Transgenic overexpression of miR-486 and sAnk1.5 does not alter glucose handling in mice.

Genetic variants that are associated with susceptibility to type 2 diabetes (T2D) are important for identification of individuals at risk and can provide insights into the molecular basis of disease. Analysis of T2D in domestic animals provides both the opportunity to improve veterinary management and breeding programs as well as to identify novel T2D risk genes. Australian-bred Burmese (ABB) cats have a 4-fold increased incidence of type 2 diabetes (T2D) compared to Burmese cats bred in the United States. This is likely attributable to a genetic founder effect. We investigated this by performing a genome-wide association scan on ABB cats. Four SNPs were associated with the ABB T2D phenotype with p values <0.005. All exons and splice junctions of candidate genes near significant single-nucleotide polymorphisms (SNPs) were sequenced, including the genes DGKG, IFG2BP2, SLC8A1, E2F6, ETV5, TRA2B and LIPH. Six candidate polymorphisms were followed up in a larger cohort of ABB cats with or without T2D and also in Burmese cats bred in America, which exhibit low T2D incidence. The original SNPs were confirmed in this cohort as associated with the T2D phenotype, although no novel coding SNPs in any of the seven candidate genes showed association with T2D. The identification of genetic markers associated with T2D susceptibility in ABB cats will enable preventative health strategies and guide breeding programs to reduce the prevalence of T2D in these cats.

Use of preclinical models to identify markers of type 2 diabetes susceptibility and novel regulators of insulin secretion – A step towards precision medicine

In obese individuals, white adipose tissue (WAT) is infiltrated by large numbers of macrophages, resulting in enhanced inflammatory responses that contribute to insulin resistance. Here we show that expression of the CXC motif chemokine ligand-14 (CXCL14), which targets tissue macrophages, is elevated in WAT of obese mice fed a high fat diet (HFD) compared with lean mice fed a regular diet. We found that HFD-fed CXCL14-deficient mice have impaired WAT macrophage mobilization and improved insulin responsiveness. Insulin-stimulated phosphorylation of Akt kinase in skeletal muscle was severely attenuated in HFD-fed CXCL14+/- mice but not in HFD-fed CXCL14-/- mice. The insulin-sensitive phenotype of CXCL14-/- mice after HFD feeding was prominent in female mice but not in male mice. HFD-fed CXCL14-/- mice were protected from hyperglycemia, hyperinsulinemia, and hypoadiponectinemia and did not exhibit increased levels of circulating retinol-binding protein-4 and increased expression of interleukin-6 in WAT. Transgenic overexpression of CXCL14 in skeletal muscle restored obesity-induced insulin resistance in CXCL14-/- mice. CXCL14 attenuated insulin-stimulated glucose uptake in cultured myocytes and to a lesser extent in cultured adipocytes. These results demonstrate that CXCL14 is a critical chemoattractant of WAT macrophages and a novel regulator of glucose metabolism that functions mainly in skeletal muscle.

Vitamin D Receptor Gene Polymorphisms and Type 2 Diabetes: A Meta-analysis

Many genetic association studies support a contribution of genetic variants in the KCNJ11-ABCC8 gene locus to type 2 diabetes (T2D) susceptibility in Caucasians. In non-Caucasian populations, however, there have been only a few association studies, and discordant results were obtained. Herein, we selected a total of 31 SNPs covering a 211.3-kb region of the KCNJ11-ABCC8 locus, characterized the patterns of linkage disequilibrium (LD) and haplotype structure, and performed a case-control association study in a Japanese population consisting of 909 T2D patients and 893 control subjects. We found significant associations between eight SNPs, including the KCNJ11 E23K and ABCC8 S1369A variants, and T2D. These disease-associated SNPs were genetically indistinguishable because of the presence of strong LD, as found previously in Caucasians. For the KCNJ11 E23K variant, the most significant association was obtained under a dominant genetic model (OR 1.32, 95% CI 1.09-1.60, P = 0.004). A meta-analysis of East Asian studies, comprising a total of 3,357 T2D patients (77.4% Japanese) and 2,836 control subjects (77.8% Japanese), confirmed the significant role of the KCNJ11 E23K variant in T2D susceptibility. Furthermore, we found evidence suggesting that the KCNJ11 E23K genotype is independently associated with higher blood-pressure levels.

Objective In the current study, we aim to investigate whether 12-week treadmill exercise alleviates insulin resistance and muscle atrophy, and to explore whether MG53 along with IR/IRS/AKT/mTOR cascade play a role in the physiopathological changes of db/db mice.&#x0D; Methods 20 db/db mice and 20 age-matched non-diabetic m/m mice were assigned to 4 groups as MC (m/m control) group, ME (m/m exercise) group, DC (db/db control) group and DE (db/db exercise) group. After an intervention of treadmill exercise of moderate intensity for 12 weeks, glucose and insulin tolerance tests, insulin resistance index (HOMA-IR, homeostasis model assessment of insulin resistance) and lipid metabolic profile were determined using blood samples. Skeletal muscles were utilized for determination of cross-sectional area (CSA), protein level detection of MG53 and insulin signaling pathway. &#x0D; Results Compared with MC mice, the AUC (areas under curve) of IPGTT (intraperitoneal glucose tolerance test) and IPITT (intraperitoneal insulin tolerance test) as well as HOMA-IR were significantly increased, and lipid parameters (serum triglyceride and total cholesterol) increased significantly in DC group. The upregulation of MG53 protein in different skeletal muscles (quadriceps, gastrocnemius and soleus muscle) could be observed in DC mice. Phosphorylated proteins of IR-β (β subunit of insulin receptor), IRS1, AKT (protein kinase B), mTOR (mammalian target of rapamycin), p70S6k and S6 ribosomal protein after acute insulin stimulation were downregulated with significance, whereas no significant difference was found in total protein levels of IR-β and AKT except IRS1 in DC group. The results of AUC of IPGTT and IPITT, HOMA-IR and serum lipid parameters in DE group were significantly decreased compared with DC group. 12-week moderate exercise was sufficient to downregulate the expression of MG53 in skeletal muscles of diabetic db/db mice. In addition, treadmill exercise-induced improvement of insulin signal transduction and insulin-dependent protein synthesis may partially account for the heavier muscle mass and larger muscle size.&#x0D; Conclusions In summary, insulin resistance and muscle atrophy of diabetic db/db mice could be effectively attenuated by 12-week moderate treadmill exercise by regulating MG53, MG53-mediated ubiquitin-dependent degradation of IRS1 and insulin signaling transduction.

Objective: Fat accumulation in liver, pancreas, skeletal muscle, and visceral bed relates to type 2 diabetes (T2D) . However, distribution of fat in these compartments is heterogenous and it is unclear if specific distribution patterns indicate high T2D risk. We therefore investigated fat distribution patterns and their link to future T2D. Methods: From 2168 individuals without diabetes undergoing computed tomography in Japan, this case-cohort study included 658 randomly selected individuals and 146 incident cases of T2D with a 6-year follow-up. Data-driven analysis (k-means) was applied to develop clusters based on fat content in liver, pancreas, muscle, and visceral bed. Hazard ratios (HRs) for association of clusters and incident T2D were estimated using weighted Cox regression. In 3 individuals without diabetes with magnetic resonance imaging and metabolic phenotyping in Germany, cluster validation with additional assessment of glycemic traits from OGTTs was conducted. Results: We identified four clusters of fat distribution: cluster 1 (Hepatic steatosis cluster) , cluster 2 (Pancreatic steatosis cluster) , cluster 3 (Myosteatosis dominant cluster) , cluster 4 (Steatopenic cluster) . Compared with Steatopenic cluster, the adjusted-HRs (95%CIs) for incident T2D were 4.02 (2.27-7.12) in Hepatic steatosis cluster, 3.38 (1.65-6.91) in Pancreatic steatosis cluster, and 1.95 (1.07-3.54) in Myosteatosis dominant cluster. The clusters were replicated in the German cohort with similar distribution of AUC-glucose to the diabetes hazard in the Japanese cohort. Insulin sensitivity and insulin secretion were different across clusters with the lowest insulin sensitivity and highest insulin secretion in Hepatic steatosis cluster. Conclusions: Extending evidence about fat accumulation in single compartments, we identified specific patterns of fat distribution with different T2D risk presumably due to differences in insulin sensitivity and insulin secretion. Disclosure H.Yamazaki: None. N.Stefan: Advisory Panel; Gilead Sciences, Inc., GlaxoSmithKline plc., Sanofi, Consultant; AstraZeneca, Intercept Pharmaceuticals, Inc., Novo Nordisk, Pfizer Inc., Speaker's Bureau; AstraZeneca, Boehringer Ingelheim International GmbH, Lilly Diabetes, Merck Sharp &amp; Dohme Corp. A.Fritsche: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk, Sanofi-Aventis Deutschland GmbH. A.L.Birkenfeld: None. R.Wagner: Advisory Panel; Akcea Therapeutics, Daiichi Sankyo, Sanofi-Aventis Deutschland GmbH, Speaker's Bureau; Lilly, Novo Nordisk, Sanofi-Aventis Deutschland GmbH. M.Heni: Advisory Panel; Boehringer Ingelheim International GmbH, Research Support; Boehringer Ingelheim International GmbH, Sanofi, Speaker's Bureau; Amryt Pharma Plc, Boehringer Ingelheim International GmbH, Novo Nordisk. S.Tauchi: None. J.Machann: None. T.Haueise: None. Y.Yamamoto: None. M.Dohke: None. N.Hanawa: None. Y.Kodama: Other Relationship; Eisai Co., Ltd. A.Katanuma: None. Funding the German Center for Diabetes Research (DZD, 01GI0925) , the state of Baden-Württemberg (32-5400/58/2, Forum Gesundheitsstandort Baden-Württemberg) , and JSPS KAKENHI Grant Number 19K16978

ObjectiveKallistatin (KST), also known as SERPIN A4, is a circulating, broadly acting human plasma protein with pleiotropic properties. Clinical studies in humans revealed reduced KST levels in obesity. The exact role of KST in glucose and energy homeostasis in the setting of insulin resistance and type 2 diabetes is currently unknown. MethodsKallistatin mRNA expression in human subcutaneous white adipose tissue (sWAT) of 47 people with overweight to obesity of the clinical trial “Comparison of Low Fat and Low Carbohydrate Diets With Respect to Weight Loss and Metabolic Effects (B-SMART)” was measured. Moreover, we studied transgenic mice systemically overexpressing human KST (hKST-TG) and wild type littermate control mice (WT) under normal chow (NCD) and high-fat diet (HFD) conditions. ResultsIn sWAT of people with overweight to obesity, KST mRNA increased after diet-induced weight loss. On NCD, we did not observe differences between hKST-TG and WT mice. Under HFD conditions, body weight, body fat and liver fat content did not differ between genotypes. Yet, during intraperitoneal glucose tolerance tests (ipGTT) insulin excursions and HOMA-IR were lower in hKST-TG (4.42 ± 0.87 AU, WT vs. 2.20 ± 0.27 AU, hKST-TG, p < 0.05). Hyperinsulinemic euglycemic clamp studies with tracer-labeled glucose infusion confirmed improved insulin sensitivity by higher glucose infusion rates in hKST-TG mice (31.5 ± 1.78 mg/kg/min, hKST-TG vs. 18.1 ± 1.67 mg/kg/min, WT, p < 0.05). Improved insulin sensitivity was driven by reduced hepatic insulin resistance (clamp hepatic glucose output: 7.7 ± 1.9 mg/kg/min, hKST-TG vs 12.2 ± 0.8 mg/kg/min, WT, p < 0.05), providing evidence for direct insulin sensitizing effects of KST for the first time. Insulin sensitivity was differentially affected in skeletal muscle and adipose tissue. Mechanistically, we observed reduced Wnt signaling in the liver but not in skeletal muscle, which may explain the effect. ConclusionsKST expression increases after weight loss in sWAT from people with obesity. Furthermore, human KST ameliorates diet-induced hepatic insulin resistance in mice, while differentially affecting skeletal muscle and adipose tissue insulin sensitivity. Thus, KST may be an interesting, yet challenging, therapeutic target for patients with obesity and insulin resistance.

Dear Editor: The fluidity of cell membranes has been described as a central link in the association of fatty acids (FAs) with susceptibility to type 2 diabetes (T2D). Decreased membrane fluidity has been connected with insulin resistance. Obesity, especially excess fat around the waist, is considered a primary cause of insulin resistance. It is well known that insulin resistance is the basis of the development of T2D. Adipose inflammation is described as a key component of the pathophysiology in obesity-related insulin resistance, T2D, and downstream complications. T2D has long been defined as a disease of inflammation. A bulk of data suggests that the pro-inflammatory cytokine interferon-γ (IFN-γ) plays a major role in the regulation of the inflammatory response that accompanies the generation of insulin resistance, obesity, and T2D. IFN-γ is regarded as one of the most significant independent predictors of metabolic syndrome. Adequate membrane fluidity is essential to cell function. The fluidity of cell membranes is strongly linked to its FA composition. The more saturated the fats are in the membrane, the more rigid the membrane. Conversely, unsaturated fatty acids (UFAs) with the kinks produced by their double bonds increase membrane fluidity. UFAs have been assessed to have profound effects onmembrane fluidity that, in turn, influences a variety of cellular functions including the properties of the insulin receptor and insulin sensitivity as well as glucose transport across membranes. Although the specific mechanism bywhich a shift from unsaturated toward saturated fatty acids in cell membranes disrupts insulin sensitivity is not well established, it has been speculated that a decrease in GLUT4 expression in adipocytes may represent a possibility. It has been found that IFN-γmarkedly reduces expression of insulin signaling proteins including the GLUT4 glucose transporter. Interestingly, theGLUT4 expression is downregulated in adipose tissue in obesity. GLUT4 is a major mediator of glucose removal from the circulation and a key regulator of whole-body glucose homeostasis. GLUT4 is one of the 13 sugar protein transporters that catalyzes hexose transport across cell membranes. Among these sugar transporters, GLUT4 is highly expressed in adipose tissue. GLUT4 in adipose tissue is indispensable for normal global glucose homeostasis, while insulin receptors in this tissue appear much less critical. Depletion of GLUT4 in either adipose tissue or skeletal muscles has been recognized to cause insulin resistance and propensity toward diabetes. Remarkable, overexpression of GLUT4 in the adipose tissue of musclespecific GLUT4-deficient mice has been shown to overcome the glucose intolerance and diabetes. It has been observed that UFAs exert an anti-inflammatory action on adipose tissue and especially on mature adipocytes. Although there is strong evidence about the beneficial effect of certain UFAs in the adequate proportion in patients with T2D, there are no concluding data in this respect. UFAs have been recognized to influence insulin metabolism, positively improving considerably insulin action. Intake of diets rich in PUFAs, particularly n-3 and n-6, has been shown to modulate the inflammatory response modifying the evolution of T2D. All these contentions led us to hypothesize that adequate UFA membrane levels may counteract the generation of insulin resistance through the induction of GLUT4 expression as a result of inhibition of IFN-γ production. Understanding the impact of the interaction between IFN-γ and GLUT4 on the onset of T2D may provide new insights into the pathogenesis and prevention of this condition. Administration of anti-IFN-γ may be a promising therapy to halt or prevent T2D, it being understood that diet and exercise are mandatory. * Raffaella Mormile raffaellamormile@alice.it

Full text Figures and data Side by side Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The consequences of aneuploidy have traditionally been studied in cell and animal models in which the extrachromosomal DNA is from the same species. Here, we explore a fundamental question concerning the impact of aneuploidy on systemic metabolism using a non-mosaic transchromosomic mouse model (TcMAC21) carrying a near-complete human chromosome 21. Independent of diets and housing temperatures, TcMAC21 mice consume more calories, are hyperactive and hypermetabolic, remain consistently lean and profoundly insulin sensitive, and have a higher body temperature. The hypermetabolism and elevated thermogenesis are likely due to a combination of increased activity level and sarcolipin overexpression in the skeletal muscle, resulting in futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) activity and energy dissipation. Mitochondrial respiration is also markedly increased in skeletal muscle to meet the high ATP demand created by the futile cycle and hyperactivity. This serendipitous discovery provides proof-of-concept that sarcolipin-mediated thermogenesis via uncoupling of the SERCA pump can be harnessed to promote energy expenditure and metabolic health. Editor's evaluation This important paper provides new insight into the effect of extra-copies of a chromosome, thus aneuploidy, on body metabolisms in mammals. The authors used various solid analyses on the metabolisms and physiology of the transgenic mouse with most of human chromosome 21 and presented convincing results to support the authors' claims. The work would be of interest to researchers who work on the physiology and biochemistry of body metabolisms in mammals. https://doi.org/10.7554/eLife.86023.sa0 Decision letter Reviews on Sciety eLife's review process Introduction The presence of an extra chromosome in mammals is generally lethal during fetal development, due to widespread cellular havoc caused by misregulated gene expression arising from gene dosage imbalance (Zhu et al., 2018). Down syndrome (DS), resulting from trisomy of chromosome 21, is one of the rare aneuploidies compatible with life although as many as 80% of trisomy 21 conceptuses miscarry (Antonarakis et al., 2020). The increased expression of ~500 transcribed sequences of human chromosome 21 (Hsa21) affects many cell types and organ systems during development and in the postnatal period (Korenberg et al., 1994; Antonarakis, 2017). Humans with trisomy 21 have cognitive deficits, altered craniofacial development, and are at significantly higher risk for congenital heart defects, hearing and vision loss, leukemia, gastrointestinal disease, and early-onset dementia (Antonarakis et al., 2020). Given the significant impact of intellectual disability on the lives of individuals with DS, research emphasis has naturally focused on the neurological deficits underpinning trisomy 21 (Potier and Reeves, 2016). In addition to developmental abnormalities associated with DS, there is an increasing awareness that adolescents and adults with DS also have an increased incidence of obesity, insulin resistance, and diabetes (Van Goor et al., 1997; Bertapelli et al., 2016; Fonseca et al., 2005). Although this was first noted in the 1960s (Milunsky and Neurath, 1968), the underlying cause for these metabolic dysregulations is mostly unknown and largely underexplored. Beyond clinical observations, limited studies have been conducted to determine the physiological underpinnings of metabolic impairments seen in DS (reviewed in Dierssen et al., 2020). Our recent study on the Ts65Dn mouse model represents the most in-depth metabolic analysis, to date, of any DS mouse model (Sarver et al., 2023). However, the segmental trisomic Ts65Dn mouse contains only ~55% of the orthologous protein-coding genes (PCGs) found on Hsa21 (Gupta et al., 2016). In addition, it contains additional trisomic genes from the centromeric region of mouse chromosome 17 (Mmu17) not found in Hsa21, thus complicating the genotype-phenotype relationships (Duchon et al., 2011; Reinholdt et al., 2011). In the past two decades, more than 20 mouse models of DS have been generated (Herault et al., 2017). Despite their utility in advancing DS research, none of these models recapitulate the full spectrum of human DS. With the exception of Tc1, all the DS mouse models are trisomic for some, but not all, of the orthologous mouse genes found in Hsa21 (Herault et al., 2017). Tc1 is the first mouse model with an independently segregating Hsa21 (O’Doherty et al., 2005). However, Tc1 mice are missing >50 of the 220 PCGs on Hsa21 due to deletion and mutations (Gribble et al., 2013). In addition, Tc1 mice show extensive mosaicism (i.e. the human chromosome is present in zygotes but is lost randomly from cells during development). As a consequence, every mouse has a unique developmental trajectory, complicating the interpretations of results obtained from Tc1 mice. To overcome the limitations of previously generated trisomic mouse models, a transchromosomic mouse model (TcMAC21) carrying an independently segregating and near-complete copy of Hsa21 was recently developed (Kazuki et al., 2020). TcMAC21 is not mosaic and contains 93% of HSA21q PCGs, and is considered the most representative mouse model of DS. Both mouse and rat that carry a non-mosaic Hsa21 recapitulate many DS phenotypes related to the central nervous system (e.g. reduced cerebellum volume, learning and memory deficit), craniofacial skeleton, and heart (Kazuki et al., 2020; Kazuki et al., 2022). The metabolic phenotype of TcMAC21, however, is unknown and has yet to be examined. The availability of the TcMAC21 mouse model has afforded a unique opportunity to address two fundamental questions: (1) what is the impact of aneuploidy on systemic metabolism; (2) what are the molecular, cellular, and physiological consequences of introducing a foreign (human) chromosome from an evolutionarily distant species into mice? Unexpectedly, we discovered that TcMAC21 mice have all the hallmarks of hypermetabolism, likely driven by a combination of hyperactivity and elevated mitochondrial respiration and futile sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) pump activity in the skeletal muscle as a consequence of endogenous sarcolipin (SLN) overexpression. Our study has provided further evidence and proof-of-concept that endogenous SLN-mediated uncoupling of the SERCA pump can be harnessed for energy dissipation, weight loss, and metabolic health. Results Human chromosome 21 genes are differentially expressed and regulated in mouse adipose tissue, liver, and skeletal muscle TcMAC21 mice carry a non-mosaic and independently segregating mouse artificial chromosome with a near-complete copy of the long arm of human chromosome 21 (Hsa21q) (Kazuki et al., 2020). The Hsa21q in TcMAC21 is comprised of ~37 Mb and 199 PCGs (Figure 1A). RNA-sequencing showed that TcMAC21 mice are capable of expressing Hsa21-derived transcripts in each of the tissues examined, and that gene expression is regulated in a tissue-specific manner. The transcriptional activity map of Hsa21 shows regions of gene expression and repression (Figure 1B). There are three large regions of Hsa21 with little or no transcription activity: 29.5–31.1 Mb, 44.5–44.8 Mb, and 45.3–46.2 Mb. The first gap of transcriptional inactivity contains the PCGs Cldn8 and Cldn17, Girk1, and 33 distinct Krtap (keratin-associated protein) genes. The second transcriptionally inactive area contains 16 Krtap genes, and the third transcriptionally inactive area contains 8 PCGs (Col6a1, Col6a2, Col18a1, Fctd, Lss, Pcbp3, Slc19a1, and Spatc1l). By filtering the transcriptional map to display expressed genes only, we highlighted all the genes with their differential expression profiles across five major metabolic tissues—brown adipose tissue (BAT), inguinal white adipose tissue (iWAT), gonadal white adipose tissue (gWAT), liver, and skeletal muscle (Figure 1C). Figure 1 Download asset Open asset Human chromosome 21 genes are differentially expressed and regulated in mouse adipose tissue, liver, and skeletal muscle. (A) Graphical representation of human chromosome 21 (Hsa21) and the entire long arm (Hsa21q) region carried by a mouse artificial chromosome in the transchromosomic mouse model (TcMAC21). Four deletions that occurred during generation of the transchromosomic mice eliminate 14/213 protein-coding genes (PCGs; 7%) and 105/487 predicted or known non-protein-coding genes (NPCGs; 22%) (Kazuki et al., 2020). (B) Global view of transcriptionally expressed and repressed PCG and NPCG regions over the entire Hsa21q across five tissues. Gray box denotes transcript that is not detected. (C) Transcriptional activity map showing only Hsa21 genes expressed by at least one tissue. Gray box denotes transcript that is not detected. (D) Overlap analysis showing shared expression of human PCGs and NPCGs across five tissues. Of the 235 unique human genes expressed by the TcMAC21 mice, 54% are PCGs and 46% are NPCGs. B, brown adipose tissue; iW, inguinal white adipose tissue; gW, gonadal white adipose tissue; L, liver; M, skeletal muscle (gastrocnemius); n.d., not detected. n=5 RNA samples per group per tissue type. Mice were on high-fat diet for 16 weeks at the time of tissue collection. One of the more striking differences in expression profile is between visceral (gonadal) and subcutaneous (inguinal) white adipose tissue (gWAT and iWAT respectively). gWAT expresses 115 human PCGs while iWAT expresses only 27. A similar pattern can be seen in the non-protein-coding genes (NPCGs). We were unable to detect any Hsa21-derived NPCGs in the iWAT, while in gWAT we observed 37. Overlap analysis was carried out to assess how similar expression profiles were between tissues (Figure 1D). Of the 126 Hsa21-derived PCGs expressed by at least one tissue, a majority (65 total) are shared between BAT, gWAT, liver, and skeletal muscle. Of the 109 Hsa21-derived NPCGs expressed by at least one tissue, the majority (53 total) are uniquely expressed by skeletal muscle. Of note, the liver uniquely expresses 6 PCGs and 2 NPCGs, gWAT 4 and 3, skeletal muscle 3 and 53, BAT 1 and 10, and iWAT 0 and 0. Together, these data indicate that Hsa21-derived transcripts are differentially expressed and regulated across major metabolic tissues in TcMAC21 mice. Hypermetabolism in TcMAC21 mice fed a standard chow Having established that Hsa21-derived transcripts are differentially expressed and regulated in mouse organs and tissues, we next asked the impact of the extra human genetic material and genes on systemic metabolism. As previously documented, TcMAC21 pups are born at the same weight as their euploid littermates (Kazuki et al., 2020). However, by 3 months of age TcMAC21 mice fed a standard chow weighed significantly less (~8.5 g) than euploid littermates, and this weight difference remained stable over time (Figure 2A). The size and body weight differences were not due to reduced plasma IGF-1 and growth hormone, as their circulating levels were in fact higher in TcMAC21 compared to euploid mice (Figure 2—figure supplement 1). Body composition analysis showed that TcMAC21 have significantly reduced absolute and relative (normalized to body weight) fat mass (Figure 2B). Although the absolute lean mass was reduced in TcMAC21 mice, the relative lean mass (normalized to body weight) was not different between genotypes (Figure 2B). Tissue collection at termination of the study also showed smaller visceral and subcutaneous fat mass and liver weight in TcMAC21 mice (Supplementary file 1). Figure 2 with 3 supplements see all Download asset Open asset Hypermetabolism in TcMAC21 mice fed a standard chow. (A) Body weights of mice fed a standard chow. (B) Body composition analysis of fat and lean mass (relative to body weight). (C) Absolute and relative (normalized to body weight) food intake over a 24 hr period. (D–F) Energy expenditure (EE) and physical activity level over 24 hr period in ad libitum chow-fed mice (D), during fasting (E), and refeeding after a fast (F). EE is normalized to lean mass in the 24 hr trace or analyzed by ANCOVA where body weight was used as a covariate. (G) Hematoxylin and eosin (H&E)-stained sections of inguinal white adipose tissue (iWAT) and adipocyte cross-sectional area (CSA) quantification. (H) Histology of gonadal white adipose tissue (gWAT) and adipocyte CSA quantification. (I) Histology of liver tissues with quantification of area covered by lipid droplets per focal plane. (J) Fasting serum triglyceride, cholesterol, non-esterified fatty acids (NEFA), β-hydroxybutyrate (ketone) levels. (K) Fasting blood glucose and insulin levels. (L) Insulin resistance index (homeostatic model assessment for insulin resistance [HOMA-IR]). (M) Glucose tolerance tests. (N) Insulin tolerance tests. Sample size for all data: euploid (n=8) and TcMAC21 (n=9). Differences in body weight were not due to reduced caloric intake, as TcMAC21 mice actually consumed the same or slightly higher amount of food despite being markedly leaner (Figure 2C and Supplementary file 2). Thus, relative to their body weight, TcMAC21 actually consumed a significantly higher amount of food than euploid controls (Figure 2C). Indirect calorimetry analysis indicated that TcMAC21 mice—regardless of the photocycle (light or dark phase) and metabolic states (ad libitum fed, fasting, refeed)—were expending ~25% more energy and were significantly more active compared to euploid controls (Figure 2D–F and Supplementary file 2). It is known that normalization to lean mass can lead to an overestimation of energy expenditure (EE) (Tschöp et al., 2012). For this reason, ANCOVA (using body weight as a covariate of EE) (Tschöp et al., 2012) were also performed. Both types of analyses suggested that TcMAC21 mice had significantly higher EE relative to euploid controls (Figure 2D–F). Despite much higher caloric intake per gram body mass, TcMAC21 mice were much leaner due to substantially elevated physical activity and EE. Hyperactivity and elevated EE were not due to altered circulating thyroid hormones, as serum triiodothyronine (T3, the active form of TH) levels were not different between chow fed TcMAC21 and euploid mice (Figure 2—figure supplement 1). Serum level of thyroxine (T4), the precursor of T3, were modestly elevated in TcMAC21 relative to euploid mice. In accordance with the lean phenotype, TcMAC21 mice had significantly smaller adipocyte cell size in both subcutaneous (inguinal) and visceral (gonadal) fat depots (Figure 2G–H), as well as significantly reduced fat accumulation in liver (Figure 2I). Fasting triglyceride, non-esterified fatty acid (NEFA), and β-hydroxybutyrate levels were not different between genotypes; fasting cholesterol, however, was higher in TcMAC21 mice (Figure 2J). Although fasting insulin levels were not different between groups, fasting blood glucose was significantly lower in TcMAC21 mice (Figure 2K). The insulin resistance index (homeostatic model assessment for insulin resistance [HOMA-IR]) along with GTT and insulin tolerance test (ITT) suggested modest improvements in insulin sensitivity in TcMAC21 relative to euploid mice (Figure 2L–N). Assessment of the pancreas showed that TcMAC21 mice have similar β-islet cross-sectional area (CSA), insulin and somatostatin (SST) content, and insulin granule and vesicle size compared to euploid controls (Figure 2—figure supplement 2). Taken together, these data indicate that chow-fed TcMAC21 mice at baseline are lean despite increased caloric intake, and this is largely due to elevated physical activity and EE. TcMAC21 mice are resistant to diet-induced obesity and metabolic dysfunction The hypermetabolic phenotypes seen in chow-fed TcMAC21 predicted that these mice would be resistant to diet-induced obesity and metabolic dysfunction. Indeed, after 8 weeks on high-fat diet (HFD), TcMAC21 mice gained only ~3 g of body weight, whereas the euploid controls gained >15 g of body weight over the same period. Consequently, TcMAC21 mice weighed ~50% less than euploid controls (Figure 3A). Consistent with the lean phenotype, the absolute and relative (normalized to body weight) fat mass were markedly reduced compared to euploid controls (Figure 3B). The weights of other organs (liver, kidney, BAT) at time of termination were also lower in TcMAC21 mice, but tibia length was not different between genotypes (Figure 3—figure supplement 1 and Supplementary file 3). Complete blood count revealed no differences in erythroid, lymphoid, and myeloid cell numbers between genotypes (Supplementary file 4). Because relative lean mass was higher in TcMAC21 compared to euploid mice (Figure 3B), the lean phenotype seen in HFD-fed TcMAC21 is largely due to reduced adiposity. Accordingly, TcMAC21 had significantly smaller adipocyte cell size in both subcutaneous (inguinal) and visceral (gonadal) fat depots, and a marked reduction in lipid accumulation in the liver (Figure 3C–E). Figure 3 with 1 supplement see all Download asset Open asset TcMAC21 mice are resistant to diet-induced obesity and metabolic dysfunction. (A) Body weights over time on a high-fat diet and representative mouse images. (B) Body composition analysis of fat and lean mass. (C) Histology of inguinal white adipose tissue (iWAT) and quantification of adipocyte cross-sectional area (CSA). (D) Histology of gonadal white adipose tissue (gWAT) and quantification of adipocyte CSA. (E) Histology of liver tissue and quantification of area covered by lipid droplets per focal plane. (F) Fasting serum triglyceride, cholesterol, non-esterified fatty acids (NEFA), β-hydroxybutyrate (ketone) levels. (G) Fasting blood glucose and insulin levels. (H) Insulin resistance index (homeostatic model assessment for insulin resistance [HOMA-IR]). (I) Glucose tolerance tests (GTTs). (J) Serum insulin levels during GTT. (K) Insulin tolerance tests (ITTs). (L) Blood glucose levels after an overnight (16 hr) fast and 1, 2, and 3 hr of food reintroduction. (M) Serum insulin levels after a 16 hr fast and 2 hr of refeeding. (N) Pancreas histology and quantification of β-islet CSA. (O–P) Pancreatic insulin and somatostatin (SST) contents (normalized to pancreatic protein input). (Q) Electron micrographs (EM) of pancreatic β-cells showing dense insulin granules and their surrounding vesicles, and quantification of insulin granule CSA, insulin vesicle CSA, and the ratio of insulin granule to insulin vesicle. n=8 euploid and 8–9 TcMAC21 mice for all graphs from A to P. n=3 euploid and 3 TcMAC21 used for EM quantification; each data point represents 1200 insulin granules and 1200 insulin vesicles quantified across six unique locations per mouse, graphs Q. Although fasting serum triglyceride, NEFA, and β-hydroxybutyrate levels were not different between genotypes, serum cholesterol was significantly lower in TcMAC21 mice (Figure 3F). Fasting glucose and insulin levels, and the insulin resistance index (HOMA-IR), were markedly lower in TcMAC21 mice relative to euploid controls (Figure 3G–H), indicative of enhanced insulin sensitivity. In glucose tolerance tests (GTTs), even though the rate of glucose disposal was similar between TcMAC21 and euploid mice, the amount of serum insulin present during GTT (time 0, 15, and 30 min) was dramatically lower in TcMAC21 (Figure 3I–J). This indicates that a substantially lower amount of insulin is sufficient to promote glucose clearance in TcMAC21 at a rate comparable to euploid mice, consistent with elevated insulin sensitivity in the peripheral tissues. Indeed, when we directly assessed insulin action via ITTs, TcMAC21 mice clearly exhibited higher insulin sensitivity as indicated by the significant differences in insulin-stimulated glucose disposal (Figure 3K). To independently confirm TcMAC21 mice are more insulin sensitive, we fasted the mice overnight (16 hr) then reintroduced them to food. Under this fasting-refeeding condition, we could clearly see the resumption of food intake was successful at increasing blood glucose in TcMAC21 (Figure 3L); however, the insulin response to food intake in TcMAC21 mice was strikingly smaller in magnitude compared to euploid controls (Figure 3M). Again, these data indicate that TcMAC21 mice are significantly more insulin sensitive since a substantially lower insulin response during fasting-refeeding is sufficient for glucose clearance at a rate comparable to euploid mice. These results prompted us to determine if there were developmental in the pancreas to reduced insulin in response to glucose or food intake, of elevated insulin sensitivity in peripheral tissues. of β-islet pancreatic insulin and content, as well as insulin granule and vesicle size not any differences between TcMAC21 and euploid mice (Figure thus out a developmental cause and in of enhanced insulin Pancreatic granule size was also not different between genotypes (Figure 2—figure supplement development of the Taken together, these data indicate that TcMAC21 mice are resistant to obesity and insulin Hypermetabolism in TcMAC21 mice fed an we to the physiological for TcMAC21 resistance to weight and insulin resistance when fed an we to out there is a in caloric TcMAC21 mice actually consumed the same or slightly higher amount of food as the euploid controls despite markedly lower body weight (Figure and Supplementary file Thus, relative to their body weight, HFD-fed TcMAC21 mice consumed a significantly higher amount of food than the euploid controls (Figure To out any dysfunction of the that we and samples of each mouse to weight, energy was different between TcMAC21 and euploid mice (Figure energy lower in TcMAC21 (Figure a modest in the of energy intake per gram body weight) was significantly higher in TcMAC21 and were similar across genotypes, the data hypermetabolism being a cause of the lean phenotype seen in TcMAC21 mice. Indeed, when we EE and physical activity of both groups, we found that TcMAC21 mice have markedly higher EE and physical activity of cycle and metabolic states (Figure and Supplementary file of the overestimation of EE when normalized to lean mass, we also ANCOVA where body weight was used a covariate (Tschöp et al., 2012). Both types of analyses suggested that TcMAC21 mice fed an had significantly higher EE relative to euploid controls (Figure The striking difference in EE was similar to TcMAC21 fed a standard chow (Figure but to an even when mice were fed an due to the availability of lipid for Figure 4 Download asset Open asset Hypermetabolism in TcMAC21 mice fed a high-fat diet (A) Absolute and relative (normalized to body weight) food intake in mice fed an (B) energy content, weight, and of TcMAC21 mice and euploid (C) Serum triiodothyronine and thyroxine levels. (D–F) Energy expenditure (EE) and activity level over 24 hr period in ad libitum HFD-fed mice (D), during fasting (E), and refeeding after a fast (F). EE is normalized to lean mass in the 24 hr trace or analyzed by ANCOVA where body weight was used as a covariate. (G) and over 3 in both the and dark (H) of mice. (I) Body weights of TcMAC21, and mice. (J) of TcMAC21, and mice. (K) histology of brown adipose tissue (L) of lipid area per focal in BAT of euploid and (M) of mouse genes known to major metabolic in Sample size for all data: euploid (n=8) and TcMAC21 (n=9). Because TcMAC21 mice a large of we the circulating levels of thyroid as are known to metabolic rate and EE et al., Both serum active and of levels were significantly higher in TcMAC21 relative to euploid mice (Figure hormone, however, was not elevated in HFD-fed mice at (Figure 2—figure supplement 1). EE was body of TcMAC21 mice would likely Indeed, of TcMAC21 were most in the dark cycle when mice are active (Figure Assessment with showed an elevated the region of TcMAC21 mice, whereas the was not different between (Figure the differences in in TcMAC21 even when compared to mice (Figure thus out body weight as the cause of generation to for body however, was not observed in chow-fed mice (Figure 2—figure supplement even though chow-fed TcMAC21 were also hyperactive and had higher EE. Consistent with the analysis of the BAT revealed a marked reduction in fat accumulation and a brown in TcMAC21 mice when compared to euploid controls (Figure due to being We metabolic genes in BAT to be in we found differences in and fat genes between the two of mice (Figure Figure supplement 1). Hypermetabolism of TcMAC21 mice is from in adipose and liver The of observed in and fat genes us to the differences in gene expression be due to and To test we conducted an RNA-sequencing analysis of BAT, liver, gWAT, and Again, to and to we found the of BAT, liver, gWAT, and iWAT in TcMAC21 to be similar to euploid with only a limited of genes expression was significantly altered (Figure Figure with supplements see all Download asset Open asset Hypermetabolism of TcMAC21 mice is from in adipose and liver expressed mouse genes both protein-coding and non-protein-coding genes in brown adipose tissue (BAT), liver, gonadal white adipose tissue (gWAT), and inguinal white adipose tissue data is relative to euploid and presented as The of genes represents all and mouse genes by for all tissues. The indicate genes and the indicate genes. (E) view and of transcriptional in BAT, liver, gWAT, and iWAT to the strikingly in the mouse across the tissues. There are only a of differentially expressed genes across tissues, with the relative of the for each tissue. Of the are protein-coding genes (PCGs; dark and are non-protein-coding genes (NPCGs; Of the are and are In only a is noted in the of all tissues of the Sample size for euploid per and TcMAC21 per There was not a tissue that had more than of significantly altered (Figure and Supplementary of the significant in gene in for the striking differences in phenotypes between TcMAC21 and euploid mice. the RNA-sequencing analyses of metabolic genes in BAT, liver, gWAT, and iWAT also showed in TcMAC21 of diet or and or (Figure supplements Together, these data indicate that TcMAC21 mice are hyperactive and hypermetabolic with elevated but these phenotypes are largely from in BAT, liver, gWAT, and overexpression in skeletal muscle TcMAC21 hypermetabolism Given the differences seen in body weight, tissue histology and lipid insulin body physical

The MBL2 gene encodes the mannose-binding lectin protein (MBL), which is secreted by the liver. Several variants of MBL2 have been found to be associated with altered serum levels and susceptibility to various chronic diseases. Defects in MBL protein polymerization that result in functional impairments and/or low serum levels may influence genetic susceptibility to type 2 diabetes (T2D) and its complications. Therefore, the present case-control study was conducted to assess the potential association of six MBL2 gene variants and haplotypes with susceptibility to T2D and its complications in Morocco. The MBL2 gene was genotyped by PCR-sequencing for the promoting, non-coding, and coding regions in 435 individuals. Our findings revealed a significant association between the heterozygous CG and homozygous recessive GG genotypes of the variant at position −221 C > G in the MBL2 gene promoter with an increased risk of T2D. Similarly, for +4 C > T in the non-coding region, statistical analysis indicates a strong association with T2D risk, particularly with the heterozygous CT and homozygous recessive TT genotypes. The LYQC haplotype is also found to be associated with T2D risk. Furthermore, the heterozygous CT genotype, and recessive T allele of the variant at position +4 C > T, and heterozygous GA genotype of codon Gly54Asp of the MBL2 gene, are associated with protection against hypertension in T2D patients. However, no association was observed between MBL2 variants and dyslipidemia in T2D patients. The study concludes that −221 C > G and +4 C > T variants of the MBL2 gene significantly contribute to T2D susceptibility in Morocco.

Analysis of candidate genes on chromosome 20q12-13.1 reveals evidence for BMI mediated association of PREX1 with type 2 diabetes in European Americans

BackgroundRed rice koji (RRK), prepared by growing Monascus species on steamed rice, has been reported to lower blood glucose levels in diabetic animal models. However, the action mechanism is not yet completely understood.ObjectiveThe objective of this study was to examine the mechanism underlying the hypoglycemic action of RRK extract in two diabetic animal models: the insulin-deficiency mice, where the insulin deficiency was induced by streptozotocin (STZ), and insulin-resistance mice, where the insulin resistance was induced by a high-fat diet (HFD).DesignLow (12.5 mg/kg body weight [BW]) and high (50.0 mg/kg BW) doses of RRK extract were orally administered to the mice for 10 successive days (0.25 mL/day/mouse). The protein expression levels of glucose transporter type 4 (GLUT4) in the skeletal muscle and glucose transporter type 2 (GLUT2) in the liver were measured. Blood glucose (BG) levels of STZ-treated mice in insulin tolerance test (ITT) and BG and insulin levels of HFD-fed mice in intraperitoneal glucose tolerance test (IPGTT) were investigated.ResultsIn the STZ-treated mice, oral administration of RRK extract lowered BG levels and food intake but increased plasma 1,5-anhydroglucitol level. Moreover, the RRK extract lowered the BG levels of STZ-treated mice as measured by ITT. In the HFD-fed mice, we confirmed that the orally administered RRK extract lowered the BG and the homeostasis model assessment index for insulin resistance. Furthermore, the RRK extract lowered the BG and insulin levels of HFD-fed mice in IPGTT. Regarding the protein levels of GLUT, the orally administered RRK extract increased the GLUT4 level in the skeletal muscle; however, the RRK extract did not alter the GLUT2 level in the liver of either the STZ-treated or the HFD-fed mice.DiscussionOur study demonstrates that RRK extract can improve impaired glucose tolerance in mouse models of diabetes by enhancing GLUT4 expression in skeletal muscle.ConclusionThese results suggest that RRK extract could potentially be a functional food for the treatment of diabetes mellitus.

Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) regulates translation of IGF2, a growth factor that plays a key role in controlling fetal growth and organogenesis including adipogenesis and pancreatic development. In Caucasians, the rs4402960 G>T polymorphism of IGF2BP2 has been shown to predispose to type 2 diabetes (T2D) in multiple populations. In this study, we tested whether rs4402960 G>T and rs11705701 G>A contribute to the development of T2D in a Russian population. Both markers were genotyped in Russian diabetic (n = 1,470) and non-diabetic patients (n = 1,447) using a Taqman allele discrimination assay. The odds ratio (OR) for the risk of developing T2D was calculated using logistic regression assuming an additive genetic model adjusted for age, sex, HbA1c, hypertension, obesity, and body mass index (BMI). Multivariate linear regression analyses were used to test genotype-phenotype correlations, and adjusted for age, sex, hypertension, obesity, and BMI. Expression of IGF2BP2 in the visceral adipose tissue was quantified using real-time PCR. The content of IGF2BP2 protein and both its isoforms (p58 and p66) in the adipose tissue was measured using Western blot analysis. There was no significant association between rs4402960 and T2D. Whereas, allele A of rs11705701 was associated with higher T2D risk (OR = 1.19, p < 0.001). Diabetic and non-diabetic carriers of genotype TT (rs4402960) had significantly increased HOMA-IR (p = 0.033 and p = 0.031, respectively). Non-diabetic patients homozygous for AA (rs11705701) had higher HOMA-IR (p = 0.04), lower HOMA-β (p = 0.012), and reduced 2-h insulin levels (p = 0.016). Non-obese individuals (diabetic and non-diabetic) homozygous for either AA (rs11705701) or TT (rs4402960) had higher levels of IGF2BP2 mRNA in the adipose tissue than other IGF2BP2 variants. Also, allele A of rs11705701 was associated with reduced amounts of the short isoform (p58) and increased levels of the long isoform (p66) of the IGF2BP2 protein in adipose tissue of non-obese diabetic and non-diabetic subjects. IGF2BP2 genetic variants contribute to insulin resistance in Russian T2D patients. The short protein isoform p58 of IGF2BP2 is likely to play an anti-diabetogenic role in non-obese individuals.

Objective Aerobic exercise training is important to prevent and cure chronic diseases such as diabetes, cardiovascular diseases and so on. Apelin has been identified as a novel myokine in recent years, and the exogenous supplementation of apelin can promote the glucose absorption, the biosynthesis of mitochondria and the oxidation of fatty acids in skeletal muscle. Intraperitoneal glucose (GTT) and insulin tolerance tests (ITT) are useful in vivo assays that provide approximations of glucose metabolism and homeostasis. The bigger area under the curve (AUC) confirmed the decreased glucose clearance, which is evaluated by GTT. However, the mechanism of apelin mediating glucose metabolism during aerobic exercise training is not clear. Our study was to investigate the differences of GTT and ITT after four weeks training between wild-type (WT) mice (C57BL/6J) and apelin Knockout (KO) mice.&#x0D; Methods Two-month-old WT and KO were divided into trained and control groups (n=8-10/group) respectively. There are four groups: WT control (WC), apelin KO control (KC), WT trained (WT), and apelin KO trained (KT). The trained groups were trained on treadmills for four weeks (six days per week and one hour per day). In order to maintain the exercise intensity, the speed is at 70%-75% VO2max with an incline of 5 degrees. The control groups were kept at a sedentary condition. after four weeks of interventions, glucose was measured at 0, 15, 30, 45, 60, 90, 120min following GTT. Glucose was also measured at 0, 30, 60, 90, 120min following ITT.&#x0D; Results (1) blood glucose levels and AUC of the KC were significantly bigger than those of WC. ITT showed that KC also had slower insulin-stimulated glucose clearance compared with the WC. (2) Following 4-week training, KT had lower blood glucose levels and AUC of the KT was significantly smaller than those of KC. KT had faster insulin-stimulated glucose clearance compared with KC.&#x0D; Conclusions Without apelin, glucose tolerance and insulin tolerance in mice will decrease. And aerobic exercise training improves them in apelin deficiency mice.