control, and rate of re-mission (euglycemic maintenance without antidiabetictherapy) within 1 year (2). Furthermore, short-term IIT wasreported to achieve long-term glycemic control in a largepercentage of patients with newly diagnosed T2DM (7-10). Although short-term IIT in patients with newly diag-nosed diabetes was investigated, the impact of this in-tervention in patients with long-standing diabetes is notwell documented (11). To the best of our knowledge, thecorrelation between pre- and post-IIT physiological stim-ulation of endogenous glucagon and GLP-1 following ameal has not been investigated.The aim of the present study was to investigate the dif-ferences in pre- and post-IIT levels of insulin, glucagon,and GLP-1 secretion following meal stimulation in pa-tients with established T2DM.

MATERIALS AND METHODSSubjectsThe study included 26 outpatients with T2DM who had beenregularly treated at the Division of Diabetes and Metabolism,Department of Internal Medicine, Tokyo Metropolitan HirooHospital. Each patient fulfilled the following inclusion criteria: 1)patients were 40-80 yr of age with T2DM >5 yr and had notbeen treated with incretin-based therapy such as GLP-1 or adipeptidyl peptidase-4 (DPP4) inhibitor; 2) all patients had a gly-cated hemoglobin (HbA1c) level >7.5% (National Glycohe-moglobin Standardization Program) and a BMI <30 kg/m2.The exclusion criteria included any major illness or endocrino-logical, cardiac, renal, gastrointestinal or severe hepatic disorder,

Key-words: Glucagon, glucagon-like peptide-1, intensive insulin therapy.

Correspondence: M. Shimodaira, MD, PhD, Department of Internal Medicine, IidaMunicipal Hospital, 438 Yawata-machi, Iida, Nagano, 395-8502, Japan.

E-mail: masanori19810813@yahoo.co.jp

Accepted March 14, 2013.

First published online April 12, 2013.

Effects of short-term intensive glycemic control on insulin,glucagon, and glucagon-like peptide-1 secretion in patientswith Type 2 diabetesM. Shimodaira1,2,3, Y. Muroya3, N. Kumagai3, K. Tsuzawa3, and K. Honda3

1Departments of Internal Medicine, Iida Municipal Hospital, Nagano; 2Division of Laboratory Medicine, Department of Pathologyand Microbiology, Nihon University School of Medicine, Tokyo; 3Departments of Internal Medicine, Tokyo Metropolitan HirooHospital, Tokyo, Japan

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prior history of gastrointestinal surgery, and severe neuropathy(associated with autonomic neuropathy) or retinopathy (prepro-liferative retinopathy or more severe stages). All patients agreedto participate in the study after receiving oral and written infor-mation. The study was conducted according to the principles ofthe Declaration of Helsinki and Tokyo Metropolitan Hiroo Hos-pital.

ProtocolAfter an overnight fast (21:00 h), the patients were examined ina seated position and a cannula was inserted into the patients’cubital vein for blood sampling, following which the patientswere instructed to consume test meals (Meal Test C®, SarayaCorporation, Osaka, Japan; 592 kcal with 75.0 g carbohydrate,8.0 g protein, and 28.5 g fat) for breakfast. Blood samples wereobtained directly from the catheters before as well as 30 and 60min after breakfast. Subsequently, the patients were hospital-ized and treated with IIT for 10-14 days. The target levels ofglycemic control were the following: fasting plasma glucose of<150 mg/dl and postprandial 2-h plasma glucose of <200mg/dl. After improvement in glycemic control, patients were in-structed to consume test meals and blood sampling was re-peated. Differences in pre- and post-IIT for the following vari-ables were quantified; serum immunoreactive insulin (IRI), serumC-peptide immunoreactivity (CPR), plasma glucagon, and plas-ma active GLP-1 levels.

AssaysBlood collection tubes were coated with ethylenediaminete-traacetic acid disodium salt (1.25 mg/ml of blood) and containedaprotinin and a DPP-4 inhibitor (10 μl/ml of blood; Linco Re-search Inc., St. Charles, MO, USA). Samples were immediatelyprocessed and kept on ice to retard peptide breakdown. Aftercentrifugation at 4 C, plasma and serum samples were keptfrozen at –20 C until analysis. Plasma glucose was measured im-mediately using the glucose oxidase method. Serum glycoal-bumin was measured by an enzymatic method using an albu-min-specific protease (ketoamine oxidase) and an albumin as-say reagent (Lucica glycated albumin-L; Asahi Kasei Pharma,Tokyo, Japan). Serum IRI levels were examined at a commerciallaboratory (SRL Inc., Tokyo, Japan) using radioimmunoassay(RIA). Plasma glucagon levels were measured by RIA using theGlucagon kit Daiichi-II (TFB Corp., Tokyo, Japan). Plasma activeGLP-1 levels were measured using an enzyme-linked im-munosorbent assay kit (Millipore Corp., Billerica, MA, USA) atSRL, Inc.

Statistical analysisInsulin resistance (IR) was quantified using the homeostasis mod-el assessment (HOMA) of IR (HOMA-IR) reported by Matthews etal. (12) and calculated as follows: HOMA-IR = [fasting glucose(mg/dl) / 18] × [fasting insulin (μIU/ml) / 22.5]. Islet β-cell functionwas evaluated by homeostasis model assessment β-cell func-tion (HOMA-β) using the following formula: 20 × fasting insulin(μIU/ml) / fasting glucose (mg/dl) − 3.5 (13); insulinogenic indexwas calculated as follows: (ΔIRI30min / ΔGlucose30min) (mIU/mmol);(IRI30min − IRI0min) / (Glucose30min − Glucose0min) to reflect early-phase insulin secretion (14). Based on the method described byVilsbøll et al. (15), the areas under the curve (AUC) from 0 to 60min were calculated using the trapezoidal rule, and t-tests forpaired data were used for comparing pre- and post-IIT variables.Statistical analysis was performed using the Statistical Package

for the Social Sciences ver. 17.0 (SPSS, Inc., Chicago, IL, USA).The results of the statistical analyses were shown as the mean±SD,unless otherwise specified. Analyses of efficacy were carried outat each time point using the Wilcoxon signed rank test againstthe baseline. Comparisons of time curves during the meal tol-erance test were analyzed by two-way repeated-measures anal-ysis of variance, followed by the Wilcoxon signed rank test ateach time measurement value; p-values <0.05 were consideredstatistically significant.

RESULTS

Twenty-six patients (15 males; mean age 63.5±6.5 yr;HbA1c levels 8.6%±0.9%; duration of diabetes 13.5±5.8yr) completed this study. The characteristics of the pa-tients are shown in Table 1.As shown in Table 2, the glycoalbumin levels decreasedsignificantly from 23.0% at pre-IIT to 19.6% at post-IIT(p<0.001). No significant differences in pre- and post-IIT

No.=26

M/F 15/11

Age (yr) 63.5±6.5

Hba1c (%) 8.6±0.9

Duration of diabetes (yr) 13.4±5.8

Duration of IIT (days) 12.0±1.8

Therapeutic measuresInsulin (%) 30.7Sulfonylureas (%) 57.7Alpha-glucosidase inhibitors (%) 57.7Biguanides (%) 34.6Thiazolidinedione (%) 26.9

Hypertension (%) 83.3

Hyperlipidemia (%) 76

Current smoking (%) 34

Alcohol drinking (%) 34

Familial history of diabetes (%) 30

Continuous variables are expressed as mean±standard deviation. Categor-ical variables are expressed as percentage. HbA1c: glycated hemoglobin;IIT: intensive insulin therapy.

Table 1 - Characteristics of study patients enrolled in the study.

Pre-IIT Post-IIT p

Glycoalbumin (%) 23.0±3.9 19.6±2.7 <0.001

BMI (kg/m2) 25.4±3.3 24.8±3.4 <0.001

SBP (mmHg) 124.5±12.1 111.4±9.9 <0.001

DBP (mmHg) 74.4±11.4 68.0±7.8 <0.001

TC (mg/dl) 184.8±29.4 166.3±27.9 0.003

HDL-C (mg/dl) 52.6±9.3 55.7±8.7 0.010

LDL-C (mg/dl) 110.1±31.2 101.2±28.8 0.180

TG (mg/dl) 118.8±40.2 111.2±24.7 0.103

hs-CRP (mg/dl) 0.13±0.15 0.10±0.16 0.190

BMI: body mass index; SBP: systolic blood pressure; DBP: diastolic bloodpressure; TC: total cholesterol; HDL-C: HDL cholesterol; LDL-C: LDLcholesterol; TG: triglyceride; hs-CRP: high sensitivity C-reactive protein.

Table 2 - Clinical characteristics of the patients at pre-intensiveinsulin therapy (IIT) and at post-IIT.

M. Shimodaira, Y. Muroya, N. Kumagai, et al.

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LDL cholesterol, triglycerides, and high-sensitivity C-re-active protein were observed and BMI and blood pres-sure significantly decreased post-IIT.The differences in insulin secretion, as well as glucose,glucagon, and GLP-1 levels during the meal tolerancetest from pre- to post-IIT are shown in Table 3. Serum IRIand CPR levels at 0, 30, and 60 min pre- and post-IITwere similar, whereas glucose levels at 0, 30, and 60 minpost-IIT significantly decreased from the baseline as fol-lows: 142.4 to 123.5 mg/dl (p<0.001), 173.9 to 148.7mg/dl (p<0.001), and 220.8 to 188.5 mg/dl (p=0.012),respectively. The post-IIT serum glucagon levels at 0 and60 min compared with pre-IIT levels decreased from105.4 to 91.6 pg/dl (p<0.001) and 119.8 to 101.6 pg/dl(p<0.001), respectively. In addition, post-IIT GLP-1 at 0,30, and 60 min significantly increased compared with pre-IIT levels. However, HOMA-β, HOMA-IR, and insulino-genic index were not significantly different following IIT.

The prandial AUC0-60min of glucagon significantly de-creased from 6755±996 to 5796±1074 (pg/dl·60 min)(p<0.001), and AUC0-30min and AUC0-60min of GLP-1 sig-nificantly increased following IIT. However, prandial AUCof CPR did not change after IIT (Table 4).

DISCUSSION

Several studies have reported elevated fasting glucagonlevels in T2DM (16, 17). Although glucagon levels typi-cally decreased after oral or intravenous glucose admin-istration in healthy individuals, glucose-induced sup-pression of glucagon secretion is markedly impaired, andmeal-induced glucagon excursions are typically exag-gerated in patients with T2DM (16). Although the regu-lation of glucagon secretion is not fully understood, thereis evidence that insulin and/or other paracrine factors inthe pancreatic islets participate in α-cell regulation (18).Another explanation for the positive association couldbe that GLP-1 may directly stimulate glucagon secretionin insulin-deficient islets (19).However, the mechanisms underlying increased glucagonsecretion in such patients are not clearly understood be-cause some studies have reported increased α-cell num-bers in T2DM (20, 21). In contrast, Henquin et al. (22) re-cently indicated that a higher proportion of α- to β-cellsin the islets was due to a decrease in the numbers of β-cells rather than an increase in those of α-cells. This im-balance may alter the normal inhibitory influence of β-cells on α-cells and lead to relative hyperglucagonemiain T2DM.In the present meal tolerance test, we used Meal Test C®

(Saraya Corporation, Osaka, Japan; 592 kcal with 75.0 gcarbohydrate, 8.0 g protein, and 28.5 g fat), which is areproduction of traditional Japanese meal with a higherratio of carbohydrates and lower ratios of fat and pro-tein. Vollmer et al. (23) reported that GLP-1 levels weresimilar in both the oral glucose tolerance test (OGTT) andmeal tolerance test in T2DM. In comparison to tradition-al OGTT, the present test meal was more physiological-ly balanced, as it contained all primary nutrients. There-fore, the present meal tolerance test may have providedan additional physiologic stimulus to assess incretin re-sponses.The aim of our study was to investigate differences be-tween pre- and post-IIT insulin, glucagon, and GLP-1 se-cretion in response to a meal tolerance test in patientswith established T2DM undergoing short-term IIT. How-ever, no significant improvements in β-cell function werenoted in these patients, as a dramatic decrease inglucagon and glucose as well as increase in GLP-1 lev-els was observed. Thus, improved glycemic control dur-ing IIT is likely orchestrated by alterations in GLP-1 andglucagon secretion.The relative contributions of improved α- and β-cell func-tion in short-term glycemic control are debatable. How-ever, the correlation between the suppression ofglucagon response to meal ingestion and improved GLP-1 secretion after IIT clearly indicated that the contribu-tion of glucagon suppression was not negligible. Abnor-malities in glucagon secretion occur in T2DM, and mostabnormalities, if not all, may actually reflect an impair-

Pre-IIT Post-IIT p

AUC of CPR (ng/ml⋅min)AUC0-30min 87.5±25.5 85.9±27.2 0.290AUC0-60min 193.8±61.2 194.5±61.9 0.450

AUC of glucagon (pg/dl⋅min)AUC0-60min 6755±996 5796±1074 <0.001

AUC of GLP-1 (pg/dl⋅min)AUC0-30min 267±127 574±245 <0.001AUC0-60min 654±281 1248±411 <0.001

Table 4 - Area under the curve (AUC) of C-peptide immunoreac-tivity (CPR), glucagon and glucagon-like peptide 1 (GLP-1) at pre-intensive insulin therapy (IIT) and post-IIT.

Pre-IIT Post-IIT p

Glucose (mg/dl)0 min 142.4±24.2 123.5±15.4 <0.00130 min 173.9±43.9 148.7±32.6 <0.00160 min 220.8±36.4 188.5±30.6 <0.001

IRI (μU/ml)0 min 13.8±7.8 12.2±7.6 0.43530 min 21.0±12.2 22.5±10.8 0.21060 min 20.3±14.6 23.2±12.5 0.061

CPR (ng/ml)0 min 2.5±0.6 2.4±0.8 0.26330 min 3.4±1.1 3.4±1.0 0.39960 min 3.7±1.3 3.8±1.4 0.052

Glucagon (pg/ml)0 min 105.4±17.0 91.6±18.6 <0.00160 min 119.8±17.4 101.6±20.4 <0.001

GLP-1 (pg/ml)0 min 2.8±1.5 4.1±1.5 <0.00130 min 6.0±3.7 15.4±10.6 <0.00160 min 6.8±2.2 10.1±2.9 <0.001

HOMA-β 65.7±33.9 75.5±42.8 0.127

HOMA-IR 4.8±3.2 3.7±2.6 0.172

Insulinogenic index 0.22±0.15 0.23±0.18 0.392

IRI: immunoreactive insulin; CPR: C-peptide immunoreactivity; HOMA:homeostasis model assessment.

Table 3 - Changes in glucose, insulin, glucagon and glucagon-like peptide 1 (GLP-1) during meal tolerance test from pre-in-tensive insulin therapy (IIT) to post-IIT.

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ment of α-cell glucose sensing (i.e., an impaired abilityof glucose to suppress glucagon secretion). Juhl et al.(23) investigated the impact of a 5-week sulfonylurea (gli-clazide) treatment on plasma glucagon in 10 T2DM pa-tients and noted a significant glucagon decrease in treat-ed patients (gliclazide vs placebo: 57±8 vs 79±10 pmol/l,p=0.011). In our study, basal and prandial insulin levelswere unchanged despite a substantial reduction inglycemia, which clearly indicated improved α-cell func-tion during short-term IIT. Therefore, we presumed thatrapid improvement in glycemic control resulted in earlyrestoration of pancreatic α-cell function.The strategy of administering short-term IIT has been wellstudied in newly diagnosed T2DM patients. Impaired β-cell function appears to be reversible, particularly in theearly stages of T2DM, when the limiting threshold for re-versibility of decreased β-cell mass has probably not beencrossed (24). Therefore, a shorter period of prior gluco-toxicity may result in the restoration of β-cell function(25). McFarlane et al. (26) reported that pancreatic β-cellfunction steadily improved during 8-12 weeks of eug-lycemia following the initial restoration of euglycemia. Inour study, the IIT duration was probably too brief to de-termine any additional advantageous effects of improvedβ-cell function, i.e., no statistical improvement in insulinsensitivity. Furthermore, patients enrolled in the presentstudy clearly had typical characteristics of T2DM com-mon among Japanese patients; i.e., substantially low in-sulin responses after the ingestion of meal without mas-sive obesity or severe insulin resistance (27). Thus, a pos-sible explanation as to why we did not find improvementsin β-cell function and insulin sensitivity may lie in patients’characteristics of relatively lower β-cell function and high-er insulin sensitivity. Further studies are required to clar-ify why insulin did not increase despite an increase inGLP-1 and a decrease in glucagon levels.Insulin is a molecule with strong anti-inflammatory prop-erties, characterized by its ability to suppress the gener-ation of a range of early proinflammatory substances (28),which have been shown to modify insulin sensitivity andsecretion. In the present study, we measured levels ofhigh sensitivity C-reactive protein (hs-CRP) as a parame-ter of inflammation; however, we could not identify a sig-nificant reduction during a 10-14-day duration of IIT.Therefore, changes in GLP-1 and glucagon secretion arethought to be independent of inflammation.This study had some limitations. First, our sample sizewas small and all patients were treated with intensivetherapy while they were hospitalized. Therefore, furtherstudies with a larger population conducted as a case-con-trol study (for example: control with basal insulin therapyonly or with diet therapy only) would be ideal. Second, inthe present study, we evaluated insulin resistance andsensitivity using a meal tolerance test; however, we couldnot identify changes in these parameters during IIT.Therefore, replication studies using the glucose clamptechnique would be important to confirm our findings,as this technique is useful to quantify first- and second-phase insulin secretion to measure β-cell function. Third,we measured only hs-CRP levels as an inflammatory pa-rameter, despite the fact that other cytokines, e.g. TNF-α or interleukins, may interfere with insulin secretion.

In conclusion, we observed decreased postprandialglucagon and increased GLP-1 levels following a mealtolerance test in Japanese patients with establishedT2DM following short-term IIT. Thus, our results providenew insights into the role of glucagon and GLP-1 secre-tion in patients with established T2DM undergoing IIT.

ACKNOWLEDGMENTSWe would like to thank the nursing staff at Tokyo Metropolitan Hiroo Hos-pital for their excellent patient care.

Declaration of interestThe authors report no conflict of interests.

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