LncRNA H19 inhibition impairs endoplasmic reticulum-mitochondria contact in hepatic cells and augments gluconeogenesis by increasing VDAC1 levels

Abstract

Inspite of exerting independent cellular functions, the endoplasmic-reticulum (ER) and the mitochondria also physically connect at specific sites termed mitochondria-associated ER membranes (MAMs) and these sites consist of several tethering proteins that play varied roles in diverse cellular processes. However, the regulation of these tethering proteins within the cell is relatively less studied. Here, we show that several MAM proteins are significantly altered in the liver during diabetes and among these, the lncRNA, H19 regulates the levels of VDAC1. Inhibition of H19 expression using H19 specific siRNA altered VDAC1, mitochondrial Ca²⁺ and oxygen consumption rate, ATP and ROS levels and enhanced ER and mitochondria coupling in Hepa 1-6 cells. While H19 inhibition did not impact lipid accumulation, levels of gluconeogenic genes were significantly increased. JNK-phosphorylation and IRS1-Ser307-phosphorylation were increased by H19 inhibition and this was associated with abrogation of insulin-stimulated AKT (Ser-473) phosphorylation and glucose uptake in Hepa 1-6 cells. While inhibition of VDAC1 expression using siRNAs and with metformin significantly rescued the effects of H19 inhibition, VDAC1 overexpression alone exerted effects similar to H19 inhibition, suggesting that VDAC1 increase mediates the adverse effects of H19. In-vivo H19 inhibition using specific siRNAs increased hepatic VDAC1, pJNK and pIRS1 (Ser307) levels and decreased AKT (Ser-473) phosphorylation in mice. These suggest an important role of the H19-VDAC1 axis in ER-mitochondria coupling and regulation of gluconeogenesis in the liver during diabetes.

Keywords: Ca(2+); Diabetes; ER-Mitochondria communication; Gluconeogenesis; JNK; Liver; ROS; lncRNA.

Graphical abstract

Fig. 1

Expression levels of MAM proteins in livers of db/ + and db/db mice. (A) Total RNA was isolated from the livers of normal (db/+) and diabetic (db/db) mice and 1 μg RNA was reverse transcribed and subjected to qRT-PCR to assess the transcript levels of MAM proteins. 18S rRNA was used as the normalization control. Hepatic tissues of normal (db/+) and diabetic (db/db) mice were lysed and 20–40 μg lysates were run on SDS-PAGE and the levels of MFN2 (B), PACS2 (C), SIGMA1R (D), VDAC1 (E), MCU (F), FACL4 (G), GRP75 (H), MFN1 (I) and IP3R1/2/3 (J) were evaluated by Western Blot analysis. β-actin or Vinculin were used as the loading controls. Densitometric analyses of the blots are shown in the panels below. Data are means of at least four animals in each group and are presented as mean ± SEM. **p < 0.01, *p < 0.05, ns: non-significant.

Fig. 2

H19 inhibition this alters levels of MAM proteins in Hepa 1–6 cells. (A) Total RNA was isolated from normal (db/+) and diabetic (db/db) mice (n = 5) livers and 1 μg RNA was reverse transcribed and subjected to qRT-PCR to assess the transcript levels of the lncRNA H19. 18S rRNA was used as the normalization control. (B) Hepa 1–6 cells were transfected with either the scramble or H19 siRNA (0.1–20 nM) and at 48h, the relative expression level of H19 was measured by Real time PCR as in “A”. (C) Total RNA was isolated from Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM) and the transcript levels of Facl4, Mfn2, Pacs2, Vdac1, Sigma1R and Mcu were evaluated by qRT-PCR. Hepa 1–6 cells transfected as in “C” were lysed and lysates (20–40 μg) were assessed for the protein levels of VDAC1 (D), MFN2 (E), FACL4 (F), PACS2 (G) SIGMA1R (H) and MCU (I) by Western Blot analysis. β-actin or Vinculin were used as the loading controls and densitometric analyses are given in the panels below. Data are presented as mean ± SEM of at least three independent replicates. ***p < 0.0001, **p < 0.01, *p < 0.05, ns: non-significant.

Fig. 3

Inhibition of H19 induces mitochondrial dysfunction in Hepa 1–6 cells. (A) Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is represented by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. (B) Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) were evaluated for total mitochondrial (B) and cytosolic (C) Ca²⁺ levels using Rhod-2 AM (1 μM) and Fluo-3 AM (1 μM), respectively, by FACS analysis. Mitochondrial ROS levels as detected by Mitosox (5 μM) (D) and total ATP (normalized to total protein) content (E) in Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) are presented. (F) OCR of Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM) for 48 h in the presence of 2 μM oligomycin (ATP synthase inhibitor), 1.25 μM FCCP (uncoupling agent) and 2 μM rotenone and 2 μM antimycin (complex I and III inhibitors, respectively). Basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production were determined as described in the “Methods” section. (G) Mitochondria was isolated from scramble and H19 siRNA transfected cells as described in the “Methods” section. OCR in isolated mitochondria (5 μg) from scramble and H19 siRNA transfected cells was performed in the presence of ADP (4 mM), oligomycin (2.5 μg/ml), FCCP (4 μM) and antimycin A (4 μM). (H) Total RNA was isolated from Hepa 1–6 cells transfected with either the scramble or H19 siRNA as in “A”, reverse transcribed and the transcript levels of genes specific to Complexes I, III, IV and V of the mitochondrial electron transport chain were evaluated by RT-PCR. 18S rRNA was taken as the normalization control. Scramble and H19 siRNA (5 nM, 48h) transfected cells were evaluated for mitochondrial membrane potential and integrity using JCI (I) and for mitochondrial number using mitochondrial short d-loop specific primers by qRT-PCR. β-2 microglobulin was used as normalization control (J). (K) Glycolytic rates in scramble and H19 siRNA transfected cells were assessed in the presence of glucose (10 mM), oligomycin (2 μM) and 2-Deoxy-d-glucose (100 mM). Glycolysis, glycolytic capacity and glycolytic reserve as determined are presented. All experiments were done thrice and data are presented are means ± SEM.**p < 0.01, *p < 0.05, ns: non-significant. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Effects of H19 inhibition on genes of fatty acid and cholesterol metabolism and gluconeogenesis in Hepa 1–6 cells. (A) Hepa 1–6 cells were transfected with either the scramble or H19 siRNA (5 nM, 48 h) and total cellular lipid content was evaluated using Bodipy. Total RNA isolated from Hepa 1–6 cells transfected as in “A” was reverse transcribed and assessed for the relative transcript expression of genes of fatty acid and cholesterol metabolism, namely Fasn, Dgat2, Hmgcr, Hmgcs, Acadl, Hadha (B) and gluconeogenesis, namely G6Pase, Pcx, Fbp1 and Pck1 (C). Cells transfected as in “A” were lysed and lysates (20–40 μg) were resolved on SDS-PAGE, transferred to nitrocellulose membranes and probed with antibodies against G6Pase (D), FBP1 (E), PCK1 (F) and PCx (G). β-actin or Vinculin were used as the loading controls and densitometric analyses are given in the panels below. All experiments were done at least three times and data are presented as means ± SEM.**p < 0.01, *p < 0.05, ns: non-significant.

Fig. 5

Overexpression of VDAC1 increases gluconeogenic genes and impairs mitochondrial function in Hepa 1–6 cells. Hepa 1–6 cells were transfected with either the empty vector or the VDAC1 overexpression clone (0.5 and 1 μg). At 48h, cells were lysed and lysates (20 μg) were probed for the levels of VDAC1 (A). (B) Hepa 1–6 cells were transfected as in “A” and on completion of incubation, cells were washed and labelled with annexin V and Propidium Iodide (PE) and analyzed for apoptosis by flow cytometry. Annexin V (FITC-A) positive cells are represented. Hepa 1–6 cells transfected with either the empty vector or the VDAC1 overexpression clone (0.5 and 1 μg) were lysed and lysates (20–40 μg) were probed to evaluate G6Pase (C), PCx (D), PCK1 (E) and FBP1 (F) protein levels by Western Blot analysis. β-actin or Vinculin were used as loading controls. Representative blots are shown and densitometric analyses (n = 3) are given in the panels below. (G) Hepa 1–6 cells transfected with either the empty vector or the VDAC1 clone (1 μg) were incubated in the presence of sodium lactate (20 mM) and sodium pyruvate (2 mM) and glucose output into the media was measured. Data was normalized to the protein content. Cells transfected as in “C” were analyzed for mitochondrial Ca²⁺ levels using Rhod-2 AM (1 μM) (H), cytosolic Ca²⁺ levels using Fluo-3 AM (1 μM) (I) and mitochondrial ROS (J). (K) Representative OCR of Hepa 1–6 cells after transfection with either the empty vector or the VDAC1 clone (1 μg) for 48h in the presence of 2 μM oligomycin, 1.25 μM FCCP and 2 μM rotenone and 2 μM antimycin and quantitative analysis of basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production. Experiments were performed thrice and data are presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05, ns: non-significant.

Fig. 6

H19 inhibition or upregulation of VDAC1 increases JNK phosphorylation and abrogates insulin signaling in Hepa 1–6 cells. Hepa 1–6 cells transfected with either the scramble or H19 siRNA (5 nM, 48h) were lysed and protein lysates were evaluated for the protein levels of p-JNK1/2/JNK1/2 (A), p-p38/p38 (B), p-ERK1/2/ERK1/2 (C) and p-IRS1(Ser307)/IRS1 (D). (E) Cells transfected as “A” were incubated in the absence or presence of insulin (10 nM, 30 min). On termination of incubation, cells were lysed and probed for the levels of p-AKT and AKT by Western Blot analysis. Hepa 1–6 cells were transfected with either the empty vector or the VDAC1 overexpression clone (0.5 and 1 μg). At 48h, cells were lysed and lysates (20–40 μg) were probed for the protein levels p-JNK1/2/JNK1/2 (F) and p-IRS1(Ser307)/IRS1(G). (H) Cells transfected as “F” were incubated in the absence or presence of insulin (10 nM, 30 min), lysed and probed for the protein levels of p-AKT and AKT. HSC70, β-actin or vinculin were used as loading controls. Representative densitometric blots are given and the densitometric analyses are given below. (I) Hepa 1–6 cells transfected with either the empty vector or the VDAC1 clone (1 μg) were incubated in the absence or presence of insulin (10 nM, 30 min) and glucose levels were measured using 2-NBDG as described in the “Methods” section. Experiments were done three times and data are presented as means ± SEM.***p < 0.001, **p < 0.01, *p < 0.05, ns: non-significant.

Fig. 7

VDAC1 inhibition rescues H19 siRNA mediated effects in Hepa1-6 cells. (A) Hepa 1–6 cells were transfected with either the scramble or VDAC1 siRNA (5–20 nM) for 48h, lysed and probed for the levels of VDAC1 by Western Blot analysis. Hepa 1–6 cells were co-transfected with H19 siRNA (5 nM) and VDAC1 siRNA (5 nM) and after 48h, the levels of PCK1 (B), G6Pase (C), FBP1 (D), p-JNK1/2/JNK1/2 (E) and p-IRS1(Ser307)/IRS1 (F) were evaluated by Western Blot analyses using specific antibodies. Vinculin or HSC70 was used as the loading control. Representative blots are shown and densitometric analyses of three blots are given in the panels below. (G) Hepa 1–6 cells transfected as in “A” were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is depicted by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. (H) OCR in the presence of 2 μM oligomycin (ATP synthase inhibitor), 1.25 μM FCCP (uncoupling agent) and 2 μM rotenone and 2 μM antimycin (complex I and III inhibitors, respectively) of Hepa 1–6 cells transfected as in “A”. Basal respiration, maximal respiration, proton leak, spare respiratory capacity and ATP production were determined as described in the “Materials and Methods” section. Experiments were done three times and data presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 8

Metformin rescues H19 siRNA mediated effects in Hepa1-6 cells. (A) Hepa 1–6 cells were incubated either without (Control) or with metformin (20–1000 μM) for 42h; after competition of incubation, total RNA was isolated and subjected to qRT-PCR using VDAC1 (A) or H19 (B) specific primers. 18S rRNA was used as the normalization control. Hepa 1–6 cells were transfected with either scramble or H19 siRNA (5 nM) in the presence or absence of metformin (1000 μM) for 42h; after competition of incubation, total RNA was isolated and subjected to qRT-PCR to determine the transcript levels of Fbp1 (C), G6Pase (D), Pcx (E) and Pck1(F). 18S rRNA was used as the normalization control. (G) Hepa 1–6 cells transfected as in “C” were subjected to in-situ proximity ligation assay using anti-VDAC1 and anti-IP3R antibodies. Representative confocal images of the cells are shown and the interaction between IP3R and VDAC1 is indicated by red dots. Nuclei are indicated by blue staining using DAPI. Quantification of IP3R-VDAC1 interactions is represented by red blobs per nucleus. Scale: 20 μm; Magnification: 60X. At least five images were captured per incubation. Each data point is the mean of three independent experiments and is presented as means ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05. ns: non-significant (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

In-vivo H19 antagonism upregulates VDAC1 and promotes JNK and IRS1 (Ser 307) phosphorylation. As described in a previous study, mice were injected (i.v) with either the scramble or H19 siRNA (3 mg/kg body weight). Total RNA was isolated from the liver tissues, reverse transcribed and the levels of H19 (A) and Vdac1 (B) were analyzed by qRT-PCR. 18S rRNA was taken as the normalization control. (C) Mice liver tissues were homogenised and the levels of VDAC1 (C), pJNK1/2/JNK1/2 (D), pIRS1(Ser307)/IRS1(E) and pAKT/AKT (F) were assessed by Western Blot analysis using specific antibodies. Vinculin, HSC70 or β-actin were used as loading controls. Each data point is the mean of values from at least three animals and are presented as means ± SEM.***p < 0.001, *p < 0.05.