Liang Sheng

NF-κB–inducing kinase (NIK) promotes hyperglycemia and glucose intolerance in obesity by augmenting glucagon action

The canonical inhibitor of nuclear factor κB kinase subunit β (IKK-β)–nuclear factor of κ light polypeptide gene enhancer in B cells 1 (NF-κB1) pathway has been well documented to promote insulin resistance; however, the noncanonical NF-κB–inducing kinase (NIK)–NF-κB2 pathway is not well understood in obesity. Additionally, the contribution of counter-regulatory hormones, particularly glucagon, to hyperglycemia in obesity is unclear. Here we show that NIK promotes glucagon responses in obesity. Hepatic NIK was abnormally activated in mice with dietary or genetic obesity. Systemic deletion of Map3k14, encoding NIK, resulted in reduced glucagon responses and hepatic glucose production (HGP). Obesity is associated with high glucagon responses, and liver-specific inhibition of NIK led to lower glucagon responses and HGP and protected against hyperglycemia and glucose intolerance in obese mice. Conversely, hepatocyte-specific overexpression of NIK resulted in higher glucagon responses and HGP. In isolated mouse livers and primary hepatocytes, NIK also promoted glucagon action and glucose production, at least in part by increasing cAMP response element-binding (CREB) stability. Therefore, overactivation of liver NIK in obesity promotes hyperglycemia and glucose intolerance by increasing the hyperglycemic response to glucagon and other factors that activate CREB.

Hepatic TRAF2 Regulates Glucose Metabolism Through Enhancing Glucagon Responses

Obesity is associated with intrahepatic inflammation that promotes insulin resistance and type 2 diabetes. Tumor necrosis factor receptor–associated factor (TRAF)2 is a key adaptor molecule that is known to mediate proinflammatory cytokine signaling in immune cells; however, its metabolic function remains unclear. We examined the role of hepatic TRAF2 in the regulation of insulin sensitivity and glucose metabolism. TRAF2 was deleted specifically in hepatocytes using the Cre/loxP system. The mutant mice were fed a high-fat diet (HFD) to induce insulin resistance and hyperglycemia. Hepatic glucose production (HGP) was examined using pyruvate tolerance tests, 2H nuclear magnetic resonance spectroscopy, and in vitro HGP assays. The expression of gluconeogenic genes was measured by quantitative real-time PCR. Insulin sensitivity was analyzed using insulin tolerance tests and insulin-stimulated phosphorylation of insulin receptors and Akt. Glucagon action was examined using glucagon tolerance tests and glucagon-stimulated HGP, cAMP-responsive element–binding (CREB) phosphorylation, and expression of gluconeogenic genes in the liver and primary hepatocytes. Hepatocyte-specific TRAF2 knockout (HKO) mice exhibited normal body weight, blood glucose levels, and insulin sensitivity. Under HFD conditions, blood glucose levels were significantly lower (by >30%) in HKO than in control mice. Both insulin signaling and the hypoglycemic response to insulin were similar between HKO and control mice. In contrast, glucagon signaling and the hyperglycemic response to glucagon were severely impaired in HKO mice. In addition, TRAF2 overexpression significantly increased the ability of glucagon or a cAMP analog to stimulate CREB phosphorylation, gluconeogenic gene expression, and HGP in primary hepatocytes. These results suggest that the hepatic TRAF2 cell autonomously promotes hepatic gluconeogenesis by enhancing the hyperglycemic response to glucagon and other factors that increase cAMP levels, thus contributing to hyperglycemia in obesity.

Lipocalin-13 Regulates Glucose Metabolism by both Insulin-Dependent and Insulin-Independent Mechanisms

ABSTRACT Insulin sensitivity is impaired in obesity, and insulin resistance is the primary risk factor for type 2 diabetes. Here we show that lipocalin-13 (LCN13), a lipocalin superfamily member, is a novel insulin sensitizer. LCN13 was secreted by multiple cell types. Circulating LCN13 was markedly reduced in mice with obesity and type 2 diabetes. Three distinct approaches were used to increase LCN13 levels: LCN13 transgenic mice, LCN13 adenoviral infection, and recombinant LCN13 administration. Restoration of LCN13 significantly ameliorated hyperglycemia, insulin resistance, and glucose intolerance in mice with obesity. LCN13 enhanced insulin signaling not only in animals but also in cultured adipocytes. Recombinant LCN13 increased the ability of insulin to stimulate glucose uptake in adipocytes and to suppress hepatic glucose production (HGP) in primary hepatocyte cultures. Additionally, LCN13 alone was able to suppress HGP, whereas neutralization of LCN13 increased HGP in primary hepatocyte cultures. These data suggest that LCN13 regulates glucose metabolism by both insulin-dependent and insulin-independent mechanisms. LCN13 and LCN13-related molecules may be used to treat insulin resistance and type 2 diabetes.

Mouse hepatocyte overexpression of NF-κB-inducing kinase (NIK) triggers fatal macrophage-dependent liver injury and fibrosis

Damaged, necrotic, or apoptotic hepatocytes release damage‐associated molecular patterns that initiate sterile inflammation, and liver inflammation drives liver injury and fibrosis. Here we identified hepatic nuclear factor kappa B (NF‐κB)‐inducing kinase (NIK), a Ser/Thr kinase, as a novel trigger of fatal liver inflammation. NIK is activated by a broad spectrum of stimuli. It was up‐regulated in injured livers in both mice and humans. In primary mouse hepatocytes, NIK overexpression stimulated, independently of cell injury and death, release of numerous chemokines and cytokines that activated bone marrow‐derived macrophages (BMDMs). BMDMs in turn secreted proapoptotic molecules that stimulated hepatocyte apoptosis. Hepatocyte‐specific expression of the NIK transgene triggered massive liver inflammation, oxidative stress, hepatocyte apoptosis, and liver fibrosis, leading to weight loss, hypoglycemia, and death. Depletion of Kupffer cells/macrophages reversed NIK‐induced liver destruction and death. Conclusion: the hepatocyte NIK‐liver immune cell axis promotes liver inflammation, injury, and fibrosis, thus driving liver disease progression. (Hepatology 2014;60:2064–2075)

Lipocalin 13 protein protects against hepatic steatosis by both inhibiting lipogenesis and stimulating fatty acid β-Oxidation

Background: Obesity is associated with lipocalin 13 deficiency and fatty liver disease. Results: Recombinant lipocalin 13 suppressed lipogenesis and promoted β-oxidation. It attenuated hepatic steatosis and insulin resistance in obese mice. Conclusion: Lipocalin 13 is a new regulator of lipogenesis and β-oxidation; its deficiency contributes to fatty liver disease. Significance: Lipocalin 13 or its related molecules have therapeutic potential in treating metabolic diseases. Obesity is associated with hepatic steatosis, partially due to increased lipogenesis and decreased fatty acid β-oxidation in the liver; however, the underlying mechanism of abnormal lipid metabolism is not fully understood. We reported previously that obesity is associated with LCN13 (lipocalin 13) deficiency. LCN13 is a lipocalin family member involved in glucose metabolism, and LCN13 deficiency appears to contribute to hyperglycemia in obese mice. Here, we show that LCN13 is also an important regulator of lipogenesis and β-oxidation in the liver. In primary hepatocytes, recombinant LCN13 directly suppressed lipogenesis and increased fatty acid β-oxidation, whereas neutralization of endogenous LCN13 had an opposite effect. Transgenic overexpression of LCN13 protected against hepatic steatosis in mice with either dietary or genetic (ob/ob) obesity. LCN13 transgenic overexpression also improved hyperglycemia, glucose intolerance, and insulin resistance in ob/ob mice. Short-term LCN13 overexpression via an adenovirus-mediated gene transfer similarly attenuated hepatic steatosis in db/db mice. LCN13 inhibited the expression of important lipogenic genes and stimulated the genes that promote β-oxidation. These results suggest that LCN13 decreases liver lipid levels by both inhibiting hepatic lipogenesis and stimulating β-oxidation. LCN13 deficiency is likely to contribute to fatty liver disease in obese mice.

Hepatic SH2B1 and SH2B2 Regulate Liver Lipid Metabolism and VLDL Secretion in Mice

SH2B1 is an SH2 and PH domain-containing adaptor protein. Genetic deletion of SH2B1 results in obesity, type 2 diabetes, and fatty liver diseases in mice. Mutations in SH2B1 are linked to obesity in humans. SH2B1 in the brain controls energy balance and body weight at least in part by enhancing leptin sensitivity in the hypothalamus. SH2B1 in peripheral tissues also regulates glucose and lipid metabolism, presumably by enhancing insulin sensitivity in peripheral metabolically-active tissues. However, the function of SH2B1 in individual peripheral tissues is unknown. Here we generated and metabolically characterized hepatocyte-specific SH2B1 knockout (HKO) mice. Blood glucose and plasma insulin levels, glucose tolerance, and insulin tolerance were similar between HKO, albumin-Cre, and SH2B1f/f mice fed either a normal chow diet or a high fat diet (HFD). Adult-onset deletion of SH2B1 in the liver either alone or in combination with whole body SH2B2 knockout also did not exacerbate HFD-induced insulin resistance and glucose intolerance. Adult-onset, but not embryonic, deletion of SH2B1 in the liver attenuated HFD-induced hepatic steatosis. In agreement, adult-onset deletion of hepatic SH2B1 decreased the expression of diacylglycerol acyltransferase-2 (DGAT2) and increased the expression of adipose triglyceride lipase (ATGL). Furthermore, deletion of liver SH2B1 in SH2B2 null mice attenuated very low-density lipoprotein (VLDL) secretion. These data indicate that hepatic SH2B1 is not required for the maintenance of normal insulin sensitivity and glucose metabolism; however, it regulates liver triacylglycerol synthesis, lipolysis, and VLDL secretion.

Herbal constituent sequoyitol improves hyperglycemia and glucose intolerance by targeting hepatocytes, adipocytes, and β-cells

The prevalence of insulin resistance and type 2 diabetes increases rapidly; however, treatments are limited. Various herbal extracts have been reported to reduce blood glucose in animals with either genetic or dietary type 2 diabetes; however, plant extracts are extremely complex, and leading compounds remain largely unknown. Here we show that 5-O-methyl-myo-inositol (also called sequoyitol), a herbal constituent, exerts antidiabetic effects in mice. Sequoyitol was chronically administrated into ob/ob mice either orally or subcutaneously. Both oral and subcutaneous administrations of sequoyitol decreased blood glucose, improved glucose intolerance, and enhanced insulin signaling in ob/ob mice. Sequoyitol directly enhanced insulin signaling, including phosphorylation of insulin receptor substrate-1 and Akt, in both HepG2 cells (derived from human hepatocytes) and 3T3-L1 adipocytes. In agreement, sequoyitol increased the ability of insulin to suppress glucose production in primary hepatocytes and to stimulate glucose uptake into primary adipocytes. Furthermore, sequoyitol improved insulin signaling in INS-1 cells (a rat β-cell line) and protected INS-1 cells from streptozotocin- or H₂O₂-induced injury. In mice with streptozotocin-induced β-cell deficiency, sequoyitol treatments increased plasma insulin levels and decreased hyperglycemia and glucose intolerance. These results indicate that sequoyitol, a natural, water-soluble small molecule, ameliorates hyperglycemia and glucose intolerance by increasing both insulin sensitivity and insulin secretion. Sequoyitol appears to directly target hepatocytes, adipocytes, and β-cells. Therefore, sequoyitol may serve as a new oral diabetes medication.

Hepatocyte TRAF3 promotes insulin resistance and type 2 diabetes in mice with obesity

Objective Metabolic inflammation is believed to promote insulin resistance and type 2 diabetes progression in obesity. TRAF3, a cytoplasmic signaling protein, has been known to mediate/modulate cytokine signaling in immune cells. The goal is to define the metabolic function of hepatic TRAF3 in the setting of obesity. Methods Hepatocyte-specific TRAF3 knockout mice were generated using the loxp/albumin-cre system. Liver TRAF3 was deleted in adult obese mice via Cre adenoviral infection. Both high fat diet-induced and genetic obesity were examined. TRAF3 levels and insulin signaling were measured by immunoblotting. Insulin sensitivity, hepatic glucose production, and glucose metabolism were examined by glucose, insulin, and pyruvate tolerance tests. Hepatic steatosis was examined by Oil red O staining of liver sections and measuring liver triacylglycerol levels. Results Liver TRAF3 levels were lower in the fasted states in normal mice, and were aberrantly higher in obese mice and in mice with streptozotocin-induced hyperglycemia. Glucose directly increased TRAF3 levels in primary hepatocytes. Hepatocyte-specific deletion of TRAF3 decreased hyperinsulinemia, insulin resistance, glucose intolerance, and hepatic steatosis in mice with either high fat diet-induced obesity or genetic obesity (ob/ob); conversely, in lean mice, adenovirus-mediated overexpression of TRAF3 in the liver induced hyperinsulinemia, insulin resistance, and glucose intolerance. Deletion of TRAF3 enhanced the ability of insulin to stimulate phosphorylation of Akt in hepatocytes, whereas overexpression of TRAF3 suppressed insulin signaling. Conclusions Glucose increases the levels of hepatic TRAF3. TRAF3 in turn promotes hyperglycemia through increasing hepatic glucose production, thus forming a glucose-TRAF3 reinforcement loop in the liver. This positive feedback loop may drive the progression of type 2 diabetes and nonalcoholic fatty liver disease in obesity.

Intracellular lipid content is a key intrinsic determinant for hepatocyte viability and metabolic and inflammatory states in mice

The liver is an essential metabolic organ. In addition to metabolizing glucose and lipids, hepatocytes also secrete various cytokines that modulate both hepatocyte metabolism and liver inflammation. Hepatocyte injury and death and liver inflammation are the major contributors to liver diseases, including nonalcoholic steatohepatitis (NASH). Anatomic locations have a profound effect on hepatocyte metabolism, and liver zonation describes the metabolic heterogeneity of hepatocytes along the portovenous axis. However, it is unclear whether hepatocyte heterogeneity is affected by intrinsic factors and whether dietary fat, a risk factor for NASH, has distinct detrimental effects on different hepatocyte subpopulations. Here, we showed that mouse livers contained both high-lipid and low-lipid subpopulations of hepatocytes. The high-lipid subpopulation was more susceptible to injury and apoptosis and produced more proinflamatrory cytokines after treatment with endotoxin and saturated fatty acids. Dietary fat consumption further increased fatty acid uptake, intracellular lipid levels, hepatocyte injury and death, and the expression of proinflammatory cytokines in the high-lipid subpopulation. In contrast, dietary fat slightly increased lipid levels, cell death, and expression of proinflammatory cytokines in the low-lipid subpopulation. The low-lipid subpopulation produced more glucose. Fat consumption further activated the gluconeogenic program in the low-lipid, but not the high-lipid, subpopulations. These data suggest that intracellular lipid content is a key intrinsic determinant for hepatocyte heterogeneity of metabolic, inflammatory, and survival states.

Thymic NF-κB-inducing kinase regulates CD4+ T cell-elicited liver injury and fibrosis in mice

BACKGROUND & AIMS The liver is an immunologically-privileged organ. Breakdown of liver immune privilege has been reported in chronic liver disease; however, the role of adaptive immunity in liver injury is poorly defined. Nuclear factor-κB-inducing kinase (NIK) is known to regulate immune tissue development, but its role in maintaining liver homeostasis remains unknown. This study aimed to assess the role of NIK, particularly thymic NIK, in regulating liver adaptive immunity. METHODS NIK was deleted systemically or conditionally using the Cre/loxp system. Cluster of differentiation [CD]4+ or CD8+ T cells were depleted using anti-CD4 or anti-CD8 antibody. Donor bone marrows or thymi were transferred into recipient mice. Immune cells were assessed by immunohistochemistry and flow cytometry. RESULTS Global, but not liver-specific or hematopoietic lineage cell-specific, deletion of NIK induced fatal liver injury, inflammation, and fibrosis. Likewise, adoptive transfer of NIK-null, but not wild-type, thymi into immune-deficient mice induced liver inflammation, injury, and fibrosis in recipients. Liver inflammation was characterized by a massive expansion of T cells, particularly the CD4+ T cell subpopulation. Depletion of CD4+, but not CD8+, T cells fully protected against liver injury, inflammation, and fibrosis in NIK-null mice. NIK deficiency also resulted in inflammation in the lung, kidney, and pancreas, but to a lesser degree relative to the liver. CONCLUSIONS Thymic NIK suppresses development of autoreactive T cells against liver antigens, and NIK deficiency in the thymus results in CD4+ T cell-orchestrated autoimmune hepatitis and liver fibrosis. Thus, thymic NIK is essential for the maintenance of liver immune privilege and liver homeostasis. LAY SUMMARY We found that global or thymus-specific ablation of the NIK gene results in fatal autoimmune liver disease in mice. NIK-deficient mice develop liver inflammation, injury, and fibrosis. Our findings indicate that thymic NIK is essential for the maintenance of liver integrity and homeostasis.