Rajiv L. Joshi

Increased Islet Cell Proliferation, Decreased Apoptosis, and Greater Vascularization Leading to β-Cell Hyperplasia in Mutant Mice Lacking Insulin

The targeted disruption of the two nonallelic insulin genes in mouse was reported previously to result in intrauterine growth retardation, severe diabetes immediately after suckling, and death within 48 h of birth. We have further used these animals to investigate the morphology and cell biology of the endocrine pancreas in late gestation and at birth when insulin is absent throughout development. Pancreatic -cells were identified by detecting the activity of the LacZ gene inserted at the Ins2 locus. A significant increase in the mean area of the islets was found at embryonic d 18.5 (E18.5) and in the newborn in Ins1/, Ins2/ animals compared with Ins1/, Ins2/ and wild-type controls, whereas the blood glucose levels were unaltered. The individual size of the -cells in the insulin-deficient fetuses was similar to controls, suggesting that the relative increase in islet size was due to an increase in cell number. Immunohistochemistry for proliferating cell nuclear antigen within the pancreatic ductal epithelium showed no differences in labeling index between insulin-deficient and control mice, and no change in the number of -cells associated with ducts, but the relative size distribution of the islets was altered so that fewer islets under 5,000 m 2 and more islets greater than 10,000m 2 were present in Ins1/, Ins2/ animals. This suggests that the greater mean islet size seen in insulin-deficient animals represented an enlargement of formed islets and was not associated with an increase in islet neogenesis. The proportional contribution of - and -cells to the islets was not altered. This was supported by an increase in the number of cells containing immunoreactive proliferating cell nuclear antigen in both islet - and -cells at E18.5 in insulin-deficient mice, and a significantly lower incidence of apoptotic cells, as determined by molecular histochemistry using the terminal deoxynucleotidyl transferase-mediated deoxy-UTP nick end labeling reaction. The density of blood vessels within sections of whole pancreas, or within islets, was determined by immunohistochemistry for the endothelial cell marker CD31 and was found to be increased 2-fold in insulin-deficient mice compared with controls at E18.5. However, no changes were found in the steady-state expression of mRNAs encoding vascular endothelial growth factor, its receptor Flk-1, IGF-I or -II, the IGF-I and insulin receptors, or insulin receptor substrates-1 or -2 in pancreata from Ins1/, Ins2/ mice compared with Ins1/, Ins2/ controls. Thus, we conclude that the relative hyperplasia of the islets in late gestation in the insulin-deficient mice was due to an increased islet cell proliferation coupled with a reduced apoptosis, which may be related to an increased vascularization of the pancreas. (Endocrinology 143: 1530 –1537, 2002)

Engrailed protects mouse midbrain dopaminergic neurons against mitochondrial complex I insults

Mice heterozygous for the homeobox gene Engrailed-1 (En1) display progressive loss of mesencephalic dopaminergic (mDA) neurons. We report that exogenous Engrailed-1 and Engrailed-2 (collectively Engrailed) protect mDA neurons from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial complex I toxin used to model Parkinsons disease in animals. Engrailed enhances the translation of nuclearly encoded mRNAs for two key complex I subunits, Ndufs1 and Ndufs3, and increases complex I activity. Accordingly, in vivo protection against MPTP by Engrailed is antagonized by Ndufs1 small interfering RNA. An association between Engrailed and complex I is further confirmed by the reduced expression of Ndufs1 and Ndufs3 in the substantia nigra pars compacta of En1 heterozygous mice. Engrailed also confers in vivo protection against 6-hydroxydopamine and α-synuclein-A30P. Finally, the unilateral infusion of Engrailed into the midbrain increases striatal dopamine content, resulting in contralateral amphetamine-induced turning. Therefore, Engrailed is both a survival factor for adult mDA neurons and a regulator of their physiological activity.

Engrailed homeoprotein recruits the adenosine A1 receptor to potentiate ephrin A5 function in retinal growth cones.

Engrailed 1 and engrailed 2 homeoprotein transcription factors (collectively Engrailed) display graded expression in the chick optic tectum where they participate in retino-tectal patterning. In vitro, extracellular Engrailed guides retinal ganglion cell (RGC) axons and synergises with ephrin A5 to provoke the collapse of temporal growth cones. In vivo disruption of endogenous extracellular Engrailed leads to misrouting of RGC axons. Here we characterise the signalling pathway of extracellular Engrailed. Our results show that Engrailed/ephrin A5 synergy in growth cone collapse involves adenosine A1 receptor activation after Engrailed-dependent ATP synthesis, followed by ATP secretion and hydrolysis to adenosine. This is, to our knowledge, the first evidence for a role of the adenosine A1 receptor in axon guidance. Based on these results, together with higher expression of the adenosine A1 receptor in temporal than nasal growth cones, we propose a computational model that illustrates how the interaction between Engrailed, ephrin A5 and adenosine could increase the precision of the retinal projection map.

Genetic manipulation of insulin signaling, action and secretion in mice Insights into glucose homeostasis and pathogenesis of type 2 diabetes

Non‐insulin‐dependent diabetes mellitus (NIDDM) is a complex heterogeneous polygenic disease characterized mainly by insulin resistance and pancreatic β‐cell dysfunction. In recent years, several genetically engineered mouse models have been developed for the study of the pathophysiological consequences of defined alterations in a single gene or in a set of candidate diabetogenes. These represent new tools that are providing invaluable insights into NIDDM pathogenesis. In this review, we highlight the lessons emerging from the study of some of the transgenic or knockout mice in which the expression of key actors in insulin signaling, action or secretion has been manipulated. In addition to contributing to our knowledge of the specific roles of individual genes in the control of glucose homeostasis, these studies have made it possible to address several crucial issues in NIDDM that have remained controversial or unanswered for a number of years.

Interaction of p85 subunit of PI 3-kinase with insulin and IGF-1 receptors analysed by using the two-hybrid system.

Interaction of the p85 subunit of PI 3‐kinase with the insulin receptor (IR) and the IGF‐1 receptor (IGF‐1R) was investigated using the two‐hybrid system by assessing for his3 and lacZ activation in S. cerevisiae. The experiments were performed with the cytoplasmic β domain (wild type or mutated) of IR and IGF‐1R and p85 or its subdomains (N + C‐SH2, N‐SH2, C‐SH2, SH3 + N‐SH2). The results of his3 activation indicated that p85, N + C‐SH2 and C‐SH2 interact with both IRβ and IGF‐1Rβ, whereas N‐SH2 and SH3 + N‐SH2 interact only with IRβ. Interaction of p85 and N + C‐SH2 with IRβ(ΔC‐43) or IGF‐1Rβ(ΔC‐43) in which the C‐terminal 43 amino acids (including the YXXM motif) were deleted, persisted. The internal binding site thus revealed was not altered by further mutating Y960/F for IR or Y950/F for IGF‐1R. Activation of lacZ upon interaction of p85 with IRβ(ΔC‐43) was 4‐fold less as compared to IRβ. This activation with p85 and IGF‐1Rβ was 4‐fold less as compared to IRβ and was somewhat increased (2‐fold) for IGE‐1Rβ(ΔC‐43). Thus, the C‐terminal domain in IGF‐1R appears to exert a negative control on binding of p85 thereby providing a possible regulatory mechanism for direct activation of the PI 3‐kinase pathway.

Progressive nigrostriatal terminal dysfunction and degeneration in the engrailed1 heterozygous mouse model of Parkinson's disease

Current research on Parkinsons disease (PD) pathogenesis requires relevant animal models that mimic the gradual and progressive development of neuronal dysfunction and degeneration that characterizes the disease. Polymorphisms in engrailed 1 (En1), a homeobox transcription factor that is crucial for both the development and survival of mesencephalic dopaminergic neurons, are associated with sporadic PD. This suggests that En1 mutant mice might be a promising candidate PD model. Indeed, a mouse that lacks one En1 allele exhibits decreased mitochondrial complex I activity and progressive midbrain dopamine neuron degeneration in adulthood, both features associated with PD. We aimed to further characterize the disease-like phenotype of these En1(+/-) mice with a focus on early neurodegenerative changes that can be utilized to score efficacy of future disease modifying studies. We observed early terminal defects in the dopaminergic nigrostriatal pathway in En1(+/-) mice. Several weeks before a significant loss of dopaminergic neurons in the substantia nigra could be detected, we found that striatal terminals expressing high levels of dopaminergic neuron markers TH, VMAT2, and DAT were dystrophic and swollen. Using transmission electron microscopy, we identified electron dense bodies consistent with abnormal autophagic vacuoles in these terminal swellings. In line with these findings, we detected an up-regulation of the mTOR pathway, concurrent with a downregulation of the autophagic marker LC3B, in ventral midbrain and nigral dopaminergic neurons of the En1(+/-) mice. This supports the notion that autophagic protein degradation is reduced in the absence of one En1 allele. We imaged the nigrostriatal pathway using the CLARITY technique and observed many fragmented axons in the medial forebrain bundle of the En1(+/-) mice, consistent with axonal maintenance failure. Using in vivo electrochemistry, we found that nigrostriatal terminals in the dorsal striatum were severely deficient in dopamine release and reuptake. Our findings support a progressive retrograde degeneration of En1(+/-) nigrostriatal neurons, akin to what is suggested to occur in PD. We suggest that using the En1(+/-) mice as a model will provide further key insights into PD pathogenesis, and propose that axon terminal integrity and function can be utilized to estimate dopaminergic neuron health and efficacy of experimental PD therapies.

IGF-1 receptor as an alternative receptor for metabolic signaling in insulin receptor-deficient muscle cells

We have derived skeletal muscle cell lines from wild‐type (wt) and insulin receptor (IR) knockout mice to unravel the metabolic potential of IGF‐1 receptor (IGF‐1R). Both wt and IR−/− myoblasts differentiated into myotubes with similar patterns of expression of muscle‐specific genes such as MyoD, myogenin and MLC1A indicating that IR is not required for this process. Binding of 125I‐IGF‐1 on wt and IR−/− myotubes was similar showing that IGF‐1R was not upregulated in the absence of IR. Stimulation of IR−/− myotubes with IGF‐1 (10−10 to 10−7 M) increased glucose uptake and incorporation into glycogen, induced IRS‐1 phosphorylation and activated PI 3‐kinase and MAP kinase, two enzymes of major signaling pathways. These effects were comparable to those obtained with wt myotubes using insulin or IGF‐1 or with IR−/− myotubes using insulin at higher concentrations. This study provides a direct evidence that IGF‐1R can represent an alternative receptor for metabolic signaling in muscle cells.

Insulin receptor-deficient cells as a new tool for dissecting complex interplay in insulin and insulin-like growth factors.

Cell systems derived from knockout mice for the insulin receptor (IR) or the IGF‐1 receptor (IGF‐1R) represent unique tools for dissecting complex interplay in the actions of insulin and insulin‐like growth factors through their cognate versus non‐cognate receptor. In this study, we used a fibroblast cell line derived from IR‐deficient mice to investigate metabolic and mitogenic effects of IGF‐1 and insulin. IGF‐1 was able to stimulate glucose uptake, glucose incorporation into glycogen and thymidine incorporation in such cells. Phosphatidylinositol 3‐kinase and mitogen‐activated protein kinase, two enzymes of major metabolic‐mitogenic signaling pathways, were activated upon stimulating these cells with IGF‐1. All these effects were also achieved when IR‐deficient cells were stimulated with insulin. Thus, IGF‐1R can represent an alternative receptor through which insulin might exert some of its effects.

Knock-in of diphteria toxin A chain gene at Ins2 locus: effects on islet development and localization of Ins2 expression in the brain.

We report here knock-in of diphteria toxin A chain (dta) gene at the Ins2 locus, using the strategy previously employed to insert lacZ under control of the Ins2 promoter. Mutant Ins2dta/+, Ins2dta/lacZ or Ins2lacZ/+ mouse pups were generated by breeding and analyzed to study the effects of toxigenetic β-cell ablation on islet development and to localize the extrapancreatic Ins2 expression site in the brain. Ins2dta/+ and Ins2dta/lacZ pups developed a severe diabetic ketoacidosis and died rapidly. Histological analysis of their pancreas revealed that β-cells completely disappeared in their islets as evidenced by loss of lacZ activity or insulin immunonostaining. β-cell ablation did not alter the size of other islet cell populations which were normal at birth, although the glucagon-cell population was reduced by 85% at embryonic day E12.5. In the brain, comparative analysis of lacZ expression in Ins2lacZ/+ and Ins2dta/lacZ mice identified the choroid plexus (CP) as a major Ins2 expression site. This finding was confirmed by RT-PCR analysis of insulin transcripts in RNAs prepared from microdissected wild-type CP. Transcripts for other key β-cell markers, with the notable exception of Pdx-1, were also found in CP RNAs. These results must revive interest in studies focused on extrapancreatic insulin gene expression.

Structural features in aminoacyl-tRNAs required for recognition by elongation factor Tu.

In bacterial polypeptide synthesis aminoacyl‐tRNA (aa‐tRNA) bound to elongation factor Tu (EF‐Tu) and GTP is part of a crucial intermediate ribonucleoprotein complex involved in the decoding of messenger RNA. The conformation and topology as well as the affinity of the macromolecules in this ternary aa‐tRNA·EF‐Tu·GTP complex are of fundamental importance for the nature of the interaction of the complex with the ribosome. The structural elements of aa‐tRNA required for interaction with EF‐Tu and GTP and the resulting functional implications are presented here.