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. Author manuscript; available in PMC: 2012 Sep 9.
Published in final edited form as: Biochem Biophys Res Commun. 2011 Aug 17;412(4):728–731. doi: 10.1016/j.bbrc.2011.08.041

Decreased Expression of ATP6V1H in Type 2 Diabetes: a Pilot Report on the Diabetes Risk Study in Mexican Americans

Melanie F Molina 1, Hui-Qi Qu 1,*, Anne R Rentfro 2, Saraswathy Nair 3, Yang Lu 1, Craig L Hanis 4, Joseph B McCormick 1, Susan P Fisher-Hoch 1,*
PMCID: PMC3176628  NIHMSID: NIHMS319145  PMID: 21871445

Abstract

Objective

Previous studies in mice and humans observed down-regulation of the gene expression of ATP6V1H associated with type 2 diabetes. This study identified prospectively changes in ATP6V1H expression before and after overt diabetes.

Methods

Expression of ATP6V1H in peripheral blood was compared pre and post development of diabetes in nine individuals.

Results

Considerable variation of ATP6V1H mRNA levels was observed between different individuals. However, within each individual the decrease in expression of ATP6V1H with the development of diabetes was highly statistically significant.

Conclusions

ATP6V1H may represent a critical molecular mechanism involved in the development of type 2 diabetes and its compilations through its important regulatory effect on vacuolar-ATPase activity.

Keywords: ATP6V1H, diabetic complications, longitudinal study, type 2 diabetes, vacuolar ATPase

Introduction

The ATPase, H+ transporting, lysosomal 50/57kDa, V1 subunit H gene (ATP6V1H) at Chr8q11.2 encodes for the V1H subunit of vacuolar ATPase (V-ATPase). The ATPase family consists of multi-subunit enzymes that hydrolyze ATP and use the energy to catalyze a variety of critical cellular processes [1]. V-ATPase was originally discovered in plant and fungal vacuoles, and subsequently found widely in a variety of eukaryotic endomembrane organelles [1]. V-ATPase belongs to the rotary ATPase family, membrane complexes that use rotary motor mechanisms to translocate ions across membranes [2]. The major function of V-ATPase is to acidify intracellular compartments by using the energy gathered from ATP hydrolysis to pump protons [3]. This function gives V-ATPase important roles in receptor-mediated endocytosis, intracellular trafficking processes and protein degradation [4].

V-ATPase has two functional domains V0 and V1, which are involved in proton translocation and ATP hydrolysis respectively [3,4,5]. The V0 domain consists of five distinct subunits: a, c, c′, c″, and d, with a stoichiometry of a1d1c4–5c′1c″1 and the function to conduct protons [1]. The V1 domain contains eight subunits (A-H, with stoichiometry A3B3C1D1E3F1G3H1), which form a peripheral complex responsible for the hydrolysis of ATP[6,7]. Of particular interest in the V1 domain is the regulatory subunit H (V1H)[4], owing to the fact that the down-regulation of its gene expression correlates with the presence of type 2 diabetes [8]. Olsson et al. observed down-regulation of ATP6V1H in human pancreatic islets from patients with type 2 diabetes[9]. The expression of ATP6V1H correlated negatively with HbA1c levels, and positively with glucose-stimulated insulin secretion [9]. To establish the dynamics of gene expression coincident with the development of diabetes, we created a nested cohort with impaired fasting glucose (the Diabetes Risk Study (DRS) cohort: fasting blood glucose ≥100 mg/dl and <126 mg/dl). This DRS cohort was selected from our larger community-recruited Cameron County Hispanic Cohort (CCHC n=2500) consisting of Mexican Americans resident on the Texas Mexico border in whom we document elevated prevalence of diabetes[10].

Methods

1. Ethics Statement

Written informed consent was obtained from each participant, and the Committee for the Protection of Human Subjects of the University of Texas Health Science Center at Houston (UTHealth) approved this study.

2. Subjects

To determine the proximal changes accompanying transition to overt type 2 diabetes we followed 297 DRS participants recruited over 3 years and examined quarterly. During this period 9 participants transitioned from pre-diabetes to overt diabetes (2006 American Diabetes Association). Three were male and 6 female; age range from 32 to 74 years of age (median 57 years). Each participant had 2 to 5 clinic visits (median 4 visits). At each visit a questionnaire and clinical examination were administered and whole blood was collected and stabilized in PAXgene Blood RNA Tubes (Qiagen) for isolation and purification of intracellular RNA.

3. Gene expression profiling

Transcriptome profiling of RNA samples from the 9 participants was performed using the Illumina HumanHT-12 Beachip (Illumina, San Diego) at the McGill University and Génome Québec Innovation Centre.

4. Data anlaysis

The Flexarray software[11] was used to analyze the microarray data. The data was normalized using the Lumi Bioconductor package[12] implemented in Flexarray. 34,694 genes were assayed on the HT-12 Beachip, and the expression of 22,251 genes were detectable. 12443 genes were removed from further analysis because of undetectable expression. As shown by the principle component analysis, the transcriptome profiles of the RNA samples were mainly clustered by each individual participant, instead of clustered by disease status. The transcriptome profile of each individual participant was obviously different. Based on this phenomenon, pairwise Z test was selected to compare the differences in gene expression within each individual. Statistical significance was corrected for multiple comparisons by Benjamini and Hochberg False Discovery Rate (FDR) using the QVALUE software[13].

Results

The transcriptome profiles of the RNA samples clustered by each individual participant highlighted the robust longitudinal design of our prospective study. Alterations in gene expression coincident with development of diabetes could not be properly assessed using a case-control design, because of significant variations between individual subjects. However, within serial specimens from each subject we were able to document decreased expression of ATP6V1H in peripheral blood with development of diabetes. Among all the genes assayed in our study, the difference of ATP6V1H expression between pre-diabetes and overt diabetes was the most significant with uncorrected P=7.18×10−11, and FDR corrected q-value=1.14×10−6. Fig. 1 shows the considerable variations observed for each of the individuals with development of diabetes as the expression of ATP6V1H decreased in each individual without exception.

Fig. 1.

Fig. 1

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The expression of ATP6V1H in each participant. Dramatic variations were observed among the different individuals, which was concordant with our PCA finding of the whole transcriptome profiles. However, compared with pre-diabetes status, the expression of ATP6V1H in diabetes decreased in each individual without exception, with pairwise Z test P=7.18×10−11. This observation on ATP6V1H expression highlighted the robust design of our longitudinal study.

Discussion

Using a unique cohort of extensively documented individuals with pre-diabetes we observed decreased expression of ATP6V1H in peripheral white blood cells with the development of clinical diabetes with a high degree of statistical significance. This novel observation casts new light on the mechanisms involved in development of frank diabetes and clues to the processes associated with onset and progress of the disease state and its complications. This study is novel in two ways, one it is a prospective longitudinal study that provides comparison of gene expression in each individual over time associated with actually progression from pre-diabetes to diabetes. Secondly it examines the changes in peripheral blood white cell expression associated with development of diabetes, which provides a highly accessible source of potential markers for development of diabetes. Because these participants were newly diagnosed with diabetes they were not under treatment for diabetes at the time of their conversion.

The V1H encoded by ATP6V1H has been shown to exercise an important regulatory effect on V-ATPase activity [14]. V1H has a highly helical structure consisting of two domains: the N-domain with seventeen consecutive α-helices (amino acids 2–352) and the C-domain with eight α-helices (amino acids 353–478) [15]. The α-helical motifs in Subunit H show structural similarity to the armadillo or HEAT motifs (an arrangement of three α-helices, each about 42 amino acids in length) found in the importin family of proteins [15]. The importin proteins bind to nuclear localization signals (NLS) on other proteins via a hydrophobic, shallow groove, facilitating protein transport from the cytosol into the nucleus [16,17]. Further studies show that the N-terminal domain is important for the ATP hydrolysis function of the V1 domain of V-ATPase, and the C-terminal domain is needed for proper communication between the V1 and V0 domains of V-ATPase [18,19,20]. In addition, it has also been shown that the C-terminal domain of V1H is necessary in inhibiting ATPase activity of free V1 domain complexes by interacting with subunit F via a cysteine residue at position 381[21]. These studies demonstrated the critical role of V1H in maintaining proper V-ATPase function.

Impaired V1H expression in diabetes may be important in the development of diabetes and its complications. The principle function of V-ATPase is to acidify intracellular compartments [3], allowing it to play an important role in receptor-mediated endocytosis, intracellular trafficking processes and protein degradation (Fig. 2)[4]. The V-ATPase enzyme is ubiquitous, and enzyme malfunction may play important roles in diabetes and its complications, e.g. increased risk of intracellular infections such as tuberculosis.

Fig. 2.

Fig. 2

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Cellular localizations and functions of V-ATPase. V-ATPase is present in a variety of intracellular organelles. In the lysosomes and central vacuoles of eukaryotic organisms, the action of the V-ATPase creates an acidic environment which facilitates the degradation of macromolecules by activating digestive enzymes [30]. In synaptic vesicles and chromaffin granules, the proton gradient generated by V-ATPase acts as a driving force, which promotes the uptake of neurotransmitters and other small molecules destined for secretion [31]. In some cases, a low pH is needed in secretory vesicles to activate enzymes which can convert hormones such as insulin from their precursor to mature form [32]. At the plasma membrane in a variety of highly specialized cells, the V-ATPase is involved in acid secretion—an important process in urine formation[5], bone resorption[33], and sperm maturation and storage [34]. The enzyme also plays an important role in the lysosomal targeting of enzymes in the Golgi apparatus, giving rise to a low pH within late endsomes that allows the enzymes to dissociate from the mannose-6-phosphate receptor [35]. Finally, in the Golgi apparatus, the acidification provided by V-ATPase ensures proper sorting of proteins to their destined location within the cell [36].

V-ATPase plays an important role in the development of insulin resistance and diabetes. The V-ATPase pumps protons into the endosome lumen, creating an acidic environment that not only enables ligand-receptor complexes (brought in from the plasma membrane) to dissociate [22], but also allows for the vesicular trafficking of released ligands from early endosomes to late endosomes[23]. These processes are important in the dissociation of insulin and its receptor [24,25]. A study conducted by Benzi et al. in 1997 compromised endosomal acidification by using an ionophore known as monensin and effectively mimicked the abnormal processing of insulin in patients with type 2 diabetes [26]. V-ATPase is also involved in the increased risk of tuberculosis in patients with diabetes. V-ATPase is involved in endocytosis through the acidification of the phagosome [27]—a critical process in intracellular microbial killing [28], especially in specialized phagocytic cells such as macrophages. Bidani et al. showed that the inhibition of V-ATPase in alveolar macrophages led to a rise in phagosomal pH and a decrease in reactive oxygen species (ROS) production, both effects compromise substantially intracellular microbial killing [29]. Mycobacterium tuberculosis (MTB) has already evolved a mechanism to resist acidification by excluding V-ATPase from the phagosome [27]. Impaired function of V-ATPase in diabetes may deteriorate the susceptibility to MTB.

This is a unique study in that we were able to follow individuals with pre-diabetes prospectively and document gene expression as they developed diabetes. Though the small numbers of individuals transitioning to diabetes limited our study, the consistency of our observations is striking and gives confidence that these are real events in the evolution of the disease. This new knowledge throws fresh light on an important molecular mechanism involved in development of diabetes, and importantly, it is consistent with a newly recognized major risk of diabetes, specifically increased susceptibility to tuberculosis.

  • This prospective study investigated gene expression in progress of diabetes in human.

  • ATP6V1H in peripheral blood decreased with progression from pre-diabetes to diabetes.

  • This observation casts new light on the mechanisms involved in diabetes and its complications.

Acknowledgments

We thank our cohort recruitment team, particularly Rocio Uribe, Elizabeth Braunstein and Julie Ramirez. We also thank Marcela Montemayor and other laboratory staff for their contribution, and Christina Villarreal for administrative support. We thank Valley Baptist Medical Center, Brownsville for providing us space for our Center for Clinical and Translational Science Clinical Research Unit. We also thank the community of Brownsville and the participants who so willingly participated in this study in their city.

This work was supported by MD000170 P20 funded from the National Center on Minority Health and Health Disparities (NCMHD), and the Centers for Translational Science Award 1U54RR023417-01 from the National Center for Research Resources (NCRR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. M.F.M. is supported by a summer student fellowship from the University of Texas at Austin. H.Q.Q is supported by intramural funding from the University of Texas School of Public Health.

Footnotes

Conflict of Interest statement: None declared.

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