Directed overexpression of insulin in Leydig cells causes a progressive loss of germ cells.

Abstract

The primary goal of this study was to determine the 5'region of the Insl3 gene that specifically targets the expression of human insulin to Leydig cells, and to explore whether the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in different mouse models of diabetes. We show here that the sequence between nucleotides -690 and +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of the human insulin transgene (Insl3-hIns). We also found that the 3'untranslated region (3'UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo. Expression analysis revealed that the temporal expression pattern of the hIns transgene in Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were able to initiate and complete spermatogenesis. However, there was a progressive and age-dependent degeneration of the germ cells that lead to male infertility with increasing age.

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Accepted Manuscript
Title: Directed overexpression of insulin in Leydig cells
causes a progressive loss of germ cells
Authors: Katayoon Shirneshan, Stefan Binder, Detlef B¨ ohm,
Stephan Wolf, Ulrich Sancken, Andreas Meinhardt, Michael
Schmid, Wolfgang Engel, Ibrahim M. Adham
PII:
DOI:
Reference:
S0303-7207(08)00291-8
doi:10.1016/j.mce.2008.07.007
MCE 6920
To appear in:
Molecular and Cellular Endocrinology
Received date:
Revised date:
Accepted date:
27-3-2008
3-7-2008
5-7-2008
Please cite this article as: Shirneshan, K., Binder, S., B¨ ohm, D., Wolf, S., Sancken,
U., Meinhardt, A., Schmid, M., Engel, W., Adham, I.M., Directed overexpression of
insulin in Leydig cells causes a progressive loss of germ cells, Molecular and Cellular
Endocrinology (2007), doi:10.1016/j.mce.2008.07.007
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peer-00532051, version 1 - 4 Nov 2010
Author manuscript, published in "Molecular and Cellular Endocrinology 295, 1-2 (2008) 79"
DOI : 10.1016/j.mce.2008.07.007

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Accepted Manuscript
Corresponding author at: Institute of Human Genetics, University of Göttingen, Heinrich-

1



Directed overexpression of insulin in Leydig cells causes a progressive loss
of germ cells
Katayoon Shirneshana, Stefan Bindera, Detlef Böhma, Stephan Wolfa, Ulrich Sanckena,
Andreas Meinhardtb, Michael Schmidc, Wolfgang Engela, Ibrahim M. Adhama, *
aInstitute of Human Genetics, University of Göttingen, D-37073 Göttingen, Germany;
bDepartment of Anatomy and Cell Biology, University of Giessen, D-35385 Giessen,
Germany; cInstitute of Human Genetics, University of Würzburg, D-97074 Würzburg,
Germany.



Keywords: Insl3; Insulin; Transgenic mice
Short Title: Insulin overexpression impairs spermatogenesis




Düker-Weg 12, D-37073 Göttingen, Germany. Tel.: +49 551 397522; fax: +49 551 399303.
E-mail address: iadham@gwdg.de (I. Adham)



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2
Abstract
The primary goal of this study was to determine the 5`region of the Insl3 gene that
specifically targets the expression of human insulin to Leydig cells, and to explore whether
the testicular proinsulin is efficiently processed to insulin that is able to rescue the diabetes in
different mouse models of diabetes. We show here that the sequence between nucleotides -
690 to +4 of mouse Insl3 promoter is sufficient to direct the Leydig cell-specific expression of
the human insulin transgene (Insl3-hIns). We also found that the 3´untranslated region
(3´UTR) of Insl3 was effective in enhancing transgene expression of the insulin in vivo.
Expression analysis revealed that the temporal expression pattern of the hIns transgene in
Leydig cells of transgenic testes is roughly the same as that of the endogenous Insl3. Despite
the Leydig cells translate human proinsulin and secrete a significant level of free C-peptide
into the serum, the Leydig cell-derived insulin is not able to overcome the diabetes in
different mouse models of diabetes, suggesting a lack of glucose sensing mechanisms in the
Leydig cells. A consequence of overexpression of the human proinsulin in Leydig cells was
the decrease of fertility of transgenic males at older ages. Germ cells in transgenic males were
able to initiate and complete spermatogenesis. However, there was a progressive and age-
dependent degeneration of the germ cells that lead to male infertility with increasing age.












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glucose levels.

3
1. Introduction

Diabetes mellitus is a group of diseases characterised by an absolute or relative insulin
deficiency and by poor glucose control (hyperglycemia) in the blood. In type I diabetes, also
known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes mellitus,
there is an absolute lack of insulin due to the progressive loss of pancreatic β-cells in the islets
of Langerhans. Despite intensive research efforts, daily insulin injection remains the only
therapy. This unsatisfactory treatment strongly affects the life style of the patients and cannot
achieve the same degree of glycemic control as provided by endogenous insulin produced
from the pancreatic β-cells. Therefore, transplantation of insulin-producing tissues and gene
therapy are potential therapeutically strategies to overcome type I diabetes. Large-scale
application of islet cell implantations to type I diabetic patients is hindered by limited
availability of insulin-producing tissues and the subsequent need for life-long
immunosuppressants (Remuzzi et al., 1994). An alternative approach is the ectopic expression
of insulin in other tissues using gene therapy. The goal is to restore and to control insulin
production in another tissue, thereby reducing the elevated blood glucose levels to a normal
range without causing hypoglycemia. For successful therapy, at least three crucial issues must
be adequately addressed. The ectopic tissues expressing engineered insulin must have
constitutive and regulated secretory pathways and express proprotein convertases PC2 and
PC1/3 that are required for proinsulin processing (Steiner, 1998). These cells must have
glucose sensing mechanisms for adequate regulation of insulin secretion according to blood
Several approaches, which have been taken in mind these crucial issues, have addressed
the feasibility of in vivo gene therapy for the delivery of insulin to diabetic patients. Target
tissues tested include liver, muscle, pituitary, exocrine pancreas and intestinal K-cells (Valera
et al., 1994; Kolodka et al., 1995; Lipes et al., 1996; Goldfine et al., 1997; Bartlett et al.,
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4
1997; Abai et al., 1999; Bochan et al., 1999; Cheung et al., 2000; Lu et al., 2005). The results
are rather disappointing.
INSL3, a member of the insulin superfamily, is synthesized as preprotein in pre- and
postnatal Leydig cells and play an essential role in growth of gubernaculums ligament during
transabdominal descent of the testis (Zimmermann et al., 1997; 1999; Balvers et al., 1998;
Nef and Parada, 1999; Adham et al., 2002; Koskimies et al., 2003). Although the structure of
circulating INSL3 is only known in bovine (Büllesbach and Schwabe, 2002), the presence of
certain conserved amino acids of the A and B chain at the N and C termini of the pro-INSL3
suggests that the mode of in vivo processing of pro-INSL3 and resulting structure of INSL3 is
similar to that of insulin and relaxin (Adham et al., 1993; Hombach-Klonisch et al., 2004). It
is known that the processing of the proinsulin occurs in the trans-Golgi by a regulated
secretory pathway (Kelly, 1985; Rhodes and Halban, 1987). Similar to islet β cells, several
endocrine cells such as intermediate lobe pituitary cells, and gastrointestinal G and K cells
efficiently process proinsulin to mature, biologically active insulin via a regulated pathway of
protein secretion (Lipes et al. 1996, Cheung et al., 2000; Lu et al., 2005), whereas in
hepatocytes, which have only a constitutive pathway of protein secretion, proinsulin
processing to insulin is extremely inefficient (Valera et al., 1994; Kolodka et al., 1995).
To assess the efficiency of the endocrine Leydig cells in processing the proinsulin to the
biological active insulin and to determine the ability of the testicular insulin to overcome the
diabetes in mice, we overexpressed insulin in the Leydig cells during pre- and postnatal
development. The rational for this study was underpinned by our previous study showing that
ectopic expression of Insl3 in β-cells rescue the cryptorchidism in Insl3-null males. These
results reveal that the β-cells efficiently processed the Insl3 gene product to the functional
hormone. To achieve our goal, the insulin was targeted using the upstream regulated sequence
of murine Insl3 gene, and transgenic mice were generated. To determine whether the
peer-00532051, version 1 - 4 Nov 2010