Featured ArticlesVolume 23 | May 2019
|Dysregulated transmethylation leading to HCC compromises redox homeostasis and glucose formationThe incidence of hepatocellular carcinoma (HCC) is increasing. Dysregulated metabolism is an important contributor to the pathogenesis of cancer. Experimental models suggest that impeding glucose production promotes HCC. The role of liver glycine N-methyltransferase (GNMT) in the relationship between glucose control and HCC is of particular interest. GNMT-null mice develop HCC by eight months of age, suggesting a causal role in tumorigenesis. Hughey et al. studied the relationship of GNMT action and glucose production to HCC formation. The results show that the lack of GNMT reduces glucose production. Reduced glucose production is coupled to impaired NAD+ synthesis and salvage. A decline in NAD+ availability may redirect the flux of glucose precursors to pathways that regulate tumorigenesis.
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Objective: The loss of liver glycine N-methyltransferase (GNMT) promotes liver steatosis and the transition to hepatocellular carcinoma (HCC). Previous work showed endogenous glucose production is reduced in GNMT-null mice with gluconeogenic precursors being used in alternative biosynthetic pathways that utilize methyl donors and are linked to tumorigenesis. This metabolic programming occurs before the appearance of HCC in GNMT-null mice. The metabolic physiology that sustains liver tumor formation in GNMT-null mice is unknown. The studies presented here tested the hypothesis that nutrient flux pivots from glucose production to pathways that incorporate and metabolize methyl groups in GNMT-null mice with HCC.
Methods: 2H/13C metabolic flux analysis was performed in conscious, unrestrained mice lacking GNMT to quantify glucose formation and associated nutrient fluxes. Molecular analyses of livers from mice lacking GNMT including metabolomic, immunoblotting, and immunochemistry were completed to fully interpret the nutrient fluxes.
Results: GNMT knockout (KO) mice showed lower blood glucose that was accompanied by a reduction in liver glycogenolysis and gluconeogenesis. NAD+ was lower and the NAD(P)H-to-NAD(P)+ ratio was higher in livers of KO mice. Indices of NAD+ synthesis and catabolism, pentose phosphate pathway flux, and glutathione synthesis were dysregulated in KO mice.
Conclusion: Glucose precursor flux away from glucose formation towards pathways that regulate redox status increase in the liver. Moreover, synthesis and scavenging of NAD+ are both impaired resulting in reduced concentrations. This metabolic program blunts an increase in methyl donor availability, however, biosynthetic pathways underlying HCC are activated.[Hide abstract]
|The pseudokinase MLKL regulates hepatic insulin sensitivity Inflammation and cell death contribute to the development of type 2 diabetes (T2D). Necroptosis, a recently characterized necrosis integrated with the extrinsic apoptosis pathway, is widely viewed as a highly proinflammatory mode of cell death. The pseudokinase MLKL is a key protein in necroptosis. However, the involvement of necroptotic markers in insulin resistance and T2D remains unclear. Xu, Du, and colleagues show that the loss of MLKL in mice prevents obesity-induced insulin resistance and glucose intolerance. These findings reveal a role of MLKL in insulin sensitivity and suggest the potential involvement of necroptotic regulators in the physiopathology of T2D.
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Objective: The mixed lineage kinase domain like (MLKL) protein, receptor interacting protein (RIPK) 1, and RIPK3 are key regulators of necroptosis, a highly pro-inflammatory mode of cell death that has been implicated in various pathological processes and human diseases. However, the role of these necroptotic regulators in diabetes remains unknown. Here we sought to delineate the role of MLKL in insulin resistance and type 2 diabetes (T2D).
Methods: We first analyzed the expression of key necroptotic regulators in obese/diabetic mouse models. We then utilized MLKL knockout (MLKL−/−) mice to evaluate the effects of MLKL on obesity-induced metabolic complications. We further determined the consequences of MLKL inhibition on hepatic insulin signaling and explored the underlying mechanism. Finally, we assessed the potential therapeutic effects of necroptotic inhibitor, necrostatin-1 (Nec-1), in ob/ob mice.
Results: In wild-type or obese mice (ob/ob, db/db, or diet-induced obesity), MLKL was increased in certain obesity-associated tissues, particularly in the liver. Whole-body deficiency of MLKL prevented obesity-induced insulin resistance and glucose intolerance. Inhibition of MLKL or other key necroptotic regulators enhanced hepatic insulin sensitivity. MLKL modulated insulin-stimulated PI(3,4,5)P3 production in liver cells but did not affect the expression of inflammatory genes in vitro and in vivo. Nec-1 administration ameliorated insulin resistance and glucose intolerance in ob/ob mice.
Conclusions: These findings reveal MLKL as a regulator of insulin sensitivity and suggest necroptotic regulators might be potential therapeutic targets for insulin resistance and T2D.[Hide abstract]
|Tim-3 aggravates podocyte injury in diabetic nephropathy Diabetic nephropathy (DN) is the most common microvascular complication of diabetes mellitus. Macrophages are the most abundant immune cells in the diabetic kidney. T cell immunoglobulin domain and mucin domain-3 (Tim-3) has diverse regulatory roles in macrophages. Yang et al. demonstrate that the immune checkpoint molecule Tim-3 accelerates podocyte injury, which aggravates the progression of DN by triggering the NF-κB/TNF-α signaling pathway in macrophages.
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Objective: Macrophage-mediated inflammation plays a significant role in the development and progression of diabetic nephropathy (DN). However, the underlying mechanisms remain unclear. Studies suggest that T cell immunoglobulin domain and mucin domain-3 (Tim-3) has complicated roles in regulating macrophage activation, but its roles in the progression of DN are still completely unknown.
Methods: We downregulated Tim-3 expression in kidney (intrarenal injection of Tim-3 shRNA expressing lentivirus or global Tim-3 knockout mice) and induced DN by streptozotocin (STZ). We analyzed the degree of renal injury, especially the podocyte injury induced by activated macrophages in vitro and in vivo. Then, we transferred different bone marrow derived macrophages (BMs) into STZ-induced Tim-3 knockdown mice to examine the effects of Tim-3 on macrophages in DN.
Results: First, we found that Tim-3 expression on renal macrophages was increased in patients with DN and in two diabetic mouse models, i.e. STZ-induced diabetic mice and db/db mice, and positively correlated with renal dysfunction of DN patients. Tim-3 deficiency ameliorated renal damage in STZ-induced diabetes with concurrent increase in protein levels of Nephrin and WT-1. Similar effects were observed in mice with Tim-3 knockdown diabetic mice. Second, adoptive transfer of Tim-3-expressing macrophages, but not Tim-3 knockout macrophages, accelerated diabetic renal injury in DN mice, suggesting a key role for Tim-3 on macrophages in the development of DN. Furthermore, we found NF-κB activation and TNF-α excretion were upregulated by Tim-3 in diabetic kidneys, and podocyte injury was associated with the Tim-3-mediated activation of the NF-κB/TNF-α signaling pathway in DN macrophages both in vivo and in vitro.
Conclusions: These results suggest that Tim-3 functions as a key regulator in renal inflammatory processes and serves as a potential therapeutic target for renal injury in DN.[Hide abstract]
|Foregut tyrosine metabolism in glucose tolerancePancreatic β-cells integrate several different external signals to finetune the release of insulin and thus accurately regulate blood glucose levels. The foregut is the major source of circulating dopamine (DA) and L-DOPA, which are synthesized from tyrosine. DA and L-DOPA might limit glucose-stimulated insulin secretion (GSIS). Korner, Cline, et al. provide proof-of-principle evidence for the existence of a postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of GSIS as proposed by the anti-incretin hypothesis.
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Objective: We hypothesized that DA and L-DOPA derived from nutritional tyrosine and the resultant observed postprandial plasma excursions of L-DOPA and DA might affect glucose tolerance via their ability to be taken-up by beta cells and inhibit glucose-stimulated β-cell insulin secretion.
Methods: To investigate a possible circuit between meal-stimulated 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine (DA) production in the GI tract and pancreatic β-cells, we: 1) mapped GI mucosal expression of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); 2) measured L-DOPA and DA content of GI mucosal tissues following meal challenges with different L-tyrosine (TYR) content, 3) determined whether meal TYR content impacts plasma insulin and glucose excursions; and 4) characterized postprandial plasma excursions of L-DOPA and DA in response to meal tyrosine content in rodents and a population of bariatric surgery patients. Next, we characterized: 1) the metabolic transformation of TYR and L-DOPA into DA in vitro using purified islet tissue; 2) the metabolic transformation of orally administrated stable isotope labeled TYR into pancreatic DA, and 3) using a nuclear medicine technique, we studied endocrine beta cells in situ release and binding of DA in response to a glucose challenge.
Results: We demonstrate in rodents that intestinal content and circulatory concentrations L-DOPA and DA, plasma glucose and insulin are responsive to the tyrosine (TYR) content of a test meal. Intestinal expression of two enzymes, Tyrosine hydroxylase (TH) and Aromatic Amino acid Decarboxylase (AADC), essential to the transformation of TYR to DA was mapped and the metabolism of metabolism of TYR to DA was traced in human islets and a rodent beta cell line in vitro and from gut to the pancreas in vivo. Lastly, we show that β cells secrete and bind DA in situ in response to glucose stimulation.
Conclusions: We provide proof-of-principle evidence for the existence of a novel postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of glucose-stimulated β-cell insulin secretion as proposed by the anti-incretin hypothesis.[Hide abstract]
|RYGB reprograms enterocyte triglyceride metabolism Bariatric surgery, such as Roux-en-Y gastric bypass (RYGB) is currently the most effective treatment for obesity and its related comorbidities. One important mechanism of action is a rapid and long-lasting reduction of plasma triglyceride (TG) levels. How RYGB decreases plasma TG levels, however, is unclear. Kaufman et al. reveal substantial RYGB-induced changes in how enterocytes handle lipids, including TG absorption, reesterification, storage in lipid droplets, and oxidation. These changes likely contribute to the RYGB-induced improvement in plasma TG levels.|
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Objective: Roux-en-Y gastric bypass (RYGB) surgery produces rapid and persistent reductions in plasma triglyceride (TG) levels associated with fewer cardiovascular events. The mechanisms of the reduction in systemic TG levels remain unclear. We hypothesized that RYGB reduces intestinal TG secretion via altered enterocyte lipid handling.
Methods: RYGB or Sham surgery was performed in diet-induced obese, insulin-resistant male Sprague–Dawley rats. First, we tested whether RYGB reduced test meal-induced TG levels in the intestinal lymph, a direct readout of enterocyte lipid secretion. Second, we examined whether RYGB modified TG enterocyte secretion at the single lipid level and in comparison to other lipid subclasses, applying mass spectrometry lipidomics to the intestinal lymph of RYGB and Sham rats (0–21 days after surgery). Third, we explored whether RYGB modulated the metabolic characteristics of primary enterocytes using transcriptional and functional assays relevant to TG absorption, reesterification, storage in lipid droplets, and oxidation.
Results: RYGB reduced overall postprandial TG concentrations compared to Sham surgery in plasma and intestinal lymph similarly. RYGB reduced lymphatic TG concentrations more than other lipid subclasses, and shifted the remaining TG pool towards long-chain, unsaturated species. In enterocytes of fasted RYGB rats, lipid uptake was transcriptionally (Fatp4, Fabp2, Cd36) and functionally reduced compared to Sham, whereas TG reesterification genes were upregulated.
Conclusion: Our results show that RYGB substantially reduces intestinal TG secretion and modifies enterocyte lipid absorption and handling in rats. These changes likely contribute to the improvements in the plasma TG profile observed after RYGB in humans.[Hide abstract]
|Brown fat organogenesis and maintenance requires AKT1 and AKT2Strategies to increase brown adipose tissue (BAT) amount and/or function are being considered as therapeutic strategies to fight metabolic diseases. Thus, understanding BAT development is of high clinical relevance. Using mouse genetics, Sanchez-Gurmaches and colleagues define distinct and overlapping functions for AKT1 and AKT2 in brown and white adipose tissue development and show that they are largely dispensable for skeletal muscle development.
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Objective: Understanding the signaling mechanisms that control brown adipose tissue (BAT) development is relevant to understanding energy homeostasis and obesity. The AKT kinases are insulin effectors with critical in vivo functions in adipocytes; however, their role in adipocyte development remains poorly understood. The goal of this study was to investigate AKT function in BAT development.
Methods: We conditionally deleted Akt1 and Akt2 either individually or together with Myf5-Cre, which targets early mesenchymal precursors that give rise to brown adipocytes. Because Myf5-Cre also targets skeletal muscle and some white adipocyte lineages, comparisons were made between AKT function in BAT versus white adipose tissue (WAT) and muscle development. We also deleted both Akt1 and Akt2 in mature brown adipocytes with Ucp1-Cre or Ucp1-CreER to investigate AKT1/2 signaling in BAT maintenance.
Results: AKT1 and AKT2 are individually dispensable in Myf5-Cre lineages in vivo for establishing brown and white adipocyte precursor cell pools and for their ability to differentiate (i.e. induce PPARγ). AKT1 and AKT2 are also dispensable for skeletal muscle development, and AKT3 does not compensate in either the adipocyte or muscle lineages. In contrast, AKT2 is required for adipocyte lipid filling and efficient downstream AKT substrate phosphorylation. Mice in which both Akt1 and Akt2 are deleted with Myf5-Cre lack BAT but have normal muscle mass, and doubly deleting Akt1 and Akt2 in mature brown adipocytes, either congenitally (with Ucp1-Cre), or inducibly in older mice (with Ucp1-CreER), also ablates BAT. Mechanistically, AKT signaling promotes adipogenesis in part by stimulating ChREBP activity.
Conclusions: AKT signaling is required in vivo for BAT development but dispensable for skeletal muscle development. AKT1 and AKT2 have both overlapping and distinct functions in BAT development with AKT2 being the most critical individual isoform. AKT1 and AKT2 also have distinct and complementary functions in BAT maintenance.[Hide abstract]
|Pirt deficiency has female-specific effects on energy metabolismBrown adipose tissue (BAT) thermogenesis increases with prolonged exposure to cold, enabling mammals to maintain core body temperature. There is evidence to suggest direct cold-sensing by brown adipocytes via transient receptor potential melastatin 8 (TRPM8). Phosphoinositide interacting regulator of TRPs (Pirt) is an endogenous regulator of TRP channels. Jall and colleagues report that Pirt deficient female mice have an increased body weight and impaired glucose tolerance. This increased susceptibility to develop obesity and glucose intolerance is abrogated in the presence of a high-fat, high-sugar diet. Also, the authors reveal that Pirt is dispensable for TRPM8-induced BAT thermogenesis in vivo.
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Objective: The contribution of brown adipose tissue (BAT) to adult human metabolic control is a topic of ongoing investigation. In context, understanding the cellular events leading to BAT uncoupling, heat production, and energy expenditure is anticipated to produce significant insight into this endeavor. The phosphoinositide interacting regulator of transient receptor potentials (Pirt) was recently put forward as a key protein regulating cold sensing downstream of the transient receptor potential melastatin 8 (TRPM8). Notably, TRPM8 has been identified as a non-canonical regulator of BAT thermogenesis. The aim of this investigation was to delineate the role of Pirt in energy homeostasis and glucose metabolism - and the possible involvement of Pirt in TRPM8-elicited energy expenditure.
Methods: To this end, we metabolically phenotyped male and female Pirt deficient (Pirt−/−) mice exposed to a low-fat chow diet or to a high-fat, high-sugar (HFHS) diet.
Results: We identified that chow-fed female Pirt−/− mice have an increased susceptibility to develop obesity and glucose intolerance. This effect is abrogated when the mice are exposed to a HFHS diet. Conversely, Pirt−/− male mice display no metabolic phenotype on either diet relative to wild-type (WT) control mice. Finally, we observed that Pirt is dispensable for TRPM8-evoked energy expenditure.
Conclusions: We here report subtle metabolic abnormalities in female, but not male, Pirt−/− mice. Future studies are required to tease out if metabolic stressors beyond dietary interventions, e.g. temperature fluctuations, are interacting with Pirt-signaling and metabolic control in a sex-specific fashion.[Hide abstract]
|RORα deletion does not affect the metabolic susceptibility to western style dietThe incidence of obesity and associated metabolic disorders increase worldwide. Accumulating evidence points towards nuclear receptor (NR) transcription factors as potential treatment targets. The retinoic acid-related orphan receptor α (RORα) is a NR that has been implicated in the regulation of a wide variety of metabolic pathways. Molinaro et al. generated a liver-specific RORα deficient mouse strain that was metabolically phenotyped on both chow and western-style diet (WSD). Their data show that the deletion of RORα in the liver does not affect glucose or lipid metabolism during WSD in mice. Further studies testing the role of liver RORα or the double RORα and RORγ deletion in different dietary settings are needed to further elucidate the role of RORα in obesity and metabolic disorders.
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Objective: The nuclear receptor superfamily is a potential target for the development of new treatments for obesity and metabolic diseases. Increasing evidence has pointed towards the retinoic acid-related orphan receptor-alpha (RORα) as an important nuclear receptor involved in several biological processes. RORα full body knockout mice display improved metabolic phenotypes on both chow and high fat (60% fat, 20% carbohydrate) diets, but also have severe behavioral abnormalities. Here we investigated the effect of hepatic RORα by generating mice with liver-specific RORα deletion to elucidate the role of this nuclear receptor on host metabolism.
Methods: 8 week-old mice with liver-specific RORα deletion and littermate controls were fed either chow or western-style diets (40% fat, 40% carbohydrate) for 12 weeks. Metabolic phenotyping was performed at the end of the dietary intervention.
Results: Here, we show that hepatic RORα deletion does not affect the metabolic susceptibility to either chow or western-style diet in terms of glucose metabolism and adiposity.
Conclusions: Our data indicate that liver deletion of RORα does not have a pivotal role in the regulation of hepatic glucose and lipid metabolism on chow or western-style diet.[Hide abstract]
|Perm1 shapes skeletal muscle responses to endurance exercise trainingSkeletal muscle plays key roles in metabolic homeostasis and whole-body energy expenditure. Endurance exercise training remodels skeletal muscle by activating signaling pathways and regulating transcription factors. Perm1, a recently identified protein, is selectively expressed in skeletal and cardiac muscles. To gain insights into the cellular function of Perm1, Cho et al. searched for Perm1 interacting proteins. They show that Perm1 regulates physical activity-dependent CaMKII and MAPK p38 signaling and is important for a subset of exercise-induced responses. Their findings suggest that Perm1 modulates excitation-transcription coupling and shapes the response to endurance exercise.
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Objective: Endurance exercise training remodels skeletal muscle, leading to increased mitochondrial content and oxidative capacity. How exercise entrains skeletal muscle signaling pathways to induce adaptive responses remains unclear. In past studies, we identified Perm1 (PGC-1 and ERR induced regulator, muscle 1) as an exercise-induced gene and showed that Perm1 overexpression elicits similar muscle adaptations as endurance exercise training. The mechanism of action and the role of Perm1 in exercise-induced responses are not known. In this study, we aimed to determine the pathway by which Perm1 acts as well as the importance of Perm1 for acute and long-term responses to exercise.
Methods: We performed immunoprecipitation and mass spectrometry to identify Perm1 associated proteins, and validated Perm1 interactions with the Ca2+/calmodulin-dependent protein kinase II (CaMKII). We also knocked down Perm1 expression in gastrocnemius muscles of mice via AAV-mediated delivery of shRNA and assessed the impact of reduced Perm1 expression on both acute molecular responses to a single treadmill exercise bout and long-term adaptive responses to four weeks of voluntary wheel running training. Finally, we asked whether Perm1 levels are modulated by diet or diseases affecting skeletal muscle function.
Results: We show that Perm1 associates with skeletal muscle CaMKII and promotes CaMKII activation. In response to an acute exercise bout, muscles with a knock down of Perm1 showed defects in the activation of CaMKII and p38 MAPK and blunted induction of regulators of oxidative metabolism. Following four weeks of voluntary training, Perm1 knockdown muscles had attenuated mitochondrial biogenesis. Finally, we found that Perm1 expression is reduced in diet-induced obese mice and in muscular dystrophy patients and mouse models.
Conclusions: Our findings identify Perm1 as a muscle-specific regulator of exercise-induced signaling and Perm1 levels as tuners of the skeletal muscle response to exercise. The decreased Perm1 levels in states of obesity or muscle disease suggest that Perm1 may link pathological states to inefficient exercise responses.[Hide abstract]