Robert M. O'Doherty

Composition and energy harvesting capacity of the gut microbiota: relationship to diet, obesity and time in mouse models

Background and Aims Increased efficiency of energy harvest, due to alterations in the gut microbiota (increased Firmicutes and decreased Bacteroidetes), has been implicated in obesity in mice and humans. However, a causal relationship is unproven and contributory variables include diet, genetics and age. Therefore, we explored the effect of a high-fat (HF) diet and genetically determined obesity (ob/ob) for changes in microbiota and energy harvesting capacity over time. Methods Seven-week-old male ob/ob mice were fed a low-fat diet and wild-type mice were fed either a low-fat diet or a HF-diet for 8 weeks (n=8/group). They were assessed at 7, 11 and 15 weeks of age for: fat and lean body mass (by NMR); faecal and caecal short-chain fatty acids (SCFA, by gas chromatography); faecal energy content (by bomb calorimetry) and microbial composition (by metagenomic pyrosequencing). Results A progressive increase in Firmicutes was confirmed in both HF-fed and ob/ob mice reaching statistical significance in the former, but this phylum was unchanged over time in the lean controls. Reductions in Bacteroidetes were also found in ob/ob mice. However, changes in the microbiota were dissociated from markers of energy harvest. Thus, although the faecal energy in the ob/ob mice was significantly decreased at 7 weeks, and caecal SCFA increased, these did not persist and faecal acetate diminished over time in both ob/ob and HF-fed mice, but not in lean controls. Furthermore, the proportion of the major phyla did not correlate with energy harvest markers. Conclusion The relationship between the microbial composition and energy harvesting capacity is more complex than previously considered. While compositional changes in the faecal microbiota were confirmed, this was primarily a feature of high-fat feeding rather than genetically induced obesity. In addition, changes in the proportions of the major phyla were unrelated to markers of energy harvest which changed over time. The possibility of microbial adaptation to diet and time should be considered in future studies.

Depletion of Liver Kupffer Cells Prevents the Development of Diet-Induced Hepatic Steatosis and Insulin Resistance

OBJECTIVE Increased activity of the innate immune system has been implicated in the pathogenesis of the dyslipidemia and insulin resistance associated with obesity and type 2 diabetes. In this study, we addressed the potential role of Kupffer cells (liver-specific macrophages, KCs) in these metabolic abnormalities. RESEARCH DESIGN AND METHODS Rats were depleted of KCs by administration of gadolinium chloride, after which all animals were exposed to a 2-week high-fat or high-sucrose diet. Subsequently, the effects of these interventions on the development of hepatic insulin resistance and steatosis were assessed. In further studies, the effects of M1-polarized KCs on hepatocyte lipid metabolism and insulin sensitivity were addressed. RESULTS As expected, a high-fat or high-sucrose diet induced steatosis and hepatic insulin resistance. However, these metabolic abnormalities were prevented when liver was depleted of KCs. In vitro, KCs recapitulated the in vivo effects of diet by increasing hepatocyte triglyceride accumulation and fatty acid esterification, and decreasing fatty acid oxidation and insulin responsiveness. To address the mechanisms(s) of KC action, we inhibited a panel of cytokines using neutralizing antibodies. Only neutralizing antibodies against tumor necrosis factor-α (TNFα) attenuated KC-induced alterations in hepatocyte fatty acid oxidation, triglyceride accumulation, and insulin responsiveness. Importantly, KC TNFα levels were increased by diet in vivo and in isolated M1-polarized KCs in vitro. CONCLUSIONS These data demonstrate a role for liver macrophages in diet-induced alterations in hepatic lipid metabolism and insulin sensitivity, and suggest a role for these cells in the etiology of the metabolic abnormalities of obesity/type 2 diabetes.

Divergent metabolic outcomes arising from targeted manipulation of the gut microbiota in diet-induced obesity

Objective The gut microbiota is an environmental regulator of fat storage and adiposity. Whether the microbiota represents a realistic therapeutic target for improving metabolic health is unclear. This study explored two antimicrobial strategies for their impact on metabolic abnormalities in murine diet-induced obesity: oral vancomycin and a bacteriocin-producing probiotic (Lactobacillus salivarius UCC118 Bac+). Design Male (7-week-old) C57BL/J6 mice (9–10/group) were fed a low-fat (lean) or a high-fat diet for 20 weeks with/without vancomycin by gavage at 2 mg/day, or with L salivarius UCC118Bac+ or the bacteriocin-negative derivative L salivarius UCC118Bac− (each at a dose of 1×109 cfu/day by gavage). Compositional analysis of the microbiota was by 16S rDNA amplicon pyrosequencing. Results Analysis of the gut microbiota showed that vancomycin treatment led to significant reductions in the proportions of Firmicutes and Bacteroidetes and a dramatic increase in Proteobacteria, with no change in Actinobacteria. Vancomycin-treated high-fat-fed mice gained less weight over the intervention period despite similar caloric intake, and had lower fasting blood glucose, plasma TNFα and triglyceride levels compared with diet-induced obese controls. The bacteriocin-producing probiotic had no significant impact on the proportions of Firmicutes but resulted in a relative increase in Bacteroidetes and Proteobacteria and a decrease in Actinobacteria compared with the non-bacteriocin-producing control. No improvement in metabolic profiles was observed in probiotic-fed diet-induced obese mice. Conclusion Both vancomycin and the bacteriocin-producing probiotic altered the gut microbiota in diet-induced obese mice, but in distinct ways. Only vancomycin treatment resulted in an improvement in the metabolic abnormalities associated with obesity thereby establishing that while the gut microbiota is a realistic therapeutic target, the specificity of the antimicrobial agent employed is critical.

A moderate increase in carnitine palmitoyltransferase 1a activity is sufficient to substantially reduce hepatic triglyceride levels

Nonalcoholic fatty liver disease (NAFLD), hypertriglyceridemia, and elevated free fatty acids are present in the majority of patients with metabolic syndrome and type 2 diabetes mellitus and are strongly associated with hepatic insulin resistance. In the current study, we tested the hypothesis that an increased rate of fatty acid oxidation in liver would prevent the potentially harmful effects of fatty acid elevation, including hepatic triglyceride (TG) accumulation and elevated TG secretion. Primary rat hepatocytes were transduced with adenovirus encoding carnitine palmitoyltransferase 1a (Adv-CPT-1a) or control adenoviruses encoding either beta-galactosidase (Adv-beta-gal) or carnitine palmitoyltransferase 2 (Adv-CPT-2). Overexpression of CPT-1a increased the rate of beta-oxidation and ketogenesis by approximately 70%, whereas esterification of exogenous fatty acids and de novo lipogenesis were unchanged. Importantly, CPT-1a overexpression was accompanied by a 35% reduction in TG accumulation and a 60% decrease in TG secretion by hepatocytes. There were no changes in secretion of apolipoprotein B (apoB), suggesting the synthesis of smaller, less atherogenic VLDL particles. To evaluate the effect of increasing hepatic CPT-1a activity in vivo, we injected lean or obese male rats with Adv-CPT-1a, Adv-beta-gal, or Adv-CPT-2. Hepatic CPT-1a activity was increased by approximately 46%, and the rate of fatty acid oxidation was increased by approximately 44% in lean and approximately 36% in obese CPT-1a-overexpressing animals compared with Adv-CPT-2- or Adv-beta-gal-treated rats. Similar to observations in vitro, liver TG content was reduced by approximately 37% (lean) and approximately 69% (obese) by this in vivo intervention. We conclude that a moderate stimulation of fatty acid oxidation achieved by an increase in CPT-1a activity is sufficient to substantially reduce hepatic TG accumulation both in vitro and in vivo. Therefore, interventions that increase CPT-1a activity could have potential benefits in the treatment of NAFLD.

Intermittent hypoxia reverses the diurnal glucose rhythm and causes pancreatic β‐cell replication in mice

Obstructive sleep apnoea (OSA) and type 2 diabetes frequently co‐exist and potentially interact haemodynamically and metabolically. However, the confounding effects of obesity have obscured the examination of any independent or interactive effects of the hypoxic stress of OSA and the hyperglycaemia of type 2 diabetes on haemodynamic and metabolic outcomes. We have developed a chronically catheterized, unhandled, lean murine model to examine the effects of intermittent hypoxic (IH) exposure and exogenous glucose infusion on the diurnal pattern of arterial blood pressure and blood glucose, as well as pancreatic β‐cell growth and function. Four experimental groups of adult male C57BL/J mice were exposed to 80 h of (1) either IH (nadir of inspired oxygen 5–6% at 60 cycles h−1 for 12 h during light period) or intermittent air (IA; control) and (2) continuous infusion of either 50% dextrose or saline (control). IH exposure during saline infusion caused a sustained increase in arterial blood pressure of 10 mmHg (P < 0.0001), reversed the normal diurnal rhythm of blood glucose (P < 0.03), doubled corticosterone levels (P < 0.0001), and increased replication of pancreatic β‐cells from 1.5 ± 0.3 to 4.0 ± 0.8% bromodeoxyuridine (BrdU)‐positive) β‐cells. The combined stimulus of IH exposure and glucose infusion attenuated the hypertension, exacerbated the reversed diurnal glucose rhythm, and produced the highest rates of apoptosis in β‐cells, without any additive effects on β‐cell replication. We conclude that, in contrast to the development of sustained hypertension, IH impaired glucose homeostasis only during periods of hypoxic exposure. IH acted as a stimulus to pancreatic β‐cell replication, but the presence of hyperglycaemia may increase the hypoxic susceptibility of β‐cells. This model will provide a basis for future mechanistic studies as well as assessing the metabolic impact of common comorbities in OSA, including obesity, insulin resistance and type 2 diabetes.

CCAAT/Enhancer-binding Protein β Deletion Reduces Adiposity, Hepatic Steatosis, and Diabetes in Leprdb/db Mice

CCAAT/enhancer-binding protein β (C/EBPβ) plays a key role in initiation of adipogenesis in adipose tissue and gluconeogenesis in liver; however, the role of C/EBPβ in hepatic lipogenesis remains undefined. Here we show that C/EBPβ inactivation in Leprdb/db mice attenuates obesity, fatty liver, and diabetes. In addition to impaired adipogenesis, livers from C/EBPβ-/- x Leprdb/db mice had dramatically decreased triglyceride content and reduced lipogenic enzyme activity. C/EBPβ deletion in Leprdb/db mice down-regulated peroxisome proliferator-activated receptor γ2 (PPARγ2) and stearoyl-CoA desaturase-1 and up-regulated PPARα independent of SREBP1c. Conversely, C/EBPβ overexpression in wild-type mice increased PPARγ2 and stearoyl-CoA desaturase-1 mRNA and hepatic triglyceride content. In FAO cells, overexpression of the liver inhibiting form of C/EBPβ or C/EBPβ RNA interference attenuated palmitate-induced triglyceride accumulation and reduced PPARγ2 and triglyceride levels in the liver in vivo. Leptin and the anti-diabetic drug metformin acutely down-regulated C/EBPβ expression in hepatocytes, whereas fatty acids up-regulate C/EBPβ expression. These data provide novel evidence linking C/EBPβ expression to lipogenesis and energy balance with important implications for the treatment of obesity and fatty liver disease.

Mice Lacking NKT Cells but with a Complete Complement of CD8+ T-Cells Are Not Protected against the Metabolic Abnormalities of Diet-Induced Obesity

The contribution of natural killer T (NKT) cells to the pathogenesis of metabolic abnormalities of obesity is controversial. While the combined genetic deletion of NKT and CD8+ T-cells improves glucose tolerance and reduces inflammation, interpretation of these data have been complicated by the recent observation that the deletion of CD8+ T-cells alone reduces obesity-induced inflammation and metabolic dysregulation, leaving the issue of the metabolic effects of NKT cell depletion unresolved. To address this question, CD1d null mice (CD1d−/−), which lack NKT cells but have a full complement of CD8+ T-cells, and littermate wild type controls (WT) on a pure C57BL/6J background were exposed to a high fat diet, and glucose intolerance, insulin resistance, dyslipidemia, inflammation, and obesity were assessed. Food intake (15.5±4.3 vs 15.3±1.8 kcal/mouse/day), weight gain (21.8±1.8 vs 22.8±1.4 g) and fat mass (18.6±1.9 vs 19.5±2.1 g) were similar in CD1d−/− and WT, respectively. As would be expected from these data, metabolic rate (3.0±0.1 vs 2.9±0.2 ml O2/g/h) and activity (21.6±4.3 vs 18.5±2.6 beam breaks/min) were unchanged by NKT cell depletion. Furthermore, the degree of insulin resistance, glucose intolerance, liver steatosis, and adipose and liver inflammatory marker expression (TNFα, IL-6, IL-10, IFN-γ, MCP-1, MIP1α) induced by high fat feeding in CD1d−/− were not different from WT. We conclude that deletion of NKT cells, in the absence of alterations in the CD8+ T-cell population, is insufficient to protect against the development of the metabolic abnormalities of diet-induced obesity.

Dendritic Cells Promote Macrophage Infiltration and Comprise a Substantial Proportion of Obesity-Associated Increases in CD11c+ Cells in Adipose Tissue and Liver

Obesity-associated increases in adipose tissue (AT) CD11c+ cells suggest that dendritic cells (DC), which are involved in the tissue recruitment and activation of macrophages, may play a role in determining AT and liver immunophenotype in obesity. This study addressed this hypothesis. With the use of flow cytometry, electron microscopy, and loss-and-gain of function approaches, the contribution of DC to the pattern of immune cell alterations and recruitment in obesity was assessed. In AT and liver there was a substantial, high-fat diet (HFD)–induced increase in DC. In AT, these increases were associated with crown-like structures, whereas in liver the increase in DC constituted an early and reversible response to diet. Notably, mice lacking DC had reduced AT and liver macrophages, whereas DC replacement in DC-null mice increased liver and AT macrophage populations. Furthermore, delivery of bone marrow–derived DC to lean wild-type mice increased AT and liver macrophage infiltration. Finally, mice lacking DC were resistant to the weight gain and metabolic abnormalities of an HFD. Together, these data demonstrate that DC are elevated in obesity, promote macrophage infiltration of AT and liver, contribute to the determination of tissue immunophenotype, and play a role in systemic metabolic responses to an HFD.

Regulation of hexokinase II activity and expression in human muscle by moderate exercise

A single bout of exercise increases the rate of insulin-stimulated glucose uptake and metabolism in skeletal muscle. Exercise also increases insulin-stimulated glucose 6-phosphate in skeletal muscle, suggesting that exercise increases hexokinase activity. Within 3 h, exercise increases hexokinase II (HK II) mRNA and activity in skeletal muscle from rats. It is not known, however, if a single bout of moderate-intensity exercise increases HK II expression in humans. The present study was undertaken to answer this question. Six subjects had percutaneous biopsies of the vastus lateralis muscle before and 3 h after a single 3-h session of moderate-intensity aerobic (60% of maximal oxygen consumption) exercise. Glycogen synthase, HK I, and HK II activities as well as HK I and HK II mRNA content were determined from the muscle biopsy specimens. The fractional velocity of glycogen synthase was increased by 446 +/- 84% after exercise (P < 0.005). Hexokinase II activity in the soluble fraction of the homogenates increased from 1.2 +/- 0.4 to 4.5 +/- 1.6 pmol.min-1.microgram-1 (P < 0.05) but was unchanged in the particulate fraction (4.3 +/- 1.3 vs. 5.3 +/- 1.5). HK I activity in neither the soluble nor particulate fraction changed after exercise. Relative to a 28S rRNA control signal, HK II mRNA increased from 0.091 +/- 0.02 to 0.195 +/- 0.037 (P < 0.05), whereas HK I mRNA was unchanged (0.414 +/- 0.061 vs. 0.498 +/- 0.134, P < 0.20). The increase in HK II activity after moderate exercise in healthy subjects could be one factor responsible for the enhanced rate of insulin-stimulated glucose uptake seen after exercise.

Whole body overexpression of PGC-1α has opposite effects on hepatic and muscle insulin sensitivity

Type 2 diabetes is characterized by fasting hyperglycemia, secondary to hepatic insulin resistance and increased glucose production. Peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha) is a transcriptional coactivator that is thought to control adaptive responses to physiological stimuli. In liver, PGC-1alpha expression is induced by fasting, and this effect promotes gluconeogenesis. To examine whether PGC-1alpha is involved in the pathogenesis of hepatic insulin resistance, we generated transgenic (TG) mice with whole body overexpression of human PGC-1alpha and evaluated glucose homeostasis with a euglycemic-hyperinsulinemic clamp. PGC-1alpha was moderately (approximately 2-fold) overexpressed in liver, skeletal muscle, brain, and heart of TG mice. In liver, PGC-1alpha overexpression resulted in increased expression of hepatocyte nuclear factor-4alpha and the gluconeogenic enzymes phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. PGC-1alpha overexpression caused hepatic insulin resistance, manifested by higher glucose production and diminished insulin suppression of gluconeogenesis. Paradoxically, PGC-1alpha overexpression improved muscle insulin sensitivity, as evidenced by elevated insulin-stimulated Akt phosphorylation and peripheral glucose disposal. Content of myoglobin and troponin I slow protein was increased in muscle of TG mice, indicating fiber-type switching. PGC-1alpha overexpression also led to lower reactive oxygen species production by mitochondria and reduced IKK/IkappaB signaling in muscle. Feeding a high-fat diet to TG mice eliminated the increased muscle insulin sensitivity. The dichotomous effect of PGC-1alpha overexpression in liver and muscle suggests that PGC-1alpha is a fuel gauge that couples energy demands (muscle) with the corresponding fuel supply (liver). Thus, under conditions of physiological stress (i.e., prolonged fast and exercise training), increased hepatic glucose production may help sustain glucose utilization in peripheral tissues.