Bas Brouwers

Impaired Islet Function in Commonly Used Transgenic Mouse Lines due to Human Growth Hormone Minigene Expression

The human growth hormone (hGH) minigene is frequently used in the derivation of transgenic mouse lines to enhance transgene expression. Although this minigene is present in the transgenes as a secondcistron, and thus not thought to be expressed, we found that three commonly used lines, Pdx1-Cre(Late), RIP-Cre, and MIP-GFP, each expressed significant amounts of hGH in pancreatic islets. Locally secreted hGH binds to prolactin receptors on β cells, activates STAT5 signaling, and induces pregnancy-like changes in gene expression, thereby augmenting pancreatic β cell mass and insulin content. In addition, islets of Pdx1-Cre(Late) mice have lower GLUT2 expression and reduced glucose-induced insulin release and are protected against the β cell toxin streptozotocin. These findings may be important when interpreting results obtained when these and other hGH minigene-containing transgenic mice are used.

Metabolic and Behavioural Phenotypes in Nestin-Cre Mice Are Caused by Hypothalamic Expression of Human Growth Hormone

The Nestin-Cre driver mouse line has mild hypopituitarism, reduced body weight, a metabolic phenotype and reduced anxiety. Although several causes have been suggested, a comprehensive explanation is still lacking. In this study we examined the molecular mechanisms leading to this compound phenotype. Upon generation of the Nestin-Cre mice, the human growth hormone (hGH) minigene was inserted downstream of the Cre recombinase to ensure efficient transgene expression. As a result, hGH is expressed in the hypothalamus. This results in the auto/paracrine activation of the GH receptor as demonstrated by the increased phosphorylation of signal transducer and activator of transcription 5 (STAT5) and reduced expression of growth hormone releasing hormone (Ghrh). Low Ghrh levels cause hypopituitarism consistent with the observed mouse growth hormone (mGH) deficiency. mGH deficiency caused reduced activation of the GH receptor and hence reduced phosphorylation of STAT5 in the liver. This led to decreased levels of hepatic Igf-1 mRNA and consequently postnatal growth retardation. Furthermore, genes involved in lipid uptake and synthesis, such as CD36 and very low-density lipoprotein receptor were upregulated, resulting in liver steatosis. In conclusion, this study demonstrates the unexpected expression of hGH in the hypothalamus of Nestin-Cre mice which is able to activate both the GH receptor and the prolactin receptor. Increased hypothalamic GH receptor signaling explains the observed hypopituitarism, reduced growth and metabolic phenotype of Nestin-Cre mice. Activation of either receptor is consistent with reduced anxiety.

Phlorizin Pretreatment Reduces Acute Renal Toxicity in a Mouse Model for Diabetic Nephropathy

Background: The diabetogenic agent streptozotocin (STZ) induces direct kidney injury, which is a setback in diabetic nephropathy (DN) research. Results: The Sglt inhibitor phlorizin reduces STZ uptake and hence toxicity in the kidneys. Conclusion: In the kidney, STZ toxicity is mediated by Sglts. Significance: Using the proposed STZ regimen, researchers can now induce DN without direct damage to proximal tubuli. Streptozotocin (STZ) is widely used as diabetogenic agent in animal models for diabetic nephropathy (DN). However, it is also directly cytotoxic to kidneys, making it difficult to distinguish between DN-related and STZ-induced nephropathy. Therefore, an improved protocol to generate mice for DN studies, with a quick and robust achievement of the diabetic state, without direct kidney toxicity is required. To investigate the mechanism leading to STZ-induced nephropathy, kidney damage was induced with a high dose of STZ. This resulted in delayed gastric emptying, at least partially caused by impaired desacyl ghrelin clearance. STZ uptake in the kidneys is to a large extent mediated by the sodium/glucose cotransporters (Sglts) because the Sglt inhibitor phlorizin could reduce STZ uptake in the kidneys. Consequently, the direct toxic effects in the kidney and the gastric dilatation were resolved without interfering with the β-cell toxicity. Furthermore, pancreatic STZ uptake was increased, hereby decreasing the threshold for β-cell toxicity, allowing for single low non-nephrotoxic STZ doses (70 mg/kg). In conclusion, this study provides novel insights into the mechanism of STZ toxicity in kidneys and suggests a more efficient regime to induce DN with little or no toxic side effects.

The AlfpCre mouse revisited: Evidence for liver steatosis related to growth hormone deficiency

With great interest we read the article by Mueller et al. on the development of steatosis and hepatocellular carcinoma in mice by disrupting hepatic growth hormone (GH) and glucocorticoid receptor signaling. They demonstrate that abrogation of hepatic signal transducer and activator of transcription 5 (Stat5) signaling caused liver steatosis by up-regulation of genes involved in lipid uptake and synthesis. However, we have concerns regarding the use of AlfpCre transgenic mice to inactivate Stat5 in the liver. While generating the AlfpCre transgene, the entire human GH (hGH) gene was inserted for its introns and polyadenylation signal. As a consequence, a polycistronic messenger RNA (mRNA) transcript encoding Cre Recombinase and hGH was transcribed in the liver, hypothalamus, and pituitary (data not shown). However, hGH was only translated in the hypothalamus and pituitary (Fig. 1A). It has previously been described that targeted expression of hGH in the hypothalamus reduces expression of hypothalamic GH releasing hormone (GHRH), leading to GH deficiency (GHD). This is also the case in AlfpCre mice in a C57Bl6/J background, as shown by quantitative real-time polymerase chain reaction (qRTPCR). In the hypothalamus, expression levels of GHRH were reduced by 70% (P< 0.01) and mouse GH messenger RNA (mRNA) in the adenopituitary by 61% (P< 0.05). AlfpCre mice also showed hypoplasia of the pituitary (Fig. 1B). Hepatic insulinlike growth factor 1 (Igf1) mRNA and Igf1 plasma protein levels were decreased by 71% (P< 0.001) and 60% (P< 0.001), respectively, and reduced phosphorylation of Stat5 in AlfpCre livers was shown by western blotting (Fig. 1C), confirming GHD in AlfpCre mice. Consequently, AlfpCre mice exhibit postnatal growth retardation (Fig. 1D), a finding that was also observed in AlfpCrec-Jun mice. Downstream of the impaired GH signaling, AlfpCre livers showed increased levels of triglycerides (TGs), free fatty acids (FFAs), and cholesterol, indicating liver steatosis (Fig. 1E). Finally, genes involved in lipid uptake and synthesis, including CD36, very low-density lipoprotein receptor, and peroxisome proliferator-activated receptor gamma, were significantly up-regulated by 2.52(P< 0.01), 5.44(P< 0.01), and

Endosome to trans-Golgi network transport of Proprotein Convertase 7 is mediated by a cluster of basic amino acids and palmitoylated cysteines

Proprotein Convertase 7 (PC7) is a Furin-like endoprotease that cleaves precursor proteins at basic amino acids. PC7 is concentrated in the trans-Golgi network (TGN) but it shuttles between the plasma membrane and the TGN depending on sequences in the cytoplasmic tail. A short region containing a three amino acids motif, P724-L725-C726, is essential and sufficient for internalization of PC7 but not for TGN localization, which requires the additional presence of the juxtamembrane region. In this study we have investigated the contribution of a cluster of basic amino acids and two reversibly palmitoylated cysteine residues to endocytic trafficking. Stable cell lines overexpressing chimeric proteins (CD25 and CD46) containing the cytoplasmic domain of PC7 in which the basic cluster alone or together with both palmitoylated cysteines are mutated showed enhanced surface expression as demonstrated by immunofluorescence experiments and surface biotinylation. The mutant proteins no longer recycled to the TGN in antibody uptake experiments and accumulated in an endosomal compartment. Recycling of wild type PC7 to the TGN is blocked by nocodazole, suggesting that PC7 shuttles to the TGN via late endosomes, similar to Furin. Unlike furin, however, PC7 was found to recycle to a region within the TGN, which is deficient in sialyltransferase, as shown by resialylation experiments. In conclusion, a novel motif, composed of a basic amino acid cluster and two palmitoylated cysteines are essential for TGN localization and endocytic trafficking.

Liver-Specific Inactivation of the Proprotein Convertase FURIN Leads to Increased Hepatocellular Carcinoma Growth.

Proprotein convertases are subtilisin-like serine endoproteases that cleave and hence activate a variety of proproteins, including growth factors, receptors, metalloproteases, and extracellular matrix proteins. Therefore, it has been suggested that inhibition of the ubiquitously expressed proprotein convertase FURIN might be a good therapeutic strategy for several tumor types. Whether this is also the case for hepatocellular carcinoma (HCC) is currently not clear. In a mouse model for HCC expression of Furin was not altered in the tumors, while those of PC7, PC5/6, and PACE4 significantly decreased, at least at some time points. To investigate the impact of Furin inhibition on the development and progression of HCC in this model, Furin was genetically ablated in the liver. Furin inactivation resulted in an increased tumor mass after 5 weeks. This was not caused by decreased apoptosis, since no differences in the apoptosis index could be observed. However, it could at least partially be explained by increased hepatocyte proliferation at 5 weeks. The tumors of the Furin knockout mice were histologically similar to those in wild type mice. In conclusion, liver-specific Furin inhibition in HCC enhances the tumor formation and will not be a good therapeutic strategy for this tumor type.

Human Growth Hormone in Transgenesis: A Growing Problem?

Temporospacial overexpression or inactivation of genes in mouse models has undeniably boosted biomedical research. This is most commonly achieved using the Cre-loxP technique, where mice expressing Cre recombinase driven by a tissue-specific or inducible promoter are bred with mice in which one or more exons of a gene of interest are flanked by LoxP sites (1). This allows for a more in-depth view of the gene function in the context of a particular time frame and tissue system, while avoiding unwanted effects due to the absence of this gene during development. However, in the recent years several pitfalls have emerged related to this technique (2–4). There have been reports of disturbed endogenous gene expression at the site of the Cre-transgene insertion (4), low or mosaic Cre expression (5,6), and expression in undesired tissues, such as unwanted Cre activity in nutrient-sensing neurons from transgenic mice that express Cre under the control of Ins2 and Pdx1 promoters (7,8). Equally disturbing is the finding that high Cre activity can cause chromosomal rearrangements, apoptosis, and decreased proliferation (9–11). Particularly for diabetes and central nervous system research, these caveats have been reviewed in detail (2,3). In this issue …

Transgenic Artifacts Caused by Passenger Human Growth Hormone

The minigene encoding human growth hormone (hGH) has been incorporated into over 300 transgenic mouse lines to improve transgene expression. However, unexpected and functional hGH expression can drastically alter physiology. We list here the mouse lines in which ectopic hGH has been confirmed, and we provide a wiki for lines awaiting analysis.