Elimination of the NLRP3-ASC inflammasome protects against chronic obesity-induced pancreatic damage

Clinical evidence that the blockade of IL-1β in type-2 diabetic patients improves glycemia is indicative of an autoinflammatory mechanism that may trigger adiposity-driven pancreatic damage. IL-1β is a key contributor to the obesity-induced inflammation and subsequent insulin resistance, pancreatic β-cell dysfunction, and the onset of type 2 diabetes. Our previous studies demonstrated that the ceramides activate the Nod-like receptor family, pyrin domain containing 3 (Nlrp3) inflammasome to cause the generation of mature IL-1β and ablation of the Nlrp3 inflammasome in diet-induced obesity improves insulin signaling. However, it remains unclear whether the post-translational processing of active IL-1β in pancreas is regulated by the NLRP3 inflammasome or whether the alternate mechanisms play a dominant role in chronic obesity-induced pancreatic β-cell exhaustion. Here we show that loss of ASC, a critical adaptor required for the assembly of the NLRP3 and absent in melanoma 2 inflammasome substantially improves the insulin action. Surprisingly, despite lower insulin resistance in the chronically obese NLRP3 and ASC knockout mice, the insulin levels were substantially higher when the inflammasome pathway was eliminated. The obesity-induced increase in maturation of pancreatic IL-1β and pancreatic islet fibrosis was dependent on the NLRP3 inflammasome activation. Furthermore, elimination of NLRP3 inflammasome protected the pancreatic β-cells from cell death caused by long-term high-fat feeding during obesity with significant increase in the size of the islets of Langerhans. Collectively, this study provides direct in vivo evidence that activation of the NLRP3 inflammasome in diet-induced obesity is a critical trigger in causing pancreatic damage and is an important mechanism of progression toward type 2 diabetes. Copyright © 2011 by The Endocrine Society

Impaired Mitochondrial Fat Oxidation Induces FGF21 in Muscle

SummaryFatty acids are the primary fuel source for skeletal muscle during most of our daily activities, and impaired fatty acid oxidation (FAO) is associated with insulin resistance. We have developed a mouse model of impaired FAO by deleting carnitine palmitoyltransferase-1b specifically in skeletal muscle (Cpt1bm−/−). Cpt1bm−/− mice have increased glucose utilization and are resistant to diet-induced obesity. Here, we show that inhibition of mitochondrial FAO induces FGF21 expression specifically in skeletal muscle. The induction of FGF21 in Cpt1b-deficient muscle is dependent on AMPK and Akt1 signaling but independent of the stress signaling pathways. FGF21 appears to act in a paracrine manner to increase glucose uptake under low insulin conditions, but it does not contribute to the resistance to diet-induced obesity

Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism

© 2015, National Academy of Sciences. All rights reserved. The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity

Genetic Deletion of DNAJB3 Using CRISPR-Cas9, Produced Discordant Phenotypes

Several pathways and/or genes have been shown to be dysregulated in obesity-induced insulin resistance (IR) and type 2 diabetes (T2D). We previously showed, for the first time, impaired expression of DNAJB3 mRNA and protein in subjects with obesity, which was concomitant with increased metabolic stress. Restoring the normal expression of DNAJB3 attenuated metabolic stress and improved insulin signaling both in vivo and in vitro, suggesting a protective role of DNAJB3 against obesity and T2D. The precise underlying mechanisms remained, however, unclear. This study was designed to confirm the human studies in a mouse model of dietary obesity-induced insulin resistance, and, if validated, to understand the underlying mechanisms. We hypothesized that mice lacking DNAJB3 would be more prone to high-fat (HF)-diet-induced increase in body weight and body fat, inflammation, glucose intolerance and insulin resistance as compared with wild-type (WT) littermates. Three DNAJB3 knockout (KO) lines were generated (KO 30, 44 and 47), using CRISPR-Cas9. Male and female KO and WT mice were fed a HF diet (45% kcal fat) for 16 weeks. Body weight was measured biweekly, and a glucose tolerance test (GTT) and insulin tolerance test (ITT) were conducted at week 13 and 14, respectively. Body composition was determined monthly by nuclear magnetic resonance (NMR). Following euthanasia, white adipose tissue (WAT) and skeletal muscle were harvested for further analyses. Compared with WT mice, male and female KO 47 mice demonstrated higher body weight and fat mass. Similarly, KO 47 mice also showed a slower rate of glucose clearance in GTT that was consistent with decreased mRNA expression of the GLUT4 gene in WAT but not in the muscle. Both male and female KO 47 mice exhibited higher mRNA levels of the pro-inflammatory marker TNF-a in WAT only, whereas increased mRNA levels of MCP1 chemokine and the ER stress marker BiP/Grp78 were observed in male but not in female KO 47 mice. However, we did not observe the same changes in the other KO lines. Taken together, the phenotype of the DNAJB3 KO 47 mice was consistent with the metabolic changes and low levels of DNAJB3 reported in human subjects. These findings suggest that DNAJB3 may play an important role in metabolic functions and glucose homeostasis, which warrants further phenotyping and intervention studies in other KO 47 and other KO mice, as well as investigating this protein as a potential therapeutic target for obesity and T2D

Transcriptome analysis of murine thymocytes reveals age-associated changes in thymic gene expression

The decline in adaptive immunity, na&#239;ve T-cell output and a contraction in the peripheral T cell receptor (TCR) repertoire with age are largely attributable to thymic involution and the loss of critical cytokines and hormones within the thymic microenvironment. To assess the molecular changes associated with this loss of thymic function, we used cDNA microarray analyses to examine the transcriptomes of thymocytes from mice of various ages ranging from very young (1 month) to very old (24 months). Genes associated with various biological and molecular processes including oxidative phosphorylation, T- and B- cell receptor signaling and antigen presentation were observed to significantly change with thymocyte age. These include several immunoglobulin chains, chemokine and ribosomal proteins, annexin A2, vav 1 and several S100 signaling proteins. The increased expression of immunoglobulin genes in aged thymocytes could be attributed to the thymic B cells which were found to be actively producing IgG and IgM antibodies. Upon further examination, we found that purified thymic T cells derived from aged but not young thymi also exhibited IgM on their cell surface suggesting the possible presence of auto-antibodies on the surface thymocytes with advancing age. These studies provide valuable insight into the cellular and molecular mechanisms associated with thymic aging.</p

Diet effects on Cpt1b^m-/- adiposity.

<p>Control mice (red dashed lines, N = 10 animals per time point on chow and N = 8 animals per time point on low fat diet) and Cpt1b^m-/- mice (black lines, N = 9 animals per time point on chow and N = 12 animals per time point on low fat diet) were monitored for 24 weeks to assess gain of Weight, Fat, and FFM. Results are compared between groups fed low fat diet (A-C) and chow diet (D-F). Asterisks indicate significance with P ≤ 0.05.</p

Examination of carnitine palmitoyltransferase 1 abundance in white adipose tissue: implications in obesity research

Carnitine palmitoyltransferase 1 (CPT1) is essential for the transport of long-chain fatty acids into the mitochondria for oxidation. Recently, it was reported that decreased CPT1b mRNA in adipose tissue was a contributing factor for obesity in rats. We therefore closely examined the expression level of in adipose tissue from mice, rats, and humans. is the predominate isoform in adipose tissue from all three species. Rat white adipose tissue has a moderate amount of mRNA, but it is very minor compared with expression in muscle. Total CPT1 activity in adipose tissue is also minor relative to other tissues. Both and mRNA were increased in gonadal fat but not inguinal fat by diet-induced obesity in mice. We also measured and expression in subcutaneous adipose tissue from human subjects with a wide range of body mass indexes (BMIs). Interestingly, expression positively correlated with BMI ( = 0.46), but there was no correlation with ( = 0.04). Our findings indicate that white adipose tissue fatty acid oxidation capacity is minor compared with that of metabolically active tissues. Furthermore, given the already low abundance of in white adipose tissue, it is unlikely that decreases in its expression can quantitatively decrease whole body energy expenditure enough to contribute to an obese phenotype

Diet has little effect on glucose clearance and muscle physiology of Cpt1b^m-/- mice.

<p>(A-B) GTT are shown for control (white circles) and Cpt1b^m-/- (black squares) mice that were fed (A) low fat diet (N = 8 animals per genotype) or (B) chow diet (N = 15 animals per genotype). (C) Levels in control (white) and Cpt1b^m-/- mice (black) of FGF21 mRNA in red quadriceps muscle (left panel), and FGF21 protein in serum; (right panel) (N = 8 animals per genotype for low fat diet and N = 10 animals per genotype for chow diet). (D) EM showing increased IMCL in soleus of Cpt1b^m-/- (right) mice relative to controls (left). (E-F) (N = 4 animals per genotype for low fat diet and N = 6 animals per genotype for chow diet) Relative levels of markers of mitochondrial biogenesis and lipid usage in control (white) and Cpt1b^m-/- (black) mouse red quadriceps muscle from mice fed (E) low fat diet or (F) chow diet. Asterisks indicate significance with P ≤ 0.05.</p