Analysis of beta-cell maturity and mitochondrial morphology in juvenile non-human primates exposed to maternal Western-style diet during development.

Introduction: Using a non-human primate (NHP) model of maternal Western-style diet (mWSD) feeding during pregnancy and lactation, we previously reported altered offspring beta:alpha cell ratio in vivo and insulin hyper-secretion ex vivo. Mitochondria are known to maintain beta-cell function by producing ATP for insulin secretion. In response to nutrient stress, the mitochondrial network within beta cells undergoes morphological changes to maintain respiration and metabolic adaptability. Given that mitochondrial dynamics have also been associated with cellular fate transitions, we assessed whether mWSD exposure was associated with changes in markers of beta-cell maturity and/or mitochondrial morphology that might explain the offspring islet phenotype. Methods: We evaluated the expression of beta-cell identity/maturity markers (NKX6.1, MAFB, UCN3) via florescence microscopy in islets of Japanese macaque pre-adolescent (1 year old) and peri-adolescent (3-year-old) offspring born to dams fed either a control diet or WSD during pregnancy and lactation and weaned onto WSD. Mitochondrial morphology in NHP offspring beta cells was analyzed in 2D by transmission electron microscopy and in 3D using super resolution microscopy to deconvolve the beta-cell mitochondrial network. Results: There was no difference in the percent of beta cells expressing key maturity markers in NHP offspring from WSD-fed dams at 1 or 3 years of age; however, beta cells of WSD-exposed 3 year old offspring showed increased levels of NKX6.1 per beta cell at 3 years of age. Regardless of maternal diet, the beta-cell mitochondrial network was found to be primarily short and fragmented at both ages in NHP; overall mitochondrial volume increased with age. In utero and lactational exposure to maternal WSD consumption may increase mitochondrial fragmentation. Discussion: Despite mWSD consumption having clear developmental effects on offspring beta:alpha cell ratio and insulin secretory response to glucose, this does not appear to be mediated by changes to beta-cell maturity or the beta-cell mitochondrial network. In general, the more fragmented mitochondrial network in NHP beta cells suggests greater ability for metabolic flexibility.

Maternal Western-style diet programs skeletal muscle gene expression in lean adolescent Japanese macaque offspring.

Early-life exposure to maternal obesity or a maternal calorically dense Western-style diet (WSD) is strongly associated with a greater risk of metabolic diseases in offspring, most notably insulin resistance and metabolic dysfunction-associated steatotic liver disease (MASLD). Prior studies in our well-characterized Japanese macaque model demonstrated that offspring of dams fed a WSD, even when weaned onto a control (CTR) diet, had reductions in skeletal muscle mitochondrial metabolism and increased skeletal muscle insulin resistance compared to offspring of dams on CTR diet. In the current study, we employed a nested design to test for differences in gene expression in skeletal muscle from lean 3-year-old adolescent offspring from dams fed a maternal WSD in both the presence and absence of maternal obesity or lean dams fed a CTR diet. We included offspring weaned to both a WSD or CTR diet to further account for differences in response to post-weaning diet and interaction effects between diets. Overall, we found that a maternal WSD fed to dams during pregnancy and lactation was the principal driver of differential gene expression (DEG) in offspring muscle at this time point. We identified key gene pathways important in insulin signaling including PI3K-Akt and MAP-kinase, regulation of muscle regeneration, and transcription-translation feedback loops, in both male and female offspring. Muscle DEG showed no measurable difference between offspring of obese dams on WSD compared to those of lean dams fed WSD. A post-weaning WSD effected offspring transcription only in individuals from the maternal CTR diet group but not in maternal WSD group. Collectively, we identify that maternal diet composition has a significant and lasting impact on offspring muscle transcriptome and influences later transcriptional response to WSD in muscle, which may underlie the increased metabolic disease risk in offspring.

Initiation of metformin in early pregnancy results in fetal bioaccumulation, growth restriction, and renal dysmorphology in a primate model.

BACKGROUND: In recent years, pragmatic metformin use in pregnancy has stretched to include prediabetes mellitus, type 2 diabetes mellitus, gestational diabetes mellitus, and (most recently) preeclampsia. However, with its expanded use, concerns of unintended harm have been raised. OBJECTIVE: This study developed an experimental primate model and applied ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry for direct quantitation of maternal and fetal tissue metformin levels with detailed fetal biometry and histopathology. STUDY DESIGN: Within 30 days of confirmed conception (defined as early pregnancy), 13 time-bred (timed-mated breeding) Rhesus dams with pregnancies designated for fetal necropsy were initiated on twice-daily human dose-equivalent 10 mg/kg metformin or vehicle control. Pregnant dams were maintained as pairs and fed either a control chow or 36% fat Western-style diet. Metformin or placebo vehicle control was delivered in various treats while the animals were separated via a slide. A cesarean delivery was performed at gestational day 145, and amniotic fluid and blood were collected, and the fetus and placenta were delivered. The fetus was immediately necropsied by trained primate center personnel. All fetal organs were dissected, measured, sectioned, and processed per clinical standards. Fluid and tissue metformin levels were assayed using validated ultrahigh performance liquid chromatography coupled to triple-quadrupole mass spectrometry in selected reaction monitoring against standard curves. RESULTS: Among 13 pregnancies at gestational day 145 with fetal necropsy, 1 dam and its fetal tissues had detectable metformin levels despite being allocated to the vehicle control group (>1 µmol metformin/kg maternal weight or fetal or placental tissue), whereas a second fetus allocated to the vehicle control group had severe fetal growth restriction (birthweight of 248.32 g [<1%]) and was suspected of having a fetal congenital condition. After excluding these 2 fetal pregnancies from further analyses, 11 fetuses from dams initiated on either vehicle control (n=4: 3 female and 1 male fetuses) or 10 mg/kg metformin (n=7: 5 female and 2 male fetuses) were available for analyses. Among dams initiated on metformin at gestational day 30 (regardless of maternal diet), significant bioaccumulation within the fetal kidney (0.78-6.06 µmol/kg; mean of 2.48 µmol/kg), liver (0.16-0.73 µmol/kg; mean of 0.38 µmol/kg), fetal gut (0.28-1.22 µmol/kg; mean of 0.70 µmol/kg), amniotic fluid (0.43-3.33 µmol/L; mean of 1.88 µmol/L), placenta (0.16-1.00 µmol/kg; mean of 0.50 µmol/kg), fetal serum (0.00-0.66 µmol/L; mean of 0.23 µmol/L), and fetal urine (4.10-174.10 µmol/L; mean of 38.5 µmol/L) was observed, with fetal levels near biomolar equivalent to maternal levels (maternal serum: 0.18-0.86 µmol/L [mean of 0.46 µmol/L]; maternal urine: 42.60-254.00 µmol/L [mean of 149.30 µmol/L]). Western-style diet feeding neither accelerated nor reduced metformin bioaccumulations in maternal or fetal serum, urine, amniotic fluid, placenta, or fetal tissues. In these 11 animals, fetal bioaccumulation of metformin was associated with less fetal skeletal muscle (57% lower cross-sectional area of gastrocnemius) and decreased liver, heart, and retroperitoneal fat masses (P<.05), collectively driving lower delivery weight (P<.0001) without changing the crown-rump length. Sagittal sections of fetal kidneys demonstrated delayed maturation, with disorganized glomerular generations and increased cortical thickness. This renal dysmorphology was not accompanied by structural or functional changes indicative of renal insufficiency. CONCLUSION: Our study demonstrates fetal bioaccumulation of metformin with associated fetal growth restriction and renal dysmorphology after maternal initiation of the drug within 30 days of conception in primates. Given these results and the prevalence of metformin use during pregnancy, additional investigation of any potential immediate and enduring effects of prenatal metformin use is warranted.

The prostaglandin E2 EP3 receptor has disparate effects on islet insulin secretion and content in ß-cells in a high-fat diet-induced mouse model of obesity.

Signaling through prostaglandin E2 EP3 receptor (EP3) actively contributes to the ß-cell dysfunction of type 2 diabetes (T2D). In T2D models, full-body EP3 knockout mice have a significantly worse metabolic phenotype than wild-type controls due to hyperphagia and severe insulin resistance resulting from loss of EP3 in extra-pancreatic tissues, masking any potential beneficial effects of EP3 loss in the ß cell. We hypothesized ß-cell-specific EP3 knockout (EP3 ßKO) mice would be protected from high-fat diet (HFD)-induced glucose intolerance, phenocopying mice lacking the EP3 effector, Gαz, which is much more limited in its tissue distribution. When fed a HFD for 16 wk, though, EP3 ßKO mice were partially, but not fully, protected from glucose intolerance. In addition, exendin-4, an analog of the incretin hormone, glucagon-like peptide 1, more strongly potentiated glucose-stimulated insulin secretion in islets from both control diet- and HFD-fed EP3 ßKO mice as compared with wild-type controls, with no effect of ß-cell-specific EP3 loss on islet insulin content or markers of replication and survival. However, after 26 wk of diet feeding, islets from both control diet- and HFD-fed EP3 ßKO mice secreted significantly less insulin as a percent of content in response to stimulatory glucose, with or without exendin-4, with elevated total insulin content unrelated to markers of ß-cell replication and survival, revealing severe ß-cell dysfunction. Our results suggest that EP3 serves a critical role in temporally regulating ß-cell function along the progression to T2D and that there exist Gαz-independent mechanisms behind its effects.NEW & NOTEWORTHY The EP3 receptor is a strong inhibitor of ß-cell function and replication, suggesting it as a potential therapeutic target for the disease. Yet, EP3 has protective roles in extrapancreatic tissues. To address this, we designed ß-cell-specific EP3 knockout mice and subjected them to high-fat diet feeding to induce glucose intolerance. The negative metabolic phenotype of full-body knockout mice was ablated, and EP3 loss improved glucose tolerance, with converse effects on islet insulin secretion and content.

Epigenetic modulation of cell fate during pancreas development.

Epigenetic modifications to DNA and its associated proteins affect cell plasticity and cell fate restrictions throughout embryonic development. Development of the vertebrate pancreas is characterized by initial is an over-lapping expression of a set of transcriptional regulators in a defined region of the posterior foregut endoderm that collectively promote pancreas progenitor specification and proliferation. As development progresses, these transcription factors segregate into distinct pancreatic lineages, with some being maintained in specific subsets of terminally differentiated pancreas cell types throughout adulthood. Here we describe the progressive stages and cell fate restrictions that occur during pancreas development and the relevant known epigenetic regulatory events that drive the dynamic expression patterns of transcription factors that regulate pancreas development. In addition, we highlight how changes in epigenetic marks can affect susceptibility to pancreas diseases (such as diabetes), adult pancreas cell plasticity, and the ability to derive replacement insulin-producing ß cells for the treatment of diabetes.