Effects of AgRP Inhibition on Energy Balance and Metabolism in Rodent Models

Activation of brain melanocortin-4 receptors (MC4-R) by α-melanocyte-stimulating hormone (MSH) or inhibition by agouti-related protein (AgRP) regulates food intake and energy expenditure and can modulate neuroendocrine responses to changes in energy balance. To examine the effects of AgRP inhibition on energy balance, a small molecule, non-peptide compound, TTP2515, developed by TransTech Pharma, Inc., was studied in vitro and in rodent models in vivo. TTP2515 prevented AgRP from antagonizing α-MSH-induced increases in cAMP in HEK 293 cells overexpressing the human MC4-R. When administered to rats by oral gavage TTP2515 blocked icv AgRP-induced increases in food intake, weight gain and adiposity and suppression of T4 levels. In both diet-induced obese (DIO) and leptin-deficient mice, TTP2515 decreased food intake, weight gain, adiposity and respiratory quotient. TTP2515 potently suppressed food intake and weight gain in lean mice immediately after initiation of a high fat diet (HFD) but had no effect on these parameters in lean chow-fed mice. However, when tested in AgRP KO mice, TTP2515 also suppressed food intake and weight gain during HFD feeding. In several studies TTP2515 increased T4 but not T3 levels, however this was also observed in AgRP KO mice. TTP2515 also attenuated refeeding and weight gain after fasting, an effect not evident in AgRP KO mice when administered at moderate doses. This study shows that TTP2515 exerts many effects consistent with AgRP inhibition however experiments in AgRP KO mice indicate some off-target effects of this drug. TTP2515 was particularly effective during fasting and in mice with leptin deficiency, conditions in which AgRP is elevated, as well as during acute and chronic HFD feeding. Thus the usefulness of this drug in treating obesity deserves further exploration, to define the AgRP dependent and independent mechanisms by which TTP2515 exerts its effects on energy balance.     

Cord blood ASP is predicted by maternal lipids and correlates with fetal birth weight

Background: The acylation stimulating protein (ASP) is a potent lipogenic adipokine that correlates with postprandial triglyceride (TG) clearance and is linked to the pathophysiology of obesity and related disorders. Objective: To investigate ASP levels in cord blood and its relation to maternal and cord blood lipid parameters and fetal birth weight. Methods and Procedures: Thirty nondiabetic pregnant women, their newborns, and thirty‐three nonpregnant controls were included in this study. Fasting maternal and cord blood ASP, TGs, nonesterified fatty acids (NEFAs), cholesterol, glucose levels, in addition to maternal BMI and fetal birth weight were measured. Results: No significant difference was found between cord blood ASP (16.3 ± 0.96 nmol/l) and ASP levels in the adult controls (15.7 ± 1.0 nmol/l). Cord blood ASP, however, was lower than maternal plasma ASP levels (25.4 ± 1.6 nmol/l, P < 0.001). Yet, lipid levels in cord blood, particularly TGs were markedly decreased compared to control and maternal TG levels (threefold and 7.4‐fold, P < 0.001 respectively). Maternal TGs significantly correlated with fetal birth weight (r = 0.54, P = 0.002). Multiple regression analysis showed that maternal TGs (β = 0.57, P = 0.01) and NEFAs (β = 0.43, P = 0.024) predicted 45% variation in cord blood ASP levels, independent of all measured maternal and cord blood parameters. Cord blood ASP showed a positive correlation with fetal birth weight (r = 0.524, P = 0.037) in neonates above average fetal birth weight of the studied population. Discussion: This is the first study investigating ASP in cord blood. We suggest that maternal hypertriglyceridemia is associated with increased fetal ASP production, thus enhancing fetal fat storage independent of maternal glucose variations in nondiabetic women.     

The osmotic activation of transporter ProP is tuned by both its C-terminal coiled-coil and osmotically induced changes in phospholipid composition

Transporter ProP of Escherichia coli (ProPEc) senses extracellular osmolality and mediates osmoprotectant uptake when it is rising or high. A replica of the ProPEc C terminus (Asp468-Arg497) forms an intermolecular alpha-helical coiled-coil. This structure is implicated in the osmoregulation of intact ProPEc, in vivo. Like that from Corynebacterium glutamicum (ProPCg), the ProP orthologue from Agrobacterium tumefaciens (ProPAt) sensed and responded to extracellular osmolality after expression in E. coli. The osmotic activation profiles of all three orthologues depended on the osmolality of the bacterial growth medium, the osmolality required for activation rising as the growth osmolality approached 0.7 mol/kg. Thus, each could undergo osmotic adaptation. The proportion of cardiolipin in a polar lipid extract from E. coli increased with extracellular osmolality so that the osmolality activating ProPEc was a direct function of membrane cardiolipin content. Group A ProP orthologues (ProPEc, ProPAt) share the C-terminal coiled-coil domain and were activated at low osmolalities. Like variant ProPEc-R488I, in which the C-terminal coiled-coil is disrupted, ProPEc derivatives that lack the coiled-coil and Group B orthologue ProPCg required a higher osmolality to activate. The amplitude of ProPEc activation was reduced 10-fold in its deletion derivatives. The coiled-coil structure is not essential for osmotic activation of ProP per se. However, it tunes Group A orthologues to osmoregulate over a low osmolality range. Coiled-coil lesions may impair both coiled-coil formation and interaction of ProPEc with amplifier protein ProQ. Cardiolipin may contribute to ProP adaptation by altering bulk membrane properties or by acting as a ProP ligand.     

Increased plasma acylation-stimulating protein correlates with hyperlipidemia at late gestation

Objectives: Obesity is often associated with negative consequences, including hyperlipidemia and insulin resistance. Weight gain during pregnancy is also associated with major lipid alterations. Fat storage is enhanced in early pregnancy. At late gestation, hyperlipidemia becomes a major manifestation. The acylation‐stimulating protein (ASP) is a potent lipogenic adipocytokine that correlates with postprandial triglyceride (TG) clearance in vivo and has been linked to hyperlipidemic disorders. The role of ASP during a normal pregnancy is unknown. The objective of this study was to investigate plasma ASP levels in correlation with the lipid profile during late gestation. Research Methods and Procedures: Seventy healthy women at late gestation and 60 non‐pregnant controls of similar age and prepregnancy BMI were included in a cross‐sectional study. Fasting plasma ASP levels and the lipid profile of all of the women were measured. Results: ASP levels were markedly elevated in the pregnant women (66%, p < 0.001). ASP levels correlated strongly with the elevated levels of TGs (r = 0.608, p < 0.000), apolipoprotein B (0.519, p < 0.000), and low‐density lipoprotein‐cholesterol (r = 0.405, p < 0.000). Multivariate analysis adjusting for BMI and age showed that changes in ASP levels at late gestation were best predicted by TG and apoB levels, accounting for 53.8% of plasma ASP variation. For the controls, ASP strongly correlated with BMI, which was the only significant predictor of ASP levels. Discussion: Gestational hormone alterations during pregnancy may affect ASP function as a lipogenic factor. Increased plasma ASP levels at late gestation and their strong correlation with parameters reflecting very low‐density lipoprotein accumulation are suggestive of ASP resistance, which may further contribute to the hyperlipidemic state, shifting energy in the form of TGs to the rapidly growing fetus.     

In silico analysis of Single Nucleotide Polymorphisms (SNPs) in human BRAF gene

BRAF gene mutations are frequently seen in both inherited and somatic diseases. However, the harmful mutations for BRAF gene have not been predicted in silico. Owing to the importance of BRAF gene in cell division, differentiation and secretion processes, the functional analysis was carried out to explore the possible association between genetic mutations and phenotypic variations. Genomic analysis of BRAF was initiated with SIFT followed by PolyPhen and SNPs&GO servers to retrieve the 85 deleterious non-synonymous SNPs (nsSNPs) from dbSNP. A total of 5 mutations i.e. c.406T>G (S136A), c.1446G>T (R462I), c.1556 A>G (K499E), c.1860 T>A (V600E) and c.2352 C>T (P764L) that are found to exert benign effects on the BRAF protein structure and function were chosen for further analysis. Protein structural analysis with these amino acid variants was performed by using I-Mutant, FOLD-X, HOPE, NetSurfP, Swiss PDB viewer, Chimera and NOMAD-Ref servers to check their solvent accessibility, molecular dynamics and energy minimization calculations. Our in silico analysis suggested that S136A and P764L variants of BRAF could directly or indirectly destabilize the amino acid interactions and hydrogen bond networks thus explain the functional deviations of protein to some extent. Screening for BRAF, S136A and P764Lvariants may be useful for disease molecular diagnosis and also to design the molecular inhibitors of BRAF pathways.     

BRAF gene: From human cancers to developmental syndromes

The BRAF gene encodes for a serine/threonine protein kinase that participates in the MAPK/ERK signalling pathway and plays a vital role in cancers and developmental syndromes (RASopathies). The current review discusses the clinical significance of the BRAF gene and other members of RAS/RAF cascade in human cancers and RAS/MAPK syndromes, and focuses the molecular basis and clinical genetics of BRAF to better understand its parallel involvement in both tumourigenesis and RAS/MAPK syndromes—Noonan syndrome, cardio-facio-cutaneous syndrome and LEOPARD syndrome.