Dipeptidyl Peptidase-4 Inhibition Ameliorates Western Diet–Induced Hepatic Steatosis and Insulin Resistance Through Hepatic Lipid Remodeling and Modulation of Hepatic Mitochondrial Function

Novel therapies are needed for treating the increasing prevalence of hepatic steatosis in Western populations. In this regard, dipeptidyl peptidase-4 (DPP-4) inhibitors have recently been reported to attenuate the development of hepatic steatosis, but the potential mechanisms remain poorly defined. In the current study, 4-week-old C57Bl/6 mice were fed a high-fat/high-fructose Western diet (WD) or a WD containing the DPP-4 inhibitor, MK0626, for 16 weeks. The DPP-4 inhibitor prevented WD-induced hepatic steatosis and reduced hepatic insulin resistance by enhancing insulin suppression of hepatic glucose output. WD-induced accumulation of hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content was significantly attenuated with DPP-4 inhibitor treatment. In addition, MK0626 significantly reduced mitochondrial incomplete palmitate oxidation and increased indices of pyruvate dehydrogenase activity, TCA cycle flux, and hepatic TAG secretion. Furthermore, DPP-4 inhibition rescued WD-induced decreases in hepatic PGC-1α and CPT-1 mRNA expression and hepatic Sirt1 protein content. Moreover, plasma uric acid levels in mice fed the WD were decreased after MK0626 treatment. These studies suggest that DPP-4 inhibition ameliorates hepatic steatosis and insulin resistance by suppressing hepatic TAG and DAG accumulation through enhanced mitochondrial carbohydrate utilization and hepatic TAG secretion/export with a concomitant reduction of uric acid production.     

Amyloid-β induces hepatic insulin resistance by activating JAK2/STAT3/SOCS-1 signaling pathway

Epidemiological studies indicate that patients with Alzheimer's disease (AD) have an increased risk of developing type 2 diabetes mellitus (T2DM), and experimental studies suggest that AD exacerbates T2DM, but the underlying mechanism is still largely unknown. This study aims to investigate whether amyloid-β (Aβ), a key player in AD pathogenesis, contributes to the development of insulin resistance, as well as the underlying mechanism. We find that plasma Aβ40/42 levels are increased in patients with hyperglycemia. APPswe/PSEN1dE9 transgenic AD model mice with increased plasma Aβ40/42 levels show impaired glucose and insulin tolerance and hyperinsulinemia. Furthermore, Aβ impairs insulin signaling in mouse liver and cultured hepatocytes. Aβ can upregulate suppressors of cytokine signaling (SOCS)-1, a well-known insulin signaling inhibitor. Knockdown of SOCS-1 alleviates Aβ-induced impairment of insulin signaling. Moreover, JAK2/STAT3 is activated by Aβ, and inhibition of JAK2/STAT3 signaling attenuates Aβ-induced upregulation of SOCS-1 and insulin resistance in hepatocytes. Our results demonstrate that Aβ induces hepatic insulin resistance by activating JAK2/STAT3/SOCS-1 signaling pathway and have implications toward resolving insulin resistance and T2DM.     

Targeted Inactivation of Kinesin-1 in Pancreatic ��-Cells In Vivo Leads to Insulin Secretory Deficiency

OBJECTIVE

Suppression of Kinesin-1 by antisense oligonucleotides, or overexpression of dominant-negative acting kinesin heavy chain, has been reported to affect the sustained phase of glucose-stimulated insulin secretion in β-cells in vitro. In this study, we examined the in vivo physiological role of Kinesin-1 in β-cell development and function.

RESEARCH DESIGN AND METHODS

A Cre-LoxP strategy was used to generate conditional knockout mice in which the Kif5b gene is specifically inactivated in pancreatic β-cells. Physiological and histological analyses were carried out in Kif5b knockout mice as well as littermate controls.

RESULTS

Mice with β-cell specific deletion of Kif5b (Kif5b^fl/−:RIP2-Cre) displayed significantly retarded growth as well as slight hyperglycemia in both nonfasting and 16-h fasting conditions compared with control littermates. In addition, Kif5b^fl/−:RIP2-Cre mice displayed significant glucose intolerance, which was not due to insulin resistance but was related to an insulin secretory defect in response to glucose challenge. These defects of β-cell function in mutant mice were not coupled with observable changes in islet morphology, islet cell composition, or β-cell size. However, compared with controls, pancreas of Kif5b^fl/−:RIP2-Cre mice exhibited both reduced islet size and increased islet number, concomitant with an increased insulin vesicle density in β-cells.

CONCLUSIONS

In addition to being essential for maintaining glucose homeostasis and regulating β-cell function, Kif5b may be involved in β-cell development by regulating β-cell proliferation and insulin vesicle synthesis.Insulin is exclusively produced and secreted from pancreatic β-cells in two distinct phases in response to elevated blood glucose levels. The first phase of insulin release is triggered by a rapid increase of intracellular calcium level leading to fusion of predocked insulin granules at the plasma membrane (1). The second phase of insulin release requires the mobilization of insulin-containing granules from the storage pool to the β-cell periphery to sustain insulin release (2). The molecular mechanism for the first phase of insulin release has been extensively investigated (1,3–5); however, little is known regarding the second phase of insulin secretion (6).Pharmacological and cytological observations suggest that dynamic turnover of tubulin and microtubules is important for regulation of intracellular transportation of insulin granules and their subsequent release from β-cells (7). Boyd et al. (8) observed a proportion of insulin-containing vesicles attached along the microtubules by double-immunostaining of primary cultured pancreatic β-cells. Furthermore, colchicine treatment does not affect the immediate release of insulin but significantly attenuates the following sustained phase of response. In addition, Suprenant and Dentler (9) demonstrated direct binding of insulin-containing granules to microtubules in vitro, and that insulin granule movement along microtubules is dependent on microtubule-associated proteins in the presence of ATP. Therefore, it was suggested that microtubules within the β-cell serve as supporting structures (railways) upon which insulin granules travel from the β-cell interior to the plasma membrane.Kinesin and dynein are two motor proteins that have been identified to translocate cargos along microtubules to opposite directions for fast transportation. Conventional kinesin (Kinesin-1) is a heterotetramer of two heavy chains (KHCs) and two light chains (KLCs). The head domain of KHC contains the ATP binding domain for generating motile force as well as a motif for interaction with microtubules, whereas the tail domain and KLC are responsible for cargo binding (10–12). In mice, three conventional kinesin heavy chain genes have been identified, including Kif5a, Kif5b, and Kif5c. Kif5b is the mouse homologue of the human ubiquitous KHC (13) and was first identified and characterized in pancreatic β-cells (14). The functions and molecular mechanism of kinesin transportation have been extensively studied in neuronal cells and tissues. However, only a few reports are related to the role of this motor protein during cargo transportation in nonneuronal mammalian cell types (15–19). Meng et al. (20) reported that suppression of Kif5b by antisense oligonucleotides inhibits both basal- and glucose-stimulated insulin secretion (GSIS) in primary mouse pancreatic β-cells. Immunocytochemistry study showed that Kif5b is colocalized with some insulin-containing vesicles in β-cell lines (MIN6 & INS-1) (18). Moreover, expression of a dominant-negative KHC motor domain (KHC^mut) strongly inhibited the sustained, but not acute, insulin secretion in response to glucose challenge (18). Besides Kif5b, other motor proteins such as myosin Va and dynein are also involved in insulin secretion (21,22).All the above studies were carried out in an in vitro model, and the physiological role of Kif5b in pancreatic β-cells has not been elucidated in vivo. Therefore, to directly explore the role of Kif5b in β-cells, we generated a conditional knockout mouse under the control of RIP2-Cre by using a Cre-LoxP recombination system.     

Induction of Cytosolic Phospholipase A2α Is Required for Adipose Neutrophil Infiltration and Hepatic Insulin Resistance Early in the Course of High-Fat Feeding

In established obesity, inflammation and macrophage recruitment likely contribute to the development of insulin resistance. In the current study, we set out to explore whether adipose tissue infiltration by neutrophils that occurs early (3 days) after initiating a high-fat diet (HFD) could contribute to the early occurrence of hepatic insulin resistance and to determine the role of cytosolic phospholipase A₂α (cPLA₂α) in this process. The 3-day HFD caused a significant upregulation of cPLA₂α in periepididymal fat and in the liver. A specific antisense oligonucleotide (AS) effectively prevented cPLA₂α induction, neutrophil infiltration into adipose tissue (likely involving MIP-2), and protected against 3-day HFD–induced impairment in hepatic insulin signaling and glucose over-production from pyruvate. To sort out the role of adipose neutrophil infiltration independent of cPLA₂α induction in the liver, mice were injected intraperitoneally with anti–intracellular adhesion molecule-1 (ICAM-1) antibodies. This effectively prevented neutrophil infiltration without affecting cPLA₂α or MIP-2, but like AS, prevented impairment in hepatic insulin signaling, the enhanced pyruvate-to-glucose flux, and the impaired insulin-mediated suppression of hepatic glucose production (assessed by clamp), which were induced by the 3-day HFD. Adipose tissue secretion of tumor necrosis factor-α (TNF-α) was increased by the 3-day HFD, but not if mice were treated with AS or ICAM-1 antibodies. Moreover, systemic TNF-α neutralization prevented 3-day HFD–induced hepatic insulin resistance, suggesting its mediatory role. We propose that an acute, cPLA₂α-dependent, neutrophil-dominated inflammatory response of adipose tissue contributes to hepatic insulin resistance and glucose overproduction in the early adaptation to high-fat feeding.Established obesity, particularly if associated with insulin resistance–related morbidities, is characterized by systemic and adipose tissue inflammation (1–3). The complexity of the adipocytokines and inflammatory cell types involved in adipose inflammation is constantly increasing, and today, most myeloid cell types have been implicated in the process, including macrophages, B cells, various T-cell classes, and even eosinophils and mast cells (4–6). In contrast, much less is known about adipose tissue and liver adaptation to a short-term high-fat diet (HFD) before overt obesity is present. Metabolically, it appears that hepatic insulin resistance may be a front-line response to a short-term (3 days) HFD (7,8), representing “physiological adaptation” and/or an early maladaptive response on the causal pathway to obesity-induced whole-body insulin resistance. It currently remains unclear to what degree this early response to an HFD involves immune cells in general and, specifically, in adipose tissue.In a previous study, we demonstrated that during the first week of initiating an HFD, adipose tissue is infiltrated by neutrophils (9). Adipose tissue protein levels of the neutrophil-specific myeloperoxidase (MPO) were increased, and correspondingly, histology detected an increased number of neutrophils within the parenchyma of adipose tissue (i.e., not restricted to blood vessels). This early appearance of neutrophils in adipose tissue was recently confirmed (10), suggesting that adipose tissue inflammation in obesity largely follows the classical inflammation paradigms of acute versus chronic inflammatory cell infiltrates, predominated first by neutrophils, then lymphocytes in the subacute period, and finally, by mononuclear macrophages when inflammation becomes chronic. Yet, the co-occurrence of increased adipose neutrophil infiltration (9,10) with the early hepatic insulin resistance (7,8) prompts the question of whether the former phenomenon is causative for the latter.Cytosolic phospholipase A₂α (cPLA₂α) has received much attention as a key regulator of inflammation. It plays a major role in the stimulus-initiated production of eicosanoids (prostaglandins and the chemoattractant leukotrienes) and platelet activating factor (11). In a previous study, we demonstrated that cPLA₂α is upregulated in vascular endothelial cells in adipose tissue of mice in response to the 3-day HFD and that it mediates the elevated expression of the endothelial intracellular adhesion molecule (ICAM-1) (12) that is used for adhesion by neutrophils and monocytes. In addition, cPLA₂α has been demonstrated to regulate superoxide generation by NADPH oxidase activation (13), thus promoting phagocyte-induced oxidative stress. Intriguingly, in humans, even a single exposure to a high-fat meal induced NADPH activation and inflammatory cascades in circulating leukocytes (14–16). These findings suggest that cPLA₂α could participate in priming/activation of circulating cells upstream in inflammatory cascades that ultimately lead to adipose tissue infiltration by neutrophils, way before obesity has developed. In the current study, we set out to reveal the role of cPLA₂α and adipose tissue neutrophil infiltration in the acute adaptation to a 3-day HFD, with emphasis on whether these early inflammatory responses could underlie the development of hepatic insulin resistance.     

Disruption of the Cereblon Gene Enhances Hepatic AMPK Activity and Prevents High-Fat Diet–Induced Obesity and Insulin Resistance in Mice

A nonsense mutation in cereblon (CRBN) causes a mild type of mental retardation in humans. An earlier study showed that CRBN negatively regulates the functional activity of AMP-activated protein kinase (AMPK) in vitro by binding directly to the α₁-subunit of the AMPK complex. However, the in vivo role of CRBN was not studied. For elucidation of the physiological functions of Crbn, a mouse strain was generated in which the Crbn gene was deleted throughout the whole body. In Crbn-deficient mice fed a normal diet, AMPK in the liver showed hyperphosphorylation, which indicated the constitutive activation of AMPK. Since Crbn-deficient mice showed significantly less weight gain when fed a high-fat diet and their insulin sensitivity was considerably improved, the functions of Crbn in the liver were primarily investigated. These results provide the first in vivo evidence that Crbn is a negative modulator of AMPK, which suggests that Crbn may be a potential target for metabolic disorders of the liver.Initially, cereblon (CRBN) was identified as a target gene for a mild type of mental retardation in humans (1) and was subsequently characterized in several different functional contexts. CRBN interacts directly with large-conductance calcium-activated potassium channels and regulates their surface expression (2). Later, CRBN was identified as a primary target for thalidomide-induced teratogenicity and as a substrate receptor for the E3 ligase complex (3). More recently, we reported that CRBN interacts directly with the α₁-subunit of AMP-activated protein kinase (AMPK) and inhibits activation of the enzyme in vitro (4).AMPK is a metabolic master switch in response to variations in cellular energy homeostasis (5). The activity of AMPK can be modulated by the phosphorylation of a threonine at position 172 (Thr172) in the α-subunit by upstream kinases such as LKB1 (6). AMPK inactivates acetyl-CoA carboxylase (ACC) via direct protein phosphorylation and suppresses expression of lipogenic genes, including fatty acid synthase (FAS), thereby inhibiting fatty acid synthesis (7,8). AMPK is implicated in the regulation of hepatic glucose and lipid metabolism, thereby affecting the energy status of the whole body (7,9). Moreover, AMPK was identified as a major pharmacological target protein for the treatment of metabolic diseases. For example, experimental animal models of type 2 diabetes and obesity show that activation of AMPK by metformin or 5-aminoimidazole-4-carboxamide ribonucleoside reduces blood glucose levels and improves lipid metabolism (10–12).Our recent study found that CRBN interacted directly with the AMPK α₁-subunit both in cultured cell lines and in vitro, and the binding sites within the two proteins were localized (4). The levels of the AMPK γ-subunit and CRBN in the AMPK complex varied in a reciprocal manner; i.e., a higher CRBN content corresponded to lower γ-subunit content. AMPK activation was reduced as its γ-subunit content was decreased by CRBN. Thus, it was proposed that CRBN may act as a negative regulator of AMPK in vivo (4). The aims of the current study were to test this hypothesis and to understand the physiological role(s) of CRBN by generating Crbn knockout (KO) mice. The results showed that AMPK activity was activated constitutively in Crbn KO mice under normal conditions and that Crbn KO mice fed a long-term high-fat diet (HFD) showed a marked improvement in their metabolic status.     

G Protein�CCoupled Receptor Kinase 2 Plays a Relevant Role in Insulin Resistance and Obesity

OBJECTIVE

Insulin resistance is associated with the pathogenesis of metabolic disorders as type 2 diabetes and obesity. Given the emerging role of signal transduction in these syndromes, we set out to explore the possible role that G protein–coupled receptor kinase 2 (GRK2), first identified as a G protein–coupled receptor regulator, could have as a modulator of insulin responses.

RESEARCH DESIGN AND METHODS

We analyzed the influence of GRK2 levels in insulin signaling in myoblasts and adipocytes with experimentally increased or silenced levels of GRK2, as well as in GRK2 hemizygous animals expressing 50% lower levels of this kinase in three different models of insulin resistance: tumor necrosis factor-α (TNF-α) infusion, aging, and high-fat diet (HFD). Glucose transport, whole-body glucose and insulin tolerance, the activation status of insulin pathway components, and the circulating levels of important mediators were measured. The development of obesity and adipocyte size with age and HFD was analyzed.

RESULTS

Altering GRK2 levels markedly modifies insulin-mediated signaling in cultured adipocytes and myocytes. GRK2 levels are increased by ∼2-fold in muscle and adipose tissue in the animal models tested, as well as in lymphocytes from metabolic syndrome patients. In contrast, hemizygous GRK2 mice show enhanced insulin sensitivity and do not develop insulin resistance by TNF-α, aging, or HFD. Furthermore, reduced GRK2 levels induce a lean phenotype and decrease age-related adiposity.

CONCLUSIONS

Overall, our data identify GRK2 as an important negative regulator of insulin effects, key to the etiopathogenesis of insulin resistance and obesity, which uncovers this protein as a potential therapeutic target in the treatment of these disorders.Insulin resistance, a diminished ability of cells to respond to the action of insulin, is a key feature associated with the pathogenesis of metabolic disorders such as type 2 diabetes and obesity (1). Alterations in any of the key components of the insulin-signaling cascade, including negative regulators, have been proposed to contribute to insulin resistance (1,2). However, the origin and precise mechanisms mediating insulin resistance in physiopathological conditions are not fully understood (3).Both aging and obesity are associated with increased risk of developing type 2 diabetes and cardiovascular disease. An increase in proinflammatory and a decrease in anti-inflammatory factors is found in the obese state and may influence glucose homeostasis and insulin sensitivity (4,5). Peripheral tissues exposed to these proinflammatory cytokines develop an insulin-resistant state (6). In fact, obesity is now being considered a chronic state of low-intensity inflammation. In this regard, the cytokine tumor necrosis factor-α (TNF-α) is highly expressed in adipose tissue of obese animals and humans, and obese mice lacking either TNF-α or its receptors show protection against developing insulin resistance. The molecular mechanisms underlying TNF-α–mediated insulin resistance have been studied in models of murine and human myocytes and adipocytes and in vivo (7–11).Insulin suppresses hepatic glucose production and regulates glucose uptake in muscle and fat through translocation of GLUT4 to the cell surface (12,13). Insulin-induced GLUT4 translocation requires at least two signals, one mediated through phosphatidylinositol 3-kinase (PI3K) and another via Gαq/11 (14) in 3T3L1 adipocytes. The activated insulin receptor can phosphorylate the G protein subunit Gαq/11, leading to activation of cdc42 and PI3K, which triggers glucose transport stimulation (14–16). Signaling of receptors via G proteins is regulated by G protein–coupled receptor kinases (GRKs), a family of seven serine/threonine protein kinases that specifically recognize and phosphorylate agonist-activated G protein–coupled receptors (GPCRs). This recruits arrestin proteins that uncouple receptors from G proteins and promote internalization. The ubiquitous GRK2 isoform has been reported to regulate other pathways independently of its GPCR phosphorylation ability (17,18). GRK2 can act as an inhibitor of insulin-mediated glucose transport stimulation in 3T3L1 adipocytes by interacting with Gαq/11 function independently of its kinase activity (19). GRK2 also inhibits basal and insulin-stimulated glycogen synthesis in mouse liver FL83B cells (20). In this context, we have investigated whether GRK2 may play a relevant physiological role in the modulation of insulin responses in vivo. GRK2 expression is increased in key tissues in different experimental models of insulin resistance, and a 50% downregulation of GRK2 levels in hemizygous GRK2^+/− mice is sufficient to protect against TNF-α, aging, or high-fat diet (HFD)–induced alterations in glucose homeostasis and insulin signaling, strongly arguing for a key role for GRK2 in the modulation of insulin sensitivity in physiological and pathological conditions.     

Nox2 NADPH Oxidase Has a Critical Role in Insulin Resistance–Related Endothelial Cell Dysfunction

Insulin resistance is characterized by excessive endothelial cell generation of potentially cytotoxic concentrations of reactive oxygen species. We examined the role of NADPH oxidase (Nox) and specifically Nox2 isoform in superoxide generation in two complementary in vivo models of human insulin resistance (endothelial specific and whole body). Using three complementary methods to measure superoxide, we demonstrated higher levels of superoxide in insulin-resistant endothelial cells, which could be pharmacologically inhibited both acutely and chronically, using the Nox inhibitor gp91ds-tat. Similarly, insulin resistance–induced impairment of endothelial-mediated vasorelaxation could also be reversed using gp91ds-tat. siRNA-mediated knockdown of Nox2, which was specifically elevated in insulin-resistant endothelial cells, significantly reduced superoxide levels. Double transgenic mice with endothelial-specific insulin resistance and deletion of Nox2 showed reduced superoxide production and improved vascular function. This study identifies Nox2 as the central molecule in insulin resistance–mediated oxidative stress and vascular dysfunction. It also establishes pharmacological inhibition of Nox2 as a novel therapeutic target in insulin resistance–related vascular disease.Insulin resistance is a multisystem disorder of energy homeostasis, cell growth, and tissue repair, which has been shown to be pivotal to the initiation and progression of type 2 diabetes (1). As a result, type 2 diabetes is characterized by a portfolio of disorders including atherosclerotic coronary artery disease, stroke, and peripheral vascular disease (2). Atherosclerosis is characterized by a deleterious change in endothelial cell phenotype, a hallmark of which is excess generation of cytotoxic concentrations of reactive oxygen species (ROS) such as superoxide and failure of endogenous vascular antioxidant systems to adequately deal with this—a scenario described as oxidative stress (3).Several studies support a role for insulin resistance in the generation of pathophysiological concentrations of ROS and the development of premature atherosclerosis (4). Our own studies in experimental models of insulin resistance at a whole-body level and specific to the endothelium demonstrated that insulin resistance per se is a substrate for increased generation of ROS and accelerated atherosclerosis (5,6). ROS are thought to promote atherosclerosis through a number of different mechanisms including but not limited to enhanced oxidation of lipoproteins, activation of proinflammatory genes, alteration of vascular smooth muscle cell phenotype, and reduction of bioavailability of the antiatherosclerotic signaling radical nitric oxide (NO).A major source of ROS to emerge over the last decade is NADPH oxidase (Nox) (7,8). Nox was originally identified in phagocytes where it exists as a multisubunit complex consisting of a membrane-bound cytochrome b₅₅₈ and at least four cytosolic subunits, which translocate to the membrane upon activation. It is now clear that the Noxs are a family of enzymes with each isoform being distinguished by the membrane-spanning catalytic Nox or Duox subunit that transfers electrons from NADPH to molecular oxygen, termed Nox1-Nox5 (8). We have provided evidence that increased Nox-derived ROS may be a unifying mechanism underlying insulin resistance–related oxidative stress and atherosclerosis (5,6). In the current study, we aimed to examine the therapeutic potential of inhibiting Nox and, specifically, Nox2 to reduce oxidative stress and improve endothelial-dependent vasodilatation in insulin resistance.     

IL-18 Inhibits TNF-α-Induced Osteoclastogenesis Possibly via a T Cell-Independent Mechanism in Synergy with IL-12 In Vivo

It has recently been reported that tumor necrosis factor (TNF)-α has the ability to accelerate osteoclastogenesis. We previously reported that the proinflammatory cytokine interleukin (IL)-18 inhibits TNF-α-mediated osteoclastogenesis in mouse bone marrow cultures. In the present study, the effect of IL-18 on TNF-α-mediated osteoclastogenesis was investigated in vivo. We administered TNF-α with or without IL-18 into the supracalvaria of mice. The number of osteoclasts in the suture of the calvaria was increased in mice administered TNF-α. The number of osteoclasts in mice administered both TNF-α and IL-18 was lower than that in mice administered TNF-α alone. We previously showed that IL-12 and IL-18 synergistically inhibit TNF-α-mediated osteoclastogenesis in vitro. To assess the ability of these two cytokines to synergistically inhibit TNF-α-induced osteoclastogenesis in vivo, mice were administered the two cytokines at doses that did not inhibit osteoclast formation. The combination of IL-12 and IL-18 markedly inhibited TNF-α-induced osteoclastogenesis in vivo. To evaluate how IL-12 and IL-18 synergistically affect TNF-α-induced osteoclastogenesis, the IL-18 receptor (IL-18R) and IL-12R expression levels were analyzed by RT-PCR in bone marrow cells cultured with IL-12 or IL-18. IL-18R mRNA was increased in cells cultured with IL-12, while IL-12R mRNA was increased in cells cultured with IL-18. In addition, IL-18 inhibited TNF-α-induced osteoclastogenesis in mice with T-cell depletion caused by anti-CD4 and anti-CD8 antibodies. The present results suggest that IL-18 may inhibit TNF-α-mediated osteoclastogenesis in vivo via a T cell-independent mechanism.     

Cyclin D2 Is Essential for the Compensatory ��-Cell Hyperplastic Response to Insulin Resistance in Rodents

OBJECTIVE

A major determinant of the progression from insulin resistance to the development of overt type 2 diabetes is a failure to mount an appropriate compensatory β-cell hyperplastic response to maintain normoglycemia. We undertook the present study to directly explore the significance of the cell cycle protein cyclin D2 in the expansion of β-cell mass in two different models of insulin resistance.

RESEARCH DESIGN AND METHODS

We created compound knockouts by crossing mice deficient in cyclin D2 (D2KO) with either the insulin receptor substrate 1 knockout (IRS1KO) mice or the insulin receptor liver-specific knockout mice (LIRKO), neither of which develops overt diabetes on its own because of robust compensatory β-cell hyperplasia. We phenotyped the double knockouts and used RT-qPCR and immunohistochemistry to examine β-cell mass.

RESULTS

Both compound knockouts, D2KO/LIRKO and D2KO/IRS1KO, exhibited insulin resistance and hyperinsulinemia and an absence of compensatory β-cell hyperplasia. However, the diabetic D2KO/LIRKO group rapidly succumbed early compared with a relatively normal lifespan in the glucose-intolerant D2KO/IRS1KO mice.

CONCLUSIONS

This study provides direct genetic evidence that cyclin D2 is essential for the expansion of β-cell mass in response to a spectrum of insulin resistance and points to the cell-cycle protein as a potential therapeutic target that can be harnessed for preventing and curing type 2 diabetes.The maintenance of an adequate and functional pancreatic β-cell mass dictates the body''s ability to compensate for insulin resistance. Recent studies in autopsy samples from humans reported expansion of β-cell mass from infants through adolescence that was largely due to increased islet size (1). Further, humans with established type 2 diabetes exhibit a deficit in β-cell mass in comparison with their nondiabetic cohorts (2,3). Interestingly, obese, nondiabetic patients express a wide range of β-cell mass that is sufficient to maintain euglycemia up to a specific threshold, and crossing the threshold correlates with impaired fasting glucose and clinical diabetes (4).While direct data in humans is lacking, studies in rodents clearly indicate that β-cell mass adaptively expands to compensate for both physiological and pathophysiological states of insulin resistance including pregnancy, onset of obesity, and high-fat feeding and after partial pancreatectomy (5–9). Although the adaptation is dependent on alterations in both the number and size of β-cells and generation of new β-cells from endogenous progenitors (10,11), recent studies point to replication as a primary mechanism for the physiological maintenance of adult β-cell mass (12) and in response to insulin resistance in both rodents and humans (9,12–15).Replication is achieved by reentry of the β-cell into the cell cycle and relies on proteins regulating the G1 phase (13,16–19). Our previous work has established that cyclin D2, a G1/S cell-cycle regulator, is necessary for the postnatal expansion of β-cell mass (13). In these studies, we observed that in the absence of cyclin D2, the diminished β-cell mass established during the neonatal remodeling period was inadequate to sufficiently respond to metabolic demand for insulin in adult mice, leading to glucose intolerance but not frank diabetes (13). While these experiments indicate that cyclin D2 is important during early postnatal expansion of β-cells for the adult mouse to achieve its optimal β-cell mass, its importance in cell expansion in response to a pathophysiological demand for insulin is not known. Considering these observations, we explored whether a limited but adequate β-cell mass that is challenged by physiological stress would fall below a functional threshold required to maintain euglycemia, and eventually to promote the development of diabetes, using cyclin D2 knockout mice. To this end, we created compound double knockouts by breeding cyclin D2 (D2KO) mice with either mice that are deficient in insulin receptor substrate 1 (IRS1KO) (20,21) or mice with a knockout of the insulin receptor specifically in liver (LIRKO) (22). These models reflect the spectrum of insulin resistance and glucose intolerance observed in humans but do not develop frank diabetes in part because of compensatory β-cell expansion that can increase from 3-fold (IRS1KO) to 30-fold (LIRKO) largely by replication of β-cells (21,23).Our results indicate that both D2KO/LIRKO and D2KO/IRS1KO double-knockout mice fail to show a β-cell compensatory response to insulin resistance, leading to overt diabetes that is secondary, in part, to a dramatic decrease in β-cell mass due to reduced β-cell replication. These data provide genetic evidence that cyclin D2 is essential for the compensatory increase in β-cell hyperplasia in response to insulin-resistant states.     

Blocking Interleukin-1β Induces a Healing-Associated Wound Macrophage Phenotype and Improves Healing in Type 2 Diabetes

Diabetes is associated with persistent inflammation and defective tissue repair responses. The hypothesis of this study was that interleukin (IL)-1β is part of a proinflammatory positive feedback loop that sustains a persistent proinflammatory wound macrophage phenotype that contributes to impaired healing in diabetes. Macrophages isolated from wounds in diabetic humans and mice exhibited a proinflammatory phenotype, including expression and secretion of IL-1β. The diabetic wound environment appears to be sufficient to induce these inflammatory phenomena because in vitro studies demonstrated that conditioned medium of both mouse and human wounds upregulates expression of proinflammatory genes and downregulates expression of prohealing factors in cultured macrophages. Furthermore, inhibiting the IL-1β pathway using a neutralizing antibody and macrophages from IL-1 receptor knockout mice blocked the conditioned medium–induced upregulation of proinflammatory genes and downregulation of prohealing factors. Importantly, inhibiting the IL-1β pathway in wounds of diabetic mice using a neutralizing antibody induced a switch from proinflammatory to healing-associated macrophage phenotypes, increased levels of wound growth factors, and improved healing of these wounds. Our findings indicate that targeting the IL-1β pathway represents a new therapeutic approach for improving the healing of diabetic wounds.Chronic wounds associated with diabetes, venous insufficiency, or pressure represent a major health problem, with millions of patients afflicted and the associated treatment costing billions of dollars per year (1). Despite the socioeconomic impact of chronic wounds, the underlying causes of impaired healing are not well-understood and effective treatments remain elusive. A common characteristic of these poorly healing wounds is a persistent inflammatory response, with prolonged accumulation of macrophages and elevated levels of proinflammatory cytokines (2–5). Translational research of the dysregulation of inflammation associated with impaired healing in diabetes should provide insight into the development of new therapeutic approaches.During normal wound healing in mice, inflammatory cells such as macrophages promote healing indirectly by killing pathogens and clearing the wound of damaged tissue, but also promote healing directly by producing growth factors that induce angiogenesis, collagen deposition, and wound closure (6–9). In contrast, during impaired healing of diabetic mice, wounds exhibit prolonged accumulation of macrophage associated with elevated levels of proinflammatory cytokines and proteases and reduced levels of various growth factors, all of which mimic chronic wounds in humans (10–12). We recently demonstrated that in wounds of diabetic mice, macrophages exhibit a sustained proinflammatory phenotype with an impaired upregulation of healing-associated factors that is observed in nondiabetic mice as healing progresses (13). However, the underlying causes of the dysregulation of macrophage in diabetic wounds remain to be elucidated.Multiple factors can influence macrophage phenotype and the actual phenotypes expressed in chronic wounds are likely determined by the balance of the proinflammatory and anti-inflammatory stimuli present in the wound environment. The proinflammatory environment observed in diabetic wounds has the potential to sustain a proinflammatory macrophage phenotype, which, in turn, would contribute to sustaining the proinflammatory environment. In fact, hyperglycemia is known to induce expression of interleukin (IL)-1β in a number of different cell types, including macrophages (14–16), and IL-1β, in turn, is known to induce a proinflammatory macrophage phenotype in part by inducing itself (17). Thus, the IL-1β pathway may be part of a positive feedback loop that sustains inflammation in chronic wounds and contributes to impaired healing. However, little is known about the actual role of IL-1β in diabetic wounds.The central hypothesis of this study is that sustained activity of the IL-1β pathway in diabetic wounds contributes to impaired healing of these wounds. The results of this study demonstrate that sustained IL-1β expression in wounds of diabetic humans and mice is associated with a proinflammatory macrophage phenotype, and that inhibiting the IL-1β pathway in wounds of diabetic mice induces the switch from proinflammatory to healing-associated macrophage phenotypes and improves healing of these wounds.     

AMP-activated Protein Kinase ��2 Subunit Is Required for the Preservation of Hepatic Insulin Sensitivity by n-3 Polyunsaturated Fatty Acids

OBJECTIVE

The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs.

RESEARCH DESIGN AND METHODS

Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2^−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F).

RESULTS

Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2^−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment.

CONCLUSIONS

Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.Naturally occurring n-3 long-chain polyunsaturated fatty acids (LC-PUFAs)—namely, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)—which are abundant in sea fish, act as hypolipidemics, reduce cardiac events, and decrease the progression of atherosclerosis [reviewed in refs (1,2).]. Studies of obese humans have also demonstrated a reduction in adiposity after n-3 LC-PUFA supplementation (3,4). In rodents fed a high-fat diet, n-3 LC-PUFAs efficiently prevented the development of obesity, hepatic steatosis, and dyslipidemia (5–8), as well as impaired glucose tolerance (8–10). However, in diabetic patients, n-3 LC-PUFAs appear to have little effect on glycemic control (3,11,12).The hypolipidemic and antiobesity effects of n-3 LC-PUFAs depend on both the suppression of lipogenesis and the increase in fatty acid oxidation in several tissues, including the liver (13,14), adipose tissue (6), and intestine (15). This metabolic switch may reduce the accumulation of toxic fatty acid derivatives, while protecting insulin signaling in the liver and muscle (9,10,16). Our previous work has documented that the preservation of whole-body insulin sensitivity by n-3 LC-PUFAs in mice fed a high-fat diet mainly reflects improved hepatic insulin sensitivity (8). The effects of n-3 LC-PUFAs and their active metabolites (17,18) are mediated by peroxisome proliferator-activated receptors (PPAR), with PPAR-α and PPAR-δ (-β) being the main targets (14,16), although PPAR-γ, liver X receptor-α, hepatic nuclear factor-4, sterol regulatory element binding protein-1c (SREBP-1c) and carbohydrate-responsive element-binding protein are also involved (16,19–21).It has been demonstrated that n-3 LC-PUFAs enhanced AMP-activated protein kinase (AMPK) activity in the liver (22), intestine (23), and adipose tissue (18,24). AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and regulatory β- and γ-subunits, with multiple isoforms identified for each subunit [α1, α2, β1, β2, γ1, γ2, and γ3; reviewed in ref (25)]. Experiments using whole-body AMPKα2 null [AMPKα2^−/−; ref (26)] mice showed the importance of the AMPKα2 subunit for whole-body insulin action, while liver-specific AMPKα2 knockout mice (27) as well as adenovirus-mediated activation of AMPKα2 in the liver (28) implicated the hepatic AMPKα2 isoform in the suppression of hepatic glucose production and maintenance of fasting blood glucose levels. Furthermore, AMPK controls metabolic fluxes in response to changing cellular energy levels, namely, the partitioning between lipid oxidation and lipogenesis (29,30).We hypothesized that the effects of n-3 LC-PUFA on insulin sensitivity and lipid metabolism in mice fed an obesogenic high-fat diet require a functional AMPKα2 isoform. To test this hypothesis in vivo, AMPKα2^−/− and wild-type mice were fed either a low-fat chow diet (Chow), a corn oil-based high-fat (cHF) diet, or cHF diet in which 15% of the lipids were replaced by n-3 LC-PUFA concentrate (cHF+F). Our results demonstrate an AMPKα2-dependent action of n-3 LC-PUFAs, in 1) the preservation of hepatic and muscle insulin sensitivity; 2) the changes in hepatic diacylglycerol content and composition; and 3) the antisteatotic effect in the liver and hypolipidemic effect under insulin-stimulated conditions, such as during hyperinsulinemic-euglycemic clamp, but not when the organism depends on lipids as substrates.