HIF-1α inhibitor PX-478 preserves pancreatic ß cell function in diabetes.

During progression of type 2 diabetes, pancreatic ß cells are subjected to sustained metabolic overload. We postulated that this state mediates a hypoxic phenotype driven by hypoxia-inducible factor-1α (HIF-1α) and that treatment with the HIF-1α inhibitor PX-478 would improve ß cell function. Our studies showed that the HIF-1α protein was present in pancreatic ß cells of diabetic mouse models. In mouse islets with high glucose metabolism, the emergence of intracellular Ca2+ oscillations at low glucose concentration and the abnormally high basal release of insulin were suppressed by treatment with the HIF-1α inhibitor PX-478, indicating improvement of ß cell function. Treatment of db/db mice with PX-478 prevented the rise of glycemia and diabetes progression by maintenance of elevated plasma insulin concentration. In streptozotocin-induced diabetic mice, PX-478 improved the recovery of glucose homeostasis. Islets isolated from these mice showed hallmarks of improved ß cell function including elevation of insulin content, increased expression of genes involved in ß cell function and maturity, inhibition of dedifferentiation markers, and formation of mature insulin granules. In response to PX-478 treatment, human islet organoids chronically exposed to high glucose presented improved stimulation index of glucose-induced insulin secretion. These results suggest that the HIF-1α inhibitor PX-478 has the potential to act as an antidiabetic therapeutic agent that preserves ß cell function under metabolic overload.

Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions.

Low oxygen levels (hypoxia) trigger a variety of adaptive responses with the Hypoxia-inducible factor 1 (HIF-1) complex acting as a master regulator. HIF-1 consists of a heterodimeric oxygen-regulated α subunit (HIF-1α) and constitutively expressed ß subunit (HIF-1ß) also known as aryl hydrocarbon receptor nuclear translocator (ARNT), regulating genes involved in diverse processes including angiogenesis, erythropoiesis and glycolysis. The identification of HIF-1 interacting proteins is key to the understanding of the hypoxia signaling pathway. Besides the regulation of HIF-1α stability, hypoxia also triggers the nuclear translocation of many transcription factors including HIF-1α and ARNT. Notably, most of the current methods used to study such protein-protein interactions (PPIs) are based on systems where protein levels are artificially increased through protein overexpression. Protein overexpression often leads to non-physiological results arising from temporal and spatial artifacts. Here we describe a modified co-immunoprecipitation protocol following hypoxia treatment using endogenous nuclear proteins, and as a proof of concept, to show the interaction between HIF-1α and ARNT. In this protocol, the hypoxic cells were harvested under hypoxic conditions and the Dulbecco's Phosphate-Buffered Saline (DPBS) wash buffer was also pre-equilibrated to hypoxic conditions before usage to mitigate protein degradation or protein complex dissociation during reoxygenation. In addition, the nuclear fractions were subsequently extracted to concentrate and stabilize endogenous nuclear proteins and avoid possible spurious results often seen during protein overexpression. This protocol can be used to demonstrate endogenous and native interactions between transcription factors and transcriptional co-regulators under hypoxic conditions.

A Notch-independent mechanism contributes to the induction of Hes1 gene expression in response to hypoxia in P19 cells.

Hes1 is a Notch target gene that plays a major role during embryonic development. Previous studies have shown that HIF-1α can interact with the Notch intracellular domain and enhance Notch target gene expression. In this study, we have identified a Notch-independent mechanism that regulates the responsiveness of the Hes1 gene to hypoxia. Using P19 cells we show that silencing the Notch DNA binding partner CSL does not prevent hypoxia-dependent upregulation of Hes1 expression. In contrast to CSL, knockdown of HIF-1α or Arnt expression prevents Hes1 induction in hypoxia. Deletion analysis of the Hes1 promoter identified a minimal region near the transcription start site that is still responsive to hypoxia. In addition, we show that mutating the GA-binding protein (GABP) motif significantly reduced Hes1 promoter-responsiveness to hypoxia or to HIF-1 overexpression whereas mutation of the hypoxia-responsive element (HRE) present in this region had no effect. Chromatin immunoprecipitation assays demonstrated that HIF-1α binds to the proximal region of the Hes1 promoter in a Notch-independent manner. Using the same experimental approach, the presence of GABPα and GABPß1 was also observed in the same region of the promoter. Loss- and gain-of-function studies demonstrated that Hes1 gene expression is upregulated by hypoxia in a GABP-dependent manner. Finally, co-immunoprecipitation assays demonstrated that HIF-1α but not HIF-2α is able to interact with either GABPα or GABPß1. These results suggest a Notch-independent mechanism where HIF-1 and GABP contribute to the upregulation of Hes1 gene expression in response to hypoxia.

Identification of an alternative mechanism of degradation of the hypoxia-inducible factor-1alpha.

The hypoxia-inducible factor-1alpha (HIF-1alpha) is a master regulator of the cellular response to decreased oxygen levels. This transcription factor is highly unstable at normal oxygen concentrations and is rapidly stabilized by hypoxia. At normoxia two specific proline residues (Pro(402) and Pro(563)) of mHIF-1alpha are hydroxylated and recognized by the von Hippel-Lindau E3 ubiquitin ligase (pVHL) complex, which upon binding mediates degradation of the protein. Previous studies have demonstrated that these two proline residues are critical for high affinity binding to pVHL. We have performed a detailed analysis of a mutant form of HIF-1alpha, where both these proline residues have been mutated, and we have uncovered a novel degradation pathway, to which the HIF-1alpha mutant protein is not resistant. Our results show that the HIF-1alpha double proline mutant undergoes ubiquitination and proteasome-dependent degradation, and retains the ability to be stabilized in response to hypoxia and CoCl(2) treatment. However in contrast to the wild-type protein, stabilization of the mutant was only observed within short periods of hypoxia exposure (1-2 h). Degradation assays in the presence of the expressed prolyl hydroxylases (PHDs) 1-3 showed that, unlike the wild-type protein, the HIF-1alpha mutant was resistant to these hydroxylases. However, experiments knocking-down expression of pVHL by RNA interference showed that the HIF-1alpha mutant is degraded and ubiquitinated by a pVHL-mediated mechanism. In conclusion, we show the first evidence of a novel mechanism of degradation of HIF-1alpha at normoxia that involves pVHL but is not mediated by PHDs 1-3 or by degradation boxes surrounding Pro(402) and Pro(563).