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Apoptosis in hypoxic human pancreatic islets correlates with HIF-1 expression¹

WOLFGANG MORITZ, FRANZISKA MEIER, DEBORAH M. STROKA, MAURO GIULIANI, PATRICK KUGELMEIER, PHILIPP C. NETT, ROGER LEHMANN^*, DANIEL CANDINAS, MAX GASSMANN^,2 and MARKUS WEBER²³

Clinic for Visceral and Transplant Surgery,
^* Division of Endocrinology and Diabetology, University Hospital Zürich, CH-8091 Zürich;
Liver Laboratories, University of Birmingham, Birmingham, UK; and
Institutes of Physiology and Veterinary Physiology, University of Zürich, CH-8057 Zürich, Switzerland

³Correspondence: Clinic for Visceral and Transplant Surgery, University Hospital Zürich, Rämistrasse 100, 8091 Zürich, Switzerland. E-mail: markus.weber{at}chi.usz.ch

SPECIFIC AIMS

Despite recent success in islet transplantation, the requirement of at least two pancreata for a successful treatment of type I diabetes poses a major limitation for a widespread applicability. Because reduced oxygen supply may account for early graft loss, we exposed a variety of ß cell lines as well as isolated rat and human islets to hypoxic conditions (1% O₂) and characterized expression of the hypoxia-inducible factor-1 (HIF-1) and its relation to the morphological alterations, particularly the appearance of apoptotic features.

PRINCIPAL FINDINGS

1. When exposed to chronic hypoxia (1% O₂), isolated rat islets show distinct features of cell death in their core region
Rat islets were isolated and exposed to either normoxic (21% O₂) or hypoxic (1% O₂) culture conditions and morphology was analyzed from H&E-stained paraffin sections. Freshly isolated islets show an uneven surface reflecting the loss of the peri-insular basal membrane as a result of the enzymatic isolation process. Otherwise, islets have a normal appearance with an intact morphology. However, when exposed to hypoxia, middle-sized and large islets start to reveal a pronounced dark core by light microscopy that corresponds to eosinophilic areas with pyknotic nuclei in H&E-stained sections. Occasionally, fragmented nuclei as signs of late-stage apoptosis were detectable. Except for a few large islets, these morphological features of advanced cell death, such as pyknotic or fragmented nuclei, were not observed in islets incubated at normoxic conditions. The thickness of the islet cortex with intact morphology roughly equals the maximal radius of islets without obvious central lesions. Such hypoxia-dependent changes in the central regions of larger islets are noticeable as early as 6 h after the isolation process and eventually result in a complete disintegration of the islet core by 48 h of hypoxic stimulation. These observations emphasize the deleterious effect of chronic hypoxia on the morphological integrity of isolated pancreatic islets.

2. Hypoxia-inducible factor-1 mRNA is expressed in several pancreatic ß cell lines and in isolated islets of human and rat origin
Reduced oxygenation is known to activate a heterodimeric transcription factor, termed HIF-1, composed of a oxygen-dependent HIF-1 subunit and a constitutive heterodimerization partner HIF-1ß (ARNT). Under normoxic conditions, HIF-1 is continuously degraded but becomes stabilized under hypoxic conditions. Therefore, HIF-1 represents a useful marker for a low-oxygen environment. The presence of HIF-1 mRNA in several ß cell lines of rat or mouse origin (RIN5AH, INS-1, and MIN6) and isolated rat and human islets, all cultivated at normoxic conditions, could be confirmed by means of reverse-transcriptase PCR. The identity of the amplified product was verified by Southern blot analysis using an internal oligomer.

3. HIF-1 protein is up-regulated and translocated into the nucleus of hypoxic MIN6 cells
A more detailed examination with MIN6 cells supports the notion that HIF-1 expression is not necessarily regulated at the transcriptional level. Northern blot analysis demonstrated no dramatic changes in HIF-1 mRNA levels in MIN6 cells when exposed for different lengths of time to hypoxia (1% O₂). However, HIF-1 target genes aldolase A and VEGF were clearly up-regulated, confirming the hypoxic culture conditions. Under normoxic conditions, HIF-1 levels were reduced by constant proteosomal degradation, whereas at restricted O₂-availability HIF-1 became stabilized and translocated into the nucleus. To see whether this also holds true for pancreatic ß cells, we exposed MIN6 cells to 1% oxygen for different periods and followed expression of HIF-1 protein by Western blot analysis. Detectable levels of HIF-1 were seen as early as 1 h; maximal levels were reached 8–24 h after induction of hypoxia (Fig. 1 A). The HIF-1 signal consists of different bands, reflecting the various phosphorylated isoforms. Nuclear extracts from normoxic control cells did not reveal detectable HIF-1 signals nor did cytosolic extracts from hypoxic cells, indicating that HIF-1 is enriched in the nuclear fraction. These results were substantiated by immunofluorescent staining of HIF-1 and subsequent visualizing by confocal laser scanning microscopy. Upon hypoxic stimulation for 24 h, HIF-1 expression markedly increased and changed from a cytoplasmic to a pronounced nuclear localization (Fig. 1B ). We were able to confirm the presence of HIF-1 DNA binding activity in nuclear extracts from hypoxic MIN6 cells and isolated human islets by means of electromobility shift assays (EMSAs). These data conclusively demonstrate a hypoxia-dependent up-regulation and nuclear accumulation of HIF-1 protein in the mouse insulinoma cell line MIN6 and isolated human islets.

View larger version (70K): [in this window] [in a new window]   Figure 1. HIF-1 protein expression and subcellular localization in MIN6 cells. A) Western blot analysis of HIF-1 protein in nuclear (NE) and cytosolic (CE) extracts of MIN6 cells incubated at 1% O2. Note the induction of HIF-1 protein over time and its nuclear distribution. Lower part of the immunoblot was analyzed for the ß cell-specific nuclear homeodomain transcription factor IPF-1, demonstrating the efficiency of the separation of nuclear from cytosolic proteins. B) Immunofluorescence analysis of HIF-1 expression in MIN6 cells incubated for 24 h at normoxia (21% O2) or hypoxia (1% O2). Inverted light/fluorescence microscopy (left panel) and confocal laser scanning microscopy (right panel) show increased HIF-1 protein expression and nuclear translocation upon hypoxic stimulation. DIC: differential interference contrast.

4. HIF-1 is expressed in areas of apoptotic cell death in hypoxic isolated human islets
Isolated human islets are as vulnerable to hypoxic damage as rat islets, as shown by areas of pyknotic nuclei in H&E-stained sections (Fig. 2 ). Regions of damaged cells are confined to areas of positive insulin staining and are not seen in nonendocrine tissue. Islets exposed to 1% oxygen for 24 h show positive HIF-1 immunoreactivity with focal distribution throughout the islet, whereas in normoxic islets no HIF-1 expression was detectable.

View larger version (100K): [in this window] [in a new window]   Figure 2. Intra-islet distribution of HIF-1 and activated caspase-3 immunoreactivity. Isolated human islets were subjected to either normoxic (21% O2) or hypoxic (1% O2) incubation conditions for 24 h. Consecutive paraffin sections were stained with H&E for insulin, HIF-1, and activated caspase-3 as indicated. Note the area of nonendocrine tissue with the intact morphological appearance and the absence of insulin, HIF-1, and activated caspase-3 (arrow). This set of pictures is representative of 3 independent human islet preparations. x400.

Comparable observations were made when isolated islets were stained for activated caspase-3, a major effector of nuclear apoptotic events. Only areas of pyknotic nuclei and positive HIF-1 staining within the endocrine tissue were positive for activated caspase-3 as well. In contrast, at normoxic conditions only a small number of cells expressed detectable levels of activated caspase-3. Nonendocrine tissue did not stain positively for HIF-1 or cleaved caspase-3 at all. Though all tissue has been challenged by hypoxia, only endocrine tissue is expressing HIF-1 at a detectable level and undergoes apoptotic cell death, as evident by the presence of activated caspase-3.

CONCLUSIONS

Several factors other than immunological events have been demonstrated to be involved in the early loss of islet graft function: mechanical and chemical stress during and after the isolation process, the loss of extracellular matrix contact, and withdrawal from trophic factors. Previous reports described morphological changes within the core region of isolated and cultured islets that partially correlated with positive TUNEL staining, indicative of apoptotic cell death.

In this study, we demonstrate for the first time that such morphological changes are a consequence of chronic hypoxia. In isolated human and rat islets, we show a strong correlation between reduced oxygen supply and the occurrence of apoptosis. To simulate hypoxia in vitro, we applied an oxygen concentration of 1%, which closely reflects the oxygen tension of transplanted islets at the liver transplantation site. Under hypoxic culture conditions, areas of pyknotic cells are observed in almost all islets, whereas at normoxic conditions only large islets (diameter250 µm) show features of advanced cell death. This suggests a diffusion-related phenomenon where larger islets are more susceptible to cell death by oxygen and probably nutrient deprivation. It is therefore conceivable that the hypoxic environment at the transplantation site accelerates the apoptotic process that islets undergo as a consequence of their devascularized state.

Therefore, shortage of oxygen supply during this early post-transplantation period may contribute substantially to the loss of transplanted islets by apoptotic events and thus to early graft nonfunction. This hypothesis has been validated by our finding that in isolated hypoxic islets, the HIF-1 subunit is up-regulated in areas where apoptosis is detectable by the presence of pyknotic nuclei, nuclear fragmentation, and the activation of caspase-3. This suggests that hypoxia plays a crucial role in the loss of functional islets mass by promoting programmed cell death. We also demonstrated that HIF-1 mRNA is indeed expressed in several murine ß cell lines (RIN5AH, INS-1, MIN6) as well as in isolated islets of human and rat origin. Cell culture experiments with MIN6 cells also show a time-dependent HIF-1 protein expression and nuclear translocation upon hypoxic stimulation. EMSAs demonstrate the presence of a HIF-1 DNA binding activity in nuclear extracts from hypoxic MIN6 cells and from isolated human islets exposed to 1% O₂. HIF-1 immunoreactivity was detectable only in hypoxic human islets, not in islets that had been exposed to normoxia. Insulin staining of adjacent tissue sections suggests that HIF-1 is expressed in ß cells and most likely in other islet cell types. We never detected HIF-1 expression in exocrine tissue. Exocrine cells also seemed to be more resistant to hypoxia than endocrine cells, showing no morphological signs of ongoing cell death or positive immunoreactivity for activated caspase-3 during hypoxic incubation.

Our observations fuel the controversial discussion of whether HIF-1 represents a proapoptotic factor. One might speculate that HIF-1 fulfills a dual function by enabling cells to adjust to a hypoxic/ischemic environment, but also permits cells that cannot comply with this kind of stress to undergo apoptosis (Fig. 3 ). Although in isolated hypoxic islets HIF-1 is expressed in areas with apoptotic features, it is unclear how this transcription factor relates to the occurrence of programmed cell death. Only more detailed colocalization studies of HIF-1 and markers of apoptosis or experiments with controlled HIF-1 expression will shed light on this controversial issue.

View larger version (68K): [in this window] [in a new window]   Figure 3. Possible Islet cell fate in the course of the isolation/transplantation procedure. Pancreatic islets experience a temporal lack of oxygen due to separation from their vascular system until revascularization of the grafted tissue takes place. During this period, HIF-1 is up-regulated and mediates the metabolic adaptation to this low-oxygen environment. Besides graft loss by immunological factors, a substantial portion of the transplanted cells is no longer able to cope with the deleterious effects of hypoxia and eventually undergoes cell death by apoptosis. Since apoptosis is an energy-dependent process, prolonged hypoxia will also result in necrosis as a consequence of depleted intracellular energy stores.

We demonstrate that cultured ß cell lines and isolated human islets express HIF-1 when exposed to oxygen concentrations comparable to those that islets experience after being transplanted into the liver. HIF-1 expression is restricted to areas of hypoxia-dependent cell death and absent in exocrine tissue. A better understanding of the molecular processes involved in hypoxia-related cell death and the involvement of HIF-1 will provide the basis for future strategies to interfere with early graft loss due to limited oxygen supply and to reduce the required islet mass for achieving insulin independence after islet transplantation.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0403fje; to cite this article, use FASEB J. (March 26, 2002) 10.1096/fj.01-0403fje

² The contribution of both senior authors was equivalent.

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