Preeclampsia is a pregnancy-specific complication with long-term negative outcomes for offspring, including increased susceptibility to type 2 diabetes (T2D) in adulthood. In a rat reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia, maternal hypertension in conjunction with intrauterine growth restriction mimicked aspects of preeclampsia and resulted in female embryonic day 19 (e19) offspring with reduced β-cell area and increased β-cell apoptosis compared with offspring of sham pregnancies. Decreased pancreatic β-cell area persisted to postnatal day 13 (PD13) in females and could influence whether T2D developed in adulthood. Macrophage changes also occurred in islets in T2D. Therefore, we hypothesized that macrophages are crucial to reduction in pancreatic β-cell area in female offspring after chronic placental ischemia. Macrophage marker CD68 mRNA expression was significantly elevated in e19 and PD13 islets isolated from female RUPP offspring compared with sham. Postnatal injections of clodronate liposomes into female RUPP and sham offspring on PD2 and PD9 significantly depleted macrophages compared with injections of control liposomes. Depletion of macrophages rescued reduced β-cell area and increased β-cell proliferation and size in RUPP offspring. Our studies suggest that the presence of macrophages is important for reduced β-cell area in female RUPP offspring and changes in macrophages could contribute to development of T2D in adulthood.

High blood pressure in pregnancy is often accompanied by intrauterine growth restriction (IUGR), and both can contribute to pathophysiology of diseases with origins early in life. The Helsinki Birth Cohort Study demonstrated gestational hypertension was associated with increased risk of type 2 diabetes (T2D) in offspring, with an even greater risk if offspring had low or high birth weight (1). A meta-analysis (2) reported 1.5 times increased risk of T2D with low birth weight <2500 g. The current study focused on identifying developmental origins of an increased risk for T2D following gestational hypertension. Our previous studies have used the reduced uteroplacental perfusion pressure (RUPP) model of chronic placental ischemia–induced hypertension in the rat to investigate developmental origins of T2D after gestational hypertension (3). In this model, mechanical reduction of uteroplacental flow at gestation day 14 (GD14) of a 21-day gestation resulted in high blood pressure in the dam and reduced fetal weight at GD19, showing similarities to preeclampsia in humans (4). In the RUPP model, we demonstrated that pancreatic β-cell area was reduced and β-cell apoptosis increased in female, but not male, rat offspring at embryonic day 19 (e19) (3). This is consistent with previous studies demonstrating offspring of RUPP dams exhibit glucose intolerance (57). Pancreatic β-cell mass is set early in life, and insufficient β-cell mass influences whether an individual develops T2D (8,9). Understanding the mechanism of regulating β-cell mass is important to designing therapeutic approaches that can prevent reduction in β-cell mass or mitigate the effects of reduction postnatally.

The role of macrophages in pancreatic β-cell mass development remains to be established. However, emerging evidence suggests that macrophages may be involved in maintaining β-cell mass and in pathophysiology of T2D. Depletion of macrophages in an adult mouse does not impair glucose tolerance or affect pancreatic insulin content (10). However, the op/op mouse (11) that lacks islet macrophages throughout development has reduced β-cell mass, suggesting that the presence of the macrophage is important in utero or postnatally for establishing normal β-cell mass. In contrast, too many macrophages may contribute to reduced β-cell mass in T2D, as suggested by studies that report increased islet macrophages in humans who have developed T2D (1214) and in animal models of T2D, including the db/db mouse and GK rat (12). In offspring of complicated pregnancies, a role for excess macrophages in causing reductions in β-cell mass during pancreatic development was demonstrated in studies by Jaeckle Santos et al. (15) and Simmons et al. (16), showing increased M2 macrophage markers precede reductions in β-cell mass in pregnancies complicated by growth restriction via bilateral uterine artery ligation (BUAL) and placental insufficiency.

Building on our previous studies in the RUPP rat model of gestational hypertension, we hypothesized that macrophages are critical to the reduction in pancreatic β-cell area and increased β-cell apoptosis seen in female rat offspring after chronic placental ischemia. To test this hypothesis, we determined whether decreased pancreatic β-cell area and increased β-cell apoptosis persisted postnatally in RUPP rat offspring and whether postnatal depletion of macrophages rescued pancreatic β-cell area. Our studies demonstrate that macrophage depletion in early postnatal life rescues the defect in β-cell area in offspring of RUPP pregnancies, pointing to the important role these innate immune cells play in the pathophysiology of T2D in offspring of complicated pregnancies.

RUPP Model of Placental Ischemia–Induced Hypertension

Timed-pregnant Sprague-Dawley rats (CD IGS strain; Charles River Laboratories, Raleigh, NC) with specified breeding weights of 215 to 225 g were housed in a temperature-controlled facility, with a 12-h light/dark cycle and tap drinking water and standard rat chow (Purina LabDiet 5001) ad libitum. All procedures were approved by the Institutional Animal Care and Use Committee of the University of Minnesota. RUPP procedures and control sham surgeries were conducted on GD14 as previously described (17), with GD0 defined as date of vaginal plug. Our previous studies (1722) have consistently shown increased blood pressure in RUPP dams with this method. In the current experiments, blood pressure was not monitored. Pups were weighed within 24 h of birth and litters culled to eight pups with preference for females. Pups were housed with birth mother until necropsy on postnatal day 13 (PD13).

Experimental Design

Delivery of clodronate liposomes intraperitoneally (i.p.) targets depletion of macrophages in the peritoneal cavity, some lymph nodes, liver, spleen, and bone marrow (23,24). The liposomes are ingested by phagocytes, and clodronate accumulates and induces cell death by apoptosis. On PD2, female pups were assigned to untreated, control liposome or 7mg/ml clodronate liposome treatment groups (Fig. 1A) (F70101-A, placebo control liposome for Clophosome-A anionic; F70101C-A, Clophosome-A anionic liposomal clodronate for macrophage depletion; FormuMax Scientific, Inc.). In general, one pup of each sex from a litter was untreated, and additional female pups from each litter received either control liposomes or clodronate liposomes. In a few instances, two offspring from the same dam received the same treatment; this is indicated in the figure legend (i.e., six pups from five litters). Pups were injected i.p. with control liposomes or clodronate liposomes on PD2 (clodronate 56 mg/kg) and PD9 (clodronate 28 mg/kg). Untreated pups were not injected.

Figure 1

Characteristics of litters and offspring after RUPP surgery with gestational hypertension. A: Experimental design. Timed-pregnant Sprague-Dawley dams underwent either RUPP or sham surgery on GD14. Dams gave birth, and pups were either untreated or injected i.p. with control or clodronate liposomes on PD2 and PD9. Pups were euthanized on PD13 and tissues collected. Islets were isolated by collagenase digestion and handpicking. B: Number of live pups per litter from dams undergoing either RUPP or sham surgery (n = 24–31 litters). *P < 0.05 by Student t test. C: GD at birth of dams undergoing either RUPP or sham surgery (n = 24–31 litters). *P < 0.05 by Student t test or nonparametric Wilcoxon test. D: Birth weight of female and male pups from dams undergoing either RUPP or sham surgery, weighed within 24 h of birth. Individual pups from 22 to 28 litters. E: Weight of PD13 female and male pups from dams undergoing either RUPP or sham surgery. Individual pups from 21 to 26 litters. Statistics for panels D and E were from mixed-model ANOVAs using individual pup values with random litter effects and random litter by sex effects as well as GD at birth. *P < 0.05 for ANOVA effects or individual contrasts. Values represent mean ± SE.

Figure 1

Characteristics of litters and offspring after RUPP surgery with gestational hypertension. A: Experimental design. Timed-pregnant Sprague-Dawley dams underwent either RUPP or sham surgery on GD14. Dams gave birth, and pups were either untreated or injected i.p. with control or clodronate liposomes on PD2 and PD9. Pups were euthanized on PD13 and tissues collected. Islets were isolated by collagenase digestion and handpicking. B: Number of live pups per litter from dams undergoing either RUPP or sham surgery (n = 24–31 litters). *P < 0.05 by Student t test. C: GD at birth of dams undergoing either RUPP or sham surgery (n = 24–31 litters). *P < 0.05 by Student t test or nonparametric Wilcoxon test. D: Birth weight of female and male pups from dams undergoing either RUPP or sham surgery, weighed within 24 h of birth. Individual pups from 22 to 28 litters. E: Weight of PD13 female and male pups from dams undergoing either RUPP or sham surgery. Individual pups from 21 to 26 litters. Statistics for panels D and E were from mixed-model ANOVAs using individual pup values with random litter effects and random litter by sex effects as well as GD at birth. *P < 0.05 for ANOVA effects or individual contrasts. Values represent mean ± SE.

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Isolation of Pancreas and Islets

On PD13, rats were anesthetized with isoflurane. A midline incision was made in the abdomen, and the abdominal aorta was isolated for bleeding into a glass tube using a 25 g × 3/4 inch SURFLO winged infusion set (cat. no. SV*25BLK; Terumo Corporation). Glucose was measured from aortic bleed using a handheld glucose meter and glucose strips (Contour 9545C and 7097C; Bayer). Blood was allowed to clot for 30 min at room temperature and then placed on ice for 30 min before centrifugation at 4°C for 20 min at 730g. Serum was separated and frozen at −80°C. Serum insulin of undiluted samples was measured using rat ultrasensitive insulin ELISA per manufacturer protocol (cat. no. 80-INSRTU-E10; ALPCO).

In selected animals, pancreata of PD13 rats were isolated, weighed, and fixed in 10% neutral buffered formalin overnight and then stored at 4°C in 70% ethanol before paraffin embedding. In other pups, pancreata were perfused via hepatic duct with 0.75 mg/mL ice-cold collagenase P (cat. no. 11213865001; Roche) (1.9 units/mg) in Hanks’ balanced salt solution (HBSS) without calcium chloride, magnesium chloride, or magnesium sulfate (cat. no. 14175-095; Gibco). The perfused pancreata were isolated and digested for 3 to 4 min at 37°C and then washed three times in cold HBSS with calcium chloride and magnesium chloride (cat. no. 14025-092; Gibco) containing 2% heat-inactivated fetal bovine serum (FBS). Large undigested fragments were screened out, and the cell suspension was poured through a 70-μm filter to trap islets. The islet suspension was then transferred to a dish with warmed islet complete media: RPMI 1640 (cat. no. 10-043-CV; Corning) (with L-glutamine and without glucose), 5 mmol/L glucose, 10% heat-inactivated FBS, and penicillin-streptomycin solution (cat. no. SV30010; HyClone) (final concentration 100 units/mL penicillin and 100 μg/mL streptomycin). Approximately 100 to 150 islets and 200 acinar were handpicked from each pancreas, washed two times with phosphate-buffered saline (PBS), resuspended in 20 μL RNALater (cat. no. R0901; Sigma-Aldrich), and frozen at −80°C until isolation of RNA. Alternatively, handpicked islets were used for flow cytometry on the day of islet isolation. The right lateral lobe of liver was frozen at −80°C until RNA was extracted.

Flow Cytometry

At PD13, before blood collection, select pups underwent peritoneal lavage with 4 × 2 mL ice-cold PBS to collect cells for flow cytometry. Spleens were harvested and mechanically dissociated in cold PBS. Mononuclear cell fractions were isolated using Histopaque 1083 (cat. no. 10831; Sigma-Aldrich) from both lavage fluid and dissociated spleens. Cells were washed twice with a buffer containing PBS, 2% heat-inactivated FBS, and 2 mmol/L EDTA before staining for flow cytometry.

Handpicked islets for flow cytometry were dissociated with 0.05% trypsin (cat. no. 15090-046; Gibco) (2.5% trypsin [10×] with no phenol red) and 0.48 mmol/L EDTA in HBSS without calcium chloride, magnesium chloride, or magnesium sulfate (cat. no. 14175-095; Gibco) for 6 min at 37°C with 10 s of vortexing every 2 min. Dispersed cells were washed twice with PBS containing 2% heat-inactivated FBS and 2 mmol/L EDTA, counted, and then stained for flow cytometry.

Macrophages were evaluated in peritoneal lavage, spleen, and islets of PD13 female rats using antibodies specific to macrophage markers CD68 and CD163. Briefly, samples containing up to 1 million cells were incubated in a 0.09% sodium azide solution in PBS with 1% heat-inactivated FBS. Fc receptors were blocked using 0.25 μg/mL purified mouse anti-rat CD32 clone D34-485 (cat. no. 550270; BD Biosciences) (0.5 mg/mL) and 50% donkey serum (D9663; Sigma-Aldrich). The surface macrophage marker CD163 was stained with 6.067 μg/mL anti-CD163 (ED2) Alexa Fluor 647 (cat. no. NBP2-39099AF647; Novus Biologicals) (0.91 mg/mL). The isotype (mouse immunoglobulin G1 control Alexa Fluor 647 conjugated; cat. no. IC002R; R&D Systems) was used as a control. From here, cells were fixed with 4% paraformaldehyde fixation solution (cat. no. 420801; BioLegend) and permeabilized with Intracellular Staining Permeabilization Wash Buffer (cat. no. 421002; BioLegend) for intracellular staining of the macrophage marker CD68. The antibody mouse anti-rat CD68:FITC (cat. no. MCA341F; BioRad) (0.1 mg) was incubated with the previously stained cells at a concentration of 2 μg/mL. The isotype mouse immunoglobulin G1κ (MOPC-21) FITC isotype control (cat. no. ab106163; Abcam) (1 mg/mL) was used as a control.

M1 macrophages were defined as CD68+ CD163 and M2 macrophages as CD68+ CD163+. For enumeration of macrophages in islets, only CD68 staining (total macrophages) was determined because of the small number of cells. Cells were analyzed using a BD Accuri C6 Plus Flow Cytometer and FlowJo software (version 10). The gating strategy is illustrated in Supplementary Fig. 1. Cells were initially gated on live singlet populations. Autofluorescent cells were identified in unused fluorescent channels and excluded from the analysis.

Measurement of Pancreatic β-Cell Ratio, Size, Proliferation, and Apoptosis

Paraffin-embedded PD13 rat pancreata were sectioned at 5-μm thickness. Five insulin-stained sections taken 200 μm apart through the depth of the pancreas were evaluated. The tissue was deparaffinized using standard CitriSolv and dehydration procedures and then pretreated as previously described (3). For β-cell area analysis, the tissue underwent primary staining with guinea pig anti–insulin (cat. no. IS002; DAKO) (1:400) overnight at 4°C. Several washes of PBS with 0.01% Tween were performed the next day before secondary staining incubation with FITC anti–guinea pig antibody (Jackson ImmunoResearch) (1:500) for 90 min at 37°C. After washes of PBS with 0.01% Tween, the slides were dipped in DAPI solution (cat. no. 62248; Thermo Fisher Scientific) according to manufacturer instructions and then cover slipped with mounting media (cat. no. H-1000; Vector Laboratories). After imaging, β-cell and pancreatic areas were measured to calculate the β-cell ratio. β-cell size was calculated by β-cell area over colocalization counts of insulin-positive and DAPI-positive cells (at least 20 islets per animal). For proliferation assessment, the addition of rabbit anti-Ki67 antibodies (cat. no. ab15580; Abcam) (1:2,500) during primary staining followed by secondary staining with anti-rabbit CY3 (Jackson ImmunoResearch) (1:400) was used. β-cell proliferation was counted when insulin-positive cells exhibited costaining of DAPI and Ki67 (at least 20 islets per animal). Apoptosis was assessed using a TUNEL kit (cat. no. S7165; MilliporeSigma) according to manufacturer instructions. Colocalization of TUNEL-positive staining and insulin-positive cells was considered positive for β-cell apoptosis. At least 2,000 insulin-positive cells were counted per animal for TUNEL and proliferation analyses. Images were taken at 10× using a motorized microscope (cat. no. BZ-X800E; Keyence). Fiji Software was used to perform image analysis for β-cell ratio, β-cell size, proliferation, and apoptosis.

Quantitative RT-PCR

Total RNA was isolated from handpicked islets using QIAshredder (cat. no. 79654; QIAGEN) followed by the RNeasy Mini Kit (cat. no. 74106; QIAGEN). cDNA was generated using random 9 primers (5′-NNNNNNNNN-3′) and 40 to 100 ng RNA per reaction using the Omniscript RT Kit (cat. no. 205113; QIAGEN). Quantitative RT-PCR reactions were generated using the QuantiTect SYBR Green PCR Kit (cat. no. 204145; QIAGEN) and carried out in a Rotor-Gene Q Thermocycler (QIAGEN) for 40 cycles. Macrophage marker CD68 mRNA expression was amplified using forward primer 5′-TCTGACCTTGCTGGTACTGC-3′ and reverse primer 5′-GAAGAGTGGCAGCCTTTTTG-3′. Profiles were analyzed using Rotor-Gene Q Series software. ΔΔCt was calculated using β-actin as a normalizer with forward primer 5′-CCTGGGTATGGAATCCTGTGGCAT-3′ and reverse primer 5′-TCTTGATCTTCATGGTGCTAGGAGCC-3′. Primers were custom ordered through Integrated DNA Technologies. Primers had been previously published (25,26) and were tested for reaction efficiency. For quantitative RT-PCR of e19 islets and acinar, RPLP0 was used as the normalizer, as previously described (3).

i.p. Glucose Tolerance Test and C-peptide Assessment

Female offspring of RUPP and sham pregnancies were weaned on PD21. Animals were fed normal rat chow and tap water ad libitum. At age 8 to 9 weeks, animals were fasted for 15 to 16 h, and an i.p. glucose tolerance test was conducted. Briefly, animals were bled by tail snip for fasting blood glucose (time 0) determined by glucometer, and blood was collected in heparinized capillary tubes and centrifuged and plasma stored at −80°C. Glucose (2 g/kg) was administered i.p. and measured at 30, 60, 90, and 120 min. C-peptide in plasma of fasted female rats age 8 to 9 weeks was determined by rat C-peptide ELISA per manufacturer protocol (cat. no. 80-CRPRT-E01; ALPCO).

Statistical Analysis

For each end point, we used one male and one female pup from each litter. In a few instances, two offspring from the same dam received the same treatment, and this is indicated in the figure legend (i.e., six pups from five litters). Data are presented as mean ± SEM. Student t test or Wilcoxon rank sum test was used for data on dams comparing RUPP and sham groups (Figs. 1B and C and 3A). For end points on pups, mixed-model ANOVAs using individual pup values with random litter effects and random litter by sex effects were used. In Fig. 1D and E, gestation age was also included in the mixed model. Statistical analysis was performed using JMP and SAS software (SAS Institute, Cary, NC), with P < 0.05 considered significant. Comparing male and female RUPP and sham offspring (Figs. 1D and E, 2, and 3B), post hoc comparisons considered were as follows: sham female versus RUPP female, sham male versus RUPP male, sham female versus sham male, and RUPP female versus RUPP male. When considering the effectiveness of clodronate treatment in female RUPP and sham offspring (Figs. 46), post hoc comparisons considered were as follows: sham control liposomes versus RUPP control liposomes, sham clodronate liposomes versus RUPP clodronate liposomes, sham control liposomes versus sham clodronate liposomes, and RUPP control liposomes versus RUPP clodronate liposomes.

Data and Resource Availability

Data generated in the current study are available from the corresponding authors on reasonable request.

Reduced Body Weight of PD13 RUPP Offspring

Our previous studies demonstrated that e19 offspring of RUPP pregnancies have growth restriction with significant reduction in body weight (3). In the current study, litters of RUPP pregnancies had fewer offspring than those of sham pregnancies (Fig. 1B), and the ratio of male to female pups did not differ (data not shown). The GD at birth for RUPP offspring was significantly greater than that for sham offspring (P < 0.05) (Fig. 1C). That is, sham pups were primarily born on GD21 and RUPP pups on GD22, with one RUPP dam delivering on each of GD20 and GD23. Birth weights of offspring were determined within 24 h of delivery. Given that GD at birth was longer for RUPP (Fig. 1C), we included GD at birth in the mixed model along with random litter and random litter by sex effects. Considering birth weight, a significant RUPP effect was detected (P = 0.008), with RUPP offspring smaller than sham offspring (Fig. 1D). For weights at PD13 and including GD in the model, offspring of RUPP dams had significantly reduced body weight compared with those of sham (RUPP effect P < 0.0001) (Fig. 1E). For both males and females, RUPP pups had smaller PD13 weights. At birth and at PD13, females consistently weighed less than males at both ages.

Reduced β-Cell Area and Increased Apoptosis in Female RUPP Offspring Persists to PD13

Our previous studies demonstrated a significant decrease in β-cell area and increased apoptosis of β-cells in female e19 fetuses from RUPP versus sham pregnancies (3). To determine if this phenotype persisted postnatally, pancreata were isolated from PD13 pups of dams undergoing either RUPP or sham surgery. The reduction in β-cell area persisted in PD13 females but not in males (Fig. 2A), as did the increase in β-cell apoptosis measured by percentage TUNEL staining (Fig. 2B). Pancreas mass did not differ in the four treatment groups (male/female and RUPP/sham) (data not shown). Nonfasting blood glucose was greater in male offspring but did not differ with dam surgery (Fig. 2C), and no significant difference was detected in nonfasting serum insulin (Fig. 2D).

Figure 2

β-cell area is reduced and apoptosis increased in PD13 female offspring of RUPP surgeries. A: β-cell/pancreas area was determined by image analysis of paraffin-embedded sections of pancreas stained for insulin in both male and female offspring of dams undergoing either RUPP or sham surgery (sham female n = 5 pups, 4 litters; RUPP female n = 6 pups, 4 litters; sham male n = 5 pups, 4 litters; RUPP male n = 6 pups, 5 litters). B: Percentage TUNEL staining (β-cell apoptosis) of colocalized insulin-positive cells and TUNEL-positive cells determined by image analysis of paraffin-embedded sections of pancreas in both male and female offspring of dams undergoing either RUPP or sham surgery (sham female n = 4 pups, 3 litters; RUPP female n = 4 pups, 4 litters; sham male n = 4 pups, 3 litters; RUPP male n = 4 pups, 4 litters). C: Blood glucose of nonfasted female and male PD13 pups from dams undergoing either RUPP or sham surgery. Individual pups from 18 to 20 litters. D: Serum insulin of nonfasted female and male PD13 pups from dams undergoing either RUPP or sham surgery (sham female n = 9 pups, 7 litters; RUPP female n = 11 pups, 9 litters; sham male n = 9 pups, 8 litters; RUPP male n = 9 pups, 9 litters. *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects in panels AD. Values represent mean ± SE.

Figure 2

β-cell area is reduced and apoptosis increased in PD13 female offspring of RUPP surgeries. A: β-cell/pancreas area was determined by image analysis of paraffin-embedded sections of pancreas stained for insulin in both male and female offspring of dams undergoing either RUPP or sham surgery (sham female n = 5 pups, 4 litters; RUPP female n = 6 pups, 4 litters; sham male n = 5 pups, 4 litters; RUPP male n = 6 pups, 5 litters). B: Percentage TUNEL staining (β-cell apoptosis) of colocalized insulin-positive cells and TUNEL-positive cells determined by image analysis of paraffin-embedded sections of pancreas in both male and female offspring of dams undergoing either RUPP or sham surgery (sham female n = 4 pups, 3 litters; RUPP female n = 4 pups, 4 litters; sham male n = 4 pups, 3 litters; RUPP male n = 4 pups, 4 litters). C: Blood glucose of nonfasted female and male PD13 pups from dams undergoing either RUPP or sham surgery. Individual pups from 18 to 20 litters. D: Serum insulin of nonfasted female and male PD13 pups from dams undergoing either RUPP or sham surgery (sham female n = 9 pups, 7 litters; RUPP female n = 11 pups, 9 litters; sham male n = 9 pups, 8 litters; RUPP male n = 9 pups, 9 litters. *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects in panels AD. Values represent mean ± SE.

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Increased Macrophage Marker CD68 in Islets of RUPP Offspring at e19 and PD13

In a bilateral uterine artery ligation model of intrauterine growth restriction, the study by Jaeckle Santos et al. (15) demonstrated increased mRNA expression for macrophage markers in a transcriptomic analysis of PD14 islets, as well as immunohistochemical evidence that CD68+ cells increased in association with the islet. We determined if islets from RUPP offspring had increased mRNA expression for CD68, suggesting a change in macrophage number. At e19, CD68 mRNA expression increased >3.5-fold in the pooled islets of fetuses collected from two RUPP litters compared with pooled islets of fetuses from two sham litters (Fig. 3A), with no significant change in the acinar. The sex of the e19 fetuses was not determined. This increase in mRNA expression of CD68 persisted in PD13 females but not males (Fig. 3B), with no significant differences in PD13 acinar tissue (data not shown). The increase in CD68 mRNA expression provided a strong rationale to deplete macrophages in the PD13 female offspring and determine the effect on β-cell area.

Figure 3

Macrophage marker CD68 mRNA expression in islets is elevated after RUPP surgery. A: CD68 mRNA expression in islets and acinar isolated from e19 digested pancreata. Values represent mean + SE of triplicate reactions from pancreata pooled from two RUPP litters or two sham litters. Fold change of CD68 relative to RPLP0 using the ΔΔCt method of quantitation relative to sham islets group. *P < 0.05 by Student t test. B: CD68 mRNA expression in islets isolated from PD13 digested pancreata. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham female group (sham female n = 10 pups, 4 litters; RUPP female n = 5 pups, 3 litters; sham male n = 6 pups, 3 litters; RUPP male n = 13 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects.

Figure 3

Macrophage marker CD68 mRNA expression in islets is elevated after RUPP surgery. A: CD68 mRNA expression in islets and acinar isolated from e19 digested pancreata. Values represent mean + SE of triplicate reactions from pancreata pooled from two RUPP litters or two sham litters. Fold change of CD68 relative to RPLP0 using the ΔΔCt method of quantitation relative to sham islets group. *P < 0.05 by Student t test. B: CD68 mRNA expression in islets isolated from PD13 digested pancreata. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham female group (sham female n = 10 pups, 4 litters; RUPP female n = 5 pups, 3 litters; sham male n = 6 pups, 3 litters; RUPP male n = 13 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects.

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Clodronate Liposomes Effectively Deplete Macrophages From Multiple Compartments in PD13 Female Offspring

Female offspring were treated on PD2 and PD9 with either control liposomes or clodronate liposomes, and the effect on macrophages was assessed by evaluating CD68 mRNA expression and number of macrophages by flow cytometry. First, we demonstrated effective depletion of macrophages in the liver of sham and RUPP offspring treated with clodronate liposomes compared with control liposomes (Fig. 4A). CD68 mRNA expression in the liver was significantly reduced after treatment with clodronate. Next, we examined the effect of control and clodronate liposomes on macrophages in the islets. Treatment with control liposomes alone significantly increased CD68 mRNA expression in both RUPP and sham offspring compared with untreated animal islets (Fig. 4B). However, treatment with clodronate liposomes reduced the CD68 mRNA expression significantly compared with control liposomes (Fig. 4B). Dispersed cells from islets collected from individual pancreata were examined by flow cytometry. No significant differences in the average number of islets collected (range 53–255) were observed among the groups of animals (data not shown). Significantly fewer macrophages defined as CD68+ cells were recovered from islets of the clodronate-treated animals (Fig. 4C), verifying macrophage depletion from this compartment. As seen in Supplementary Fig. 2A, the total number of cells recovered from the islets was significantly reduced by the clodronate treatment, whereas the number of nonmacrophages was unchanged. In islets, total macrophages were defined as CD68+ cells. The fraction of cells that were macrophages was very small and was unaffected by clodronate treatment.

Figure 4

Clodronate treatment effectively depletes CD68+ cells in liver and islets of PD13 female offspring. Offspring were treated i.p. on PD2 and PD9 with clodronate liposomes or control liposomes. A: CD68 mRNA expression in liver from offspring of dams undergoing either RUPP or sham surgery. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham control liposome group (sham control liposomes n = 3 pups, 3 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 4 pups, 3 litters; RUPP clodronate liposomes n = 10 pups, 8 litters). B: CD68 mRNA expression in islets isolated from digested pancreata. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham untreated group (sham untreated n = 10 pups, 4 litters; RUPP untreated n = 5 pups, 3 litters; sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 9 pups, 9 litters). C: Flow cytometric analysis of total CD68+ macrophages collected from dispersed islets of pancreata from offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects.

Figure 4

Clodronate treatment effectively depletes CD68+ cells in liver and islets of PD13 female offspring. Offspring were treated i.p. on PD2 and PD9 with clodronate liposomes or control liposomes. A: CD68 mRNA expression in liver from offspring of dams undergoing either RUPP or sham surgery. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham control liposome group (sham control liposomes n = 3 pups, 3 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 4 pups, 3 litters; RUPP clodronate liposomes n = 10 pups, 8 litters). B: CD68 mRNA expression in islets isolated from digested pancreata. Values represent mean + SE of the fold change of CD68 relative to β-actin using ΔΔCt method of quantitation relative to sham untreated group (sham untreated n = 10 pups, 4 litters; RUPP untreated n = 5 pups, 3 litters; sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 9 pups, 9 litters). C: Flow cytometric analysis of total CD68+ macrophages collected from dispersed islets of pancreata from offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 7 pups, 7 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by sex effects.

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The effect of clodronate treatment on macrophages in the peritoneal cavity and spleen was also assessed using flow cytometry. Total macrophages were defined as CD68+ cells, with the M1 macrophage subset as CD68+ CD163 and the M2 macrophage subset as CD68+ CD163+. As shown in Fig. 5, as expected, clodronate treatment significantly reduced the M1, M2, and total number of macrophages recovered from the peritoneal lavage and spleen.

Figure 5

Clodronate treatment effectively depletes macrophages in peritoneal lavage and spleen of PD13 female offspring. A: Flow cytometric analysis of cells collected from peritoneal lavage. Log of M1, M2, and total macrophages in PD13 offspring after control liposome or clodronate liposome treatment (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 8 pups, 8 litters; sham clodronate liposomes n = 6 pups, 5 litters; RUPP clodronate liposomes n = 11 pups, 9 litters). B: Flow cytometric analysis of cells collected from mechanically dissociated spleen. Log of M1, M2, and total macrophages in PD13 offspring after control liposome or clodronate liposome treatment (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 5 litters; RUPP clodronate liposomes n = 8 pups, 6 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by treatment effects.

Figure 5

Clodronate treatment effectively depletes macrophages in peritoneal lavage and spleen of PD13 female offspring. A: Flow cytometric analysis of cells collected from peritoneal lavage. Log of M1, M2, and total macrophages in PD13 offspring after control liposome or clodronate liposome treatment (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 8 pups, 8 litters; sham clodronate liposomes n = 6 pups, 5 litters; RUPP clodronate liposomes n = 11 pups, 9 litters). B: Flow cytometric analysis of cells collected from mechanically dissociated spleen. Log of M1, M2, and total macrophages in PD13 offspring after control liposome or clodronate liposome treatment (sham control liposomes n = 6 pups, 6 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 5 litters; RUPP clodronate liposomes n = 8 pups, 6 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by treatment effects.

Close modal

Treatment with clodronate liposomes also had a minor effect on total cell and nonmacrophage recovery from lavage and spleen, as indicated by the significant treatment effect in these compartments (Supplementary Fig. 2B and C). Some of this decrease in cells recovered was due to loss of mononuclear cells that do not stain with CD68 (nonmacrophages). The fraction of macrophages recovered was significantly decreased in the lavage (Supplementary Fig. 2B), where macrophages constitute a large portion of the cell population. The fraction of macrophages in the spleen was not significantly changed (Supplementary Fig. 2C).

Treatment With Clodronate Liposomes Rescues β-Cell Area in Female PD13 RUPP Offspring

As shown in Fig. 6A, the control liposome treatment increased β-cell area in sham offspring compared with untreated sham offspring. Even with this increase, the reduction in β-cell area was still evident in PD13 female offspring of RUPP versus sham surgeries treated with control liposomes. Taken together with data on increased CD68 expression in the control liposome sham offspring (Fig. 4B), these data suggest that increasing macrophages in the islet improves β-cell area, but placental ischemia during pregnancy in the RUPP offspring still reduces β-cell area. Notably, clodronate liposome treatment rescued β-cell area in RUPP offspring compared with control liposome treatment, suggesting a critical role for macrophages in the reduction in β-cell area seen after placental ischemia in the RUPP offspring.

Figure 6

β-cell area of PD13 female RUPP offspring is rescued by macrophage depletion with clodronate liposomes. Offspring were treated i.p. on PD2 and PD9 with clodronate liposomes or control liposomes. A: β-cell/pancreas area was determined by image analysis of paraffin-embedded sections of pancreas stained for insulin in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham untreated n = 5 pups, 4 litters; RUPP untreated n = 6 pups, 4 litters; sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). β-cell/pancreas area in untreated female offspring is reproduced from Fig. 2A for ease of comparison. B: Percentage TUNEL staining (β-cell apoptosis) of colocalized insulin-positive cells and TUNEL-positive cells determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 3 pups, 3 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). C: Percentage Ki67 staining (β-cell proliferation) of colocalized insulin-positive cells and Ki67-positive cells determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). Representative images showing colocalization of insulin, Ki67, and DAPI. D and E: Average islet size (D) and β-cell size (E) determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 4 pups, 4 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 4 pups, 4 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by treatment effects.

Figure 6

β-cell area of PD13 female RUPP offspring is rescued by macrophage depletion with clodronate liposomes. Offspring were treated i.p. on PD2 and PD9 with clodronate liposomes or control liposomes. A: β-cell/pancreas area was determined by image analysis of paraffin-embedded sections of pancreas stained for insulin in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham untreated n = 5 pups, 4 litters; RUPP untreated n = 6 pups, 4 litters; sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). β-cell/pancreas area in untreated female offspring is reproduced from Fig. 2A for ease of comparison. B: Percentage TUNEL staining (β-cell apoptosis) of colocalized insulin-positive cells and TUNEL-positive cells determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 3 pups, 3 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). C: Percentage Ki67 staining (β-cell proliferation) of colocalized insulin-positive cells and Ki67-positive cells determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 5 pups, 5 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 6 pups, 6 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). Representative images showing colocalization of insulin, Ki67, and DAPI. D and E: Average islet size (D) and β-cell size (E) determined by image analysis of paraffin-embedded sections of pancreas in PD13 female offspring of dams undergoing either RUPP or sham surgery (sham control liposomes n = 4 pups, 4 litters; RUPP control liposomes n = 5 pups, 5 litters; sham clodronate liposomes n = 4 pups, 4 litters; RUPP clodronate liposomes n = 7 pups, 7 litters). *P < 0.05 for individual contrast after mixed-model ANOVA using individual pup values with random litter effects and random litter by treatment effects.

Close modal

Because clodronate treatment is potentially toxic if it escapes the liposomes, we also evaluated the effect of liposomes with or without clodronate on PD13 body weight, nonfasting blood glucose, and pancreas and spleen weight compared with untreated animals (Supplementary Fig. 3). Treatment with control or clodronate liposomes did not significantly affect PD13 body weight or pancreas mass (Supplementary Fig. 3A and C). As expected, body weight was still lower in RUPP versus sham, as indicated by the significant RUPP effect (P = 0.006). Injection of control liposomes or clodronate liposomes significantly affected nonfasting blood glucose, with the primary difference being low blood glucose in sham animals treated with clodronate (Supplementary Fig. 3B). Spleen weight was significantly reduced by clodronate treatment, presumably because of the effectiveness of clodronate in removing phagocytic cells from the rat (Supplementary Fig. 3D).

Rescue of β-Cell Area Is Associated With β-Cell Hypertrophy

β-cell area can be affected by the rates of apoptosis and proliferation, as well as by total number of islets and islet and β-cell size. As in untreated animals (Fig. 2B), apoptosis was measured in insulin-positive cells of treated animals via TUNEL staining (Fig. 6B). Unexpectedly, no change in β-cell apoptosis was detected in RUPP versus sham animals when treated with control or clodronate liposomes. With control liposome treatment, RUPP offspring did not exhibit the increased apoptosis seen in untreated animals. These data suggest that clodronate does not reverse β-cell area by decreasing apoptosis of β-cells. The rate of proliferation of β-cells was determined by Ki67 staining, as shown in Fig. 6C. Female offspring of RUPP pregnancies had reduced β-cell proliferation compared with sham offspring treated with control liposomes, suggesting decreased proliferation contributed to the reduction in β-cell area. Treatment with clodronate liposomes also reduced β-cell proliferation in sham offspring, which recovered in the RUPP offspring, suggesting an important effect of macrophages in sustaining normal β-cell proliferation. Loss of macrophages in the RUPP offspring was important in restoring β-cell proliferation.

Changes in β-cell area could also result from changes in average islet or β-cell size. No differences in β-cell number were detected in RUPP or sham offspring treated with control or clodronate liposomes (data not shown). However, reductions in islet size and β-cell size were detected in RUPP offspring compared with sham in animals treated with control liposomes (Fig. 6D and E). Moreover, β-cell size was significantly increased in clodronate-treated offspring compared with control liposome–treated offspring, both in sham and in RUPP (Fig. 6E), suggesting that rescue of β-cell area was in part due to β-cell hypertrophy.

Influence of Early Postnatal Macrophage Depletion on Glucose Phenotypes in Adult Female Offspring

Female pups were weaned at PD21 and evaluated at age 8 to 9 weeks. Fasting blood glucose and C-peptide were determined in female RUPP and sham offspring that were untreated or had been treated with control liposomes or clodronate liposomes on PD2 and PD9. As seen in Supplementary Fig. 4A, offspring age 8 to 9 weeks of sham pregnancies treated with control liposomes had increased fasting blood glucose compared with the other groups, whereas concentration of C-peptide in plasma did not differ across the six different treatment groups (Supplementary Fig. 4A). Glucose tolerance was assessed by i.p. glucose tolerance test, and comparison of area under the curve for the treatment groups did not show any significant differences (Supplementary Fig. 4A, lower panel). The time course of glucose changes was evaluated, and no significant difference in glucose tolerance was detected comparing untreated RUPP and sham offspring (Supplementary Fig. 4B, top panel) or comparing untreated sham offspring with sham offspring treated with control or clodronate liposomes (Supplementary Fig. 4B, middle panel). However, improved glucose tolerance was observed at 60 min in RUPP offspring treated with control liposomes compared with untreated RUPP offspring or RUPP offspring treated with clodronate liposomes (Supplementary Fig. 4B, lower panel).

Pancreatic β-cell area determined early in life can influence whether T2D develops in adulthood (8,9). We have demonstrated that reduced β-cell area persists postnatally in female offspring after chronic placental ischemia in pregnancy. Our current study has defined a critical role for macrophages in the maintenance of this reduced β-cell area because treatment of animals with clodronate liposomes rescued β-cell area. This rescued β-cell area was associated with increased β-cell proliferation and β-cell hypertrophy. These data are the first to demonstrate that depletion of macrophages during early postnatal life can rescue pancreatic β-cell area after an ischemic pregnancy. Therefore, interventions to affect macrophage location and function after an adverse pregnancy could potentially program development of a normal β-cell area.

An increased risk of T2D has been demonstrated in the offspring of hypertensive pregnancies with and without low birth weight (1). In the rat RUPP model, our studies (1719,21,22) and studies of others (5,27) have consistently demonstrated increased blood pressure in the dams and reduced fetal weight at e19. In addition, a study by Intapad et al. (5) has demonstrated clear reductions in birth weight of offspring from RUPP versus sham pregnancies (4.99 vs. 6.14 g). In our study, the birth weight of RUPP offspring was significantly lower than that of sham animals (females 6.9 vs. 6.7 g; males 7.2 vs. 7.0 g) and was more apparent at PD13, but this was clearly not of the same magnitude as that reported by Intapad et al. (5). GD at birth might explain some of these differences, because in our study, RUPP pregnancies were significantly longer than sham pregnancies. Another contributing factor could be documented differences in Sprague-Dawley rats of different origins (28,29), because we used CD rats from Charles River Laboratories, whereas Intapad et al. (5,6) consistently used Sprague-Dawley rats purchased from Harlan/Envigo. Our studies also consistently showed a significant difference in male and female birth weight, which was not evident in the studies by Intapad et al. (5,6).

Our previous studies reported no differences in circulating insulin at e19 despite a reduction in β-cell area and increased β-cell apoptosis (3). At PD13, persistent reduction in β-cell area and significant β-cell apoptosis continue to leave circulating insulin unaffected. Instead, a uniform level of insulin is maintained despite these pancreatic deficiencies. The lack of change in circulating insulin suggests that PD13 may be too early for defects in insulin secretion to be apparent, even in the presence of smaller β-cell area.

In the adult mouse, studies by Calderon et al. (30) and others have demonstrated that each pancreatic islet contains five to 10 macrophages, primarily M1, in close proximity to blood vessels and β-cells. In contrast to macrophages in the exocrine tissue of the pancreas, islet macrophages are long lived and self replicating and not replaced by circulating monocytes. The role of macrophages in the pancreas has been studied in a variety of settings, including in animal models of T2D that have reduced β-cell mass (31). In a study by de Vos et al. (32), macrophages coincubated with encapsulated adult rat islets significantly inhibited glucose-stimulated insulin secretion in vitro. A more significant impairment of insulin secretion by encapsulated islets was evident when macrophages were activated by LPS. In adult mice receiving a high-fat diet, Ying et al. (33,34) showed that glucose-stimulated insulin secretion is decreased, and depletion of macrophages ex vivo restores it to normal. In our study, depletion of macrophages in multiple compartments restored β-cell area to normal in female offspring from RUPP pregnancies.

Limited studies have involved the role of macrophages in influencing β-cell area during development and after adverse pregnancies. Simmons et al. (16) used a rat BUAL model starting at e19 (term 22 days) that results in IUGR. After 1 day of uterine artery ligation, β-cell mass was not immediately affected (16), but mRNA expression associated with M2 macrophages and Th2 lymphocytes was upregulated at e19 (15). At PD14, upregulation of M2 macrophage markers was still evident. By 15 weeks, β-cell mass was reduced, and rats were hyperglycemic. Glucose intolerance was evident even at age 1 week (16). In this comprehensive study, Simmons et al. restored β-cell mass in adult offspring using anti-Th2 cytokine interleukin-4 treatment postnatally, pointing to the importance of the immune system and this cytokine, but the role of the macrophage was not investigated. This study and studies in the adult pancreas provide a strong rationale for our hypothesis that macrophages are important for the reduction in β-cell area seen after placental ischemia.

Although they are different models, both the BUAL model and the RUPP model result in IUGR in the offspring, and the RUPP model also results in hypertension in the dam during pregnancy. BUAL stops blood flow via the uterine arteries, whereas the compensatory blood flow to the fetus via the ovarian arteries is still operative. In the RUPP model, blood flow is restricted via the aorta/uterine artery, with the compensatory blood flow through the ovarian artery also limited. Also, the RUPP model disrupts perfusion of the uteroplacental unit from GD14 until birth, whereas the BUAL model reduces blood flow from GD18 or GD19 until birth. The IUGR phenotype in our RUPP model is not as robust as in the BUAL model, and hypertension in the BUAL model has not been reported or evaluated. In addition, our studies have monitored sex of the offspring in the analyses. Thus, the models have similarities but are different and may reveal a different mechanism leading to reduced β-cell area depending on the duration and extent of interference of blood flow to the uteroplacental unit.

In our study, RUPP surgery increased apoptosis in insulin-associated cells of female offspring at PD13, and we found that β-cell proliferation was decreased in control liposome–treated RUPP offspring. Thus, reduced β-cell area may occur by a combination of decreased proliferation and increased apoptosis of β-cells. Macrophages have been shown to be important cells for clearing apoptotic β-cells (35), and there is evidence that clearance of the apoptotic β-cell is compromised in T2D. In T2D, it is also thought that reduced β-cell number rather than size is a major contributor to β-cell loss (36). However, β-cell hyperplasia may also occur to compensate for an insulin-resistant state (37), and adult RUPP offspring were previously reported as glucose intolerant (57).

Our studies with adult female offspring age 8 to 9 weeks indicate no significant changes in glucose tolerance in RUPP versus sham offspring, despite neonatal changes in reduced β-cell area and increased CD68 suggesting increased macrophages. This lack of effect of RUPP surgery on glucose tolerance in the adult offspring is consistent with the studies by Intapad et al. (5,6) examining oral glucose tolerance, where changes in glucose tolerance in female offspring were only apparent at age 6 and 12 months. However, a study by Heltemes et al. (7) demonstrated reduced glucose tolerance in RUPP offspring when evaluating intravenous administration of glucose in RUPP compared with normal pregnant offspring (no sham surgery) of rats age 9 weeks of unreported sex. The changes in glucose tolerance are also complicated by the report that offspring of sham surgery pregnancies have an intermediate glucose tolerance phenotype compared with those undergoing BUAL and a normal pregnancy with no surgery (38).

Control liposomes are ingested by phagocytes but do not induce cell death and may temporarily suppress phagocytosis (24). Thus, the liposome vehicle for clodronate may also have an effect, and this was clearly noted in our study. Control liposomes themselves increased CD68 mRNA expression in islets compared with untreated animals (Fig. 4B). Control liposomes did not affect the decrease in β-cell area in RUPP offspring, but the increased β-cell apoptosis noted in untreated female RUPP offspring (Fig. 2B) was not detected after control liposome treatment (Fig. 6B).

In PD13 offspring, control liposome treatment increased β-cell area and CD68 mRNA expression compared with untreated animals. These data suggest that increased macrophages from control liposome treatment resulted in greater β-cell area in both RUPP and sham offspring. In our study, increased CD68 expression was used as a potential indicator of increased macrophages. Clearly the message for CD68 is dependent on the number of macrophages, but increased macrophage activation may also increase the message for CD68 (39). Both M1 and M2 macrophages express CD68, so monitoring this protein does not provide information regarding macrophage subtype. In our study, control liposome treatment itself in the neonatal period resulted in adult sham animals that had hyperglycemia compared with RUPP and adult RUPP animals that had improved glucose tolerance compared with sham. The differential effect of control liposomes and/or clodronate on sham versus RUPP outcomes may be related to the activation state and location of the macrophages in the normal sham pregnancy versus the complicated RUPP pregnancy. Future studies will be focused on evaluating the location and/or activation state of the macrophages in the islet as related to rescue of β-cell area and glucose tolerance with aging.

In our study, β-cell size significantly increased with clodronate treatment in RUPP offspring, suggesting that the rescue of β-cell area was in part due to hypertrophy of the β-cells after macrophage depletion. Our previous study of e19 offspring of RUPP and sham pregnancies demonstrated a trend toward a reduction in mTORC1 in RUPP offspring (3), and mTORC1 has been implicated as an important regulator of β-cell size (40). Therefore, future investigations will evaluate changes in mTORC1 as a possible explanation for the change in β-cell size. Our study is also limited by the fact that we sampled a single time point at PD13, and changes in proliferation, apoptosis, and/or β-cell size are dynamic over an extended time course in the development of T2D.

As an initial step toward a deeper look at the mechanism, we have examined serum cytokine panel in the serum of RUPP and sham male and female offspring at PD13. Our preliminary results indicated that the cytokine fractalkine was significantly increased in the serum of RUPP versus sham offspring (data not shown). Fractalkine is associated with preeclampsia (41) and known to be chemotactic for macrophages, in addition to being produced by islets (4244). It will be important in the future to directly test the role of fractalkine in RUPP islets. Our initial studies also demonstrated that the complement component C3 is increased in the serum of PD13 RUPP animals compared with sham and decreased in RUPP islets (45). The innate immune complement component C3 has also been demonstrated to be an important contributor to β-cell integrity in the islet (46), in addition to being produced by the macrophage. Therefore, continued studies will evaluate components of innate immunity that may contribute to the role of the macrophage in influencing β-cell area.

Treatment with clodronate liposomes has been used experimentally in many different systems to deplete macrophages from numerous compartments (23,24,47), including pancreatic islets in mice (48). Macrophages are the primary phagocyte targeted, with minimal effects noted on neutrophils and some studies showing effects on dendritic cells (24,48). Calderon et al. (49) monitored dendritic cells in mice and saw no change with clodronate liposome treatment. We cannot discount an effect of clodronate on other phagocytes without further evaluation of other phagocytic cells in the islets and other immune cell compartments. Liposomes target the macrophage because of their primary role in phagocytosis. In addition, if clodronate escapes from liposomes with death of the phagocyte, it is readily cleared by the kidney. Our flow cytometric results confirmed significant depletion of macrophages in the peritoneal cavity, spleen, and liver, with a reduction also occurring in the islets. Some other effects of clodronate liposomes were also evident, because animals treated with clodronate liposomes had significantly fewer cells in the peritoneal lavage, including macrophages and nonmacrophages, compared with animals treated with control liposomes (Supplementary Fig. 2). Clodronate liposomes decreased spleen weight, likely as a result of resident phagocyte death, and also had an effect on PD13 pup weight. In sham offspring, clodronate liposomes decreased β-cell proliferation, increased β-cell size, and decreased nonfasting blood glucose. These data suggest that in offspring of a normal pregnancy, macrophages are influencing β-cell development and maintenance of basal glucose.

Sex differences in fasting blood glucose have been reported in humans, with males having higher fasting blood glucose than females (5052). With fasting insulin, some studies have reported higher insulin in males than females (50), whereas others have not detected a difference (52). Previous studies by others have reported differences in male and female blood glucose in adult fasted animals, with some noting that females have higher concentrations of blood glucose than males (53), others noting males have higher fasting blood glucose than females (54), and others reporting no detectable difference (55). Although nonfasting blood glucose in male offspring at PD13 was higher than in female offspring, serum insulin differences were not statistically significant. Certainly, numerous metabolic differences between males and females, such as conversion of glucose to glycogen and fat deposition, may account for the differences and would need to be further explored (53,56).

Overall, our study is the first to demonstrate that depletion of macrophages during early postnatal life can rescue the pancreatic β-cell area deficits after an ischemic pregnancy. Clearly, interventions to affect macrophage location and function after an adverse pregnancy could potentially program development of normal β-cell area. Our continued studies will assess the influence of reduced β-cell area on β-cell function and the mechanism by which macrophages affect the development and/or maintenance of T2D.

This article contains supplementary material online at https://doi.org/10.2337/figshare.20995165.

Acknowledgments. The authors thank Dr. Cara Hegg, Whiteside Institute, University of Minnesota Duluth, for her advice and expertise in flow analysis and Dr. Ramkumar Mohan for technical support.

Funding. This study was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, grant R21 HD100840.

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. K.M.R., M.A.C., M.E.H., and J.F.R. performed the animal studies, with collection of tissue samples, islets, and acinar. K.M.R., A.M.M., and C.F.L. developed flow cytometric methods for rat macrophages and conducted flow staining and analysis. K.M.R., R.R.R., and J.F.R. performed the primary analysis of the data. K.M.R., E.U.A., and J.F.R. designed the experiments and drafted the manuscript, which was reviewed, edited, and approved by all the authors. B.A. performed immunohistochemistry and insulin assays. M.A.C. performed the C-peptide assay. E.U.A. and J.F.R. are the guarantors of this work and, as such, had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.

Prior Presentation. Parts of this study were presented in abstract form at the Experimental Biology Meeting 2022, Philadelphia, PA, 2–5 April 2022.

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