Mercedes N. Munkonda

Circulating Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Regulates VLDLR Protein and Triglyceride Accumulation in Visceral Adipose Tissue

Objective—Proprotein convertase subtilisin/kexin 9 (PCSK9) promotes the degradation of the low-density lipoprotein receptor (LDLR), and its gene is the third locus implicated in familial hypercholesterolemia. Herein, we investigated the role of PCSK9 in adipose tissue metabolism. Methods and Results—At 6 months of age, Pcsk9−/− mice accumulated ≈80% more visceral adipose tissue than wild-type mice. This was associated with adipocyte hypertrophy and increased in vivo fatty acid uptake and ex vivo triglyceride synthesis. Moreover, adipocyte hypertrophy was also observed in Pcsk9−/− Ldlr−/− mice, indicating that the LDLR is not implicated. Rather, we show here by immunohistochemistry that Pcsk9−/− males and females exhibit 4- and ≈40-fold higher cell surface levels of very-low-density lipoprotein receptor (VLDLR) in perigonadal depots, respectively. Expression of PCSK9 in the liver of Pcsk9−/− females reestablished both circulating PCSK9 and normal VLDLR levels. In contrast, specific inactivation of PCSK9 in the liver of wild-type females led to ≈50-fold higher levels of perigonadal VLDLR. Conclusion—In vivo, endogenous PCSK9 regulates VLDLR protein levels in adipose tissue. This regulation is achieved by circulating PCSK9 that originates entirely in the liver. PCSK9 is thus pivotal in fat metabolism: it maintains high circulating cholesterol levels via hepatic LDLR degradation, but it also limits visceral adipogenesis likely via adipose VLDLR regulation.

C5L2 receptor disruption enhances the development of diet-induced insulin resistance in mice

INTRODUCTION Acylation stimulating protein (ASP) is a hormone secreted by the adipose tissue that has been shown to increase triglyceride storage and glucose transport in adipocytes. These effects are mediated by C5L2 receptor, which has also been associated with inflammatory effects. C5L2 deficient mice on a low-fat diet are hyperphagic yet lean due to increased energy expenditure. The present study assessed insulin sensitivity and metabolic and inflammatory changes in C5L2KO mice vs WT in diet-induced obesity. METHODS We placed C5L2KO and WT mice on a diabetogenic diet for 12 weeks and examined in vivo and ex vivo metabolism. RESULTS C5L2KO mice on a diabetogenic diet exhibit decreased insulin sensitivity. Whole body substrate partitioning is evidenced through increased glucose uptake by the liver and decreased uptake by adipose tissue and skeletal muscle. Lipid content of both liver and skeletal muscle was higher in C5L2KO mice vs WT. Furthermore, elevated levels of macrophage markers were found in adipose tissue, liver and skeletal muscle of C5L2KO mice vs WT. Several inflammatory cytokines such as IL-6, MIP-1α and KC were also elevated in plasma of C5L2KO mice vs WT. CONCLUSIONS Overall, we demonstrated that C5L2KO mice fed a diabetogenic diet develop more severe insulin resistance than WT mice through altered substrate partitioning, ectopic fat deposition and a pro-inflammatory phenotype.

Characterization of a monoclonal antibody as the first specific inhibitor of human NTP diphosphohydrolase‐3

The study and therapeutic modulation of purinergic signaling is hindered by a lack of specific inhibitors for NTP diphosphohydrolases (NTPDases), which are the terminating enzymes for these processes. In addition, little is known of the NTPDase protein structural elements that affect enzymatic activity and which could be used as targets for inhibitor design. In the present study, we report the first inhibitory monoclonal antibodies specific for an NTPDase, namely human NTPDase3 (EC 3.6.1.5), as assessed by ELISA, western blotting, flow cytometry, immunohistochemistry and inhibition assays. Antibody recognition of NTPDase3 is greatly attenuated by denaturation with SDS, and abolished by reducing agents, indicating the significance of the native conformation and the disulfide bonds for epitope recognition. Using site‐directed chemical cleavage, the SDS‐resistant parts of the epitope were located in two fragments of the C‐terminal lobe of NTPDase3 (i.e. Leu220–Cys347 and Cys347–Pro485), which are both required for antibody binding. Additional site‐directed mutagenesis revealed the importance of Ser297 and the fifth disulfide bond (Cys399–Cys422) for antibody binding, indicating that the discontinuous inhibitory epitope is located on the extracellular C‐terminal lobe of NTPDase3. These antibodies inhibit recombinant NTPDase3 by 60–90%, depending on the conditions. More importantly, they also efficiently inhibit the NTPDase3 expressed in insulin secreting human pancreatic islet cells in situ. Because insulin secretion is modulated by extracellular ATP and purinergic receptors, this finding suggests the potential application of these inhibitory antibodies for the study and control of insulin secretion.

Adiponectin and Leptin Metabolic Biomarkers in Chinese Children and Adolescents

Objective. To evaluate leptin and adiponectin as biomarkers of metabolic syndrome (MS) risk factors even in nonobese children/adolescents. Methods. Serum leptin, adiponectin, leptin:adiponectin ratio, lipids, glucose, and insulin concentrations as well as body size parameters and pubertal development were evaluated in a large population of Chinese children/adolescents (n = 3505, 6–18 years, 1722 girls and 1783 boys). Results. Leptin concentration increased while adiponectin decreased with obesity, both were influenced by pubertal development. Central obesity had an additive effect on leptin levels (above obesity alone). Leptin/adiponectin increased 8.4-fold and 3.2-fold in overweight/obesity, and 15.8- and 4.5-fold with obesity plus MS, in early and late puberty, respectively. Even in normal weight children/adolescents, higher leptin and lower adiponectin concentrations associated with increased risk profile. Conversely, overweight/obese with lower leptin or higher adiponectin concentrations had a less compromised metabolic profile. Conclusion. Leptin, adiponectin, and leptin:adiponectin ratio are informative biomarkers for obesity, central obesity, MS, and abnormal metabolic profile even in normal weight children/adolescents.

Recombinant acylation stimulating protein administration to C3-/- mice increases insulin resistance via adipocyte inflammatory mechanisms.

Background Complement 3 (C3), a key component of the innate immune system, is involved in early inflammatory responses. Acylation stimulating protein (ASP; aka C3adesArg), a C3 cleavage product, is produced in adipose tissue and stimulates lipid storage. We hypothesized that, depending on the diet, chronic ASP administration in C3−/− mice would affect lipid metabolism and insulin sensitivity via an adaptive adipose tissue inflammatory response. Methodology/Principal Findings C3−/− mice on normal low fat diet (ND) or high fat diet (HFD) were chronically administered recombinant ASP (rASP) for 25 days via an osmotic mini-pump. While there was no effect on food intake, there was a decrease in activity, with a relative increase in adipose tissue weight on ND, and a shift in adipocyte size distribution. While rASP administration to C3−/− mice on a ND increased insulin sensitivity, on a HFD, rASP administration had the opposite effect. Specifically, rASP administration in C3−/− HFD mice resulted in decreased gene expression of IRS1, GLUT4, SREBF1 and NFκB in muscle, and decreased C5L2 but increased JNK, CD36, CD11c, CCR2 and NFκB gene expression in adipose tissue as well as increased secretion of proinflammatory cytokines (Rantes, KC, MCP-1, IL-6 and G-CSF). In adipose tissue, although IRS1 and GLUT4 mRNA were unchanged, insulin response was reduced. Conclusion The effects of chronic rASP administration are tissue and diet specific, rASP administration enhances the HFD induced inflammatory response leading to an insulin-resistant state. These results suggest that, in humans, the increased plasma ASP associated with obesity and cardiovascular disease could be an additional factor directly contributing to development of metabolic syndrome, insulin resistance and diabetes.

Fluorescent N2,N3-ε-Adenine Nucleoside and Nucleotide Probes: Synthesis, Spectroscopic Properties, and Biochemical Evaluation†

N1,N6‐ethenoadenine, ε‐A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these ε‐A analogues lack the H‐bonding capability of adenine. To improve the fluorescence characteristics while preserving the H‐bonding pattern required for molecular recognition, we designed a novel probe: N2,N3‐etheno‐adenosine, (N2,N3‐ε‐A). Here, we describe four novel syntheses of the target ε‐nucleoside and related analogues. These methods are short, facile, and provide the product regiospecifically. In addition, we report the absorption and emission spectra of N2,N3‐ε‐A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N2,N3‐ε‐A in water is observed at 420 nm (ϕ=0.03, excitation at 290 nm). The biochemical relevance of the new probe was evaluated with respect to the P2Y1 receptor and NTPDases 1 and 2. N2,N3‐ε‐ATP was found to be almost equipotent with ATP at the P2Y1 receptor and was hydrolyzed by NTPDases 1 and 2 at about 80 % of the rate of ATP. Furthermore, protein binding does not seem to shift the fluorescence of N2,N3‐ε‐ATP. Based on the fluorescence and full recognition by ATP‐binding proteins, we propose N2,N3‐ε‐ATP and related nucleo(s)tides as unique probes for the investigation of adenine nucleo(s)tide‐binding proteins as well as for other biochemical applications.

The effects of acylation stimulating protein supplementation VS antibody neutralization on energy expenditure in wildtype mice

BackgroundAcylation stimulating protein (ASP) is an adipogenic hormone that stimulates triglyceride (TG) synthesis and glucose transport in adipocytes. Previous studies have shown that ASP-deficient C3 knockout mice are hyperphagic yet lean, as they display increased oxygen consumption and fatty acid oxidation compared to wildtype mice. In the present study, antibodies against ASP (Anti-ASP) and human recombinant ASP (rASP) were tested in vitro and in vivo. Continuous administration for 4 weeks via osmotic mini-pump of Anti-ASP or rASP was evaluated in wildtype mice on a high-fat diet (HFD) to examine their effects on body weight, food intake and energy expenditure.ResultsIn mature murine adipocytes, rASP significantly stimulated fatty acid uptake (+243% vs PBS, P < 0.05) while Anti-ASP neutralized the rASP response. Mice treated with Anti-ASP showed elevated energy expenditure (P < 0.0001), increased skeletal muscle glucose oxidation (+141%, P < 0.001), reduced liver glycogen (-34%, P < 0.05) and glucose-6-phosphate content (-64%, P = 0.08) compared to control mice. There was no change in body weight, food intake, fasting insulin, adiponectin, CRP or TG levels compared to controls. Interestingly, HFD mice treated with rASP showed the opposite phenotype with reduced energy expenditure (P < 0.0001) and increased body weight (P < 0.05), cumulative food intake (P < 0.0001) and liver glycogen content (+59%, P < 0.05). Again, there was no change in circulating insulin, adiponectin, CRP or TG levels, however, plasma free fatty acids were reduced (-48%, P < 0.05).ConclusionIn vitro, Anti-ASP effectively neutralized ASP stimulated fatty acid uptake. In vivo, Anti-ASP treatment increased whole body energy utilization while rASP increased energy storage. Therefore, ASP is a potent anabolic hormone that may also be a mediator of energy expenditure.

Paradoxical Glucose-Sensitizing yet Proinflammatory Effects of Acute ASP Administration in Mice

Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processes in vivo were evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-α in plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, the in vivo and in vitro experiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects.

C5aR and C5L2 act in concert to balance immunometabolism in adipose tissue

Recent studies suggested that the immunometabolic receptors; C5aR and C5L2, constitutively self-associate into homo-/heterodimers and that acylation stimulating protein (ASP/C3adesArg) or C5a treatment of adipocytes increased their colocalization. The present study evaluates the C5aR contribution in adipocytes to the metabolic and immune responses elicited by ligand stimulation. The effects of C5a, ASP, and insulin on cytokine production, triglyceride synthesis (TGS), and key signaling pathways were evaluated in isolated primary adipocytes and cultured 3T3-L1 differentiated adipocytes. In addition, mRNA expression of IRS1 and PGC1α was compared in adipose tissue samples from WT vs. C5aRKO mice. Both C5a and ASP directly increased MCP-1 (238±4%; P<0.001, and 377±2% vs. basal 100%; P<0.001, respectively) and KC (413±11%; P<0.001, and 529±16%; P<0.001 vs. basal 100%, respectively) secretion, TGS (131±1%; P<0.001, and 152±6%; P<0.001, vs. basal 100% respectively), and Akt/NFκB phosphorylation pathways in adipocytes. However, in C5aRKO adipocytes, C5a effects were disrupted, while stimulatory effects of ASP were mostly maintained. Addition of C5a completely blocked ASP signaling and activity in both C5aRKO and WT adipocytes as well as 3T3-L1 adipocytes. Furthermore, C5aRKO adipocytes revealed impaired insulin stimulation of cytokine production, with partial impairment of signaling and TGS stimulation, consistent with decreased IRS1 and PGC1α mRNA expression in adipose tissue. These observations indicate the importance of C5aR in adipose tissue metabolism and immunity, which may be regulated through heterodimerization with C5L2.

8‐BuS‐ATP derivatives as specific NTPDase1 inhibitors

Ectonucleotidases control extracellular nucleotide levels and consequently, their (patho)physiological responses. Among these enzymes, nucleoside triphosphate diphosphohydrolase‐1 (NTPDase1), −2, −3 and −8 are the major ectonucleotidases responsible for nucleotide hydrolysis at the cell surface under physiological conditions, and NTPDase1 is predominantly located at the surface of vascular endothelial cells and leukocytes. Efficacious inhibitors of NTPDase1 are required to modulate responses induced by nucleotides in a number of pathological situations such as thrombosis, inflammation and cancer.