Adipocyte lipin 1 is positively associated with metabolic health in humans and regulates systemic metabolism in mice.

Dysfunctional adipose tissue is believed to promote the development of hepatic steatosis and systemic insulin resistance, but many of the mechanisms involved are still unclear. Lipin 1 catalyzes the conversion of phosphatidic acid to diacylglycerol (DAG), the penultimate step of triglyceride synthesis, which is essential for lipid storage. Herein we found that adipose tissue LPIN1 expression is decreased in people with obesity compared to lean subjects and low LPIN1 expression correlated with multi-tissue insulin resistance and increased rates of hepatic de novo lipogenesis. Comprehensive metabolic and multi-omic phenotyping demonstrated that adipocyte-specific Lpin1-/- mice had a metabolically-unhealthy phenotype, including liver and skeletal muscle insulin resistance, hepatic steatosis, increased hepatic de novo lipogenesis, and transcriptomic signatures of nonalcoholic steatohepatitis that was exacerbated by high-fat diets. We conclude that adipocyte lipin 1-mediated lipid storage is vital for preserving adipose tissue and systemic metabolic health and its loss predisposes mice to nonalcoholic steatohepatitis.

Mitochondrial Pyruvate Carrier Inhibition Attenuates Hepatic Stellate Cell Activation and Liver Injury in a Mouse Model of Metabolic Dysfunction-associated Steatotic Liver Disease.

Hepatic stellate cells (HSC) are non-parenchymal liver cells that produce extracellular matrix comprising fibrotic lesions in chronic liver diseases. Prior work demonstrated that mitochondrial pyruvate carrier (MPC) inhibitors suppress HSC activation and fibrosis in a mouse model of metabolic dysfunction-associated steatohepatitis (MASH). In the present study, pharmacologic or genetic inhibition of the MPC in HSC decreased expression of markers of activation in vitro. MPC knockdown also reduced the abundance of several intermediates of the TCA cycle, and diminished α-ketoglutarate played a key role in attenuating HSC activation by suppressing hypoxia inducible factor-1α signaling. On high fat diets, mice with HSC-specific MPC deletion exhibited reduced circulating transaminases, numbers of HSC, and hepatic expression of markers of HSC activation and inflammation compared to wild-type mice. These data suggest that MPC inhibition modulates HSC metabolism to attenuate activation and illuminate mechanisms by which MPC inhibitors could prove therapeutically beneficial for treating MASH.

Mitochondrial pyruvate carrier inhibition initiates metabolic crosstalk to stimulate branched chain amino acid catabolism.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) has emerged as a therapeutic target for treating insulin resistance, type 2 diabetes, and nonalcoholic steatohepatitis (NASH). We evaluated whether MPC inhibitors (MPCi) might correct impairments in branched chain amino acid (BCAA) catabolism, which are predictive of developing diabetes and NASH. METHODS: Circulating BCAA concentrations were measured in people with NASH and type 2 diabetes, who participated in a recent randomized, placebo-controlled Phase IIB clinical trial to test the efficacy and safety of the MPCi MSDC-0602K (EMMINENCE; NCT02784444). In this 52-week trial, patients were randomly assigned to placebo (n = 94) or 250 mg MSDC-0602K (n = 101). Human hepatoma cell lines and mouse primary hepatocytes were used to test the direct effects of various MPCi on BCAA catabolism in vitro. Lastly, we investigated how hepatocyte-specific deletion of MPC2 affects BCAA metabolism in the liver of obese mice and MSDC-0602K treatment of Zucker diabetic fatty (ZDF) rats. RESULTS: In patients with NASH, MSDC-0602K treatment, which led to marked improvements in insulin sensitivity and diabetes, had decreased plasma concentrations of BCAAs compared to baseline while placebo had no effect. The rate-limiting enzyme in BCAA catabolism is the mitochondrial branched chain ketoacid dehydrogenase (BCKDH), which is deactivated by phosphorylation. In multiple human hepatoma cell lines, MPCi markedly reduced BCKDH phosphorylation and stimulated branched chain keto acid catabolism; an effect that required the BCKDH phosphatase PPM1K. Mechanistically, the effects of MPCi were linked to activation of the energy sensing AMP-dependent protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) kinase signaling cascades in vitro. BCKDH phosphorylation was reduced in liver of obese, hepatocyte-specific MPC2 knockout (LS-Mpc2-/-) mice compared to wild-type controls concomitant with activation of mTOR signaling in vivo. Finally, while MSDC-0602K treatment improved glucose homeostasis and increased the concentrations of some BCAA metabolites in ZDF rats, it did not lower plasma BCAA concentrations. CONCLUSIONS: These data demonstrate novel cross talk between mitochondrial pyruvate and BCAA metabolism and suggest that MPC inhibition leads to lower plasma BCAA concentrations and BCKDH phosphorylation by activating the mTOR axis. However, the effects of MPCi on glucose homeostasis may be separable from its effects on BCAA concentrations.

Monoacylglycerol O-acyltransferase 1 lowers adipocyte differentiation capacity in vitro but does not affect adiposity in mice.

OBJECTIVE: Monoacylglycerol O-acyltransferase 1 (Mogat1), a lipogenic enzyme that converts monoacylglycerol to diacylglycerol, is highly expressed in adipocytes and may regulate lipolysis by re-esterifying fatty acids released during times when lipolytic rates are low. However, the role of Mogat1 in regulating adipocyte fat storage during differentiation and diet-induced obesity is relatively understudied. METHODS: Here, adipocyte-specific Mogat1 knockout mice were generated and subjected to a high-fat diet to determine the effects of Mogat1 deficiency on diet-induced obesity. Mogat1 floxed mice were also used to develop preadipocyte cell lines wherein Mogat1 could be conditionally knocked out to study adipocyte differentiation in vitro. RESULTS: In preadipocytes, it was found that Mogat1 knockout at the onset of preadipocyte differentiation prevented the accumulation of glycerolipids and reduced the differentiation capacity of preadipocytes. However, the loss of adipocyte Mogat1 did not affect weight gain or fat mass induced by a high-fat diet in mice. Furthermore, loss of Mogat1 in adipocytes did not affect plasma lipid or glucose concentrations or insulin tolerance. CONCLUSIONS: These data suggest Mogat1 may play a role in adipocyte differentiation in vitro but not adipose tissue expansion in response to nutrient overload in mice.

Multiple antisense oligonucleotides targeted against monoacylglycerol acyltransferase 1 (Mogat1) improve glucose metabolism independently of Mogat1.

OBJECTIVE: Monoacylglycerol acyltransferase (MGAT) enzymes catalyze the synthesis of diacylglycerol from monoacylglycerol. Previous work has suggested the importance of MGAT activity in the development of obesity-related hepatic insulin resistance. Indeed, antisense oligonucleotide (ASO)-mediated knockdown of Mogat1 mRNA, which encodes MGAT1, reduced hepatic MGAT activity and improved glucose tolerance and insulin resistance in high-fat diet (HFD)-fed mice. However, recent work has suggested that some ASOs may have off-target effects on body weight and metabolic parameters via activation of the interferon alpha/beta receptor 1 (IFNAR-1) pathway. METHODS: Mice with whole-body Mogat1 knockout or a floxed allele for Mogat1 to allow for liver-specific Mogat1-knockout (by either a liver-specific transgenic or adeno-associated virus-driven Cre recombinase) were generated. These mice were placed on an HFD, and glucose metabolism and insulin sensitivity were assessed after 16 weeks on diet. In some experiments, mice were treated with control scramble or Mogat1 ASOs in the presence or absence of IFNAR-1 neutralizing antibody. RESULTS: Genetic deletion of hepatic Mogat1, either acutely or chronically, did not improve hepatic steatosis, glucose tolerance, or insulin sensitivity in HFD-fed mice. Furthermore, constitutive Mogat1 knockout in all tissues actually exacerbated HFD-induced obesity, insulin sensitivity, and glucose intolerance on an HFD. Despite markedly reduced Mogat1 expression, liver MGAT activity was unaffected in all knockout mouse models. Mogat1 overexpression in hepatocytes increased liver MGAT activity and TAG content in low-fat-fed mice but did not cause insulin resistance. Multiple Mogat1 ASO sequences improved glucose tolerance in both wild-type and Mogat1 null mice, suggesting an off-target effect. Hepatic IFNAR-1 signaling was activated by multiple Mogat1 ASOs, but its blockade did not prevent the effects of either Mogat1 ASO on glucose homeostasis. CONCLUSION: These results indicate that genetic loss of Mogat1 does not affect hepatic MGAT activity or metabolic homeostasis on HFD and show that multiple Mogat1 ASOs improve glucose metabolism through effects independent of targeting Mogat1 or activation of IFNAR-1 signaling.

Nutritional modulation of heart failure in mitochondrial pyruvate carrier-deficient mice.

The myocardium is metabolically flexible; however, impaired flexibility is associated with cardiac dysfunction in conditions including diabetes and heart failure. The mitochondrial pyruvate carrier (MPC) complex, composed of MPC1 and MPC2, is required for pyruvate import into the mitochondria. Here we show that MPC1 and MPC2 expression is downregulated in failing human and mouse hearts. Mice with cardiac-specific deletion of Mpc2 (CS-MPC2-/-) exhibited normal cardiac size and function at 6 weeks old, but progressively developed cardiac dilation and contractile dysfunction, which was completely reversed by a high-fat, low-carbohydrate ketogenic diet. Diets with higher fat content, but enough carbohydrate to limit ketosis, also improved heart failure, while direct ketone body provisioning provided only minor improvements in cardiac remodelling in CS-MPC2-/- mice. An acute fast also improved cardiac remodelling. Together, our results reveal a critical role for mitochondrial pyruvate use in cardiac function, and highlight the potential of dietary interventions to enhance cardiac fat metabolism to prevent or reverse cardiac dysfunction and remodelling in the setting of MPC deficiency.

CD36 deficiency impairs the small intestinal barrier and induces subclinical inflammation in mice.

BACKGROUND & AIMS: CD36 has immuno-metabolic actions and is abundant in the small intestine on epithelial, endothelial and immune cells. We examined the role of CD36 in gut homeostasis using mice null for CD36 (CD36KO) and with CD36 deletion specific to enterocytes (Ent-CD36KO) or endothelial cells (EC-CD36KO). METHODS: Intestinal morphology was evaluated using immunohistochemistry and electron microscopy (EM). Intestinal inflammation was determined from neutrophil infiltration and expression of cytokines, toll-like receptors and COX-2. Barrier integrity was assessed from circulating lipopolysaccharide (LPS) and dextran administered intragastrically. Epithelial permeability to luminal dextran was visualized using two photon microscopy. RESULTS: The small intestines of CD36KO mice fed a chow diet showed several abnormalities including extracellular matrix (ECM) accumulation with increased expression of ECM proteins, evidence of neutrophil infiltration, inflammation and compromised barrier function. EM showed shortened desmosomes with decreased desmocollin 2 expression. Systemically, leukocytosis and neutrophilia were present together with 80% reduction of anti-inflammatory Ly6Clow monocytes. Bone marrow transplants supported the primary contribution of non-hematopoietic cells to the inflammatory phenotype. Specific deletion of endothelial but not of enterocyte CD36 reproduced many of the gut phenotypes of germline CD36KO mice including fibronectin deposition, increased interleukin 6, neutrophil infiltration, desmosome shortening and impaired epithelial barrier function. CONCLUSIONS: CD36 loss results in chronic neutrophil infiltration of the gut, impairs barrier integrity and systemically causes subclinical inflammation. Endothelial cell CD36 deletion reproduces the major intestinal phenotypes. The findings suggest an important role of the endothelium in etiology of gut inflammation and loss of epithelial barrier integrity.

Perilipin 5-Driven Lipid Droplet Accumulation in Skeletal Muscle Stimulates the Expression of Fibroblast Growth Factor 21.

Perilipin 5 (PLIN5) is a lipid droplet protein and is highly expressed in oxidative tissue. Expression of the PLIN5 gene is regulated by peroxisome proliferator-activated receptor-α, fasting, and exercise. However, the effect of increased muscle PLIN5 expression on whole-body energy homeostasis remains unclear. To examine this, we developed a mouse line with skeletal muscle PLIN5 overexpression (MCK-Plin5). We show that MCK-Plin5 mice have increased energy metabolism and accumulate more intramyocellular triacylglycerol but have normal glucose and insulin tolerance. MCK-Plin5 mice fed high-fat chow manifest lower expression of inflammatory markers in their liver and increased expression of "browning" factors in adipose tissue. This muscle-driven phenotype is, at least in part, mediated by myokines; the MCK-Plin5 mice have 80-fold higher FGF21 gene expression in muscle and increased serum FGF21 concentration. The increase in FGF21 occurs mainly in muscles with a predominance of fast-twitch fibers, suggesting that fiber type-specific lipid storage may be part of the mechanism conferring metabolic protection in MCK-Plin5 mice. In conclusion, upregulating the PLIN5 level in skeletal muscle drives expression of the FGF21 gene in fast-twitch fibers and is metabolically protective. These findings provide insight into the physiology of PLIN5 and the potential contribution of its upregulation during exercise.

Perilipin 1 moves between the fat droplet and the endoplasmic reticulum.

Perilipin 1, unlike the other perilipins, is thought to be restricted to the fat droplet. We reassessed its cellular distribution using the fat droplet marker CGI-58 in OP9 and 3T3-L1 adipocyte lines and in brown adipose tissue (BAT). As expected, we found perilipin 1 in the fat droplet-enriched floating fraction from centrifuged adipocyte or BAT homogenates. However, about half of perilipin 1 was suspended in the cytosol/infranate or pelleted with cellular membranes. In these fractionations, most of the fat droplet-associated protein CGI-58 was in the floating fraction. In BAT and OP9 adipocytes about a third of perilipin 1 pellets, compared with a much smaller fraction of CGI-58. Co-imaging perilipin 1 and smooth endoplasmic reticulum (ER) markers reveals both ER and fat droplet associated perilipin 1 in OP9 adipocytes. Consistent with these observations, perilipin 1 overexpressed in COS7 cells mostly fractionates with cellular membranes and imaging shows it on the ER. In 3T3-L1 adipocytes almost half of perilipin 1 floats, half is suspended as infranate and small amounts pellet. Finally, driving rapid fat droplet synthesis in OP9 adipocytes increases the intensity of perilipin 1 on fat droplets, while decreasing non-fat droplet immunolabeling. Confirming the morphological findings, fractionation shows perilipin 1 moving from the pelleted to the floated fractions. In conclusion, this study documents an expanded intracellular distribution for perilipin 1 and its movement from ER to fat droplet during lipid synthesis.

A single centrifugation method for isolating fat droplets from cells and tissues.

Fat droplets (FDs) have important roles in cellular energy regulation. Isolating FDs from either cells or tissue continues to be important for studying these organelles. Here, we describe a procedure wherein whole homogenates of cultured cells or tissue are fractionated with a single centrifugation step in a standard microcentrifuge. This procedure reproducibly yields three fractions highly enriched in either FDs, soluble cellular components, or sedimentable organelles/membranes.

Diacylglycerol enrichment of endoplasmic reticulum or lipid droplets recruits perilipin 3/TIP47 during lipid storage and mobilization.

Fatty acid-induced triacylglycerol synthesis produces triacylglycerol droplets with a protein coat that includes perilipin 3/TIP47 and perilipin 4/S3-12. This study addresses the following two questions. Where do lipid droplets emerge, and how are their coat proteins recruited? We show that perilipin 3- and perilipin 4-coated lipid droplets emerge along the endoplasmic reticulum (ER). Blocking membrane trafficking with AlF(4)(-) during fatty acid-induced triacylglycerol synthesis drove perilipin 3 to the tubular ER. Forskolin, which like AlF(4)(-) activates adenylate cyclase, did not redistribute perilipin 3, but when added together with AlF(4)(-) perilipin 3 was recruited to lipid droplets rather than the ER. Thus inhibiting trafficking with AlF(4)(-) redistributed perilipin 3 differently under conditions of triacylglycerol synthesis (fatty acid addition) versus hydrolysis (forskolin) suggesting a shared acylglycerol-mediated mechanism. We tested whether diacylglycerol (DG), the immediate precursor of triacylglycerol and its first hydrolytic product, affects the distribution of perilipin 3. Stabilizing DG with the DG lipase inhibitor RHC80267 enhanced the perilipin 3 recruited to lipid droplets and raised DG levels in this fraction. Treating cells with a membrane-permeable DG recruited perilipin 3 to the ER. Stabilizing DG, by blocking its hydrolysis with RHC80267 or its acylation with triacsin C, enhanced recruitment of perilipin 3 to the ER. Expressing the ER enzyme DGAT1, which removes DG by converting it to triacylglycerol, attenuated perilipin 3 DG-induced ER recruitment. Membrane-permeable DG also drove perilipin 4 and 5 onto the ER. Together the data suggest that these lipid droplet proteins are recruited to DG-enriched membranes thereby linking lipid coat proteins to the metabolic state of the cell.