1. Volume 26, Issue 8, Pages 2212-2226.e7 (February 2019)
Loss of Hepatic Oscillatory Fed microRNAs Abrogates Refed Transition and Causes Liver Dysfunctions 
Babukrishna Maniyadath, Tandrika Chattopadhyay, Srikant Verma, Sujata Kumari, Prineeta Kulkarni, Kushal Banerjee, Asmitha Lazarus, Saurabh S. Kokane, Trupti Shetty, Krishanpal Anamika, Ullas Kolthur-Seetharam 
Cell Reports 
Volume 26, Issue 8, Pages e7 (February 2019)
DOI: /j.celrep
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2. Cell Reports 2019 26, 2212-2226.e7DOI: (10.1016/j.celrep.2019.01.087)
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3. Figure 1 miRnome of Fed Mice Liver
(A) Hierarchical clustering and heatmap of microRNAs expressed in ad libitum fed mice liver, normalized to the total read count and expressed on a log2 scale (n = 2). Global color scale shows maximum (red) and minimum (green) microRNA expression levels.
(B) Number of annotated and novel microRNAs.
(C) Loci mapping of microRNAs to intergenic, exonic, and intronic regions.
(D) qRT-PCR quantification of microRNAs, which showed high, moderate, and low counts or expression in the NGS (n = 4–7). Data are represented as mean ± SEM.
(E) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways related to the validated targets of microRNAs expressed in the liver.
See also Data S1 and S2.
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4. Figure 2 MicroRNAs Expressed in the Liver Regulate PGC1α and SIRT1
(A) Schematic representation of human PGC1a 3′ UTR with corresponding target sites for microRNAs.
(B) Luciferase assay in HEK293T cells expressing indicated microRNAs and PGC1a 3′ UTR (n = 4). Representative result from n = 4, N = 2.
(C) Schematic representation of human SIRT1 3′ UTR with corresponding target sites for microRNAs.
(D) Luciferase assay in HEK293T cells expressing microRNAs and human SIRT1 3′ UTR constructs (n = 6).
(E and F) Immunoblot of endogenous SIRT1 and PGC1α from HEK293T cells overexpressing indicated microRNAs (E) and HEK293 cells treated with LNA-based anti-microRNAs, as indicated (F) (n = 2, N = 3).
(G) Quantification of transcripts downstream to SIRT1-PGC1α from HEK293 cells overexpressing microRNAs, as indicated (n = 4).
(H) PGC1α/PPRE-dependent transcription upon overexpression of indicated microRNAs as measured by PPRE-luciferase assay (n = 6).
Data are represented as mean ± SEM and analyzed by the Student’s t test and ANOVA. A value of p ≤ 0.05 was considered statistically significant. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
See also Figure S1, Table S1, and Data S3 and S4.
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5. Figure 3
MicroRNAs Exert Network-Level Control over Mitochondrial Functions
(A) Schematic indicating predicted genes co-targeted by microRNAs.
(B) Heatmap indicating changes in expression of endogenous proteins under microRNA overexpression; expressed as fold change relative to controls in respective immunoblots (n = 2, N = 3).
(C–E) OCR in HEK293 cells overexpressing microRNAs. Representative graph of basal OCR and upon oligomycin, carbonyl-cyanide-p-trifluromethoxyphenylhydrazone (FCCP), and rotenone treatments (C) (n = 3–4), comparative analyses of basal OCR (D), and ATP production (E) (n = 9–12).
(F–H) OCR in HEK293 cells treated with LNA-based anti-microRNAs. Representative graph of basal OCR and upon oligomycin, FCCP, and rotenone treatments (F) (n = 3–4), and comparative analyses of basal OCR (G) and ATP production (H) (n = 9–12).
(I and J) OCR in HEK293 cells overexpressing miR-221/222, PGC1α, and SIRT3, as indicated, and comparative analyses of basal respiration (I) and ATP production (J) (n = 3–4, N = 2).
Data are represented as mean ± SEM and analyzed by the Student’s t test. A value of p ≤ 0.05 was considered statistically significant. #, ∗p ≤ 0.05; ##, ∗∗p ≤ 0.01; ∗∗∗p ≤
See also Figures S2 and S3 and Table S2.
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6. Figure 4
Dynamic Alterations in Hepatic Mature MicroRNAs and RISC Association of MicroRNA-mRNAs during Fed-Fast-Refed Transitions
(A) Quantification of hepatic mature microRNAs from mice fasted for 24 h and fed or refed ad libitum, as indicated (n = 8).
(B and C) Levels of indicated RISC-associated microRNAs (B) and fed- and fasted-responsive target mRNAs (C) from fed-fast-refed livers; time points as in (A) (n = 6).
(D) Schematic showing dynamic switch in RISC-associated fed-fasted microRNA-mRNA pairs in the liver during physiological transitions.
(E) Quantification of abundant mature microRNAs, selected from the NGS dataset, from fed-fast-refed livers (n = 4–7).
(F) Heatmap of fed- and fasted-responsive microRNA levels from quantifications in (E) and Figure S4F, and pathways regulated by their predicted targets.
(G) Levels of indicated RISC-associated microRNAs from fed-fast-refed livers (n = 6).
Data are represented as mean ± SEM and analyzed by the Student’s t test and ANOVA. A value of p ≤ 0.05 was considered statistically significant. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
See also Figure S4.
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7. Figure 5
Pri-/Pre-microRNA Expression Constitutes an Anticipatory Mechanism
(A and B) Quantification of pri-microRNA (n = 8) levels (A) and pre-microRNA (n = 8) levels (B) from fed-fast-refed livers; time points as indicated.
(C) Quantification of indicated hepatic pri- and pre-microRNAs from livers of control and TRF mice at ZT5, ZT8, and ZT11 (n = 3–6).
(D and E) Quantification of pri-microRNA (n = 7–8) levels (D) and pre-microRNA (n = 7–8) levels (E) from hepatocytes treated with high glucose (25 mM), low glucose (5 mM), no glucose (NG), or NG + glucagon (100 nM) as indicated.
Data are represented as mean ± SEM and analyzed by the Student’s t test and ANOVA. A value of p ≤ 0.05 was considered statistically significant. #, ∗p ≤ 0.05; ##, ∗∗p ≤ 0.01; ###, ∗∗∗p ≤ (∗p control versus TRF; #p ZT5 versus ZT8 and ZT8 versus ZT11).
See also Figure S5.
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8. Figure 6
Fed MicroRNAs Regulate Hepatic Fat Utilization and Whole-Body Energetics
(A) Schematic illustrating the design of the microRNA-sponge construct and experimental paradigm to scavenge hepatic fed microRNAs for indicated assays.
(B–D) Palmitate-induced mitochondrial OCR (FAO) in primary hepatocytes isolated from control and microRNA-sponge-injected mice (B and C) (n = 3–4, N = 2) and in HepG2 cells overexpressing indicated microRNAs (D) (n = 3, N = 2).
(E) Respiratory exchange ratio of control and microRNA-sponge-injected mice over a 24-h normal light-dark cycle (n = 4).
Data are represented as mean ± SEM and analyzed by the Student’s t test and ANOVA. A value of p ≤ 0.05 was considered statistically significant. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
See also Figure S6.
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9. Figure 7
Loss of Hepatic Fed MicroRNAs Affects Liver Physiology and Impairs Refed Transition
(A) Schematic illustrating the experimental paradigm to scavenge hepatic fed microRNAs for assays, as indicated.
(B) Blood glucose levels upon 6 hours of fasting (n = 6).
(C) Pyruvate tolerance test.
(D) Area under the curve for (C) (n = 4–5, N = 2).
(E–G) Hepatic gluconeogenic potential (n = 6). Serum-to-liver glucose levels (E). Glucose levels in starved livers of control and microRNA-sponge-injected mice (F). Pyruvate levels in starved livers of control and microRNA-sponge-injected mice (G).
(H) Blood glucose levels post-glucose infusion in fasted mice, as indicated (n = 5–6).
(I) Immunoblots of liver lysates, from mice fasted for 6 hours or after 2 hours of glucose infusion, showing anabolic and catabolic marker proteins or their phosphorylations, as indicated (n = 2, N = 2).
(J) Quantification of endogenous transcripts in liver from mice fasted for 6 h or after 2 h of glucose infusion, as indicated (n = 3, N = 2).
Data are represented as mean ± SEM and analyzed by the Student’s t test. A value of p ≤ 0.05 was considered statistically significant. ∗p ≤ 0.05; ∗∗p ≤ 0.01; ∗∗∗p ≤
See also Figure S7.
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