The role of C16:0 ceramide in the development of obesity and type 2 diabetes: CerS6 inhibition as a novel therapeutic approach.

OBJECTIVE: Ectopic fat deposition is associated with increased tissue production of ceramides. Recent genetic mouse studies suggest that specific sphingolipid C16:0 ceramide produced by ceramide synthase 6 (CerS6) plays an important role in the development of insulin resistance. However, the therapeutic potential of CerS6 inhibition not been demonstrated. Therefore, we pharmacologically investigated the selective ablation of CerS6 using antisense oligonucleotides (ASO) in obese insulin resistance animal models. METHODS: We utilized ASO as therapeutic modality, CerS6 ASO molecules designed and synthesized were initially screened for in-vitro knock-down (KD) potency and cytotoxicity. ASOs with >85% inhibition of CerS6 mRNA were selected for further investigations. Most promising ASOs verified for in-vivo KD efficacy in healthy mice. CerS6 ASO (AAGATGAGCCGCACC) was found most active with hepatic reduction of CerS6 mRNA expression. Prior to longitudinal metabolic studies, we performed a dose titration target engagement analysis with CerS6 ASO in healthy mice to select the optimal dose. Next, we utilized leptin deficiency ob/ob and high fat diet (HFD) induced obese mouse models for pharmacological efficacy study. RESULTS: CerS6 expression were significantly elevated in the liver and brown adipose, this was correlated with significantly elevated C16:0 ceramide concentrations in plasma and liver. Treatment with CerS6 ASO selectively reduced CerS6 expression by ∼90% predominantly in the liver and this CerS6 KD resulted in a significant reduction of C16:0 ceramide by about 50% in both liver and plasma. CerS6 KD resulted in lower body weight gain and accompanied by a significant reduction in whole body fat and fed/fasted blood glucose levels (1% reduction in HbA1c). Moreover, ASO-mediated CerS6 KD significantly improved oral glucose tolerance (during oGTT) and mice displayed improved insulin sensitivity. Thus, CerS6 appear to play an important role in the development of obesity and insulin resistance. CONCLUSIONS: Our investigations identified specific and selective therapeutic valid ASO for CerS6 ablation in in-vivo. CerS6 should specifically be targeted for the reduction of C16:0 ceramides, that results in amelioration of insulin resistance, hyperglycemia and obesity. CerS6 mediated C16:0 ceramide reduction could be a potentially attractive target for the treatment of insulin resistance, obesity and type 2 diabetes.

Genetic Nicotinamide N-Methyltransferase (Nnmt) Deficiency in Male Mice Improves Insulin Sensitivity in Diet-Induced Obesity but Does Not Affect Glucose Tolerance.

Antisense oligonucleotide knockdown (ASO-KD) of nicotinamide N-methyltransferase (NNMT) in high-fat diet (HFD)-fed mice has been reported to reduce weight gain and plasma insulin levels and to improve glucose tolerance. Using NNMT-ASO-KD or NNMT knockout mice (NNMT-/-), we tested the hypothesis that Nnmt deletion protects against diet-induced obesity and its metabolic consequences in males and females on obesity-inducing diets. We also examined samples from a human weight reduction (WR) study for adipose NNMT (aNNMT) expression and plasma 1-methylnicotinamide (MNAM) levels. In Western diet (WD)-fed female mice, NNMT-ASO-KD reduced body weight, fat mass, and insulin level and improved glucose tolerance. Although NNMT-/- mice fed a standard diet had no obvious phenotype, NNMT-/- males fed an HFD showed strongly improved insulin sensitivity (IS). Furthermore, NNMT-/- females fed a WD showed reduced weight gain, less fat, and lower insulin levels. However, no improved glucose tolerance was observed in NNMT-/- mice. Although NNMT expression in human fat biopsy samples increased during WR, corresponding plasma MNAM levels significantly declined, suggesting that other mechanisms besides aNNMT expression modulate circulating MNAM levels during WR. In summary, upon NNMT deletion or knockdown in males and females fed different obesity-inducing diets, we observed sex- and diet-specific differences in body composition, weight, and glucose tolerance and estimates of IS.

Peptide Optimization at the Drug Discovery-Development Interface: Tailoring of Physicochemical Properties Toward Specific Formulation Requirements.

Physicochemical properties of peptides need to be compatible with the manufacturing process and formulation requirements to ensure developability toward the commercial drug product. This aspect is often disregarded and only evaluated late in discovery, imposing a high risk for delays in development, increased costs, and finally for the project in general. Here, we report a case study of early physicochemical peptide characterization and optimization of dual glucagon-like peptide 1/glucagon receptor agonists toward specific formulation requirements. Aggregation issues which were observed at acidic pH in the presence of phenolic preservatives could be eliminated by modification of the peptide sequence, and chemical stability issues were significantly improved by addition of stabilizing formulation excipients. We describe structural, analytical, and biophysical characterization in different compositions to analyze the effect of pH and formulation excipients on physical and chemical stability. Molecular models have been generated to rationalize peptide stability behavior based on computed physicochemical descriptors and interactions with excipients. To conclude these studies, a general roadmap is proposed how to assess and optimize early physicochemical peptide properties in a sophisticated way by combining experimental and in silico profiling to provide stable peptide drugs under relevant formulation conditions at the end of discovery.

A small molecule inhibitor of Nicotinamide N-methyltransferase for the treatment of metabolic disorders.

Nicotinamide N-methyltransferase (NNMT) is a cytosolic enzyme that catalyzes the transfer of a methyl group from the co-factor S-adenosyl-L-methionine (SAM) onto the substrate, nicotinamide (NA) to form 1-methyl-nicotinamide (MNA). Higher NNMT expression and MNA concentrations have been associated with obesity and type-2 diabetes. Here we report a small molecule analog of NA, JBSNF-000088, that inhibits NNMT activity, reduces MNA levels and drives insulin sensitization, glucose modulation and body weight reduction in animal models of metabolic disease. In mice with high fat diet (HFD)-induced obesity, JBSNF-000088 treatment caused a reduction in body weight, improved insulin sensitivity and normalized glucose tolerance to the level of lean control mice. These effects were not seen in NNMT knockout mice on HFD, confirming specificity of JBSNF-000088. The compound also improved glucose handling in ob/ob and db/db mice albeit to a lesser extent and in the absence of weight loss. Co-crystal structure analysis revealed the presence of the N-methylated product of JBSNF-000088 bound to the NNMT protein. The N-methylated product was also detected in the plasma of mice treated with JBSNF-000088. Hence, JBSNF-000088 may act as a slow-turnover substrate analog, driving the observed metabolic benefits.

A liver stress-endocrine nexus promotes metabolic integrity during dietary protein dilution.

Dietary protein intake is linked to an increased incidence of type 2 diabetes (T2D). Although dietary protein dilution (DPD) can slow the progression of some aging-related disorders, whether this strategy affects the development and risk for obesity-associated metabolic disease such as T2D is unclear. Here, we determined that DPD in mice and humans increases serum markers of metabolic health. In lean mice, DPD promoted metabolic inefficiency by increasing carbohydrate and fat oxidation. In nutritional and polygenic murine models of obesity, DPD prevented and curtailed the development of impaired glucose homeostasis independently of obesity and food intake. DPD-mediated metabolic inefficiency and improvement of glucose homeostasis were independent of uncoupling protein 1 (UCP1), but required expression of liver-derived fibroblast growth factor 21 (FGF21) in both lean and obese mice. FGF21 expression and secretion as well as the associated metabolic remodeling induced by DPD also required induction of liver-integrated stress response-driven nuclear protein 1 (NUPR1). Insufficiency of select nonessential amino acids (NEAAs) was necessary and adequate for NUPR1 and subsequent FGF21 induction and secretion in hepatocytes in vitro and in vivo. Taken together, these data indicate that DPD promotes improved glucose homeostasis through an NEAA insufficiency-induced liver NUPR1/FGF21 axis.

Association of nicotinamide-N-methyltransferase mRNA expression in human adipose tissue and the plasma concentration of its product, 1-methylnicotinamide, with insulin resistance.

AIMS/HYPOTHESIS: Nicotinamide-N-methyltransferase (NNMT) was recently shown to be upregulated in mouse models of insulin resistance and obesity. So far, it is unknown whether NNMT is regulated in human disease. We have explored the hypothesis that white adipose tissue (WAT) NNMT expression and plasma 1-methylnicotinamide (MNA) concentration are increased in human insulin resistance and type 2 diabetes. METHODS: NNMT expression and plasma MNA were analysed in three groups of individuals: (1) 199 patients undergoing abdominal surgery; (2) 60 individuals on a 12-week exercise programme and (3) 55 patients on a two-step bariatric surgery programme. RESULTS: Patients with manifest type 2 diabetes have a significantly (approximately twofold) higher NNMT expression both in omental and subcutaneous WAT compared with controls. Notably, plasma MNA correlated significantly with WAT NNMT expression in patients with type 2 diabetes (women, r = 0.59, p < 0.001; men, r = 0.61, p < 0.001) but not in healthy control individuals. In insulin-resistant individuals, there was an inverse correlation between insulin sensitivity and plasma MNA (r = 0.44, p = 0.01) or adipose tissue NNMT mRNA (r = 0.64, p < 0.001). The latter association was confirmed in a second cohort (n = 60, r = 0.78, p < 0.001). Interventions improving insulin sensitivity--exercise and bariatric surgery--were associated with a significant (p < 0.001) reduction in WAT NNMT expression. Bariatric surgery was also associated with a significant decrease in plasma MNA. CONCLUSIONS/INTERPRETATION: We demonstrate that WAT NNMT expression is regulated in human insulin resistance and type 2 diabetes and that plasma MNA correlates with increased tissue NNMT expression and the degree of insulin resistance, making it a potential biomarker for loss of insulin sensitivity.

Aminopeptidase N (CD13) is a molecular target of the cholesterol absorption inhibitor ezetimibe in the enterocyte brush border membrane.

Intestinal cholesterol absorption is an important regulator of serum cholesterol levels. Ezetimibe is a specific inhibitor of intestinal cholesterol absorption recently introduced into medical practice; its mechanism of action, however, is still unknown. Ezetimibe neither influences the release of cholesterol from mixed micelles in the gut lumen nor the transfer of cholesterol to the enterocyte brush border membrane. With membrane-impermeable Ezetimibe analogues we could demonstrate that binding of cholesterol absorption inhibitors to the brush border membrane of small intestinal enterocytes from the gut lumen is sufficient for inhibition of cholesterol absorption. A 145-kDa integral membrane protein was identified as the molecular target for cholesterol absorption inhibitors in the enterocyte brush border membrane by photoaffinity labeling with photoreactive Ezetimibe analogues (Kramer, W., Glombik, H., Petry, S., Heuer, H., Schafer, H. L., Wendler, W., Corsiero, D., Girbig, F., and Weyland, C. (2000) FEBS Lett. 487, 293-297). The 145-kDa Ezetimibe-binding protein was purified by three different methods and sequencing revealed its identity with the membrane-bound ectoenzyme aminopeptidase N ((alanyl)aminopeptidase; EC 3.4.11.2; APN; leukemia antigen CD13). The enzymatic activity of APN was not influenced by Ezetimibe (analogues). The uptake of cholesterol delivered by mixed micelles by confluent CaCo-2 cells was partially inhibited by Ezetimibe and nonabsorbable Ezetimibe analogues. Preincubation of confluent CaCo-2 cells with Ezetimibe led to a strong decrease of fluorescent APN staining with a monoclonal antibody in the plasma membrane. Independent on its enzymatic activity, aminopeptidase N is involved in endocytotic processes like the uptake of viruses. Our findings suggest that binding of Ezetimibe to APN from the lumen of the small intestine blocks endocytosis of cholesterol-rich membrane microdomains, thereby limiting intestinal cholesterol absorption.

The calpain inhibitor MDL 28170 prevents inflammation-induced neurofilament light chain breakdown in the spinal cord and reduces thermal hyperalgesia.

Since long-term hyperexcitability of nociceptive neurons in the spinal cord has been suggested to be caused and maintained by changes of protein expression we assessed protein patterns in lumbar spinal cord during a zymosan induced paw inflammation employing two-dimensional (2D) gel electrophoresis. 2D PAGE revealed a time-dependent breakdown of scaffolding proteins one of which was neurofilament light chain (NFL) protein, which has been previously found to be important for axonal architecture and transport. Nociception induced breakdown of NFL in the spinal cord and dorsal root ganglias was prevented by pretreatment of the animals with a single dose of the specific inhibitor of the protease calpain (MDL-28170) which has been shown to be the primary protease involved in neurofilament degradation in neurodegenerative diseases. Treatment with the calpain inhibitor also provided anti-inflammatory and anti-hyperalgesic effects in the zymosan-induced paw inflammation model irrespective of whether the drug was administered systemically (i.p.) or delivered onto the lumbar spinal cord. This suggests that the activation of calpain is involved in the sensitization of nociceptive neurons what is partly due to neurofilament breakdown but cleavage of other calpain substrates may also be involved. Our results indicate that inhibition of pathological calpain activity may present an interesting novel drug target in the treatment of pain and inflammation.