Robert M. O’Doherty

Inhibition or deletion of the lipopolysaccharide receptor Toll-like receptor-4 confers partial protection against lipid-induced insulin resistance in rodent skeletal muscle

Aims/hypothesisA role for increased activity of the innate immune system in the pathogenesis of insulin resistance is supported by a number of studies. The current study assessed the potential role of the lipopolysaccharide receptor known as Toll-like receptor-4 (TLR-4), a component of the innate immune system, in mediating lipid-induced insulin resistance in skeletal muscle.MethodsThe effects of TLR-4 inhibition/deletion on lipid-induced insulin resistance was determined in skeletal muscle of TLR-4 null mice in vivo and in rat L6 myotubes in vitro.ResultsIn mice, acute hyperlipidaemia induced skeletal muscle insulin resistance, but a deletion of TLR-4 conferred significant protection against these effects. In L6 myotubes, inhibition of TLR-4 activity substantially reduced the capacity of the saturated fatty acid palmitate to induce insulin resistance. Importantly, palmitate activated the nuclear factor κB (NFκB) pathway in L6 myotubes and mouse skeletal muscle, and these effects were blocked by inhibition of TLR-4 in L6 myotubes and absence of TLR-4 in skeletal muscle. Furthermore, inhibition of the NFκB pathway downstream of TLR-4 in L6 myotubes also protected against the induction of insulin resistance by palmitate.Conclusions/interpretationInhibition or absence of TLR-4 confers protection against the detrimental effects of lipids on skeletal muscle insulin action, and these effects are associated with a prevention of the activation of the NFκB pathway by lipids. Importantly, inhibition of the NFκB pathway in myotubes downstream of TLR-4 also protects against lipid-induced insulin resistance, suggesting a mechanism by which reduced TLR-4 activity confers beneficial effects on insulin action.

ChREBP Mediates Glucose-Stimulated Pancreatic β-Cell Proliferation

Glucose stimulates rodent and human β-cell replication, but the intracellular signaling mechanisms are poorly understood. Carbohydrate response element-binding protein (ChREBP) is a lipogenic glucose-sensing transcription factor with unknown functions in pancreatic β-cells. We tested the hypothesis that ChREBP is required for glucose-stimulated β-cell proliferation. The relative expression of ChREBP was determined in liver and β-cells using quantitative RT-PCR (qRT-PCR), immunoblotting, and immunohistochemistry. Loss- and gain-of-function studies were performed using small interfering RNA and genetic deletion of ChREBP and adenoviral overexpression of ChREBP in rodent and human β-cells. Proliferation was measured by 5-bromo-2′-deoxyuridine incorporation, [3H]thymidine incorporation, and fluorescence-activated cell sorter analysis. In addition, the expression of cell cycle regulatory genes was measured by qRT-PCR and immunoblotting. ChREBP expression was comparable with liver in mouse pancreata and in rat and human islets. Depletion of ChREBP decreased glucose-stimulated proliferation in β-cells isolated from ChREBP−/− mice, in INS-1–derived 832/13 cells, and in primary rat and human β-cells. Furthermore, depletion of ChREBP decreased the glucose-stimulated expression of cell cycle accelerators. Overexpression of ChREBP amplified glucose-stimulated proliferation in rat and human β-cells, with concomitant increases in cyclin gene expression. In conclusion, ChREBP mediates glucose-stimulated proliferation in pancreatic β-cells.

Impact of dietary fatty acids on metabolic activity and host intestinal microbiota composition in C57BL/6J mice

Different dietary fat and energy subtypes have an impact on both the metabolic health and the intestinal microbiota population of the host. The present study assessed the impact of dietary fat quality, with a focus on dietary fatty acid compositions of varying saturation, on the metabolic health status and the intestinal microbiota composition of the host. C57BL/6J mice (n 9-10 mice per group) were fed high-fat (HF) diets containing either (1) palm oil, (2) olive oil, (3) safflower oil or (4) flaxseed/fish oil for 16 weeks and compared with mice fed low-fat (LF) diets supplemented with either high maize starch or high sucrose. Tissue fatty acid compositions were assessed by GLC, and the impact of the diet on host intestinal microbiota populations was investigated using high-throughput 16S rRNA sequencing. Compositional sequencing analysis revealed that dietary palm oil supplementation resulted in significantly lower populations of Bacteroidetes at the phylum level compared with dietary olive oil supplementation (P< 0·05). Dietary supplementation with olive oil was associated with an increase in the population of the family Bacteroidaceae compared with dietary supplementation of palm oil, flaxseed/fish oil and high sucrose (P< 0·05). Ingestion of the HF-flaxseed/fish oil diet for 16 weeks led to significantly increased tissue concentrations of EPA, docosapentaenoic acid and DHA compared with ingestion of all the other diets (P< 0·05); furthermore, the diet significantly increased the intestinal population of Bifidobacterium at the genus level compared with the LF-high-maize starch diet (P< 0·05). These data indicate that both the quantity and quality of fat have an impact on host physiology with further downstream alterations to the intestinal microbiota population, with a HF diet supplemented with flaxseed/fish oil positively shaping the host microbial ecosystem.

Sex-Specific Effect of Estrogen Sulfotransferase on Mouse Models of Type 2 Diabetes

Estrogen sulfotransferase (EST), the enzyme responsible for the sulfonation and inactivation of estrogens, plays an important role in estrogen homeostasis. In this study, we showed that induction of hepatic Est is a common feature of type 2 diabetes. Loss of Est in female mice improved metabolic function in ob/ob, dexamethasone-, and high-fat diet–induced mouse models of type 2 diabetes. The metabolic benefit of Est ablation included improved body composition, increased energy expenditure and insulin sensitivity, and decreased hepatic gluconeogenesis and lipogenesis. This metabolic benefit appeared to have resulted from decreased estrogen deprivation and increased estrogenic activity in the liver, whereas such benefit was abolished in ovariectomized mice. Interestingly, the effect of Est was sex-specific, as Est ablation in ob/ob males exacerbated the diabetic phenotype, which was accounted for by the decreased islet β-cell mass and failure of glucose-stimulated insulin secretion in vivo. The loss of β-cell mass in ob/ob males deficient in Est was associated with increased macrophage infiltration and inflammation in white adipose tissue. Our results revealed an essential role of EST in energy metabolism and the pathogenesis of type 2 diabetes. Inhibition of EST, at least in females, may represent a novel approach to manage type 2 diabetes.

Antimicrobials: Strategies for targeting obesity and metabolic health?

Obesity is associated with a number of serious health consequences, including type 2 diabetes, cardiovascular disease and a variety of cancers among others and has been repeatedly shown to be associated with a higher risk of mortality. The relatively recent discovery that the composition and metabolic activity of the gut microbiota may affect the risk of developing obesity and related disorders has led to an explosion of interest in this distinct research field. A corollary of these findings would suggest that modulation of gut microbial populations can have beneficial effects with respect to controlling obesity. In this addendum, we summarize our recent data, showing that therapeutic manipulation of the microbiota using different antimicrobial strategies may be a useful approach for the management of obesity and metabolic conditions. In addition, we will explore some of the mechanisms that may contribute to microbiota-induced susceptibility to obesity and metabolic diseases.Obesity is associated with a number of serious health consequences, including type 2 diabetes, cardiovascular disease and a variety of cancers among others and has been repeatedly shown to be associated with a higher risk of mortality. The relatively recent discovery that the composition and metabolic activity of the gut microbiota may affect the risk of developing obesity and related disorders has led to an explosion of interest in this distinct research field. A corollary of these findings would suggest that modulation of gut microbial populations can have beneficial effects with respect to controlling obesity. In this addendum, we summarize our recent data, showing that therapeutic manipulation of the microbiota using different antimicrobial strategies may be a useful approach for the management of obesity and metabolic conditions. In addition, we will explore some of the mechanisms that may contribute to microbiota-induced susceptibility to obesity and metabolic diseases.

Detailed molecular analysis of the induction of the L-PK gene by glucose

Glucose has powerful effects on gene expression and participates in the fasted-to-fed transition of the liver. However, the molecular mechanism of glucose-regulated gene expression has not been completely described. In the present study, we performed a detailed analysis of the molecular events of the insulin-independent glucose response of the liver-type pyruvate kinase (L-PK) gene. L-PK mRNA was increased by glucose at the transcriptional level as determined by real-time RT-PCR, mRNA stability measurements, and nuclear run-on assays. LY294002 and LY303511 inhibited the glucose response of the L-PK gene at the transcriptional level. Histones H3 and H4 associated with the L-PK gene promoter were hyperacetylated and HNF4alpha was constitutively bound in low and high glucose. Treatment with 20mM glucose increased recruitment of ChREBP, additional HNF4alpha, and RNA polymerase II. Glucose-stimulated the phosphorylation of the C-terminal domain of RNA polymerase II, with increased Ser5 phosphorylation near the transcription start site and increased Ser2 phosphorylation near the termination signal. LY294002 and LY303511 blocked the recruitment of RNA polymerase II to the L-PK gene, reducing the rate of transcription. The results of these studies demonstrate fundamental details of the molecular mechanism of glucose activated gene expression.

Acetylation of mitochondrial proteins by GCN5L1 promotes enhanced fatty acid oxidation in the heart

Lysine acetylation is a reversible posttranslational modification and is particularly important in the regulation of mitochondrial metabolic enzymes. Acetylation uses acetyl-CoA derived from fuel metabolism as a cofactor, thereby linking nutrition to metabolic activity. In the present study, we investigated how mitochondrial acetylation status in the heart is controlled by food intake and how these changes affect mitochondrial metabolism. We found that there was a significant increase in cardiac mitochondrial protein acetylation in mice fed a long-term high-fat diet and that this change correlated with an increase in the abundance of the mitochondrial acetyltransferase-related protein GCN5L1. We showed that the acetylation status of several mitochondrial fatty acid oxidation enzymes (long-chain acyl-CoA dehydrogenase, short-chain acyl-CoA dehydrogenase, and hydroxyacyl-CoA dehydrogenase) and a pyruvate oxidation enzyme (pyruvate dehydrogenase) was significantly upregulated in high-fat diet-fed mice and that the increase in long-chain and short-chain acyl-CoA dehydrogenase acetylation correlated with increased enzymatic activity. Finally, we demonstrated that the acetylation of mitochondrial fatty acid oxidation proteins was decreased after GCN5L1 knockdown and that the reduced acetylation led to diminished fatty acid oxidation in cultured H9C2 cells. These data indicate that lysine acetylation promotes fatty acid oxidation in the heart and that this modification is regulated in part by the activity of GCN5L1.NEW & NOTEWORTHY Recent research has shown that acetylation of mitochondrial fatty acid oxidation enzymes has greatly contrasting effects on their activity in different tissues. Here, we provide new evidence that acetylation of cardiac mitochondrial fatty acid oxidation enzymes by GCN5L1 significantly upregulates their activity in diet-induced obese mice.

Bone marrow lympho-myeloid malfunction in obesity requires precursor cell-autonomous TLR4

Obesity, a prevalent condition in adults and children, impairs bone marrow (BM) function. However, the underlying mechanisms are unclear. Here, we show that obese mice exhibit poor emergency immune responses in a toll-like receptor 4 (TLR4)-dependent manner. Canonical myeloid genes (Csf1r, Spi1, Runx1) are enhanced, and lymphoid genes (Flt3, Tcf3, Ebf1) are reduced. Using adoptive transfer and mixed BM chimera approaches we demonstrate that myeloid>lymphoid bias arises after 6 weeks of high-fat diet and depends on precursor cell-autonomous TLR4. Further, lean mice exposed to the TLR4 ligand lipopolysaccharide (LPS) at doses similar to that detectable in obese serum recapitulates BM lympho-myeloid alterations. Together, these results establish a mechanistic contribution of BM cell-intrinsic TLR4 to obesity-driven BM malfunction and demonstrate the importance of LPS. Our findings raises important questions about the impact of maternal obesity and endotoxemia to fetal hematopoiesis, as fetal immune precursors are also sensitive to TLR4 signals.Obesity can affect bone marrow cell differentiation and the generation of myeloid and lymphoid cells. Here, the authors show that diet and obesity, as well as low-dose lipopolysaccharide, can alter Toll-like receptor 4 signaling bone marrow cells to skew the myeloid-lymphoid homeostasis in mice.

Chapter 14 Insulin resistance, inflammation, and the IKK/IκB/NF-κB pathway

Overview Insulin resistance is a characteristic of human obesity, and a primary risk factor for the development of type II diabetes (T2DM). However, despite tremendous effort and great progress in a number of areas of research, the biochemical mechanisms underlying the pathogenesis of insulin resistance in obesity remain poorly understood. In short, defects in insulin signaling, alterations in the expression and/or activity of a variety of adipokines and cytokines, dyslipidemia/dysregulated lipid metabolism, and most recently, increased intracellular stress and activation of inflammatory pathways have each been implicated as important contributory mechanisms in insulin resistance. In all likelihood it is the interplay of these factors, combined with genetic predisposition and environmental influences that results in the full metabolic manifestation of insulin resistance. This chapter accentuates a bias and recent progress in one area of research that implicates increased activity of the pro-inflammatory IKK/I κ B/NF- κ B (NF- κ B) pathway in the pathogenesis of insulin resistance. The purpose of the chapter is to (i) review seminal studies addressing the potential role of increased activity of the NF- κ B pathway in decreasing insulin action; (ii) discuss the potential relevance of these studies to human obesity; (iii) address putative mechanisms of activation of the NF- κ B pathway in obesity and how activation of the NF- κ B activity may reduce insulin action; and (iv) briefly discuss the potential of targeting activity of the NF- κ B pathway in the treatment of insulin resistance. Because of the narrow focus of the subject matter in this chapter, the reader is referred to a number of other excellent studies that address the potential detrimental effects on insulin action of activity of other inflammatory pathways [1], endoplasmic reticulum (ER) stress [2], and mitochondrial dysfunction [3,4].

Kupffer cells facilitate the acute effects of leptin on hepatic lipid metabolism

Leptin has potent effects on lipid metabolism in a number of peripheral tissues. In liver, an acute leptin infusion (~120 min) stimulates hepatic fatty acid oxidation (~30%) and reduces triglycerides (TG, ~40%), effects that are dependent on phosphoinositol-3-kinase (PI3K) activity. In the current study we addressed the hypothesis that leptin actions on liver-resident immune cells are required for these metabolic effects. Myeloid cell-specific deletion of the leptin receptor (ObR) in mice or depletion of liver Kupffer cells (KC) in rats in vivo prevented the acute effects of leptin on liver lipid metabolism, while the metabolic effects of leptin were maintained in mice lacking ObR in hepatocytes. Notably, liver TG were elevated in both lean and obese myeloid cell ObR, but the degree of obesity and insulin resistance induced by a high-fat diet was similar to control mice. In isolated primary hepatocytes (HEP), leptin had no effects on HEP lipid metabolism and only weakly stimulated PI3K. However, the coculture of KC with HEP restored leptin action on HEP fatty acid metabolism and stimulation of HEP PI3K. Notably, leptin stimulated the release from KC of a number of cytokines. However, the exposure of HEP to these cytokines individually [granulocyte macrophage colony-stimulating factor, IL-1α, IL-1β, IL-6, IL-10, and IL-18] or in combination had no effects on HEP lipid metabolism. Together, these data demonstrate a role for liver mononuclear cells in the regulation of liver lipid metabolism by leptin.