Cross Tissue Trait-Pathway Network Reveals the Importance of Oxidative Stress and Inflammation Pathways in Obesity-Induced Diabetes in Mouse

Complex disorders often involve dysfunctions in multiple tissue organs. Elucidating the communication among them is important to understanding disease pathophysiology. In this study we integrate multiple tissue gene expression and quantitative trait measurements of an obesity-induced diabetes mouse model, with databases of molecular interaction networks, to construct a cross tissue trait-pathway network. The animals belong to two strains of mice (BTBR or B6), of two obesity status (obese or lean), and at two different ages (4 weeks and 10 weeks). Only 10 week obese BTBR animals are diabetic. The expression data was first utilized to determine the state of every pathway in each tissue, which is subsequently utilized to construct a pathway co-expression network and to define trait-relevant and trait-linking pathways. Among the six tissues profiled, the adipose contains the largest number of trait-linking pathways. Among the eight traits measured, the body weight and plasma insulin level possess the most number of relevant and linking pathways. Topological analysis of the trait-pathway network revealed that the glycolysis/gluconeogenesis pathway in liver and the insulin signaling pathway in muscle are of top importance to the information flow in the network, with the highest degrees and betweenness centralities. Interestingly, pathways related to metabolism and oxidative stress actively interact with many other pathways in all animals, whereas, among the 10 week animals, the inflammation pathways were preferentially interactive in the diabetic ones only. In summary, our method offers a systems approach to delineate disease trait relevant intra- and cross tissue pathway interactions, and provides insights to the molecular basis of the obesity-induced diabetes.     

Abdominal obesity in BTBR male mice is associated with peripheral but not hepatic insulin resistance

Insulin resistance is a common feature of obesity. BTBR mice have more fat mass than most other inbred mouse strains. On a chow diet, BTBR mice have elevated insulin levels relative to the C57BL/6J (B6) strain. Male F1 progeny of a B6 x BTBR cross are insulin resistant. Previously, we reported insulin resistance in isolated muscle and in isolated adipocytes in this strain. Whereas the muscle insulin resistance was observed only in male F1 mice, adipocyte insulin resistance was also present in male BTBR mice. We examined in vivo mechanisms of insulin resistance with the hyperinsulinemic euglycemic clamp technique. At 10 wk of age, BTBR and F1 mice had a >30% reduction in whole body glucose disposal primarily due to insulin resistance in heart, soleus muscle, and adipose tissue. The increased adipose tissue mass and decreased muscle mass in BTBR and F1 mice were negatively and positively correlated with whole body glucose disposal, respectively. Genes involved in focal adhesion, actin cytoskeleton, and inflammation were more highly expressed in BTBR and F1 than in B6 adipose tissue. The BTBR and F1 mice have higher levels of testosterone, which may be related to the pathological changes in adipose tissue that lead to systemic insulin resistance. Despite profound peripheral insulin resistance, BTBR and F1 mice retained hepatic insulin sensitivity. These studies reveal a genetic difference in body composition that correlates with large differences in peripheral insulin sensitivity.     

Dysregulation of microRNA-125a contributes to obesity-associated insulin resistance and dysregulates lipid metabolism in mice

Obesity is associated with an increased risk of developing insulin resistance (IR) and type 2 diabetes (T2D). A diverse group of factors including miRNA has been implicated in the pathogenesis of these two metabolic conditions, although underlying molecular mechanisms involved are not well defined. Here, we provide evidence that hepatic miR-125a levels are diminished in both genetic as well as dietary mouse models of obesity. Overexpression of miR-125a enhanced insulin signaling and attenuated cellular lipid accumulation in HepG2 cells and Hepa1–6 cells. Likewise, treatment of mice with ago-miR-125a increased insulin sensitivity, similar to overexpression of miR-125a, whereas treatment of mice with antago-miR-125a blunted the insulin sensitivity. Furthermore, overexpression of miR-125a in mice previously fed a high-fat diet (HFD), significantly improved insulin sensitivity, and attenuated obesity-linked hepatic steatosis and hepatocyte lipid accumulation. In addition, we show that ELOVL fatty acid elongase 6 (Elovl6) is a direct target of miR-125a, and participates in miR-125a mediated regulation of insulin sensitivity and lipid metabolism. These data led us to conclude that dysregulated miR-125a expression augments the development of obesity-induced IR and that miR-125a might serve as a therapeutic target for the development of new drug(s) in the clinical management of metabolic diseases.     

Positional cloning of a type 2 diabetes quantitative trait locus; tomosyn-2, a negative regulator of insulin secretion

We previously mapped a type 2 diabetes (T2D) locus on chromosome 16 (Chr 16) in an F2 intercross from the BTBR T (+) tf (BTBR) Lep(ob/ob) and C57BL/6 (B6) Lep(ob/ob) mouse strains. Introgression of BTBR Chr 16 into B6 mice resulted in a consomic mouse with reduced fasting plasma insulin and elevated glucose levels. We derived a panel of sub-congenic mice and narrowed the diabetes susceptibility locus to a 1.6 Mb region. Introgression of this 1.6 Mb fragment of the BTBR Chr 16 into lean B6 mice (B6.16(BT36-38)) replicated the phenotypes of the consomic mice. Pancreatic islets from the B6.16(BT36-38) mice were defective in the second phase of the insulin secretion, suggesting that the 1.6 Mb region encodes a regulator of insulin secretion. Within this region, syntaxin-binding protein 5-like (Stxbp5l) or tomosyn-2 was the only gene with an expression difference and a non-synonymous coding single nucleotide polymorphism (SNP) between the B6 and BTBR alleles. Overexpression of the b-tomosyn-2 isoform in the pancreatic β-cell line, INS1 (832/13), resulted in an inhibition of insulin secretion in response to 3 mM 8-bromo cAMP at 7 mM glucose. In vitro binding experiments showed that tomosyn-2 binds recombinant syntaxin-1A and syntaxin-4, key proteins that are involved in insulin secretion via formation of the SNARE complex. The B6 form of tomosyn-2 is more susceptible to proteasomal degradation than the BTBR form, establishing a functional role for the coding SNP in tomosyn-2. We conclude that tomosyn-2 is the major gene responsible for the T2D Chr 16 quantitative trait locus (QTL) we mapped in our mouse cross. Our findings suggest that tomosyn-2 is a key negative regulator of insulin secretion.     

MsrA Overexpression Targeted to the Mitochondria,but Not Cytosol,Preserves Insulin Sensitivity in Diet-Induced Obese Mice

There is growing evidence that oxidative stress plays an integral role in the processes by which obesity causes type 2 diabetes. We previously identified that mice lacking the protein oxidation repair enzyme methionine sulfoxide reductase A (MsrA) are particularly prone to obesity-induced insulin resistance suggesting an unrecognized role for this protein in metabolic regulation. The goals of this study were to test whether increasing the expression of MsrA in mice can protect against obesity-induced metabolic dysfunction and to elucidate the potential underlying mechanisms. Mice with increased levels of MsrA in the mitochondria (TgMito MsrA) or in the cytosol (TgCyto MsrA) were fed a high fat/high sugar diet and parameters of glucose homeostasis were monitored. Mitochondrial content, markers of mitochondrial proteostasis and mitochondrial energy utilization were assessed. TgMito MsrA, but not TgCyto MsrA, mice remain insulin sensitive after high fat feeding, though these mice are not protected from obesity. This metabolically healthy obese phenotype of TgMito MsrA mice is not associated with changes in mitochondrial number or biogenesis or with a reduction of proteostatic stress in the mitochondria. However, our data suggest that increased mitochondrial MsrA can alter metabolic homeostasis under diet-induced obesity by activating AMPK signaling, thereby defining a potential mechanism by which this genetic alteration can prevent insulin resistance without affecting obesity. Our data suggest that identification of targets that maintain and regulate the integrity of the mitochondrial proteome, particular against oxidative damage, may play essential roles in the protection against metabolic disease.     

Hyperglycemia, maturity-onset obesity, and insulin resistance in NONcNZO10/LtJ males, a new mouse model of type 2 diabetes

As a new mouse model of obesity-induced diabetes generated by combining quantitative trait loci from New Zealand Obese (NZO/HlLt) and Nonobese Nondiabetic (NON/LtJ) mice, NONcNZO10/LtJ (RCS10) male mice developed type 2 diabetes characterized by maturity onset obesity, hyperglycemia, and insulin resistance. To metabolically profile the progression to diabetes in preobese and obese states, a 2-h hyperinsulinemic euglycemic clamp was performed and organ-specific changes in insulin action were assessed in awake RCS10 and NON/LtJ (control) males at 8 and 13 wk of age. Prior to development of obesity and attendant increases in hepatic lipid content, 8-wk-old RCS10 mice developed insulin resistance in liver and skeletal muscle due to significant decreases in insulin-stimulated glucose uptake and GLUT4 expression in muscle. Transition to an obese and hyperglycemic state by 13 wk of age exacerbated insulin resistance in skeletal muscle, liver, and heart associated with organ-specific increases in lipid content. Thus, this polygenic mouse model of type 2 diabetes, wherein plasma insulin is only modestly elevated and obesity develops with maturity yet insulin action and glucose metabolism in skeletal muscle and liver are reduced at an early prediabetic age, should provide new insights into the etiology of type 2 diabetes.     

Alpha-Ketoisocaproate-induced hypersecretion of insulin by islets from diabetes-susceptible mice

Most patients at risk for developing type 2 diabetes are hyperinsulinemic. Hyperinsulinemia may be a response to insulin resistance, but another possible abnormality is insulin hypersecretion. BTBR mice are insulin resistant and hyperinsulinemic. When the leptin(ob) mutation is introgressed into BTBR mice, they develop severe diabetes. We compared the responsiveness of lean B6 and BTBR mouse islets to various insulin secretagogues. The transamination product of leucine, alpha-ketoisocaproate (KIC), elicited a dramatic insulin secretory response in BTBR islets. The KIC response was blocked by methyl-leucine or aminooxyacetate, inhibitors of branched-chain amino transferase. When dimethylglutamate was combined with KIC, the fractional insulin secretion was identical in islets from both mouse strains, predicting that the amine donor is rate-limiting for KIC-induced insulin secretion. Consistent with this prediction, glutamate levels were higher in BTBR than in B6 islets. The transamination product of glutamate, alpha-ketoglutarate, elicited insulin secretion equally from B6 and BTBR islets. Thus formation of alpha-ketoglutarate is a requisite step in the response of mouse islets to KIC. alpha-Ketoglutarate can be oxidized to succinate. However, succinate does not stimulate insulin secretion in mouse islets. Our data suggest that alpha-ketoglutarate may directly stimulate insulin secretion and that increased formation of alpha-ketoglutarate leads to hyperinsulinemia.     

Tsc2, a positional candidate gene underlying a quantitative trait locus for hepatic steatosis

Nonalchoholic fatty liver disease (NAFLD) is the most common cause of liver dysfunction and is associated with metabolic diseases, including obesity, insulin resistance, and type 2 diabetes. We mapped a quantitative trait locus (QTL) for NAFLD to chromosome 17 in a cross between C57BL/6 (B6) and BTBR mouse strains made genetically obese with the Lep(ob/ob) mutation. We identified Tsc2 as a gene underlying the chromosome 17 NAFLD QTL. Tsc2 functions as an inhibitor of mammalian target of rapamycin, which is involved in many physiological processes, including cell growth, proliferation, and metabolism. We found that Tsc2(+/-) mice have increased lipogenic gene expression in the liver in an insulin-dependent manner. The coding single nucleotide polymorphism between the B6 and BTBR strains leads to a change in the ability to inhibit the expression of lipogenic genes and de novo lipogenesis in AML12 cells and to promote the proliferation of Ins1 cells. This difference is due to a different affinity of binding to Tsc1, which affects the stability of Tsc2.     

Identification of a common lupus disease-associated microRNA expression pattern in three different murine models of lupus

Background

Recent reports have shown that microRNAs (miRNAs) regulate vital immunological processes and have emerged as key regulators of immune system development and function. Therefore, it is important to determine miRNA dysregulation and its pathogenic contribution in autoimmune diseases, an aspect not adequately addressed thus far.

Methodology/Principal Findings

In this study, we profiled miRNA expressions in splenic lymphocytes from three murine lupus models (MRL-lpr, B6-lpr and NZB/W_F1) with different genetic background by miRNA microarray assays and Real-time RT-PCR. Despite the genetic differences among these three lupus stains, a common set of dysregulated miRNAs (miR-182-96-183 cluster, miR-31, and miR-155) was identified in splenocytes when compared with age-matched control mice. The association of these miRNAs with the disease was highlighted by our observation that this miRNA expression pattern was evident in NZB/W mice only at an age when lupus disease is manifested. Further, we have shown that the miRNA dysregulation in MRL-lpr mice was not simply due to the activation of splenocytes. By Real-time RT-PCR, we confirmed that these miRNAs were upregulated in both purified splenic B and T cells from MRL-lpr mice. miR-127 and miR-379, which were greatly upregulated in splenocytes from lpr mice, were moderately increased in diseased NZB/W mice. In addition, Real-time RT-PCR revealed that miR-146a, miR-101a, and miR-17-92 were also markedly upregulated in splenic T, but not B cells from MRL-lpr mice.

Conclusions/Significance

The identification of common lupus disease-associated miRNAs now forms the basis for the further investigation of the pathogenic contribution of these miRNAs in autoimmune lupus, which will advance our knowledge of the role of miRNAs in autoimmunity. Given that miRNAs are conserved, with regard to both evolution and function, our observation of a common lupus disease-associated miRNA expression pattern in murine lupus models is likely to have significant pathogenic, diagnostic, and/or therapeutic implications in human lupus.     

Cellular and molecular players in adipose tissue inflammation in the development of obesity-induced insulin resistance

There is increasing evidence showing that inflammation is an important pathogenic mediator of the development of obesity-induced insulin resistance. It is now generally accepted that tissue-resident immune cells play a major role in the regulation of this obesity-induced inflammation. The roles that adipose tissue (AT)-resident immune cells play have been particularly extensively studied. AT contains most types of immune cells and obesity increases their numbers and activation levels, particularly in AT macrophages (ATMs). Other pro-inflammatory cells found in AT include neutrophils, Th1 CD4 T cells, CD8 T cells, B cells, DCs, and mast cells. However, AT also contains anti-inflammatory cells that counter the pro-inflammatory immune cells that are responsible for the obesity-induced inflammation in this tissue. These anti-inflammatory cells include regulatory CD4 T cells (Tregs), Th2 CD4 T cells, and eosinophils. Hence, AT inflammation is shaped by the regulation of pro- and anti-inflammatory immune cell homeostasis, and obesity skews this balance towards a more pro-inflammatory status. Recent genetic studies revealed several molecules that participate in the development of obesity-induced inflammation and insulin resistance. In this review, the cellular and molecular players that participate in the regulation of obesity-induced inflammation and insulin resistance are discussed, with particular attention being placed on the roles of the cellular players in these pathogeneses. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Systematic evaluation of microRNA processing patterns in tissues, cell lines, and tumors

Very little is known regarding regulation of microRNA (miRNA) biogenesis in normal tissues, tumors, and cell lines. Here, we profiled the expression of 225 precursor and mature miRNAs using real-time PCR and compared the expression levels to determine the processing patterns. RNA from 22 different human tissues, 37 human cancer cell lines, and 16 pancreas and liver tissues/tumors was profiled. The relationship between precursor and mature miRNA expression fell into the following four categories: (1) a direct correlation exists between the precursor and mature miRNA expression in all cells/tissues studied; (2) direct correlation of the precursor and mature miRNA exists, yet the expression is restricted to specific cell lines or tissues; (3) there is detectable expression of mature miRNA in certain cells and tissues while the precursor is expressed in all or most cells/tissues; or (4) both precursor and mature miRNA are not expressed. Pearson correlation between the precursor and mature miRNA expression was closer to one for the tissues but was closer to zero for the cell lines, suggesting that processing of precursor miRNAs is reduced in cancer cell lines. By using Northern blotting, we show that many of these miRNAs (e.g., miR-31, miR-105 and miR-128a) are processed to the precursor, but in situ hybridization analysis demonstrates that these miRNA precursors are retained in the nucleus. We provide a database of the levels of precursor and mature miRNA in a variety of cell types. Our data demonstrate that a large number of miRNAs are transcribed but are not processed to the mature miRNA.     

SOCS-3 inhibits insulin signaling and is up-regulated in response to tumor necrosis factor-alpha in the adipose tissue of obese mice

SOCS (suppressor of cytokine signaling) proteins are inhibitors of cytokine signaling involved in negative feedback loops. We have recently shown that insulin increases SOCS-3 mRNA expression in 3T3-L1 adipocytes. When expressed, SOCS-3 binds to phosphorylated Tyr(960) of the insulin receptor and prevents Stat 5B activation by insulin. Here we show that in COS-7 cells SOCS-3 decreases insulin-induced insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation and its association with p85, a regulatory subunit of phosphatidylinositol-3 kinase. This mechanism points to a function of SOCS-3 in insulin resistance. Interestingly, SOCS-3 expression was found to be increased in the adipose tissue of obese mice, but not in the liver and muscle of these animals. Two polypeptides known to be elevated during obesity, insulin and tumor necrosis factor-alpha (TNF-alpha), induce SOCS-3 mRNA expression in mice. Insulin induces a transient expression of SOCS-3 in the liver, muscle, and the white adipose tissue (WAT). Strikingly, TNF-alpha induced a sustained SOCS-3 expression, essentially in the WAT. Moreover, transgenic ob/ob mice lacking both TNF receptors have a pronounced decrease in SOCS-3 expression in the WAT compared with ob/ob mice, providing genetic evidence for a function of this cytokine in obesity-induced SOCS-3 expression. As SOCS-3 appears as a TNF-alpha target gene that is elevated during obesity, and as SOCS-3 antagonizes insulin-induced IRS-1 tyrosine phosphorylation, we suggest that it is a player in the development of insulin resistance.     

Using hippocampal microRNA expression differences between mouse inbred strains to characterise miRNA function

Micro-RNAs (miRNAs) are short, single-stranded, noncoding RNAs that are involved in the regulation of protein-coding genes at the level of messenger RNA (mRNA). They are involved in the regulation of numerous traits, including developmental timing, apoptosis, immune function, and neuronal development. To better understand how the expression of the miRNAs themselves is regulated, we looked for miRNA expression differences among four mouse inbred strains, A/J, BALB/cJ, C57BL/6J, and DBA/2J, in one tissue, the hippocampus. A total of 166 miRNA RT-PCR assays were used to screen RNA pools for each strain. Twenty miRNA species that were markedly different between strains were further investigated using eight individual samples per strain, and 11 miRNAs showed significant differences across strains (p < 0.05). This is the first observation of miRNA expression differences across inbred mice strains. We conducted an in silico correlation analysis of the expression of these differentially expressed miRNAs with phenotype data and mRNA expression to better characterise the effects of these miRNAs on both phenotype and the regulation of mRNA expression. This approach has allowed us to nominate miRNAs that have potential roles in anxiety, exploration, and learning and memory.     

The NLRP3 inflammasome instigates obesity-induced inflammation and insulin resistance

The emergence of chronic inflammation during obesity in the absence of overt infection or well-defined autoimmune processes is a puzzling phenomenon. The Nod-like receptor (NLR) family of innate immune cell sensors, such as the nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3 (Nlrp3, but also known as Nalp3 or cryopyrin) inflammasome are implicated in recognizing certain nonmicrobial originated 'danger signals' leading to caspase-1 activation and subsequent interleukin-1β (IL-1β) and IL-18 secretion. We show that calorie restriction and exercise-mediated weight loss in obese individuals with type 2 diabetes is associated with a reduction in adipose tissue expression of Nlrp3 as well as with decreased inflammation and improved insulin sensitivity. We further found that the Nlrp3 inflammasome senses lipotoxicity-associated increases in intracellular ceramide to induce caspase-1 cleavage in macrophages and adipose tissue. Ablation of Nlrp3 in mice prevents obesity-induced inflammasome activation in fat depots and liver as well as enhances insulin signaling. Furthermore, elimination of Nlrp3 in obese mice reduces IL-18 and adipose tissue interferon-γ (IFN-γ) expression, increases naive T cell numbers and reduces effector T cell numbers in adipose tissue. Collectively, these data establish that the Nlrp3 inflammasome senses obesity-associated danger signals and contributes to obesity-induced inflammation and insulin resistance.