Vered Gazit

Repetitive hypoglycemia in young rats impairs hippocampal long-term potentiation.

Mechanisms underlying cognitive dysfunction in young diabetic children are poorly understood, and may include synaptic dysfunction from insulin-induced hypoglycemia. We developed a model of repetitive insulin-induced hypoglycemia in young rats and examined hippocampal long-term potentiation, an electrophysiologic assay of synaptic plasticity, 3–5 d after the last hypoglycemic event. Three hypoglycemic events between postnatal d 21–25 produced modest cortical (17 ± 2.9 dead neurons per section in parasagittal cortex), but not hippocampal, neuron death quantified by Fluoro-Jade B staining. There was no change in neurogenesis in the hippocampal dentate granule cell region by quantification of bromodeoxyuridine incorporation. Although normal baseline hippocampal synaptic responses were elicited from hippocampal slices from hypoglycemic animals, long-term synaptic potentiation could not be induced in hippocampal slices from rats subjected to hypoglycemia. These results suggest that repetitive hypoglycemia in the developing brain can cause selective impairment of synaptic plasticity in the absence of cell death, and without complete disruption of basal synaptic transmission. We speculate that impaired synaptic plasticity in the hippocampus caused by repetitive hypoglycemia could underlie memory and cognitive deficits observed in young diabetic children, and that cortical neuron death caused by repetitive hypoglycemia in the developing brain may contribute to other neurologic, cognitive, and psychological problems sometimes encountered in diabetic children.

p21 is Required for Dextrose-Mediated Inhibition of Mouse Liver Regeneration

The inhibitory effect of dextrose supplementation on liver regeneration was first described more than 4 decades ago. Nevertheless, the molecular mechanisms responsible for this observation have not been elucidated. We investigated these mechanisms using the partial hepatectomy model in mice given standard or 10% dextrose (D10)‐supplemented drinking water. The results showed that D10‐treated mice exhibited significantly reduced hepatic regeneration compared with controls, as assessed by hepatocellular bromodeoxyuridine (BrdU) incorporation and mitotic frequency. D10 supplementation did not suppress activation of hepatocyte growth factor (HGF), induction of transforming growth factor alpha (TGF‐α) expression, or tumor necrosis factor alpha–interleukin‐6 cytokine signaling, p42/44 extracellular signal‐regulated kinase (ERK) activation, immediate early gene expression, or expression of CCAAT/enhancer binding protein beta (C/EBPβ), but did augment expression of the mito‐inhibitory factors C/EBPα, p21Waf1/Cip1, and p27Kip1. In addition, forkhead box M1 (FoxM1) expression, which is required for normal liver regeneration, was suppressed by D10 treatment. Finally, D10 did not suppress either FoxM1 expression or hepatocellular proliferation in p21 null mice subjected to partial hepatectomy, establishing the functional significance of these events in mediating the effects of D10 on liver regeneration. Conclusion: These data show that the inhibitory effect of dextrose supplementation on liver regeneration is associated with increased expression of C/EBPα, p21, and p27, and decreased expression of FoxM1, and that D10‐mediated inhibition of liver regeneration is abrogated in p21‐deficient animals. Our observations are consistent with a model in which hepatic sufficiency is defined by homeostasis between the energy‐generating capacity of the liver and the energy demands of the body mass, with liver regeneration initiated when the functional liver mass is no longer sufficient to meet such demand. (HEPATOLOGY 2009.)

Liver regeneration is impaired in lipodystrophic fatty liver dystrophy mice

We previously reported that mice subjected to partial hepatectomy exhibit rapid development of hypoglycemia followed by transient accumulation of fat in the early regenerating liver. We also showed that disrupting these metabolic alterations results in impaired liver regeneration. The studies reported here were undertaken to further characterize and investigate the functional importance of changes in systemic adipose metabolism during normal liver regeneration. The results showed that a systemic catabolic response is induced in each of two distinct, commonly used experimental models of liver regeneration (partial hepatectomy and carbon tetrachloride treatment), and that this response occurs in proportion to the degree of induced hepatic insufficiency. Together, these observations suggest that catabolism of systemic adipose stores may be essential for normal liver regeneration. To test this possibility, we investigated the hepatic regenerative response in fatty liver dystrophy (fld) mice, which exhibit partial lipodystrophy and have diminished peripheral adipose stores. The results showed that the development of hypoglycemia and hepatic accumulation of fat was attenuated and liver regeneration was impaired following partial hepatectomy in these animals. The fld mice also exhibited increased hepatic p21 expression and diminished plasma levels of the adipose‐derived hormones adiponectin and leptin, which have each been implicated as regulators of liver regeneration. Conclusion: These data suggest that the hypoglycemia that develops after partial hepatectomy induces systemic lipolysis followed by accumulation of fat derived from peripheral stores in the early regenerating liver, and that these events may be essential for initiation of normal liver regeneration. (HEPATOLOGY 2010)

Analysis of the role of hepatic PPARγ expression during mouse liver regeneration

Mice subjected to partial hepatectomy (PH) develop hypoglycemia, followed by increased systemic lipolysis and hepatic fat accumulation, prior to onset of hepatocellular proliferation. Strategies that disrupt these metabolic events inhibit regeneration. These observations suggest that alterations in metabolism in response to hepatic insufficiency promote liver regeneration. Hepatic expression of the peroxisome proliferator‐activated receptor gamma (PPARγ) influences fat accumulation in the liver. Therefore, the studies reported here were undertaken to assess the effects of disruption of hepatic PPARγ expression on hepatic fat accumulation and hepatocellular proliferation during liver regeneration. The results showed that liver regeneration was not suppressed, but rather modestly augmented in liver‐specific PPARγ null mice maintained on a normal diet. These animals also exhibited accelerated hepatic cyclin D1 expression. Because hepatic PPARγ expression is increased in experimental models of fatty liver disease in which liver regeneration is impaired, regeneration in liver‐specific PPARγ null mice with chronic hepatic steatosis was also examined. In contrast to the results described above, disruption of hepatic PPARγ expression in mice with diet‐induced hepatic steatosis resulted in significant suppression of hepatic regeneration. Conclusion: The metabolic and hepatocellular proliferative responses to PH are modestly augmented in liver‐specific PPARγ null mice, thus providing additional support for a metabolic model of liver regeneration. Furthermore, regeneration is significantly impaired in liver‐specific PPARγ null mice in the setting of diet‐induced chronic steatosis, suggesting that pharmacological strategies to augment hepatic PPARγ activity might improve regeneration of the fatty liver. (HEPATOLOGY 2012)

The Influence of Skeletal Muscle on the Regulation of Liver:Body Mass and Liver Regeneration

The relationship between liver and body mass is exemplified by the precision with which the liver:body mass ratio is restored after partial hepatic resection. Nevertheless, the compartments, against which liver mass is so exquisitely regulated, currently remain undefined. In the studies reported here, we investigated the role of skeletal muscle mass in the regulation of liver:body mass ratio and liver regeneration via the analysis of myostatin-null mice, in which skeletal muscle is hypertrophied. The results showed that liver mass is comparable and liver:body mass significantly diminished in the null animals compared to age-, sex-, and strain-matched controls. In association with these findings, basal hepatic Akt signaling is decreased, and the expression of the target genes of the constitutive androstane receptor and the integrin-linked kinase are dysregulated in the myostatin-null mice. In addition, the baseline expression levels of the regulators of the G1-S phase cell cycle progression in liver are suppressed in the null mice. The initiation of liver regeneration is not impaired in the null animals, although it progresses toward the lower liver:body mass set point. The data show that skeletal muscle is not the body component against which liver mass is positively regulated, and thus they demonstrate a previously unrecognized systemic compartmental specificity for the regulation of liver:body mass ratio.

The apical sorting signal for human GLUT9b resides in the N-terminus

The two splice variants of human glucose transporter 9 (hGLUT9) are targeted to different polarized membranes. hGLUT9a traffics to the basolateral membrane, whereas hGLUT9b traffics to the apical region. This study examines the sorting mechanism of these variants, which differ only in their N-terminal domain. Mutating a di-leucine motif unique to GLUT9a did not affect targeting. Chimeric proteins were made using GLUT1, a basolaterally targeted transporter, and GLUT3, an apically targeted protein whose signal lies in the C-terminus. Overexpression of the chimeric proteins in polarized cells demonstrates that the N-terminus of hGLUT9b contains a signal capable of redirecting GLUT1 to the apical membrane. The N-terminus of hGLUT9a, however, does not contain a basolateral signal sufficient enough to redirect GLUT3. Portions of the GLUT9a N-terminus were substituted with corresponding portions of the GLUT9b N-terminus to determine the motif responsible for apical targeting. The first 16 amino acids were not found to be a sufficient apical signal. The last ten amino acids of the N-termini differ only in amino-acid class at one location. In the B-form, leucine, a hydrophobic residue, is substituted for lysine, a basic residue, found in the A-form. However, mutation of the leucine in hGLUT9b to a lysine resulted in retention of the apical signal. We therefore believe the apical signal exists as an interplay between the final ten amino acids of the N-terminus and another motif within the protein such as the intracellular loop or other motifs within the N-terminus.

Epimorphin Regulates the Intestinal Stem Cell Niche via Effects on the Stromal Microenvironment

Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin ( Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission, and small bowel length in vivo. Stem cell-derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim-/- and WT mice. Enteroids cocultured with ISEMFS had increased growth and crypt-like budding compared with enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared with cocultures with wild-type (WT) ISEMFs. In primary crypt cultures, Epim-/- enteroids had significantly increased surface area and budding compared with WTs. However, stem cell assays comparing the number of Epim-/- vs. WT colony-forming units after first passage showed no differences in the absence of ISEMF support. Epim-/- vs. WT ISEMFs had increased Wnt4 expression, and addition of Wnt4 to WT cocultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment. NEW & NOTEWORTHY The role of subepithelial intestinal myofibroblasts (ISEMFs) in the gut stem cell niche is controversial. We provide novel evidence supporting ISEMFs as important niche contributors. We show that the in vivo intestinal effects of deletion of myofibroblast Epim can be recapitulated in crypt stem cell cultures in vitro. ISEMFs support cocultured stem cell proliferation and enteroid growth, and these effects are augmented by deletion of Epim, a syntaxin that regulates myofibroblast cell secretion.