Featured ArticlesVolume 5 | No. 11 | November 2016
|Inhibition of citrate cotransporter Slc13a5/mINDY by RNAi improves hepatic insulin sensitivityThe SLC13A5 gene is the mammalian ortholog of the Drosophila melanogaster "I’m not dead, yet" (INDY) gene, in which its mutation is linked to a life-span extension. The data of Brachs and colleagues demonstrate that the mammalian homolog of INDY (mINDY) is involved in the regulation of liver fat metabolism. Its inhibition by a liver-specific siRNA is a reasonable approach to prevent fatty liver disease in C57BL/6J mice on a Western diet but has no effect on body weight. The data support that effects on hepatic lipid accumulation occur independent of body weight changes or whole-body fat content. The authors suggest that non-liver mINDY effects may mediate potential effects on body weight. |
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Objective: Non-alcoholic fatty liver disease is a world-wide health concern and risk factor for cardio-metabolic diseases. Citrate uptake modifies intracellular hepatic energy metabolism and is controlled by the conserved sodium-dicarboxylate cotransporter solute carrier family 13 member 5 (SLC13A5, mammalian homolog of INDY: mINDY). In Drosophila melanogaster and Caenorhabditis elegans INDY reduction decreased whole-body lipid accumulation. Genetic deletion of Slc13a5 in mice protected from diet-induced adiposity and insulin resistance. We hypothesized that inducible hepatic mINDY inhibition should prevent the development of fatty liver and hepatic insulin resistance.
Methods: Adult C57BL/6J mice were fed a Western diet (60% kcal from fat, 21% kcal from carbohydrate) ad libitum. Knockdown of mINDY was induced by weekly injection of a chemically modified, liver-selective siRNA for 8 weeks. Mice were metabolically characterized and the effect of mINDY suppression on glucose tolerance as well as insulin sensitivity was assessed with an ipGTT and a hyperinsulinemic-euglycemic clamp. Hepatic lipid accumulation was determined by biochemical measurements and histochemistry.
Results: Within the 8 week intervention, hepatic mINDY expression was suppressed by a liver-selective siRNA by over 60%. mINDY knockdown improved hepatic insulin sensitivity (i.e. insulin-induced suppression of endogenous glucose production) of C57BL/6J mice in the hyperinsulinemic-euglycemic clamp. Moreover, the siRNA-mediated mINDY inhibition prevented neutral lipid storage and triglyceride accumulation in the liver, while we found no effect on body weight.
Conclusions: We show that inducible mINDY inhibition improved hepatic insulin sensitivity and prevented diet-induced non-alcoholic fatty liver disease in adult C57BL6/J mice. These effects did not depend on changes of body weight or body composition.[Hide abstract]
|Glucose-6-phosphate dehydrogenase contributes to the regulation of glucose uptakeSkeletal muscle insulin resistance is an early metabolic defect that precedes hyperglycemia and marked weight gain in response to high-fat diet. Lee-Young and colleagues identify a novel and previously unidentified role for glucose-6-phosphate dehydrogenase (G6PDH) in skeletal muscle. Skeletal muscle G6PDH is defective across multiple disease states in animals and humans, with an underlying pathology of impaired glucose tolerance. G6PDH is directly regulated by skeletal muscle specific neuronal nitric oxide synthase (nNOSµ) activity, and this interaction appears to play a role in the regulation of insulin-independent glucose uptake. |
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Objective: The development of skeletal muscle insulin resistance is an early physiological defect, yet the intracellular mechanisms accounting for this metabolic defect remained unresolved. Here, we have examined the role of glucose-6-phosphate dehydrogenase (G6PDH) activity in the pathogenesis of insulin resistance in skeletal muscle.
Methods: Multiple mouse disease states exhibiting insulin resistance and glucose intolerance, as well as obese humans defined as insulin-sensitive, insulin-resistant, or pre-diabetic, were examined.
Results: We identified increased glucose-6-phosphate dehydrogenase (G6PDH) activity as a common intracellular adaptation that occurs in parallel with the induction of insulin resistance in skeletal muscle and is present across animal and human disease states with an underlying pathology of insulin resistance and glucose intolerance. We observed an inverse association between G6PDH activity and nitric oxide synthase (NOS) activity and show that increasing NOS activity via the skeletal muscle specific neuronal (n)NOSμ partially suppresses G6PDH activity in skeletal muscle cells. Furthermore, attenuation of G6PDH activity in skeletal muscle cells via (a) increased nNOSμ/NOS activity, (b) pharmacological G6PDH inhibition, or (c) genetic G6PDH inhibition increases insulin-independent glucose uptake.
Conclusions: We have identified a novel, previously unrecognized role for G6PDH in the regulation of skeletal muscle glucose metabolism.[Hide abstract]
|Intestinal CREBH overexpression prevents high-cholesterol diet-induced hypercholesterolemiaCyclic AMP-responsive element-binding protein H (CREBH) is a basic leucine zipper domain transcriptional factor of the CREB/activating transcription factor family. The study of Kikuchi and colleagues suggests that intestinal CREBH functions as a metabolic regulator to attenuate diet-induced hypercholesterolemia and cholelithiasis by decreasing expression of the transporter gene Niemann-Pick C1-like 1 (Npc1l1). It is known that hepatic CREBH has the potential to ameliorate hypertriglyceridemia. This study identifies intestinal CREBH as a possible therapeutic target for the treatment of hypercholesterolemia and related metabolic diseases.|
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Objective: The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by Creb3l3) is highly expressed in the liver and small intestine. Hepatic CREBH contributes to glucose and triglyceride metabolism by regulating fibroblast growth factor 21 (Fgf21) expression. However, the intestinal CREBH function remains unknown.
Methods: To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions.
Results: Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of Niemann–Pick C1-like 1 (Npc1l1), a rate-limiting transporter mediating intestinal cholesterol absorption, was reduced in the small intestine of CREBH Tg mice. Adenosine triphosphate-binding cassette transporter A1 (Abca1), Abcg5/8, and scavenger receptor class B, member 1 (Srb1) expression levels were also reduced in CREBH Tg mice. Promoter assays revealed that CREBH directly regulates Npc1l1 expression. Conversely, CREBH null mice exhibited higher intestinal Npc1l1 expression, elevated plasma and hepatic cholesterol, and lower fecal output.
Conclusions: Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia.[Hide abstract]
|Suppressing hyperinsulinemia prevents obesity but causes rapid onset of diabetesMutation of the gene encoding leptin in mice (Lepob/ob) results in a deficiency of leptin and increases in hyperphagia and adiposity. The study of D’souza et al. indicates that reducing insulin gene dosage attenuates hyperinsulinemia and prevents obesity in Lepob/ob mice. However, curtailing hyperinsulinemia in Lepob/ob mice also resulted in the development of hyperglycemia. In the absence of leptin, sufficient insulin levels are required to prevent diabetes.|
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Objective: Hyperinsulinemia is commonly associated with obesity. Mice deficient in the adipose-derived hormone leptin (Lepob/ob) develop hyperinsulinemia prior to onset of obesity and glucose intolerance. Whether the excess of circulating insulin is a major contributor to obesity and impaired glucose homeostasis in Lepob/ob mice is unclear. It has been reported previously that diet-induced obesity in mice can be prevented by reducing insulin gene dosage. In the present study, we examined the effects of genetic insulin reduction in Lepob/ob mice on circulating insulin, body composition, and glucose homeostasis.
Leptin expressing (Lepwt/wt) mice lacking 3 insulin alleles were crossed with Lepob/ob mice to generate Lepob/ob and Lepwt/wt littermates lacking 1 (
We found that in young Lepob/ob mice, loss of 2 or 3 insulin alleles reduced plasma insulin levels by 75–95% and attenuated body weight gain by 50–90% compared to Ins1+/+;Ins2+/−;Lepob/ob mice. This corresponded with ~30% and ~50% reduced total body fat in
Conclusions: Taken together, our findings indicate that hyperinsulinemia is required for excess adiposity in Lepob/ob mice and sufficient insulin production is necessary to maintain euglycemia in the absence of leptin.[Hide abstract]
|Depot-specific differences in angiogenic capacity of adipose tissue in differential susceptibility to diet-induced obesityTo suppress the rapidly growing prevalence of obesity, several treatments have been suggested, including the modulation of angiogenesis in adipose tissue (AT). Here, Song et al. investigate whether angiogenic capacity is depot-specific by analyzing angiogenic sprouting of epididymal and inguinal fats from diet-induced obese and diet-resistant mice fed a high-fat diet (HFD). This study indicates that, compared to subcutaneous fat, visceral fat has higher angiogenic capability in response to HFD-induced over-nutrition.|
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Objective: Adipose tissue (AT) expansion requires AT remodeling, which depends on AT angiogenesis. Modulation of AT angiogenesis could have therapeutic promise for the treatment of obesity. However, it is unclear how the capacity of angiogenesis in each adipose depot is affected by over-nutrition. Therefore, we investigated the angiogenic capacity (AC) of subcutaneous and visceral fats in lean and obese mice.
Methods: We compared the AC of epididymal fat (EF) and inguinal fat (IF) using an angiogenesis assay in diet-induced obese (DIO) mice and diet-resistant (DR) mice fed a high-fat diet (HFD). Furthermore, we compared the expression levels of genes related to angiogenesis, macrophage recruitment, and inflammation using RT-qPCR in the EF and IF of lean mice fed a low-fat diet (LFD), DIO mice, and DR mice fed a HFD.
Results: DIO mice showed a significant increase in the AC of EF only at 22 weeks of age compared to DR mice. The expression levels of genes related to angiogenesis, macrophage recruitment, and inflammation were significantly higher in the EF of DIO mice than in those of LFD mice and DR mice, while expression levels of genes related to macrophages and their recruitment were higher in the IF of DIO mice than in those of LFD and DR mice. Expression of genes related to angiogenesis (including Hif1a, Vegfa, Fgf1, Kdr, and Pecam1), macrophage recruitment, and inflammation (including Emr1, Ccr2, Itgax, Ccl2, Tnf, and Il1b) correlated more strongly with body weight in the EF of HFD-fed obese mice compared to that of IF.
Conclusions: These results suggest depot-specific differences in AT angiogenesis and a potential role in the susceptibility to diet-induced obesity.[Hide abstract]
|Modulation of ambient temperature promotes inflammation and initiates atherosclerosisThe molecular mechanisms that regulate atherosclerosis initiation and progression are not fully understood. The emergence of ambient temperature as a potential variable in disease modeling has already gained interest in the atherosclerosis field. Giles, Ramkhelawon and colleagues show, for the first time, that thermoneutral housing (at 29-32°C) in combination with obesogenic Western diet promotes mild induction of atherosclerosis in wild type mice. This report describes a novel, inflammatory murine model for atherosclerosis in the absence of previously utilized genetic manipulations. This approach represents a significant step forward in atherosclerosis modeling.|
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Objective: Obesity and obesity-associated inflammation is central to a variety of end-organ sequelae including atherosclerosis, a leading cause of death worldwide. Although mouse models have provided important insights into the immunopathogenesis of various diseases, modeling atherosclerosis in mice has proven difficult. Specifically, wild-type (WT) mice are resistant to developing atherosclerosis, while commonly used genetically modified mouse models of atherosclerosis are poor mimics of human disease. The lack of a physiologically relevant experimental model of atherosclerosis has hindered the understanding of mechanisms regulating disease development and progression as well as the development of translational therapies. Recent evidence suggests that housing mice within their thermoneutral zone profoundly alters murine physiology, including both metabolic and immune processes. We hypothesized that thermoneutral housing would allow for augmentation of atherosclerosis induction and progression in mice.
Methods: ApoE−/− and WT mice were housed at either standard (TS) or thermoneutral (TN) temperatures and fed either a chow or obesogenic “Western” diet. Analysis included quantification of (i) obesity and obesity-associated downstream sequelae, (ii) the development and progression of atherosclerosis, and (iii) inflammatory gene expression pathways related to atherosclerosis.
Results: Housing mice at TN, in combination with an obesogenic “Western” diet, profoundly augmented obesity development, exacerbated atherosclerosis in ApoE−/− mice, and initiated atherosclerosis development in WT mice. This increased disease burden was associated with altered lipid profiles, including cholesterol levels and fractions, and increased aortic plaque size. In addition to the mild induction of atherosclerosis, we similarly observed increased levels of aortic and white adipose tissue inflammation and increased circulating immune cell expression of pathways related to adverse cardiovascular outcome.
Conclusions: In sum, our novel data in WT C57Bl/6 mice suggest that modulation of a single environmental variable, temperature, dramatically alters mouse physiology, metabolism, and inflammation, allowing for an improved mouse model of atherosclerosis. Thus, thermoneutral housing of mice shows promise in yielding a better understanding of the cellular and molecular pathways underlying the pathogenesis of diverse diseases.[Hide abstract]
|Osteopontin is a key player for local adipose tissue macrophage proliferation in obesityAn increasing number of studies describe adipose tissue macrophage accumulation as the main driver of obesity-associated inflammation. Osteopontin (OPN) is a secreted glycoprotein involved in a wide variety of physiological and pathological conditions, including inflammatory processes. Tardelli et al. show that OPN-treated human monocytes outnumber controls while diminishing apoptosis. Furthermore, OPN enhances proliferation rates also in in vitro differentiated macrophages, thereby pointing to a novel mechanism that might trigger and maintain low-grade inflammation in obesity.|
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Objective: Recent findings point towards an important role of local macrophage proliferation also in obesity-induced adipose tissue inflammation that underlies insulin resistance and type 2 diabetes. Osteopontin (OPN) is an inflammatory cytokine highly upregulated in adipose tissue (AT) of obese and has repeatedly been shown to be functionally involved in adipose-tissue inflammation and metabolic sequelae. In the present work, we aimed at unveiling both the role of OPN in human monocyte and macrophage proliferation as well as the impact of OPN deficiency on local macrophage proliferation in a mouse model for diet-induced obesity.
Methods: The impact of recombinant OPN on viability, apoptosis, and proliferation was analyzed in human peripheral blood monocytes and derived macrophages. Wild type (WT) and OPN knockout mice (SPP1KO) were compared with respect to in vivo adipose tissue macrophage and in vitro bone marrow-derived macrophage (BMDM) proliferation.
Results: OPN not only enhanced survival and decreased apoptosis of human monocytes but also induced proliferation similar to macrophage colony stimulating factor (M-CSF). Even in fully differentiated monocyte-derived macrophages, OPN induced a proliferative response. Moreover, proliferation of adipose tissue macrophages in obese mice was detectable in WT but virtually absent in SPP1KO. In BMDM, OPN also induced proliferation while OPN as well as M-CSF-induced proliferation was similar in WT and SPP1KO.
Conclusions: These data confirm that monocytes and macrophages not only are responsive to OPN and migrate to sites of inflammation but also they survive and proliferate more in the presence of OPN, a mechanism also strongly confirmed in vivo. Therefore, secreted OPN appears to be an essential player in AT inflammation, not only by driving monocyte chemotaxis and macrophage differentiation but also by facilitating local proliferation of macrophages.[Hide abstract]
|Critical role for adenosine receptor A2a in beta-cell proliferationType 1 and type 2 diabetes are characterized by a shortage of functional beta-cells. One potentially curative strategy would be to stimulate remaining beta-cells to proliferate. Here, Schulz et al. show that adenosine signaling through the A2a receptor is required for compensatory beta-cell proliferation in mice during pregnancy and is sufficient to promote proliferation of mouse beta-cells in vitro. Their work suggests that adenosine signaling has an important role in generating beta-cell homeostasis, a feature that could be therapeutically exploited to increase the number of beta-cells in people with diabetes.|
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Objective: Pharmacological activation of adenosine signaling has been shown to increase β-cell proliferation and thereby β-cell regeneration in zebrafish and rodent models of diabetes. However, whether adenosine has an endogenous role in regulating β-cell proliferation is unknown. The objective of this study was to determine whether endogenous adenosine regulates β-cell proliferation — either in the basal state or states of increased demand for insulin — and to delineate the mechanisms involved.
Methods: We analyzed the effect of pharmacological adenosine agonists on β-cell proliferation in in vitro cultures of mouse islets and in zebrafish models with β- or δ-cell ablation. In addition, we performed physiological and histological characterization of wild-type mice and mutant mice with pancreas- or β-cell-specific deficiency in Adora2a (the gene encoding adenosine receptor A2a). The mutant mice were used for in vivo studies on the role of adenosine in the basal state and during pregnancy (a state of increased demand for insulin), as well as for in vitro studies of cultured islets.
Results: Pharmacological adenosine signaling in zebrafish had a stronger effect on β-cell proliferation during β-cell regeneration than in the basal state, an effect that was independent of the apoptotic microenvironment of the regeneration model. In mice, deficiency in Adora2a impaired glucose control and diminished compensatory β-cell proliferation during pregnancy but did not have any overt phenotype in the basal state. Islets isolated from Adora2a-deficient mice had a reduced baseline level of β-cell proliferation in vitro, consistent with our finding that UK432097, an A2a-specific agonist, promotes the proliferation of mouse β-cells in vitro.
Conclusions: This is the first study linking endogenously produced adenosine to β-cell proliferation. Moreover, we show that adenosine signaling via the A2a receptor has an important role in compensatory β-cell proliferation, a feature that could be harnessed pharmacologically for β-cell expansion and future therapeutic development for diabetes.[Hide abstract]