Int J Clin Exp Med 2019;12(5):4840-4849www.ijcem.com /ISSN:1940-5901/IJCEM0085428

Original ArticleElectrophysiological mechanisms of calcium Channel-mediated pancreatic islet β cell hyposecretion of insulin in rats born small for gestational age

Yan-Yan Jin1*, Xu-Pin Xie2*, Meng-Zao He3, Zhen-Yong Wu4, Ying Shen4, Jian-Fang Zhu5, Jian-Hua Mao1, Li Liang5

1Children’s Hospital of Zhejiang University School of Medicine, Hangzhou, China; 2Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine, Hangzhou, China; 3Hangzhou Women’s Hospital, Hang-zhou, China; 4Department of Neurobiology, Zhejiang University School of Medicine, Hangzhou, China; 5The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. *Equal contributors.

Received July 30, 2018; Accepted February 12, 2019; Epub May 15, 2019; Published May 30, 2019

Abstract: The aim of this study was to investigate whether the effects of intrauterine malnourishment on pancreatic islet β cells in rats will last in postnatal life. Pancreatic tissue slice technique was used to obtain β cells. Whole-cell patch-clamp technique was used to record characteristics of calcium channels and cell membrane capacitance (Cm). The Cm of β cells from rats born small for gestational age (SGA) was significantly reduced. Cm was 5.69 ± 0.55 fF/Pf in the 4th week and 4.05 ± 1.04 fF/Pf in the 8th week. There were no statistical differences found in calcium current densities (Id). However, the reversal potential was much lower, inactivation curve was shifted left, and half-maximal voltage (V50) was changed in SGA rats. Present data suggests that decreased β cell secretion lasts in SGA postnatal life and is probably caused by changed calcium channel kinetics.

Keywords: Small for gestational age, pancreatic islet β cell, calcium channel, secretion, pancreatic slice, patch-clamp

Introduction

Evidence from epidemiological observations suggest that infants born small for gestational age (SGA) tend to develop metabolic syndrome (MS) or type 2 diabetes mellitus (T2DM) in adult life [1]. Stimulus or insult at a critical stage of growth and development, during early periods of life, can result in altered development of the endocrine pancreas structure and resett- ing of the physiological system. Thus, long-term changes in function may be induced [2-4]. Animal models show that maternal food res- triction during pregnancy results in reduced birth weight, impaired islet cell development, decreased islet volume, and reduction of β cell mass, due to diminished proliferation and en- hanced apoptosis in offspring [5-7]. Other re- search has shown that β cell secretory funct- ion is deficient in each developmental stage of low birth weight offspring and that the effects on insulin secretion are more significant than effects on β cell mass [7-9]. Pancreatic islet β

cell dysfunction in SGA infants is a very impor-tant and decisive element in adult-onset meta-bolic disease, though mechanisms are not well understood.

Electrical activity and ion channels of the β cell play a central role in stimulus-secretion cou-pling [10]. Calcium channels that open and close directly control intracellular calcium (Ca2+) levels and, in turn, regulate insulin release. As the most active ion in pancreatic β cells, Ca2+ is also a key regulator in glucose-stimulated insu-lin secretion [11]. Changes in time and space of free Ca2+ concentrations ([Ca2+]i) in the cyto-plasm constitute important physiological and pathological processes in calcium signaling cells [10-12].

A previous work demonstrated that pancreas insulin concentrations decrease in each devel-opmental stage after birth and that β cell mass and insulin expression decrease in rat offspring from undernourished mothers [8, 9]. Moreover,

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4841 Int J Clin Exp Med 2019;12(5):4840-4849

it was found that decreased single β cell secre-tion in SGA rats is caused by decreased expres-sion of calcium channels and reduced calcium currents in neonatal rats [13]. However, it was also found that protein expression of calcium channels compensated with age [9]. Therefore, the present study aimed to determine whether the effects of intrauterine malnourishment on pancreatic islet β cell calcium channel kinetics and secretion properties last in postnatal life. At present, there are no available papers con-cerning the electrophysiological properties of β cells in SGA rats.

Materials and methods

All animal procedures were conducted in accor-dance with the Animal Experimentation Ethics Committee of Zhejiang University. The SGA rat model was identified using protocols reported previously [13]. Briefly, virgin female Sprague Dawley rats (250-270 g) were mated with male Sprague Dawley rats (≥ 300 g). Pregnant ani-mals were housed individually. Control rats were provided with normal rat chow, containing 22.5% protein, 57.0% carbohydrates, 3.9% fat, 8.0% cellulose, 1.0% minerals, 5.0% vitamins (mixed), and 2.5% water. Restricted rats were given a 50% reduction in caloric intake. All rats had free access to tap water.

After delivery, newborn male pups from stan-dard diet mothers were weighed. Male pups that had a birth weight within ± 1 standard deviation (SD) of the mean were defined as appropriate for the gestational age (AGA) group. Male pups from food-restricted mothers were assigned to the SGA group, corresponding to -2 SD of the mean body weight of control pups. Both SGA and AGA newborn male rats were fed normally until the 4th week and 8th week.

Pancreatic slice preparation

Pancreatic slices were prepared, according to a previous work [14-16]. The animals were killed by cervical dislocation. Abdominal cavities were opened and the entry of the bile duct into the duodenum was localized and clamped, ensur-ing blockage of the bile duct behind the pan-creas. Warm (37°C) low gelling temperature agarose (1.9% wt/vol, Seaplaque GTG agarose, BMA Products) was injected into the distally clamped bile duct. After cooling with ice-cold extracellular solution (ECS), the injected and

hardened pancreas was then extracted. It was inserted into a small dish filled with warm (37°C) agarose, then immediately cooled-down on ice. A small cube was cut out of the splenic part of the agarose-embedded tissue, glued (Super Glue, ND Industries, Troy, Mich., USA) onto a vibrotome plate (VT 1000 S, Leica, Nussloch, Germany), and cut into 140 μm-thick slices, with a speed of 0.05 mm s-1 at 70 Hz. During slicing and storage, the tissue slices were kept in ice-cold ECS bubbled continuously with carbogen.

Electrophysiology

Tissue slices were placed in a submerged chamber and perfused at 1.5 mL/min with ECS, preheated for 15 minutes in a 37°C water bath. They were then bubbled with 95% O2 and 5% CO2 (pH 7.3). Cells were visualized with an upright microscope (Nikon Eclipse E600FN, Nikon, Tokyo, Japan; Zeiss Axioskop 2 FS, Carl Zeiss, Oberkochen, Germany) and a mounted charge-coupled device (CCD) camera with 5x digital amplification (Cohu, San Diego, CA). β cells from the second or third layer of select- ed islets were used for whole-cell recording. Whole-cell responses were filtered at 3 kHz and digitized at 10 kHz, using either a SWAM IIC patch-clamp amplifier (Celica, Ljubljana, Slov- enia) or an EPC10 amplifier (HEKA, Lambrecht, Germany). Patch pipettes were pulled (P-97, Sutter Instruments, Novato, CA) from borosili-cate glass capillaries (ITEM#B150-86-10; Su- tter Instruments, Novato, CA) and fire-polished immediately before use. Resistance of the pipettes ranged from 4-7 MΩ in a CsCl-based solution. To measure calcium currents, elec-trodes were filled with a solution containing (in mM): 125 CsCl, 40 HEPES, 2 MgCl2, 20 TEA-Cl, 0.05 EGTA, and 2 ATPNa2 (pH 7.2 with CsOH).

Exocytosis detection

All experiments were conducted at 32-34°C. β cells were voltage-clamped at a holding poten-tial of -80 mV. Simulated action potential bursts for stimulation were constructed by a computer from an action-potential template that was pre-recorded and applied to β cells under whole-cell voltage-clamping.

A membrane capacitance technique was used to record secretion in the fF range, estimated according to the Lindau-Neher technique [17]. Briefly, a 10-mV peak-to-peak 800 Hz sine wave

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4842 Int J Clin Exp Med 2019;12(5):4840-4849

was added to the holding potential by an EPC-10 amplifier (HEKA). Ten cycles were averaged for each data point. The extracellular solution contained (in mM): 118 NaCl, 20 tetraethylam-monium-Cl (TEA-Cl), 5.6 KCl, 1.2 MgCl2, 5 HEPES (pH 7.4 with NaOH), 2.6 CaCl2, and 5 D-glucose. The pipette solution contained (in mM): 125 potassium glutamate, 10 KCl, 10 NaCl, 1 MgCl2, 0.05 EGTA, 3 Mg-ATP, 0.1 cAMP, and 5 HEPES (adjusted to pH 7.1 using KOH). To calculate the mean incremental change in cell membrane capacitance (ΔCm) in train-stimula-tion experiments, data points obtained over 12.5 ms, before depolarization, were averaged and set as the baseline value using Matlab (Math Works, Natick, MA). Data points obtained over 500 ms, after depolarization, were aver-aged and set as the cell membrane capaci-tance (Cm) value.

Data analysis

Cells were excluded from the study if series resistance varied by > 15% over the course of the experiment. Data from β cells with rapid endocytotic steps were also excluded. Results are presented as mean ± standard error of the mean (SEM). Body weight is presented as mean ± SD. Offline analysis was conducted using Excel (Microsoft, OR), Igor Pro (WaveMetrics, Lake Oswego, OR), and GraphPad Prism (Gra- phPad Software, San Diego, CA). Significance was tested using Student’s t-test. Differences are considered significant at P < 0.05 (*) or P < 0.01 (**). Activation curves (Figure 5B) were fit-ted with the Boltzmann function:

G/Gmax = [1-exp (Vm-V50)/k]-1.....................(1)

Peak currents were converted to conductances (G) using the formula G = I/(Vm-Vrev), where Vm is the membrane voltage of depolarization puls-es and Vrev is the calculated calcium reversal potential. Gmax is the maximal conductance, V50 is the half-maximal voltage, Vm is the membrane voltage of depolarization pulses, and k is the slope factor. Inactivation curves (Figure 6B) were fitted with the Boltzmann function:

I/Imax = [1-exp (-Vm+V50)/k]-1......................(2)

I is the calcium current (ICa), Imax is the maxi-mum ICa, V50 is the voltage of half steady-state inactivation, and k is the slope factor.

Results

β cell exocytosis and insulin release decreased in the SGA group

Rat slice preparation protocol, originated by Speier and Rupnik, was used to study the elec-trophysiology of rat β cells in the current research [14-16]. Figure 1A and 1B show the morphology of islet slices and β cells, respec-tively, from adult Sprague-Dawley rats. Whole-cell patch-clamp recordings were made to study β cell exocytosis and insulin release. β cells were identified according to cell capaci-tance (almost > 5 pF). Voltage-gated Na+ cur-rents at hyperpolarizing potential and gap junc-tion currents were present in β cells (Figure 1C). Changes in cell capacitance occur when secretory granules are inserted into or are removed from the plasma membrane. These changes have been used to describe proper-ties of secretion in pancreatic β cells. To obtain accurate cell capacitance measurements, it is necessary to exclude the effects of the cell membrane area. Therefore, mean incremental changes in Cm (ΔCm) was used to represent secretion. A previous work indicated that plas-ma insulin concentrations and insulin mRNAs are affected by intrauterine nutrition [8, 9] and single β cell secretion is decreased in newborn SGA rats [13]. Thus, the current study exam-ined exocytosis induced by train depolarization stimuli. Figure 2A and 2B show representative Cm recordings obtained in 4th and 8th week adult rats, respectively. Original ΔCm data are presented in Figure 2C. At the 4th week, the SGA group had a ΔCm of 5.69 ± 0.55 fF/pF (n = 13), which was obviously lower than that of the AGA group (8.27 ± 0.85 fF/pF, n = 9), as pre-sented in Figure 2D. At the 8th week, the ΔCm of the SGA group was 4.05 ± 1.04 fF/pF (n = 8), which was significantly lower than that of the control group (7.56 ± 1.59 fF/pF, n = 6). Moreover, the ΔCm of 8th week SGA rats was less than that of 4th week SGA rats, although there was no significant difference (P = 0.07). Additionally, the ΔCm between 4th and 8th week AGA rats showed no significant differences (P > 0.05). Data from the control group are in rea-sonable agreement with previous reports, as expected from the cell surface area. Present data suggests that exocytosis from a single β cell is impaired in adult SGA rats and may grad-ually progress with time.

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4843 Int J Clin Exp Med 2019;12(5):4840-4849

Figure 1. A. Shows the morphology of islet slices of adult SD rat, the scale bar is 0.5 cm. The islet slice is indicated by an arrow. B. Shows the β cells of slices, the scale bar is 10 µm, the patch pipette is indicated by a thick arrow, the β cell is indicated by a thin arrow. C. Identification of pancreatic islet β cells.

Effects of SGA on voltage-activated calcium current

Exocytosis of insulin-containing granules is [Ca2+] i-dependent [12, 18]. A previous work has shown that expression of calcium channels and calcium currents is decreased in neonatal SGA rats [9, 13]. Furthermore, protein expres-sion of calcium channels compensated with age [9]. To investigate whether voltage activat-ed calcium currents were changed in adult SGA animals, this study set voltage pulse depolar-izations from -100 mV to 0 mV to record L+T-type current and -40 mV to 0 mV to record L-type current. To increase accuracy, peak cal-cium current densities were reported as a nor-malized value divided by cell capacitance (pF). Figure 3 shows the peak calcium channel cur-rent density of the 4th week and 8th week SGA and AGA groups. There were no statistical dif-ferences between the SGA group and AGA group at the 4th week or 8th week. There were no statistical differences between the 4th week and 8th week in AGA or SGA groups. Data sug-gests that voltage-activated calcium currents may be restored to normal in adult SGA ani-mals, consistent with previous research [9].

Effects of SGA on the I-V curve of voltage-activated calcium channels

The current study tested whe- ther the I-V relationship of volt-age-activated calcium chan-nels is affected in adult SGA rats. Voltage-activated calcium currents showed no obvious differences between the 4th week and 8th week in AGA or SGA groups. Thus, 4th week rats were chosen to study the characteristics of calcium ch- annels. The membrane was depolarized from a holding potential of -80 mV with 10 mV incremental steps from -60 to +60 mV, for a period of 300 ms. Normalized I-V curves of AGA and SGA groups are shown in Figure 4A. ICa was detect-able at test pulse potentials with membrane potentials more positive than -60 mV. This was then increased to the

maximum as the test pulse potential reached -10 to 0 mV. It was decreased when the poten-tial was more positive than 0 mV, then finally reversed at +30 to 40 mV, consistent with pre-vious work [18, 19]. Figure 4B shows no signifi-cant differences between the peak current density voltage of the AGA group (5.98 ± 0.63 pA/pF, n = 18) and SGA group (5.62 ± 0.74 pA/pF, n = 18). However, as shown in Figure 4C, there were apparent differences detected in the reversal potential between AGA and SGA groups. The reversal potential was 36.47 ± 2.88 mV in the AGA group and 29.21 ± 1.76 mV in the SGA group (P < 0.05). Present data, once again, demonstrates that peak voltage-activat-ed calcium current was not affected by SGA, but that calcium channels in SGA rats close earlier than in AGA rats.

Effects of SGA on voltage-activated calcium steady-state activation

The current study compared steady-state acti-vation of voltage-activated calcium channels in 4th week AGA and SGA groups. Currents, evoked by depolarizing voltage steps from -80 mV to potentials between -60 mV and +60 mV in 10

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4844 Int J Clin Exp Med 2019;12(5):4840-4849

Figure 2. A and B. Show the representative Cm recordings obtained in 4th week and 8th week adult rat, respectively. C. Original ΔCm data from AGA and SGA group, which directly shows βcell ΔCm from 4th and 8th week SGA was lower than AGA separately. D. Comparison of β cell ΔCm from 4th week and 8th week adult rat groups; ΔCm was 5.69 ± 0.55 fF/pF in 4th week SGA group, 8.27 ± 0.85 fF/pF in 4th week AGA group, 4.05 ± 1.04 fF/pF in 8th week SGA group, 7.56 ± 1.59 fF/pF in 8th week AGA group (4th week AGA group n = 9, 4th week SGA group n = 14, 8th week AGA group n = 9, 8th week SGA group n = 10); data are shown as x ± S.E. (*P < 0.05).

mV increments, were used to create voltage dependence of steady-state activation curves (Figure 5A). For analysis, individual ICa was normalized to the corresponding maximum cur-rent value. All data was fit with Equation (1) and found that the steady-state activation curve was not significantly shifted, as shown in Figure 5B. In addition, voltage dependence of activa-tion and conductance were not significantly dif-ferent between AGA and SGA groups. The SGA group showed similar characteristics (time course and voltage dependence) as those observed in the AGA group (Figure 5B and 5C). Slope factors (k) were also calculated (Figure 5D), but no differences were observed between

AGA and SGA groups. This may suggest that the opening speed or ease of Ca2+ channel gating is not affected by intrauterine malnourishment.

Effects of SGA on voltage-activated calcium steady-state inactivation

The current study tested the inactivation of Ca2+ channels in 4th week AGA and SGA groups. A double-pulse protocol was used to construct steady-state inactivation curves, as shown in Figure 6A. From a holding potential of -80 mV, 5 ms steps (P1) from -60 mV to 60 mV were applied in 10 mV increments at a frequency of 0.2 Hz to activate calcium channels. Each of

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4845 Int J Clin Exp Med 2019;12(5):4840-4849

Figure 3. Calcium channel current density of 4th week and 8th week of SGA and AGA group (4th week AGA group: L+T type n = 20, L type n = 12, T type n = 12; 4th week SGA group: L+T type n = 20, L type n = 18, T type n = 18; 8th week AGA group: L+T type n = 6, L type n = 6, T type n = 6; 8th week SGA group: L+T type n = 5, L type n = 5, T type n = 5).

Figure 4. A. Normalized I-V curves of 4th week AGA and SGA groups. B. Peak current density voltage in the AGA (5.98 ± 0.63 pA/pF, n = 18) and SGA (5.62 ± 0.74 pA/pF, n = 18) groups. C. Reversal potential of AGA and SGA

groups (AGA: 36.47 ± 2.88 mV, n = 21; SGA: 29.21 ± 1.76 mV, n = 18; *P < 0.05).

these steps was followed by a constant pulse (P2) to 0 mV to record outward tail current. Inactivation curves were fitted by Equation (2). Shapes of the inactivation curves were simi-lar between AGA and SGA groups, as shown in Figure 6B. However, the ICa inactivation curve of the SGA group was shifted to the left. Furthermore, V50 was -28.08 ± 1.549 in the SGA group, which was greater than that of the AGA group (P < 0.01, Figure 6C), suggesting that Ca2+ channels were inacti-vated rapidly and easily in SGA rats. Slope factors (k) were not affected (Figure 6D).

Discussion

Infants born small for gesta-tional age (SGA) due to intra-uterine malnourishment are closely related to incidence of MS and T2DM, as a high preva-lence of these diseases are

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4846 Int J Clin Exp Med 2019;12(5):4840-4849

Figure 5. A. The protocol for creating voltage dependence of steady-state activation curves. B. The current-voltage curves were normalized for a better appreciation of the shift. Currents were converted to conductance and fit was achieved according to the Boltzmann function (AGA group n = 12, SGA group n = 11), which was not significantly shifted in 4th week SGA group. C. Statistics of V50s in 4th week AGA and SGA groups. V50 in AGA group was -23.93 ± 1.99. V50 in SGA group was -26.96 ± 1.53. D. Statistics of slope factors (k). in 4th week AGA and SGA groups. k value in AGA group was 8.52 ± 1.19. k value in SGA group was 8.18 ± 1.16.

observed when SGA children reach adulthood [1-4]. Intrauterine nutrient limitation occurs at an early and critical stage of pancreas islet development, potentially inducing permanent changes in tissue structure or function [20, 21]. Of the complex and diverse pathogenesis, pan-creatic islet dysfunction is a very important ele-ment that links SGA with adult metabolic dis-eases [21]. The current study used a SGA rat model created under maternal calorie restric-tion during fetal development, as described previously [13].

Some studies have revealed that malnutrition during pregnancy influences the development and functional maturation of β cells in the fetal pancreas, causing intrinsic alterations in islets (such as impaired β cell programming, de- creased β cell mass, halted expansion of β-cell

masses, and increased apoptosis with aging) and a decrease in plasma insulin concentra-tions [7-9, 20, 21]. A previous work showed that pancreas insulin concentrations are decreased in each developmental stage after birth and that β cell mass and insulin expression is decreased in rat offspring from undernour-ished mothers [8]. Moreover, it was found that single β cell secretion was decreased in neona-tal SGA rats [13]. In the present work, using pancreatic tissue slices combined with whole-cell patch-clamp recording to obtain data in a more physiological state, results showed that the membrane capacitance of β cells from SGA rats was less than that of AGA rats, both in the 4th week and 8th week. In addition, the mem-brane capacitance of 8th week SGA rats was less than that of 4th week SGA rats, although no statistical significance was indicated. Based on

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4847 Int J Clin Exp Med 2019;12(5):4840-4849

Figure 6. SGA modulates ICa inactivation. A. The protocol for creating voltage dependence of steady-state inactiva-tion curves. B. ICa is fitted with Eq. (2), which was shifted left in 4th week SGA group. (AGA group n = 15, SGA group n = 12). C. Statistics of V50s in 4th week AGA and SGA groups. V50 in AGA group was -22.53 ± 1.005. V50 in SGA group was -28.08 ± 1.549. **P < 0.01. D. Statistics of slope factors (k) in 4th week AGA and SGA groups. k value in AGA group was 9.8 ± 0.61. k value in SGA group was 8.56 ± 0.55.

these data, it was considered that single β cell exocytosis is decreased in adult SGA rats and may gradually progress with time. This study further confirms that β cell-secretion defects are important in the pathological progression from SGA to MS.

Insulin secretion from the β-cell is a complex process. It is precisely controlled by a molecu-lar network with voltage-gated calcium chan-nels (VGCCs) as pivotal elements [10]. Voltage-gated, transient, and inward ICa is a common mechanism of initiation of Ca2+ signaling, asso-ciated with a variety of cellular responses [22-24]. The opening of VGCCs leads to a large increase of Ca2+ in microdomains near the plas-ma membrane, stimulating secretory granule trafficking and triggering insulin exocytosis

[25]. Using whole-cell patch-clamp recording, this study found that L+T-type, L-type, and T-type calcium current densities showed no sig-nificant differences in the 4th week and/or the 8th week of the SGA group and AGA group. This is consistent with a previous research in which protein expression of calcium channels was normal in adult rats [9]. Therefore, it was con-cluded that the quantity of calcium channels affected by intrauterine nutrition possibly com-pensates with age.

Next, the current study tested whether calcium characteristics were changed in the SGA group. The depolarization-evoked movement of the voltage sensors leads to conformational chang-es and/or physical repositioning of two intrinsic structures, namely, activation and inactivation

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4848 Int J Clin Exp Med 2019;12(5):4840-4849

gates [10, 13, 26]. Activation curves are com-monly used to reflect the opening speed or ease of channel gating, which involves two important parameters: V50 and k. V50 reflects the likelihood that a channel will activate, with greater values indicating a lower likelihood of activation. Moreover, k reflects the speed of activation, with larger values indicating slower speeds [10, 11, 24, 27, 28]. Present data shows that the activation curve, V50, and k were not changed in the SGA group.

Inactivation curves are commonly used to reflect inactivated speed or ease of channel gating. Rapid and complete inactivation of the ICa is a crucial step in terminating Ca2+ influx and cellular response [10, 23-29]. This study found that the inactivation curve was shifted left and V50 was changed in SGA rats, indicat-ing that the channels are easily inactivated. Similarly, normalized I-V curves showed that reversal potential was affected in the SGA group, indicating that the calcium channel closed earlier. However, k was not changed. Thus, it was proposed that the opening speed or ease of Ca2+ channel gating was not affected by intrauterine malnourishment. However, Ca2+ channels were inactivated rapidly and easily and closed earlier than in normal rats. The present work did not answer how intrauterine malnourishment affects calcium channel kinet-ics, it only described the phenomenon. Fur- ther experiments are necessary to explore the mechanisms.

In conclusion, present results reveal that de- creased β cell secretion caused by intrauter- ine malnourishment lasts in postnatal life. It tends to aggravate with time. The quantity of calcium channels was normal in adult SGA rats, but the kinetics of calcium channels were changed, possibly explaining the decrease in insulin secretion in adult life.

Acknowledgements

Yan-Yan Jin wrote the manuscript and researc- hed data. Xu-Pin Xie researched data and con-tributed to discussion. Meng-Zao He researched data. Zhen-Yong Wu and Ying Shen contributed to design and reviewed the manuscript. Jian-Fang Zhu contributed to discussion. Li Liang reviewed/edited the manuscript. Jian-Hua Mao contributed to discussion and reviewed/edited the manuscript. The authors declare that they do not have any conflicts of interest involving this work. This research was supported by the

Natural Science Foundation of Zhejiang Pro- vince (LQ18H050001, LQ19H310002) and National Natural Science Foundation of China (81170733). Prof. Li Liang is the guarantor of this work and, as such, had full access to all the data in the study. He takes responsibility for the integrity of the data and the accuracy of data analysis.

Disclosure of conflict of interest

None.

Address correspondence to: Li Liang, The First Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. E-mail: zdliangli@zju.edu.cn; Jian-Hua Mao, Children’s Hospital of Zhe- jiang University School of Medicine, Hangzhou, China. E-mail: maojh88@zju.edu.cn

References

[1] Huang YT, Lin HY, Wang CH, Su BH, Lin CC. As-sociation of preterm birth and small for gesta-tional age with metabolic outcomes in children and adolescents: a population-based cohort study from Taiwan. Pediatr Neonatol 2018; 59: 147-153.

[2] Jennings RE, Berry AA, Strutt JP, Gerrard DT, Hanley NA. Human pancreas development. De-velopment 2015; 142: 3126-3137.

[3] Marciniak A, Patro-Małysza J, Kimber-Trojnar Ż, Marciniak B, Oleszczuk J, Leszczyńska-Gorzel- ak B. Fetal programming of the metabolic syn-drome. Taiwan J Obstet Gynecol 2017; 56: 133-138.

[4] St-Pierre J, Laurent L, King S, Vaillancourt C. Effects of prenatal maternal stress on sero-tonin and fetal development. Placenta 2016; 48: S66-S71.

[5] De Oliveira JC, Gomes RM, Miranda RA, Barella LF, Malta A, Martins IP, Franco CC, Pavanello A, Torrezan R, Natali MR, Lisboa PC, Mathias PC, de Moura EG. Protein restriction during the last third of pregnancy malprograms the neuroen-docrine axes to induce metabolic syndrome in adult male rat offspring. Endocrinology 2016; 157: 1799-1812.

[6] Martins IP, de Oliveira JC, Pavanello A, Matius-so CCI, Previate C, Tófolo LP, Ribeiro TA, da Sil-va Franco CC, Miranda RA, Prates KV, Alves VS, Francisco FA, de Moraes AMP, de Freitas Math-ias PC, Malta A. Protein-restriction diet during the suckling phase programs rat metabolism against obesity and insulin resistance exacer-bation induced by a high-fat diet in adulthood. J Nutr Biochem 2018; 57: 153-161.

[7] Boehmer BH, Limesand SW, Rozance PJ. The impact of IUGR on pancreatic islet develop-

mailto:zdliangli@zju.edu.cnmailto:zdliangli@zju.edu.cnmailto:maojh88@zju.edu.cn

Electrophysiological mechanisms of β cell hyposecretion in SGA rats

4849 Int J Clin Exp Med 2019;12(5):4840-4849

[20] Meas T, Deghmoun S, Alberti C, Carreira E, Ar-moogum P, Chevenne D, Lévy-Marchal C. Inde-pendent effects of weight gain and fetal pro-gramming on metabolic complications in adults born small for gestational age. Diabeto-logia 2010; 53: 907-13.

[21] Gatford KL, Simmons RA. Prenatal program-ming of insulin secretion in intrauterine growth restriction. Clin Obstet Gynecol 2013; 56: 520-8.

[22] Gao J, Zhong X, Ding Y, Bai T, Wang H, Wu H, Liu Y, Yang J, Zhang Y. Inhibition of voltage-gated potassium channels mediates uncarbox-ylated osteocalcin-regulated insulin secretion in rat pancreatic β cells. Eur J Pharmacol 2016; 777: 41-48.

[23] García-Delgado N, Velasco M, Sánchez-Soto C, Díaz-García CM, Hiriart M. Calcium channels in postnatal development of rat pancreatic beta cells and their role in insulin secretion. Front Endocrinol (Lausanne) 2018; 9: 40.

[24] Yang SN, Berggren PO. Beta-cell CaV channel regulation in physiology and pathophysiology. Am J Physiol Endocrinol Metab 2005; 288: E16-28.

[25] Pedersen MG, Cortese G, Eliasson L. Mathe-matical modeling and statistical analysis of calcium-regulated insulin granule exocytosis in β-cells from mice and humans. Prog Biophys Mol Biol 2011; 107: 257-64.

[26] Catterall WA, Wisedchaisri G, Zheng N. The chemical basis for electrical signaling. Nat Chem Biol 2017; 13: 455-463.

[27] Enyeart JJ, Enyeart JA. Adrenal fasciculata cells express T-type and rapidly and slowly activat-ing L-type Ca2+ channels that regulate cortisol secretion. Am J Physiol Cell Physiol 2015; 308: C899-918.

[28] Zhu MM, Sun XD, Chen XD, Xiao H, Duan ML, Xu JG. Impact of gabapentin on neuronal high voltage activated Ca2+ channel properties of injured side axotomized and adjacent unin-jured dorsal root ganglions in a rat model of spinal nerve ligation. Exp Ther Med 2017; 13: 851-860.

[29] Kim JG, Sung DJ, Kim HJ, Park SW, Won KJ, Kim B, Shin HC, Kim KS, Leem CH, Zhang YH, Cho H, Bae YM. Impaired inactivation of L-type icaas a potential mechanism for variable ar-rhythmogenic liability of HERG K+ channel blocking drugs. PLoS One 2016; 11: e0149198.

ment and β-cell function. J Endocrinol 2017; 235: R63-R76.

[8] Wang X, Liang L, Du L. The effects of intrauter-ine undernutrition on pancreas ghrelin and in-sulin expression in neonate rats. J Endocrinol 2007; 194: 121-129.

[9] Xu YP, Liang L, Wang XM. The levels of Pdx1/insulin, Cacna1c and Cacna1d, and β-cell mass in a rat model of intrauterine undernutri-tion. J Matern Fetal Neonatal Med 2011; 24: 437-43.

[10] Marabita F, Islam MS. Expression of transient receptor potential channels in the purified hu-man pancreatic β-cells. Pancreas 2017; 46: 97-101.

[11] Llanos P, Contreras-Ferrat A, Barrientos G, Va-lencia M, Mears D, Hidalgo C. Glucose-depen-dent insulin secretion in pancreatic β-cell is-lets from male rats requires Ca2+ release via ROS-stimulated ryanodine receptors. PLoS One 2015; 10: e0129238.

[12] Rorsman P, Braun M. Regulation of insulin se-cretion in human pancreatic islets. Annu Rev Physiol 2013; 75: 155-79.

[13] Jin YY, He MZ, Wu ZY, Huang K, Shen Y, Liang L, Mao JH. Dysregulation of calcium channels de-creases parasecretion in pancreatic β-cells in rats born small for gestational age. Growth Factors 2016; 34: 159-165.

[14] Speier S, Rupnik M. A novel approach to in situ characterization of pancreatic beta-cells. Pflugers Arch 2003; 446: 553-558.

[15] Wu ZY, Zhu LJ, Zou N, Bombek LK, Shao CY, Wang N, Wang XX, Liang L, Xia J, Rupnik M, Shen Y. AMPA receptors regulate exocytosis and insulin release in pancreatic β cells. Traffic 2012; 13: 1124-39.

[16] Zou N, Wu X, Jin YY, He MZ, Wang XX, Su LD, Rupnik M, Wu ZY, Liang L, Shen Y. ATP regu-lates sodium channel kinetics in pancreatic is-let beta cells. J Membr Biol 2013; 246: 101-7.

[17] Lindau M. High resolution electrophysiological techniques for the study of calcium-activated exocytosis. Biochim Biophys Acta 2012; 1820: 1234-42.

[18] Rorsman P, Ramracheya R, Rorsman NJ, Zhang Q. ATP-regulated potassium channels and voltage-gated calcium channels in pancre-atic alpha and beta cells: similar functions but reciprocal effects on secretion. Diabetologia 2014; 57: 1749-61.

[19] Bokvist K, Eliasson L, Ammälä C, Renström E, Rorsman P. Co-localization of L-type Ca2+ chan-nels and insulin-containing secretory granules and its significance for the initiation of exocyto-sis in mouse pancreatic B-cells. EMBO J 1995; 14: 50-7.