Metformin and dipeptidase peptidyl-4 inhibitor differentially modulate the intestinal microbiota and plasma metabolome of metabolically dysfunctional mice

Objectives: Recent evidence indicates that gut microbiota is altered considerably by a variety of commonly prescribed medications. This study assessed the impact of 2 antidiabetic therapeutics on gut microbiota and markers of cardiometabolic disease in metabolically dysfunctional mice. Methods: C57BL/6 mice were fed a high-fat diet for 24 weeks while receiving 1 of 2 antidiabetic therapeutics—metformin or dipeptidase peptidyl-4 (DPP-4) inhibitor, PKF-275-055—for the final 12 weeks. Mice were assessed for weight gain, glucose and cholesterol metabolism, and adiposity. In addition, cecal microbiota was analyzed by 16S compositional sequencing, and plasma metabolome was analyzed by liquid chromatography with tandem mass spectrometry. Results: Both therapeutics had similar metabolic effects, attenuating mesenteric adiposity and improving cholesterol metabolism and insulin sensitivity. However, multivariate analyses of microbiota and metabolomics data revealed clear divergence of the therapeutic groups. Although both metformin and PKF-275-055 mice displayed significantly decreased Firmicutes/Bacteroidetes ratios, only metformin harboured metabolic health-associated Akkermansia, Parabacteroides and Christensenella. Paradoxically, metformin also reduced α diversity, a metric frequently associated with host metabolic fitness. PKF-275-055 mice displayed elevated levels of butyrate-producing Ruminococcus and acetogen Dorea, with reduced levels of certain plasma sphingomyelin, phosphatidylcholine and lysophosphatidylcholine entities. In turn, metformin reduced levels of acylcarnitines, a functional group associated with systemic metabolic dysfunction. Finally, several associations were identified between metabolites and altered taxa. Conclusions: This study represents the first direct comparison of the microbiota-modifying effects of metformin and a DPP-4 inhibitor, and proposes several putative microbial targets both in terms of novel therapeutic development and adverse effect prevention

The chemical basis of serine palmitoyltransferase inhibition by myriocin

Sphingolipids (SLs) are essential components of cellular membranes formed from the condensation of L-serine and a long-chain acyl thioester. This first step is catalyzed by the pyridoxal-5'-phosphate (PLP)-dependent, enzyme serine palmitoyltransferase (SPT) which is a promising therapeutic target. The fungal natural product myriocin is a potent inhibitor of SPT and is widely used to block SL biosynthesis despite a lack of a detailed understanding of its molecular-mechanism. By combining spectroscopy, mass spectrometry, X-ray crystallography, and kinetics, we have characterized the molecular details of SPT inhibition by myriocin. Myriocin initially forms an external aldimine with PLP at the active site, and a structure of the resulting co-complex explains its nanomolar affinity for the enzyme. This co-complex then catalytically degrades via an unexpected 'retro-aldol-like' cleavage mechanism to a C18 aldehyde which in turn acts as a suicide inhibitor of SPT by covalent modification of the essential catalytic lysine. This surprising dual mechanism of inhibition rationalizes the extraordinary potency and longevity of myriocin inhibition.</p

Nutrient regulation of energy metabolism in relation to obesity and type 2 diabetes

Over the past decades many western countries have witnessed an epidemic rise in obesity, insulin resistance and type 2 diabetes. There clearly is a link between increased fat storage and decreased insulin sensitivity leading to type 2 diabetes, but the mechanistic explanations have been difficult to pinpoint biochemically and genetically. In this thesis we have observed that there likely are differences in the way skeletal muscle myotubes, derived from type 2 diabetic or healthy controls metabolize fatty acids and respond towards various lipids. We have observed that T2D myotubes on average have a reduced capacity for mitochondrial fatty acids oxidation as compared to myotubes derived from healthy donors. During periods of high fatty acid load, this difference in oxidative capacity may either result in ectopic storage of intramyocellular triacylglycerols imTAG or an increased level of fatty acid metabolic intermediates that may further interfere with insulin signaling. Preincubation of myotubes with eicosapentaenoic acid (EPA) resulted in enhanced fatty acid uptake and storage of imTAG, but also reduced levels of total intracellular acyl-CoA with improved glucose oxidation and unchanged insulin mediated glucose uptake. Preincubetion with the sulfur modified fatty acid analogue tetradecylthioacetic acid (TTA) improved mitochondrial fatty acid oxidation in both T2D and control myotubes, and may thus help alleviate this metabolic difference in T2D. TTA given as a supplement to rodents fed a high fat diet for 7 weeks resulted in 40% reduced weight gain and similar marked reductions in white adipose tissue mass. TTA has previously been shown to activate all peroxisomal proliferator activated receptors (PPARs), and the reduced weight gain possibly resulted because of hepatic PPARα activation, increasing energy expenditure through some unknown mechanism likely involving ectopic uncoupling protein 3 (ucp3) expression

Implication of ceramide 1-phosphate in obesity-associated processes. Role of PEMT

220 p.Obesity is the most common metabolic disease in developed nations and has become a global epidemic in recent years. Therefore, understanding the molecular mechanisms that regulate obesity-associated processes, such as cell migration, adipogenesis and inflammation, can be a crucial step for developing novel therapeutic strategies to control obesity and obesity-related pathologies. It is well known that sphingolipid content in tissue undergoes dramatic alterations in metabolic diseases suggesting that these lipids might mediate the pathology associated with metabolic disease. In this thesis, we demonstrate that C1P enhances macrophage migration, an action that requires the activation of matrix metalloprotease- 2 and -9. We also provide evidence suggesting that adipogenesis is associated with an increase in CERK protein expression and activity. In addition, we demonstrate that exogenous C1P inhibits adipocyte differentiation of 3T3-L1 cells, as confirmed by a reduction in triglyceride accumulation and a reduction in the expression of adipocyte specific genes. This action of C1P implicates the activation of extracellular signal-regulated kinases (ERK1/2). We have also found that lack of phosphatidylethanolamine N-methyltransferase (PEMT), the enzyme responsible for phosphatidylcholine biosynthesis in liver and whose expression is blocked by C1P, attenuates obesity-associated inflammation. This occurs by decreasing both, the number of classically activated M1 proinflammatory macrophages and pro-inflammatory cytokine levels in adipose tissue. Moreover, we also demonstrate that PEMT over expression induces macrophage migration, an action that requires the activation of PI3K/Akt1/mTOR pathway. These findings may help to develop new therapeutic strategies for the treatment of obesity and obesity-related diseases

Implication of ceramide 1-phosphate in obesity-associated processes. Role of PEMT

220 p.Obesity is the most common metabolic disease in developed nations and has become a global epidemic in recent years. Therefore, understanding the molecular mechanisms that regulate obesity-associated processes, such as cell migration, adipogenesis and inflammation, can be a crucial step for developing novel therapeutic strategies to control obesity and obesity-related pathologies. It is well known that sphingolipid content in tissue undergoes dramatic alterations in metabolic diseases suggesting that these lipids might mediate the pathology associated with metabolic disease. In this thesis, we demonstrate that C1P enhances macrophage migration, an action that requires the activation of matrix metalloprotease- 2 and -9. We also provide evidence suggesting that adipogenesis is associated with an increase in CERK protein expression and activity. In addition, we demonstrate that exogenous C1P inhibits adipocyte differentiation of 3T3-L1 cells, as confirmed by a reduction in triglyceride accumulation and a reduction in the expression of adipocyte specific genes. This action of C1P implicates the activation of extracellular signal-regulated kinases (ERK1/2). We have also found that lack of phosphatidylethanolamine N-methyltransferase (PEMT), the enzyme responsible for phosphatidylcholine biosynthesis in liver and whose expression is blocked by C1P, attenuates obesity-associated inflammation. This occurs by decreasing both, the number of classically activated M1 proinflammatory macrophages and pro-inflammatory cytokine levels in adipose tissue. Moreover, we also demonstrate that PEMT over expression induces macrophage migration, an action that requires the activation of PI3K/Akt1/mTOR pathway. These findings may help to develop new therapeutic strategies for the treatment of obesity and obesity-related diseases

Insulinorésistance musculaire induite par les céramides : étude des mécanismes d'action et de l'implication du transporteur CERT

Obesity and type 2 diabetes are associated with a sedentary lifestyle and a diet rich in fat. Indeed, saturated fatty acids accumulate in non-adipose tissue such as skeletal muscle to generate lipids called ceramides (CER). My thesis project was divided into two parts with the objective to prevent CER to act. We have shown that, depending on the structure of the plasma membrane, CER alter the insulin signaling pathway by targeting PKB, a key insulin signalling protein, via a PKCζ pathway in L6 myotubes and a PP2A pathway in C2C12 myotubes. We also demonstrated that CER affect insulin sensitivity via the PKCζ pathway in human muscle cells. Once CER generated in the endoplasmic reticulum (ER), they are transported to the Golgi by a carrier called CERT to be metabolized into sphingomyelin (SM). Studies have shown that the transformation of CER into SM could be a crucial step to prevent CER to act. In several muscle insulin resistance models, expression of CERT is decreased and we demonstrated the importance of the transport of ceramide from the ER to the Golgi by inhibiting artificially the activity or the expression of CERT. In contrast, overexpression of CERT enhances insulin sensitivity in muscle cells in lipotoxiques conditions. Our results show that CERT plays a crucial role in mechanisms leading to the development of muscle insulin resistance since its presence is essential for maintaining normal traffic of CER between the ER and the Golgi.L'obésité et le diabète de type 2 sont associés à la sédentarité et à une alimentation riche en graisses. En effet, les acides gras saturés s'accumulent dans les tissus non adipeux, comme les muscles squelettiques pour générer des lipides appelés céramides (CER). Mon projet de thèse s'est articulé en deux parties dont l'objectif est d'empêcher les CER d'agir. Nous avons montré que, selon la structure de la membrane plasmique, les CER altèrent la voie de signalisation insulinique en ciblant la PKB, protéine clef de la voie insulinique, via la voie PKC? dans les myotubes L6 et la voie PP2A dans les myotubes C2C12. Nous avons aussi mis en évidence que les CER altèrent la sensibilité à l'insuline via la voie PKC? dans les cellules musculaires humaines. Une fois les CER produits au niveau du réticulum endoplasmique (RE), ils sont transportés au Golgi par un transporteur CERT pour y être métabolisés en sphingomyéline (SM) et des études ont montré que la transformation des CER en SM pouvait être une étape cruciale pour empêcher les CER d'agir. Dans plusieurs modèles d'insulino-résistance musculaire, l'expression de CERT est diminuée et nous avons démontré l'importance du transport des céramides du RE vers le Golgi en inhibant artificiellement l'activité ou l'expression de CERT. A l'opposé, la surexpression de CERT améliore la sensibilité à l'insuline dans les cellules musculaires dans des conditions lipotoxiques. Nos résultats montrent que CERT joue un rôle crucial dans les mécanismes conduisant au développement de l'insulinorésistance musculaire puisque sa présence est essentielle pour le maintien d'un trafic normal des CER entre le RE et le golgi

Studies on the structure, mechanism and inhibition of serine palmitoyltransferase

Sphingolipids and ceramides are essential components of cellular membranes and important signalling molecules. Because of a growing appreciation for their diverse biological roles, understanding of the biosynthesis and regulation of sphingolipids has recently become a key goal in drug discovery. Serine palmitoyltransferase (SPT) is a pyridoxal 5′-phosphate (PLP)-dependent enzyme that catalyses the condensation between L-serine and a long-chain acyl thioester such as palmitoyl-CoA (C16-CoA). This first step in sphingolipid biosynthesis is conserved in all organisms studied to date, from microbes to man. The fungal natural product myriocin is a potent inhibitor of SPT; however, the molecular details of inhibition are not fully understood. Myriocin contains a long alkyl chain and a polar head group thus it displays features of both SPT substrates. Therefore, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic of a key transition state of the catalytic mechanism. Through a combination of UV-vis spectroscopy, mass spectrometry, x-ray crystallography and enzyme inhibition assays it has been possible to study the interaction between S. paucimobilis SPT and myriocin. I have shown that myriocin initially forms an inhibitory PLP:myriocin aldimine complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is susceptible to unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This aldehyde is then capable of covalently modifying the active site lysine265, forming a second (suicide) inhibitory complex and rendering the enzyme catalytically inactive. Substitution of the active site lysine produced SPT K265A, an inactive enzyme that did not catalyse the breakdown of the PLP:myriocin complex. However, the determination of the crystal structure of the SPT K265A:PLP-myriocin complex revealed that the myriocin had undergone decarboxylation. Nevertheless, this SPT:PLP:decarboxymyriocin structure revealed details about myriocin’s mechanism of inhibition for the first time. The novel mechanism of myriocin degradation has implications on the structure activity relationship (SAR) and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and further expands the range of reactions catalysed by this important enzyme. As well as inhibition studies the structure of bacterial SPT was also examined by preparing an N-terminally truncated S. paucimobilis SPT. This version, shortened by 21 amino acids, was ~5-fold slower than the wild-type enzyme and suggests that the N-terminus may play a role in catalysis. Additional work has been undertaken to study an unusual membrane-bound viral SPT, composed of two naturally fused open reading frames (SPT2-SPT1) with the proposed SPT2 domain at the N-terminus and the SPT1 domain at the C-terminus. To study soluble mimics of this interesting fusion I prepared a bacterial S. paucimobilis SPT fused wild-type and mutant construct and isolated a fused SPT2-SPT1 with what appears to be single PLPbinding site

Ceramide Metabolism Regulates A Neuronal Nadph Oxidase Influencing Neuron Survival During Inflammation

Thesis (Ph.D.) University of Alaska Fairbanks, 2009Inflammation is a major component of acute and chronic pathologies of the central nervous system, including psychiatric disorders. Microglia respond to pathogens, injury, and toxins by secreting inflammatory mediators including pro-inflammatory cytokines in an event known as neuroinflammation. This thesis research investigated a link between neuroinflammation and oxidative stress, and ultimately neurodegeneration. The cytokine tumor necrosis factor alpha was shown to stimulate a neuronal NADPH oxidase (NOX), specifically by stimulating the production of ceramide and ceramide-1-phosphate via Mg 2+-neutral sphingomyelinase (Mg2+-nSMase) and ceramide kinase. Intriguingly, glucosylceramide blocked NOX activation, linking ceramide neutralization directly to a decline in oxidative stress. Most importantly, NOX activity interfered with actin and sphingosine kinase-1 via oxidation, demonstrating a positive and detrimental feedback mechanism that impedes neuronal survival pathways. Interestingly, crude extracts from wild Alaskan bog blueberries showed the ability to interfere with Mg2+-nSMase, demonstrating a specific neuroprotective property of the berry. Altogether, this thesis research defined a key neuronal pathway linking inflammation to oxidative stress via ceramide metabolism, potentially allowing for future therapeutic development to improve neuronal function and survival

Interaction of diet and adipose glucocorticoid action in metabolic syndrome

Metabolic syndrome, a combination of co-associated features including visceral obesity, dyslipidemia, hypertension, insulin resistance and increased risk of cardiovascular disease, has reached epidemic levels. Recent evidence has indicated a pathogenic role for elevated glucocorticoid (GC) levels within adipose tissue specifically, in obesity. Levels of the glucocorticoid regenerating enzyme 11β- hydroxysteroid dehydrogenase type 1 (11β-HSD-1), which converts inactive GC into active forms within cells, are elevated in adipose tissues of obese humans. Transgenic overexpression of 11β-HSD1 in adipose tissue recapitulates metabolic syndrome in mice, including visceral obesity, perhaps because glucocorticoid receptor (GR) levels are higher in visceral than subcutaneous adipose tissue. Therefore, increased adipose GC action, especially in visceral adipose tissue, might explain the striking similarities between idiopathic metabolic syndrome and rare Cushing’s syndrome of plasma GC excess. One of the major contributors to obesity and metabolic syndrome is consumption of excessive amounts of dietary fat. Specifically, saturated fats are associated with obesity and insulin resistance whereas unsaturated fats are reported to ameliorate some aspects of metabolic disease. Interestingly, chronic high fat feeding in mice decreases adipose 11β-HSD1 expression, usually without changing expression in liver. The reduced glucocorticoid regeneration in adipose tissue may ameliorate metabolic disease. This study investigated the effect of diets enriched in saturated or unsaturated fats on adipose tissue glucocorticoid action, focusing on 11β-HSD1 and glucocorticoid receptor expression. Two studies were carried out, one with pair-feeding (to control low-fat diet) and one with ab libitum feeding. In both studies, a diet high in saturated fat (stearate) resulted in significant weight loss whereas a diet consisting of 58% mixed fats resulted in significant weight gain. In the ab libitium study (but not in pair-fed animals) both mono-unsaturated (oleic) and poly-unsaturated (safflower) fat-enriched diets induced weight gain and hyperinsulinaemia and lowered levels of adipose11β-HSD1 when compared to control diet. These data suggest that diets high in saturated fats elevate adipose tissue and liver 11β-HSD1 levels which might contribute detrimentally to, or offset, any improvements in the systemic metabolic profile. Conversely, unsaturated fats cause an adipose-specific down-regulation of 11β-HSD1. In addition, genetic evidence from human and animal studies has shown that regulation of glucocorticoid production and receptor density may be an important factor determining visceral adiposity. The role of adipocyte GR levels in determining visceral adiposity was investigated. Two types of transgenic mice were generated with altered GR levels in adipocytes using the adipocyte-specific aP2 promoter; one with a “sense” rat GR cDNA (2 independent lines; D- and B-FSG) and one with an “anti-sense” rat GR-cDNA (GR-5). Initial characterisation of D- and B-FSG mice showed expression of rat GR mRNA in both female and male transgenic mice with D-FSG showing a higher copy number than B-FSG. In D-FSG transgenic mice, female mice of the F1 generation had greater adipose tissue mass than non-transgenic littermates. A 6-month high fat study was then carried out on line D-FSG. However, no differences in body weight, food intake, adipose tissue weight or blood pressure were found in either males or females. Further investigations of the levels of rat GR mRNA expression within various adipose tissues showed variable transgene expression between different generations of B- and D-FSG lines and even between adipose tissues within the same mouse. In contrast, following 6-months high fat diet, female mice under-expressing GR within adipose tissues (GR-5) showed a significant reduction in body weight and subcutaneous adipose tissue weight compared to their non-transgenic littermates. These data suggest that adipose GR density is an important determinant of visceral adiposity, in a sex-specific manner. Collectively, the data in this thesis support a role for GR density and GC metabolism in adipose tissue as important determinants of adiposity in mice. Drugs that manipulate these targets are of therapeutic interest