Hepatic acetyl CoA links adipose tissue inflammation to hepatic insulin resistance and type 2 diabetes.

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulin's ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.

Aifm2, a NADH Oxidase, Supports Robust Glycolysis and Is Required for Cold- and Diet-Induced Thermogenesis.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or ß-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

Transcriptional regulation of hepatic lipogenesis.

Fatty acid and fat synthesis in the liver is a highly regulated metabolic pathway that is important for very low-density lipoprotein (VLDL) production and thus energy distribution to other tissues. Having common features at their promoter regions, lipogenic genes are coordinately regulated at the transcriptional level. Transcription factors, such as upstream stimulatory factors (USFs), sterol regulatory element-binding protein 1C (SREBP1C), liver X receptors (LXRs) and carbohydrate-responsive element-binding protein (ChREBP) have crucial roles in this process. Recently, insights have been gained into the signalling pathways that regulate these transcription factors. After feeding, high blood glucose and insulin levels activate lipogenic genes through several pathways, including the DNA-dependent protein kinase (DNA-PK), atypical protein kinase C (aPKC) and AKT-mTOR pathways. These pathways control the post-translational modifications of transcription factors and co-regulators, such as phosphorylation, acetylation or ubiquitylation, that affect their function, stability and/or localization. Dysregulation of lipogenesis can contribute to hepatosteatosis, which is associated with obesity and insulin resistance.

A role of DNA-PK for the metabolic gene regulation in response to insulin.

Fatty acid synthase (FAS) is a central enzyme in lipogenesis and transcriptionally activated in response to feeding and insulin signaling. The transcription factor USF is required for the activation of FAS transcription, and we show here that USF phosphorylation by DNA-PK, which is dephosphorylated by PP1 in response to feeding, triggers a switch-like mechanism. Under fasting conditions, USF-1 is deacetylated by HDAC9, causing promoter inactivation. In contrast, feeding induces the recruitment of DNA-PK to USF-1 and its phosphorylation, which then allows recruitment of P/CAF, resulting in USF-1 acetylation and FAS promoter activation. DNA break/repair components associated with USF induce transient DNA breaks during FAS activation. In DNA-PK-deficient SCID mice, feeding-induced USF-1 phosphorylation/acetylation, DNA breaks, and FAS activation leading to lipogenesis are impaired, resulting in decreased triglyceride levels. Our study demonstrates that a kinase central to the DNA damage response mediates metabolic gene activation.

Cold-inducible Zfp516 activates UCP1 transcription to promote browning of white fat and development of brown fat.

Uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis and, upon cold exposure, is induced in brown adipose tissue (BAT) and subcutaneous white adipose tissue (iWAT). Here, by high-throughput screening using the UCP1 promoter, we identify Zfp516 as a transcriptional activator of UCP1 as well as PGC1α, thereby promoting a BAT program. Zfp516 itself is induced by cold and sympathetic stimulation through the cAMP-CREB/ATF2 pathway. Zfp516 directly binds to the proximal region of the UCP1 promoter, not to the enhancer region where other transcription factors bind, and interacts with PRDM16 to activate the UCP1 promoter. Although ablation of Zfp516 causes embryonic lethality, knockout embryos still show drastically reduced BAT mass. Overexpression of Zfp516 in adipose tissue promotes browning of iWAT even at room temperature, increasing body temperature and energy expenditure and preventing diet-induced obesity. Zfp516 may represent a future target for obesity therapeutics.

Phosphorylation and recruitment of BAF60c in chromatin remodeling for lipogenesis in response to insulin.

Fatty acid and triglyceride synthesis is induced in response to feeding and insulin. This lipogenic induction involves coordinate transcriptional activation of lipogenic enzymes, including fatty acid synthase and glycerol-3-phosphate acyltransferase. We recently reported the importance of USF-1 phosphorylation and subsequent acetylation in insulin-induced lipogenic gene activation. Here, we show that Brg1/Brm-associated factor (BAF) 60c is a specific chromatin remodeling component for lipogenic gene transcription in liver. In response to insulin, BAF60c is phosphorylated at S247 by atypical PKCζ/λ, which causes translocation of BAF60c to the nucleus and allows a direct interaction of BAF60c with USF-1 that is phosphorylated by DNA-PK and acetylated by P/CAF. Thus, BAF60c is recruited to form the lipoBAF complex to remodel chromatin structure and to activate lipogenic genes. Consequently, BAF60c promotes lipogenesis in vivo and increases triglyceride levels, demonstrating its role in metabolic adaption to activate the lipogenic program in response to feeding and insulin.

Epigenetic dynamics of the thermogenic gene program of adipocytes.

Brown adipose tissue (BAT) is a metabolically beneficial organ capable of burning fat by dissipating chemical energy into heat, thereby increasing energy expenditure. Moreover, subcutaneous white adipose tissue can undergo so-called browning/beiging. The recent recognition of the presence of brown or beige adipocytes in human adults has attracted much attention to elucidate the molecular mechanism underlying the thermogenic adipose program. Many key transcriptional regulators critical for the thermogenic gene program centering on activating the UCP1 promoter, have been discovered. Thermogenic gene expression in brown adipocytes rely on co-ordinated actions of a multitude of transcription factors, including EBF2, PPARγ, Zfp516 and Zc3h10. These transcription factors probably integrate into a cohesive network for BAT gene program. Moreover, these transcription factors recruit epigenetic factors, such as LSD1 and MLL3/4, for specific histone signatures to establish the favorable chromatin landscape. In this review, we discuss advances made in understanding the molecular mechanism underlying the thermogenic gene program, particularly epigenetic regulation.

Overexpression of Pref-1 in pancreatic islet ß-cells in mice causes hyperinsulinemia with increased islet mass and insulin secretion.

Preadipocyte factor-1 (Pref-1) is made as a transmembrane protein containing EGF-repeats at the extracellular domain that can be cleaved to generate a biologically active soluble form. Pref-1 is found in islet ß-cells and its level has been reported to increase in neonatal rat islets upon growth hormone treatment. We found here that Pref-1 can promote growth of pancreatic tumor derived AR42J cells. To examine Pref-1 function in pancreatic islets in vivo, we generated transgenic mouse lines overexpressing the Pref-1/hFc in islet ß-cells using rat insulin II promoter (RIP). These transgenic mice exhibit an increase in islet mass with higher proportion of larger islets in pancreas compared to wild-type littermates. This is in contrast to pancreas from Pref-1 null mice that show higher proportion of smaller islets. Insulin expression and insulin secretion from pancreatic islets from RIP-Pref-1/hFc transgenic mice are increased also. Thus, RIP-Pref-1/hFc transgenic mice show normal glucose levels but with higher plasma insulin levels in both fasting and fed conditions. These mice show improved glucose tolerance. Taken together, we conclude Pref-1 as a positive regulator of islet ß-cells and insulin production.

Pref-1 in brown adipose tissue: specific involvement in brown adipocyte differentiation and regulatory role of C/EBPδ.

Pref-1 (pre-adipocyte factor-1) is known to play a central role in regulating white adipocyte differentiation, but the role of Pref-1 in BAT (brown adipose tissue) has not been analysed. In the present study we found that Pref-1 expression is high in fetal BAT and declines progressively after birth. However, Pref-1-null mice showed unaltered fetal development of BAT, but exhibited signs of over-activation of BAT thermogenesis in the post-natal period. In C/EBP (CCAAT/enhancer-binding protein) α-null mice, a rodent model of impaired fetal BAT differentiation, Pref-1 was dramatically overexpressed, in association with reduced expression of the Ucp1 (uncoupling protein 1) gene, a BAT-specific marker of thermogenic differentiation. In brown adipocyte cell culture models, Pref-1 was mostly expressed in pre-adipocytes and declined with brown adipocyte differentiation. The transcription factor C/EBPδ activated the Pref-1 gene transcription in brown adipocytes, through binding to the proximal promoter region. Accordingly, siRNA (small interfering RNA)-induced C/EBPδ knockdown led to reduced Pref-1 gene expression. This effect is consistent with the observed overexpression of C/EBPδ in C/EBPα-null BAT and high expression of C/EBPδ in brown pre-adipocytes. Dexamethasone treatment of brown pre-adipocytes suppressed Pref-1 down-regulation occurring throughout the brown adipocyte differentiation process, increased the expression of C/EBPδ and strongly impaired expression of the thermogenic markers UCP1 and PGC-1α [PPARγ (peroxisome-proliferator-activated receptor γ) co-activator-α]. However, it did not alter normal fat accumulation or expression of non-BAT-specific genes. Collectively, these results specifically implicate Pref-1 in controlling the thermogenic gene expression program in BAT, and identify C/EBPδ as a novel transcriptional regulator of Pref-1 gene expression that may be related to the specific role of glucocorticoids in BAT differentiation.

AIFM2 Is Required for High-Intensity Aerobic Exercise in Promoting Glucose Utilization.

Skeletal muscle is a major regulator of glycemic control at rest, and glucose utilization increases drastically during exercise. Sustaining a high glucose utilization via glycolysis requires efficient replenishment of NAD+ in the cytosol. Apoptosis-inducing mitochondrion-associated factor 2 (AIFM2) was previously shown to be a NADH oxidoreductase domain-containing flavoprotein that promotes glycolysis for diet and cold-induced thermogenesis. Here, we find that AIFM2 is selectively and highly induced in glycolytic extensor digitorum longus (EDL) muscle during exercise. Overexpression (OE) of AIFM2 in myotubes is sufficient to elevate the NAD+-to-NADH ratio, increasing the glycolytic rate. Thus, OE of AIFM2 in skeletal muscle greatly increases exercise capacity, with increased glucose utilization. Conversely, muscle-specific Aifm2 depletion via in vivo transfection of hairpins against Aifm2 or tamoxifen-inducible haploinsufficiency of Aifm2 in muscles decreases exercise capacity and glucose utilization in mice. Moreover, muscle-specific introduction of NDE1, Saccharomyces cerevisiae external NADH dehydrogenase (NDE), ameliorates impairment in glucose utilization and exercise intolerance of the muscle-specific Aifm2 haploinsufficient mice. Together, we show a novel role for AIFM2 as a critical metabolic regulator for efficient utilization of glucose in glycolytic EDL muscles.

Lifespan prolonging mechanisms and insulin upregulation without fat accumulation in long-lived reproductives of a higher termite.

Kings and queens of eusocial termites can live for decades, while queens sustain a nearly maximal fertility. To investigate the molecular mechanisms underlying their long lifespan, we carried out transcriptomics, lipidomics and metabolomics in Macrotermes natalensis on sterile short-lived workers, long-lived kings and five stages spanning twenty years of adult queen maturation. Reproductives share gene expression differences from workers in agreement with a reduction of several aging-related processes, involving upregulation of DNA damage repair and mitochondrial functions. Anti-oxidant gene expression is downregulated, while peroxidability of membranes in queens decreases. Against expectations, we observed an upregulated gene expression in fat bodies of reproductives of several components of the IIS pathway, including an insulin-like peptide, Ilp9. This pattern does not lead to deleterious fat storage in physogastric queens, while simple sugars dominate in their hemolymph and large amounts of resources are allocated towards oogenesis. Our findings support the notion that all processes causing aging need to be addressed simultaneously in order to prevent it.

Signaling Pathways Regulating Thermogenesis.

Obesity, an excess accumulation of white adipose tissue (WAT), has become a global epidemic and is associated with complex diseases, such as type 2 diabetes and cardiovascular diseases. Presently, there are no safe and effective therapeutic agents to treat obesity. In contrast to white adipocytes that store energy as triglycerides in unilocular lipid droplet, brown and brown-like or beige adipocytes utilize fatty acids (FAs) and glucose at a high rate mainly by uncoupling protein 1 (UCP1) action to uncouple mitochondrial proton gradient from ATP synthesis, dissipating energy as heat. Recent studies on the presence of brown or brown-like adipocytes in adult humans have revealed their potential as therapeutic targets in combating obesity. Classically, the main signaling pathway known to activate thermogenesis in adipocytes is ß3-adrenergic signaling, which is activated by norepinephrine in response to cold, leading to activation of the thermogenic program and browning. In addition to the ß3-adrenergic signaling, numerous other hormones and secreted factors have been reported to affect thermogenesis. In this review, we discuss several major pathways, ß3-adrenergic, insulin/IGF1, thyroid hormone and TGFß family, which regulate thermogenesis and browning of WAT.

Corrigendum: Signaling Pathways Regulating Thermogenesis.

[This corrects the article DOI: 10.3389/fendo.2021.595020.].

Aging-dependent regulatory cells emerge in subcutaneous fat to inhibit adipogenesis.

Adipose tissue mass and adiposity change throughout the lifespan. During aging, while visceral adipose tissue (VAT) tends to increase, peripheral subcutaneous adipose tissue (SAT) decreases significantly. Unlike VAT, which is linked to metabolic diseases, including type 2 diabetes, SAT has beneficial effects. However, the molecular details behind the aging-associated loss of SAT remain unclear. Here, by comparing scRNA-seq of total stromal vascular cells of SAT from young and aging mice, we identify an aging-dependent regulatory cell (ARC) population that emerges only in SAT of aged mice and humans. ARCs express adipose progenitor markers but lack adipogenic capacity; they secrete high levels of pro-inflammatory chemokines, including Ccl6, to inhibit proliferation and differentiation of neighboring adipose precursors. We also found Pu.1 to be a driving factor for ARC development. We identify an ARC population and its capacity to inhibit differentiation of neighboring adipose precursors, correlating with aging-associated loss of SAT.

Characterization of desnutrin functional domains: critical residues for triacylglycerol hydrolysis in cultured cells.

Murine desnutrin/human ATGL is a triacylglycerol (TAG) hydrolase with a predicted catalytic dyad within an alpha-beta hydrolase fold in the N-terminal region. In humans, mutations resulting in C-terminal truncation cause neutral lipid storage disease with myopathy. To identify critical functional domains, we measured TAG breakdown in cultured cells by mutated or truncated desnutrin. In vitro, C-terminally truncated desnutrin displayed an even higher apparent V(max) than the full-length form without changes in K(m), which may be explained by our finding of an interaction between the C- and N-terminal domains. In live cells, however, C-terminally truncated adenoviral desnutrin had lower TAG hydrolase activity. We investigated a role for the phosphorylation of C-terminal S406 and S430 residues but found that these were not necessary for TAG breakdown or lipid droplet localization in cells. The predicted N-terminal active sites, S47 and D166, were both critical for TAG hydrolysis in live cells and in vitro. We also identified two overlapping N-terminal motifs that predict lipid substrate binding domains, a glycine-rich motif (underlined) and an amphipathic alpha-helix (bold) within amino acid residues 10-24 (ISFAGCGFLGVYHIG). G14, F17, L18, and V20, but not G16 and G19, were important for TAG hydrolysis, suggesting a potential role for the amphipathic alpha-helix in TAG binding. This study identifies for the first time critical sites in the N-terminal region of desnutrin and reveals the requirement of the C-terminal region for TAG hydrolysis in cultured cells.

Pref-1 (preadipocyte factor 1) activates the MEK/extracellular signal-regulated kinase pathway to inhibit adipocyte differentiation.

Preadipocyte factor 1 (Pref-1) is found in preadipocytes but is absent in adipocytes. Pref-1 is made as a transmembrane protein but is cleaved to generate a biologically active soluble form. Although Pref-1 inhibition of adipogenesis has been well studied in vitro and in vivo, the signaling pathway for Pref-1 is not known. Here, by using purified soluble Pref-1 in Pref-1 null mouse embryo fibroblasts (MEF), we show that Pref-1 increases MEK/extracellular signal-regulated kinase (ERK) phosphorylation in a time- and dose-dependent manner. Compared to wild-type MEF, differentiation of Pref-1 null MEF into adipocytes is enhanced, as judged by lipid accumulation and adipocyte marker expression. Both wild-type and Pref-1 null MEF show a transient burst of ERK phosphorylation upon addition of adipogenic agents. Wild-type MEF show a significant, albeit lower, second increase in ERK phosphorylation peaking at day 2. This ERK phosphorylation, corresponding to Pref-1 abundance, is absent during differentiation of Pref-1 null MEF. Prevention of this second increase in ERK1/2 phosphorylation in wild-type MEF by the MEK inhibitor PD98059 or by transient depletion of ERK1/2 via small interfering RNA-enhanced adipocyte differentiation. Furthermore, treatment of Pref-1 null MEF with Pref-1 restores this ERK phosphorylation, resulting in inhibition of adipocyte differentiation primarily by preventing peroxisome proliferator-activated receptor gamma2 induction. However, in the presence of PD98059 or depletion of ERK1/2, exogenous Pref-1 cannot inhibit adipocyte differentiation in Pref-1 null MEF. We conclude that Pref-1 activates MEK/ERK signaling, which is required for Pref-1 inhibition of adipogenesis.

The Role of Phospholipase A(2)-derived Mediators in Obesity.

Obesity has become an epidemic and its prevalence is increasing exponentially. A great deal of focus has been given to understanding the molecular processes that regulate obesity. The characterization of phospholipase A(2)s, especially adipose-specific PLA(2), have lead to a proposed role of their downstream products in the progression of obesity and obesity related disorders. This review summarizes recent developments in the role of PLA(2) and their downstream effects in the development of metabolic disorders.

Epigenetic Regulation of Hepatic Lipogenesis: Role in Hepatosteatosis and Diabetes.

Hepatosteatosis, which is frequently associated with development of metabolic syndrome and insulin resistance, manifests when triglyceride (TG) input in the liver is greater than TG output, resulting in the excess accumulation of TG. Dysregulation of lipogenesis therefore has the potential to increase lipid accumulation in the liver, leading to insulin resistance and type 2 diabetes. Recently, efforts have been made to examine the epigenetic regulation of metabolism by histone-modifying enzymes that alter chromatin accessibility for activation or repression of transcription. For regulation of lipogenic gene transcription, various known lipogenic transcription factors, such as USF1, ChREBP, and LXR, interact with and recruit specific histone modifiers, directing specificity toward lipogenesis. Alteration or impairment of the functions of these histone modifiers can lead to dysregulation of lipogenesis and thus hepatosteatosis leading to insulin resistance and type 2 diabetes.

Histone demethylase JMJD1C is phosphorylated by mTOR to activate de novo lipogenesis.

Fatty acid and triglyceride synthesis increases greatly in response to feeding and insulin. This lipogenic induction involves coordinate transcriptional activation of various enzymes in lipogenic pathway, including fatty acid synthase and glycerol-3-phosphate acyltransferase. Here, we show that JMJD1C is a specific histone demethylase for lipogenic gene transcription in liver. In response to feeding/insulin, JMJD1C is phosphorylated at T505 by mTOR complex to allow direct interaction with USF-1 for recruitment to lipogenic promoter regions. Thus, by demethylating H3K9me2, JMJD1C alters chromatin accessibility to allow transcription. Consequently, JMJD1C promotes lipogenesis in vivo to increase hepatic and plasma triglyceride levels, showing its role in metabolic adaption for activation of the lipogenic program in response to feeding/insulin, and its contribution to development of hepatosteatosis resulting in insulin resistance.

Dot1l interacts with Zc3h10 to activate Ucp1 and other thermogenic genes.

Brown adipose tissue is a metabolically beneficial organ capable of dissipating chemical energy into heat, thereby increasing energy expenditure. Here, we identify Dot1l, the only known H3K79 methyltransferase, as an interacting partner of Zc3h10 that transcriptionally activates the Ucp1 promoter and other BAT genes. Through a direct interaction, Dot1l is recruited by Zc3h10 to the promoter regions of thermogenic genes to function as a coactivator by methylating H3K79. We also show that Dot1l is induced during brown fat cell differentiation and by cold exposure and that Dot1l and its H3K79 methyltransferase activity is required for thermogenic gene program. Furthermore, we demonstrate that Dot1l ablation in mice using Ucp1-Cre prevents activation of Ucp1 and other target genes to reduce thermogenic capacity and energy expenditure, promoting adiposity. Hence, Dot1l plays a critical role in the thermogenic program and may present as a future target for obesity therapeutics.