Impaired glucose homeostasis in transgenic mice expressing the human transient neonatal diabetes mellitus locus, TNDM

Abstract

Transient neonatal diabetes mellitus (TNDM) is a rare inherited diabetic syndrome apparent in the first weeks of life and again during early adulthood. The relative contributions of reduced islet beta cell number and impaired beta cell function to the observed hypoinsulinemia are unclear. The inheritance pattern of this imprinted disorder implicates overexpression of one or both genes within the TNDM locus: ZAC, which encodes a proapoptotic zinc finger protein, and HYMAI, which encodes an untranslated mRNA. To investigate the consequences for pancreatic function, we have developed a high-copy transgenic mouse line, TNDM29, carrying the human TNDM locus. TNDM29 neonates display hyperglycemia, and older adults, impaired glucose tolerance. Neonatal hyperglycemia occurs only on paternal transmission, analogous to paternal dependence of TNDM in humans. Embryonic pancreata of TNDM29 mice showed reductions in expression of endocrine differentiation factors and numbers of insulin-staining structures. By contrast, beta cell mass was normal or elevated at all postnatal stages, whereas pancreatic insulin content in neonates and peak serum insulin levels after glucose infusion in adults were reduced. Expression of human ZAC and HYMAI in these transgenic mice thus recapitulates key features of TNDM and implicates impaired development of the endocrine pancreas and beta cell function in disease pathogenesis.

Figure 1

Structure and expression of human TNDM transgene. (A) Human PAC RP3-340H11 contains the entire ZAC and HYMAI genes. The nine exons of ZAC are indicated as bars above the line, the 2 exons of HYMAI below the line; arrows indicate the start sites for and direction of transcription. Restriction sites BsiWI or SfiI for linearization of the PAC for microinjection are indicated. Transmission and integrity of the PAC in transgenic mice was assessed by PCR using the markers indicated; transgene copy number was estimated using the probe indicated. (B) Scheme for the RT-PCR assay for simultaneous detection of human ZAC (h) and mouse Zac (m) mRNAs. RT-PCR products were digested with StyI, which specifically cleaves the human product. (C) RT-PCR of ZAC and Zac in RNA from neonatal tissues of line TNDM29 hemizygous mice after paternal transmission of the transgene. Control lanes are nontransgenic mouse brain RNA (mouse) and human fetal brain RNA (human). (D) RT-PCR of ZAC and Zac RNA in the adult tissues from line TNDM29 and TNDM35 hemizygotes after paternal transmission. (E) RT-PCR of human HYMAI in the neonatal tissues of TNDM29 hemizygotes. Amplification of ZAC RNA in the same cDNA samples was used as an amplification control.

Figure 2

In situ hybridization of human and mouse TNDM locus genes in transgenic embryos. Sagittal sections of viscera from 14.5-dpc TNDM29 transgenic (A–D) and wild-type (E–H) embryos hybridized with human ZAC (A and G), mouse Zac (C and E), human HYMAI (B and H), and mouse Hymai (D and F) antisense riboprobes. Arrows indicate pancreas. ad, adrenal gland; kid, kidney; li, liver; st, stomach. Scale bar: 200 μm (A–H). Sagittal sections of head and neck from 14.5-dpc TNDM29 transgenic embryos labeled with human ZAC (I) and human HYMAI (J) antisense riboprobes. Note the labeling in brain and muscle. Scale bar: 350 μm (I and J).

Figure 3

In situ hybridization of human and mouse TNDM locus genes in pancreatic islets. Cryostat sections of pancreas from TNDM29 (A–F) and wild-type (G–J) mice of the indicated postnatal ages hybridized with antisense probes for human ZAC (A, C, and I), mouse Zac (E, G), human HYMAI (B, D, and J) and mouse Hymai (F and H). Arrows indicate pancreatic islets; arrowheads indicate exocrine parts. Note that the labeling for human ZAC and HYMAI is heavier in islets and weaker in the exocrine pancreas (A–D), whereas the opposite is true for Zac and Hymai (E–H). (I and J) Hybridization of human ZAC and HYMAI probes to nontransgenic sections as negative control. Scale bars: 70 μm (A and B); 90 μm (C–J).

Figure 4

Blood glucose, pancreatic insulin content, and serum insulin in TNDM29 mice. (A) Whole-blood glucose concentrations in mice at the indicated ages. (B) Total pancreatic insulin content. (C) Serum insulin concentration. Juvenile and adult measurements were performed on fasted animals. All results are expressed as mean ± SEM (n = 6–12); *P < 0.05 transgenic versus wild-type control group by unpaired, two-tailed Student’s t test and Mann Whitney U test.

Figure 5

Glucose and insulin tolerance in TNDM29 mice. Glucose tolerance tests (A–C): glucose concentration determined in whole blood at the indicated times following intraperitoneal injection of glucose in neonate (A), juvenile (B), and adult (C) TNDM29 and wild-type mice. Serum insulin concentrations (D–F) at the indicated time points of the glucose tolerance test in neonates (D), juveniles (E), and adults (F). Insulin tolerance tests (G–I): glucose concentration determined in whole blood at the indicated times following intraperitoneal injection of insulin in neonates (G), juvenile (H), and adult (I) TNDM29 and wild-type mice. Euglycemic hyperinsulinemic clamp studies in TNDM29 and wild-type mice (J): whole-body glucose turnover rate (TO), whole-body glycolysis rate (Glycol) and whole-body glycogen synthesis (GlnSynth) during the test are shown. All results are expressed as mean ± SEM (n = 12–20, in more than 3 separate experiments, except for the clamp studies, where n = 5–6). *P < 0.05, **P < 0.01 transgenic versus control group by unpaired, two-tailed Student’s t test and Mann Whitney U test, as appropriate.

Figure 6

Insulin secretion from isolated pancreatic islets. Insulin secretion was measured in islets isolated form neonatal pancreata (postnatal days 2–3, n = 10–15, in 3 separate experiments). Islets were cultured overnight in 11 mM glucose, and then insulin secretion was determined after 25 minutes’ treatment in 3 mM glucose, 17 mM glucose, or 17 mM glucose plus 0.1 μM PACAP. Results are presented as nanograms of insulin secreted per milligram total islet protein (A) and as fold increase in insulin secretion (B).

Figure 7

Immunohistochemical and morpho-metric analysis of postnatal pan-creata. H&E staining of paraffin-embedded pancreas sections from juvenile wild-type (A) and TNDM29 littermates (B). Insulin immunohistochemistry at the indicated ages in wild-type (C, E, and G) and TNDM29 (D, F, and H) littermates. Volume density (I) for insulin-positive structures at the indicated ages (the embryonic data from Figure 8 are included for comparison; n = 5 pancreata per group). Representative islets stained for glucagon from juvenile wild-type (J) and TNDM29 pancreata (K) and (L) measure of proportion of islets showing infiltration of glucagon-positive cells (as defined in Methods; n = 5 pancreata per group). Results are expressed as mean ± SEM; *P < 0.05 **P < 0.01 transgenic versus control group by unpaired, two-tailed Student’s t test. Scale bars: 50 μm (A and B); 200 μm (C–H); 60 μm (J and K).

Figure 8

Immunohistochemistry for hormone-producing cells in embryonic pancreata. Sagittal sections from 14.5-dpc embryos showing pancreas stained for insulin (A and B), glucagon (C and D), somatostatin (SOM; E and F), and PP (G and H) in TNDM29 (B, D, F, and H) and wild-type (A, C, E, and G) littermates. (I) Volume density for the positive structures (n = 3–5 embryos per group). Results are expressed as mean ± SEM; *P < 0.05, **P < 0.01 transgenic versus control group by unpaired, two-tailed Student’s t test. Scale bars: 150 μm (A–D); 40 μm (E–H).

Figure 9

Immunohistochemistry for differentiation markers in embryonic pancreata. Sagittal sections from 14.5-dpc embryos showing pancreas stained for PDX-1 (A and B), Ngn3 (C and D), and PAX6 (E and F). (G) Intensity of PDX-1 staining in pancreas determined in wild-type and TNDM29 sections (n = 4 embryos per group). (H) Volume density for the positive structures (n = 3 embryos per group) of Ngn3 and PAX6. Results are expressed as mean ± SEM; *P < 0.05 transgenic versus control group by unpaired, two-tailed Student’s t test. Scale bar: 80 μm (A–F).