Rat neonatal beta cells lack the specialised metabolic phenotype of mature beta cells

Abstract

Aims/hypothesis: Fetal and neonatal beta cells have poor glucose-induced insulin secretion and only gain robust glucose responsiveness several weeks after birth. We hypothesise that this unresponsiveness is due to a generalised immaturity of the metabolic pathways normally found in beta cells rather than to a specific defect.

Methods: Using laser-capture microdissection we excised beta cell-enriched cores of pancreatic islets from day 1 (P1) neonatal and young adult Sprague-Dawley rats in order to compare their gene-expression profiles using Affymetrix U34A microarrays (neonatal, n = 4; adult, n = 3).

Results: Using dChip software for analysis, 217 probe sets for genes/38 expressed sequence tags (ESTs) were significantly higher and 345 probe sets for genes/33 ESTs significantly lower in beta cell-enriched cores of neonatal islets compared with those of adult islets. Among the genes lower in the neonatal beta cells were key metabolic genes including mitochondrial shuttles (malate dehydrogenase, glycerol-3-phosphate dehydrogenase and glutamate oxalacetate transaminase), pyruvate carboxylase and carnitine palmitoyl transferase 2. Differential expression of these enzyme genes was confirmed by quantitative PCR on RNA from isolated neonatal (P2 until P28) and adult islets and with immunostaining of pancreas. Even by 28 days of age some of these genes were still expressed at lower levels than in adults.

Conclusions/interpretation: The lack of glucose responsiveness in neonatal islets is likely to be due to a generalised immaturity of the metabolic specialisation of pancreatic beta cells.

Fig. 1

Heat map display of the differentially expressed genes in laser-captured enriched beta cells from neonatal (P1) and adult pancreases. Using dChip analysis with LCB cutoff of 2 and p<0.050, adult beta cells had 345 genes and 33 ESTs more highly expressed than in neonates, while neonatal beta cells had 217 genes and 38 ESTs more highly expressed than in adult

Fig. 2

Metabolic pathways involved in glucose-stimulated insulin secretion in beta cells. Based on dChip analysis of microarray data of enriched beta cell samples from neonatal vs adult samples, genes with lower expression in the neonates (adult/neonatal ≤−1.2) are in blue and those with higher expression in neonates (neonatal/adult≥1.2) are in red; unchanged in black. Asp, aspartate; AST, aspartate aminotransferase; ACLY, ATP citrate lyase; CPT1A, carnitine palmitoyl transferase 1; CPT2, carnitine palmitoyl transferase 2; FASN, fatty acid synthase; Glu, glutarate; GPD1, glycerol-3-phosphate dehydrogenase 1; GPD2, glycerol-3-phosphate dehydrogenase 2; MDH1, malate dehydrogenase 1; MDH2, malate dehydrogenase 2; ME1, malic enzyme 1; OAA, oxaloacetic acid; P, phosphate; PC, pyruvate carboxylase; PDH, pyruvate dehydrogenase; PK, pyruvate kinase; αKG, α-ketoglutarate

Fig. 3

qPCR confirmation of selected metabolic genes. The differential expression of selected metabolic genes was confirmed by qPCR in islets isolated from neonatal P2 (black bars, n=4 samples of islets pooled from a litter) and P7 rats (grey bars, n=3 samples of islets pooled from a litter); data are expressed relative to isolated adult islets (dotted line, n=5). One-way ANOVA with Tukey post hoc analysis. **p<0.01; ***p<0.001

Fig. 4

Immunostaining of PK and mitochondrial glycerol-3-phosphate dehydrogenase 2 (GPD2). By immunostaining, the protein levels of PK (top panels, VIP as chromagen) and GPD2 (bottom panels, insulin red, GPD2 green, overlap yellow) reflect the mRNA expression through the neonatal time period

Fig. 5

Time course of expression of selected metabolic genes over the first postnatal month by qPCR. The genes encoding the metabolic enzymes have different patterns of expression through the first postnatal month: (a) pyruvate carboxylase (black diamond), malic enzyme 1 (cytosolic) (white circle); (b) malate dehydrogenase 1 (cytosolic) (black square), glutamate oxalacetate transaminase 1 (cytosolic) (white triangle), glycerol phosphate dehydrogenase 2 (mitochondrial) (black circle) (c) carnitine palmitoyltransferase 1 (white circle), carnitine palmitoyltransferase 2 (black triangle); (d) pyruvate kinase (Pklr isoform) (white circle). All but Cpt1 are expressed at 20% or less than adult levels during the first week. Data expressed relative to pooled isolated adult islets (n=5). Student's t test for comparison of expression between weeks. *p<0.05, **p<0.01, ***p<0.001. ANOVA for repeated measures throughout the time course, p<0.001 in all comparisons, except for Pk