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. 2022 Jan 1;163(1):bqab226.
doi: 10.1210/endocr/bqab226.

Isoform-specific Roles of Prolyl Hydroxylases in the Regulation of Pancreatic β-Cell Function

Monica Hoang  1 , Emelien Jentz  1 , Sarah M Janssen  1 , Daniela Nasteska  2   3 , Federica Cuozzo  2   3 , David J Hodson  2   3 , A Russell Tupling  4 , Guo-Hua Fong  5 , Jamie W Joseph  1
Affiliations

Isoform-specific Roles of Prolyl Hydroxylases in the Regulation of Pancreatic β-Cell Function

Monica Hoang et al. Endocrinology. .

Abstract

Pancreatic β-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated β-cell-specific knockout (KO) mice for all 3 isoforms of PHD (β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the β-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for β-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both β-PHD1 KO and β-PHD3 KO had elevated β-cell apoptosis and reduced β-cell mass. Isolated islets from β-PHD1 KO and β-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in β-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, β-PHD1 KO and β-PHD3 KO mice had defective β-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.

Keywords: ARNT/HIF1β; HIF1α; PHD; cell metabolism; hypoxia; insulin release; insulin secretion; islet; metabolism; pancreatic β-cell; prolyl hydroxylases.

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Figures

Figure 1.
Figure 1.
Effects of DMOG (200 µg/g body weight) on in vivo glucose homeostasis and dynamic islet insulin secretion from male C57BL/6N mice. (A) Blood glucose levels during an ipGTT (n = 12 mice per experimental group). (B) Plasma insulin levels (n = 12 mice per experimental group). (C) Insulin tolerance test (n = 12 mice per experimental group). (D) Islets were stimulated with low glucose (LG, 2 mM) for 10 minutes followed by high glucose (HG, 16.7 mM) plus or minus 5 µM DMOG for 35 minutes followed by HG + KCl (30 mM) for 20 minutes (n = 10 per experimental group; each n represents 25 islets). (E) AUC of D. Data are mean ± SEM. P < .05, **P < .01, and ***P < .001.
Figure 2.
Figure 2.
Mitochondrial respiration in response to DMOG in INS1 832/13 cells. (A) OCR was measured in response to low glucose (2 mM), high glucose (Glucose, 16.7 mM) plus or minus DMOG (0-1000 µM), oligomycin (Oligo, 10 µM), dinitrophenol (DNP, 50 µM) plus pyruvate (Pyr, 20 mM), and rotenone (Rot, 5 µM) plus myxothiazol (Myx, 5 µM). (B) AUC of A. (C) ATP turnover (HG OCR minus oligomycin OCR. (D) Nonmitochondrial oxygen consumption (rotenone plus myxothiazol OCR). Data are mean ± SEM (n = 10, each n represents 1 well). *P < .05, **P < .01, ***P < .001, ****P < .0001.
Figure 3.
Figure 3.
Mitochondrial respiration in response to DMOG in male islets from C57BL/6N mice. (A) Islet OCR in response to low glucose (2 mM), high glucose (Glucose, 16.7 mM) plus or minus DMOG (0-10 µM), oligomycin (Oligo, 20 µM), DNP (100 µM) plus dimethylmalate (DMM, 10 mM) plus dimethyl αketoglutarate (DMαKG, 10 mM), and rotenone (Rot, 10 µM) plus myxothiazol (Myx,10 µM). (B) AUC of A. Data are mean ± SEM (n = 10, each n represents 1 well containing 50 islets). *P < .05.
Figure 4.
Figure 4.
PHD mRNA expression (A) in male PHD1, PHD2, and PHD3 control (wt) and β-cell-specific PHD1-3 knockout (β-PHD1-3 KO) mice (n = 6, each n represents a group of 100 islets). Gene expression was corrected by an internal control gene (cyclophilin) and then expressed as percent control. Western blotting (B and C) (expression was normalized to total protein loaded on the gel and then expressed as a percent of control) (n = 6, each n represents a group of 100 islets). Immunofluorescent staining (D) of PHD1, PHD2, and PHD3 in control (wt) and β-cell-specific PHD1-3 KO (β-PHD1-3 KO) male mice (white scale bar 10 µm). Green = insulin, red = PHD1, PHD2, or PHD3, blue = nuclei (n = 4, each n represents 1 mouse pancreas, 4 mice were used per experimental group).
Figure 5.
Figure 5.
In vivo glucose homeostasis in male control (wt), β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice. (A) Blood glucose levels during an intraperitoneal glucose tolerance test (ipGTT). (B) AUC of A. (C) plasma insulin during the ipGTT in A. (D) AUC of C. (E) Insulin tolerance test (ITT). (F) AUC of E. (G) body weight. Data are mean ± SEM (n = 10, each n represents 1 mouse, 10 mice were used per experimental group). *P < .05, ***P < .001.
Figure 6.
Figure 6.
Indirect calorimetry measurement of whole-body bioenergetics in male control (wt) and β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice. (A) total activity, (B) respiratory exchange ratio (RER), (D) whole-body lipid oxidation, (E) whole-body carbohydrate oxidation. Data are mean ± SEM (n = 10, each n represents 1 mouse, 10 mice were used per experimental group). *P < .05.
Figure 7.
Figure 7.
β-Cell proliferation (A), apoptosis (B), and α- and β-cell mass (C) in male control (wt), β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO mice. (C) α-Cell mass white bars and β-cell mass black bars. Data are mean ± SEM (n = 6-9 mice, each n represents 1 mouse pancreas, each experimental group had an n between 6 and 9, from these a minimum 3000 insulin-positive cells were counted per genotype). *P < .05, **P < .01, ***P < .001. White scale bar 10 µm. Green = insulin, red = Ki67 or cleaved caspase 3, blue = nuclei.
Figure 8.
Figure 8.
Islet insulin secretion and nucleotides from male control (wt), β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO islets. (A) Islet insulin secretion in response to low glucose (2 mM; LG), high glucose (10 mM; HG), LG or HG plus 10 mM dimethylmalate (DMM) and dimethyl α-ketoglutarate (DMαKG), and LG or HG plus 30 mM potassium chloride (KCl) and 200 µM diazoxide (D). Mean ± SEM (n = 24-43 groups of 10 islets per treatment per genotype, islets were obtained from 5 to 7 mice per genotype). *P < .05 LG control vs LG PHD KO, #P < .05 HG control vs HG PHD KO, xP < .05 LG plus KCl plus diazoxide control vs LG plus KCl plus diazoxide PHD KO, °P < .05 HG plus KCl plus diazoxide control vs HG plus KCl plus diazoxide PHD KO, +P < .05 LG plus DMM plus DMαKG control vs LG plus DMM plus DMαKG PHD KO, •P < .05, •••P < .001 HG plus DMM plus DMαKG control vs HG plus DMM plus DMαKG PHD KO. (B) Islet ATP/ADP ratio (C) and NADPH/NADP+ ratio from male control (wt), β-PHD1 KO, β-PHD2 KO, and β-PHD3 KO islets. Low glucose (LG, 2 mM); high glucose (HG, 10 mM)(n = 10-14 groups of 25 islets per treatment per genotype, islets were obtained from 4-6 mice per genotype). *P < .05 HG wt vs HG PHD KO islets. (D) Gene expression from male islets (n = 8 groups of 100 islets per treatment per genotype, islets were obtained from 6 or 7 mice per genotype). Gene expression was corrected by an internal control (cyclophilin) and then expressed as percent control. Data are mean ± SEM.
Figure 9.
Figure 9.
PHD1 and PHD3 regulated insulin secretion. The loss of PHD1 and PHD3 is associated with reduced GLUT2, PKM2, and ACC2 and the elevation of PDK1. The loss of GLUT2 and PKM2 lowers glycolysis, and the increased PDK1 inhibits pyruvate dehydrogenase (PDH), which reduces glucose oxidation, ATP/ADP, and insulin secretion. The loss of ACC2 can reduce malonyl CoA levels allowing CPT1 mediated shunting of anaplerotic substrates to β-oxidation. This would lower the β-cells ability to generate NADPH/NADP+ leading to an inhibition of insulin secretion.
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