Sex- and strain-specific effects of mitochondrial uncoupling on age-related metabolic diseases in high-fat diet-fed mice.

Mild uncoupling of oxidative phosphorylation is an intrinsic property of all mitochondria and may have evolved to protect cells against the production of damaging reactive oxygen species. Therefore, compounds that enhance mitochondrial uncoupling are potentially attractive anti-aging therapies; however, chronic ingestion is associated with a number of unwanted side effects. We have previously developed a controlled-release mitochondrial protonophore (CRMP) that is functionally liver-directed and promotes oxidation of hepatic triglycerides by causing a subtle sustained increase in hepatic mitochondrial inefficiency. Here, we sought to leverage the higher therapeutic index of CRMP to test whether mild mitochondrial uncoupling in a liver-directed fashion could reduce oxidative damage and improve age-related metabolic disease and lifespan in diet-induced obese mice. Oral administration of CRMP (20 mg/[kg-day] × 4 weeks) reduced hepatic lipid content, protein kinase C epsilon activation, and hepatic insulin resistance in aged (74-week-old) high-fat diet (HFD)-fed C57BL/6J male mice, independently of changes in body weight, whole-body energy expenditure, food intake, or markers of hepatic mitochondrial biogenesis. CRMP treatment was also associated with a significant reduction in hepatic lipid peroxidation, protein carbonylation, and inflammation. Importantly, long-term (49 weeks) hepatic mitochondrial uncoupling initiated late in life (94-104 weeks), in conjugation with HFD feeding, protected mice against neoplastic disorders, including hepatocellular carcinoma (HCC), in a strain and sex-specific manner. Taken together, these studies illustrate the complex variation of aging and provide important proof-of-concept data to support further studies investigating the use of liver-directed mitochondrial uncouplers to promote healthy aging in humans.

Insight into nucleophilic fragmentation mechanisms by glutamic acid side chain in singly protonated glutathione and related peptidyl ions.

Fragmentation mechanisms of the singly protonated glutathione (γ-ECG) and its synthetic analogue peptides (ECG and PPECG) have been investigated by liquid chromatography tandem-mass spectrometry and theoretical calculations. In the mass spectra, similar fragmentation patterns were observed for γ-ECG and ECG, but a completely different one was found in the case of PPECG. The E-C amide bond cleavage is the predominant pathway for the fragmentation of γ-ECG and ECG, whereas the additional N-terminal prolyl residues in PPECG significantly suppress the E-C amide bond cleavage. Theoretical calculations reveal that the fragmentation efficiencies of the E-C bonds in the protonated γ-ECG and ECG are much higher than that in the protonated PPECG, being attributed to their lower barriers of the potential energy; clearly the introduction of two prolyl residues can increase substantially the potential energy barrier. In the proposed mechanism, the protonated E-C amide bonds in the three peptides are first weakened followed by a nucleophilic addition by the glutamyl carboxyl oxygen atom in side chain, leading to the breaking of the E-C amide bonds. However, the processes of E-C bond fragmentation for three protonated analogs were not collaborative. Protonated amide bonds first fragment, then the nucleophilic addition by the side chain of glutamyl carboxyl oxygen atom takes places. On the other hand, the prolyl residues in PPECG can largely diminish the nucleophilic addition, resulting in a much lower efficiency of its E-C amide bond breaking. Distance analysis indicates that breaking the E-C amide bonds in the protonated γ-ECG, ECG, and PPECG ions could not occur without the assistance from the nucleophilic attack, highlighting an asynchronous collaborative process in the bond breakings.

Obesity-associated, but not obesity-independent, tumors respond to insulin by increasing mitochondrial glucose oxidation.

Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association remain under debate. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we applied and validated a stable isotope method to measure the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose) in tumor cells. Using this method, we found that three tumor cell lines associated with obesity (colon cancer [MC38], breast cancer [4T1], and prostate cancer [TRAMP-C3] cells) increase VPDH/VCS in response to physiologic concentrations of insulin. In contrast, three tumor cell lines that are not associated with obesity (melanoma [YUMM1.7], B cell lymphoma [BCL1 clone 5B1b], and small cell lung cancer [NCI-H69] cells) exhibited no oxidative response to insulin. The observed increase in glucose oxidation in response to insulin correlated with a dose-dependent increase in cell division in obesity-associated tumor cell lines when grown in insulin, whereas no alteration in cell division was seen in tumor types not associated with obesity. These data reveal that a shift in substrate preference in the setting of physiologic insulin may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity.

Uncoupling Hepatic Oxidative Phosphorylation Reduces Tumor Growth in Two Murine Models of Colon Cancer.

Obesity is associated with colon cancer pathogenesis, but the underlying mechanism is actively debated. Here, we confirm that diet-induced obesity promotes tumor growth in two murine colon cancer models and show that this effect is reversed by an orally administered controlled-release mitochondrial protonophore (CRMP) that acts as a liver-specific uncoupler of oxidative phosphorylation. This agent lowered circulating insulin, and the reduction of tumor growth was abrogated by an insulin infusion raising plasma insulin to the level of high-fat-fed mice. We also demonstrate that hyperinsulinemia increases glucose uptake and oxidation in vivo in tumors and that CRMP reverses these effects. This study provides evidence that perturbations of whole-organism energy balance or hepatic energy metabolism can influence neoplastic growth. Furthermore, the data show that glucose uptake and utilization by cancers in vivo are not necessarily constitutively high but rather may vary according to the hormonal milieu.

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.

Role of patatin-like phospholipase domain-containing 3 on lipid-induced hepatic steatosis and insulin resistance in rats.

UNLABELLED: Genome-wide array studies have associated the patatin-like phospholipase domain-containing 3 (PNPLA3) gene polymorphisms with hepatic steatosis. However, it is unclear whether PNPLA3 functions as a lipase or a lipogenic enzyme and whether PNPLA3 is involved in the pathogenesis of hepatic insulin resistance. To address these questions we treated high-fat-fed rats with specific antisense oligonucleotides to decrease hepatic and adipose pnpla3 expression. Reducing pnpla3 expression prevented hepatic steatosis, which could be attributed to decreased fatty acid esterification measured by the incorporation of [U-(13) C]-palmitate into hepatic triglyceride. While the precursors for phosphatidic acid (PA) (long-chain fatty acyl-CoAs and lysophosphatidic acid [LPA]) were not decreased, we did observe an ∼20% reduction in the hepatic PA content, ∼35% reduction in the PA/LPA ratio, and ∼60%-70% reduction in transacylation activity at the level of acyl-CoA:1-acylglycerol-sn-3-phosphate acyltransferase. These changes were associated with an ∼50% reduction in hepatic diacylglycerol (DAG) content, an ∼80% reduction in hepatic protein kinase Cε activation, and increased hepatic insulin sensitivity, as reflected by a 2-fold greater suppression of endogenous glucose production during the hyperinsulinemic-euglycemic clamp. Finally, in humans, hepatic PNPLA3 messenger RNA (mRNA) expression was strongly correlated with hepatic triglyceride and DAG content, supporting a potential lipogenic role of PNPLA3 in humans. CONCLUSION: PNPLA3 may function primarily in a lipogenic capacity and inhibition of PNPLA3 may be a novel therapeutic approach for treatment of nonalcoholic fatty liver disease-associated hepatic insulin resistance.

Characterization of the hyperphagic response to dietary fat in the MC4R knockout mouse.

Defective melanocortin signaling causes hyperphagic obesity in humans and the melanocortin-4 receptor knockout mouse (MC4R(-/-)). The human disease most commonly presents, however, as haploinsufficiency of the MC4R. This study validates the MC4R(+/-) mouse as a model of the human disease in that, like the MC4R(-/-), the MC4R(+/-) mouse also exhibits a sustained hyperphagic response to dietary fat. Furthermore, both saturated and monounsaturated fats elicit this response. N-acylphosphatidylethanolamine (NAPE) is a signaling lipid induced after several hours of high-fat feeding, that, if dysregulated, might explain the feeding behavior in melanocortin obesity syndrome. Remarkably, however, MC4R(-/-) mice produce elevated levels of NAPE and are fully responsive to the anorexigenic activity of NAPE and oleoylethanolamide. Interestingly, additional differences in N-acylethanolamine (NAE) biochemistry were seen in MC4R(-/-) animals, including reduced plasma NAE levels and elevated hypothalamic levels of fatty acid amide hydrolase expression. Thus, while reduced expression of NAPE or NAE does not explain the high-fat hyperphagia in the melanocortin obesity syndrome, alterations in this family of signaling lipids are evident. Analysis of the microstructure of feeding behavior in response to dietary fat in the MC4R(-/-) and MC4R(+/-) mice indicates that the high-fat hyperphagia involves defective satiation and an increased rate of food intake, suggesting defective satiety signaling and enhanced reward value of dietary fat.

Fasting hyperglycemia is not associated with increased expression of PEPCK or G6Pc in patients with Type 2 Diabetes.

Fasting hyperglycemia in patients with type 2 diabetes mellitus (T2DM) is attributed to increased hepatic gluconeogenesis, which has been ascribed to increased transcriptional expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase, catalytic (G6Pc). To test this hypothesis, we examined hepatic expression of these 2 key gluconeogenic enzymes in 2 rodent models of fasting hyperglycemia and in patients with T2DM. In rats, high-fat feeding (HFF) induces insulin resistance but a robust beta-cell response prevents hyperglycemia. Fasting hyperglycemia was induced in the first rat model by using nicotinamide and streptozotocin to prevent beta-cell compensation, in combination with HFF (STZ/HFF). In a second model, control and HFF rats were infused with somatostatin, followed by portal vein infusion of insulin and glucagon. Finally, the expression of these enzymes was measured in liver biopsy samples obtained from insulin sensitive, insulin resistant, and untreated T2DM patients undergoing bariatric surgery. Rats treated with STZ/HFF developed modest fasting hyperglycemia (119 +/- 4 vs. 153 +/- 6 mg/dL, P < 0.001) and increased rates of endogenous glucose production (EGP) (4.6 +/- 0.6 vs. 6.9 +/- 0.6 mg/kg/min, P = 0.02). Surprisingly, the expression of PEPCK or G6Pc was not increased. Matching plasma insulin and glucagon with portal infusions led to higher plasma glucoses in the HFF rats (147 +/- 4 vs. 161 +/- 4 mg/dL, P = 0.05) with higher rates of EGP and gluconeogenesis. However, PEPCK and G6Pc expression remained unchanged. Finally, in patients with T2DM, hepatic expression of PEPCK or G6Pc was not increased. Thus, in contrast to current dogma, these data demonstrate that increased transcriptional expression of PEPCK1 and G6Pc does not account for increased gluconeogenesis and fasting hyperglycemia in patients with T2DM.

N-acylphosphatidylethanolamine, a gut- derived circulating factor induced by fat ingestion, inhibits food intake.

N-acylphosphatidylethanolamines (NAPEs) are a relatively abundant group of plasma lipids of unknown physiological significance. Here, we show that NAPEs are secreted into circulation from the small intestine in response to ingested fat and that systemic administration of the most abundant circulating NAPE, at physiologic doses, decreases food intake in rats without causing conditioned taste aversion. Furthermore, (14)C-radiolabeled NAPE enters the brain and is particularly concentrated in the hypothalamus, and intracerebroventricular infusions of nanomolar amounts of NAPE reduce food intake, collectively suggesting that its effects may be mediated through direct interactions with the central nervous system. Finally, chronic NAPE infusion results in a reduction of both food intake and body weight, suggesting that NAPE and long-acting NAPE analogs may be novel therapeutic targets for the treatment of obesity.