Ube4A maintains metabolic homeostasis and facilitates insulin signaling in vivo.

OBJECTIVE: Defining the regulators of cell metabolism and signaling is essential to design new therapeutic strategies in obesity and NAFLD/NASH. E3 ubiquitin ligases control diverse cellular functions by ubiquitination-mediated regulation of protein targets, and thus their functional aberration is associated with many diseases. The E3 ligase Ube4A has been implicated in human obesity, inflammation, and cancer. However, its in vivo function is unknown, and no animal models are available to study this novel protein. METHODS: A whole-body Ube4A knockout (UKO) mouse model was generated, and various metabolic parameters were compared in chow- and high fat diet (HFD)-fed WT and UKO mice, and in their liver, adipose tissue, and serum. Lipidomics and RNA-Seq studies were performed in the liver samples of HFD-fed WT and UKO mice. Proteomic studies were conducted to identify Ube4A's targets in metabolism. Furthermore, a mechanism by which Ube4A regulates metabolism was identified. RESULTS: Although the body weight and composition of young, chow-fed WT and UKO mice are similar, the knockouts exhibit mild hyperinsulinemia and insulin resistance. HFD feeding substantially augments obesity, hyperinsulinemia, and insulin resistance in both sexes of UKO mice. HFD-fed white and brown adipose tissue depots of UKO mice have increased insulin resistance and inflammation and reduced energy metabolism. Moreover, Ube4A deletion exacerbates hepatic steatosis, inflammation, and liver injury in HFD-fed mice with increased lipid uptake and lipogenesis in hepatocytes. Acute insulin treatment resulted in impaired activation of the insulin effector protein kinase Akt in liver and adipose tissue of chow-fed UKO mice. We identified the Akt activator protein APPL1 as a Ube4A interactor. The K63-linked ubiquitination (K63-Ub) of Akt and APPL1, known to facilitate insulin-induced Akt activation, is impaired in UKO mice. Furthermore, Ube4A K63-ubiquitinates Akt in vitro. CONCLUSION: Ube4A is a novel regulator of obesity, insulin resistance, adipose tissue dysfunction and NAFLD, and preventing its downregulation may ameliorate these diseases.

The IP6K Inhibitor LI-2242 Ameliorates Diet-Induced Obesity, Hyperglycemia, and Hepatic Steatosis in Mice by Improving Cell Metabolism and Insulin Signaling.

Obesity and nonalcoholic fatty liver disease (NAFLD) are global health concerns, and thus, drugs for the long-term treatment of these diseases are urgently needed. We previously discovered that the inositol pyrophosphate biosynthetic enzyme IP6K1 is a target in diet-induced obesity (DIO), insulin resistance, and NAFLD. Moreover, high-throughput screening (HTS) assays and structure-activity relationship (SAR) studies identified LI-2242 as a potent IP6K inhibitor compound. Here, we tested the efficacy of LI-2242 in DIO WT C57/BL6J mice. LI-2242 (20 mg/kg/BW daily, i.p.) reduced body weight in DIO mice by specifically reducing the accumulation of body fat. It also improved glycemic parameters and reduced hyperinsulinemia. LI-2242-treated mice displayed reduced the weight of various adipose tissue depots and an increased expression of metabolism- and mitochondrial-energy-oxidation-inducing genes in these tissues. LI-2242 also ameliorated hepatic steatosis by reducing the expression of genes that enhance lipid uptake, lipid stabilization, and lipogenesis. Furthermore, LI-2242 enhances the mitochondrial oxygen consumption rate (OCR) and insulin signaling in adipocytes and hepatocytes in vitro. In conclusion, the pharmacologic inhibition of the inositol pyrophosphate pathway by LI-2242 has therapeutic potential in obesity and NAFLD.

Whole Body Ip6k1 Deletion Protects Mice from Age-Induced Weight Gain, Insulin Resistance and Metabolic Dysfunction.

(1) Background: We previously demonstrated that disruption of IP6K1 improves metabolism, protecting mice from high-fat diet-induced obesity, insulin resistance, and non-alcoholic fatty liver disease and steatohepatitis. Age-induced metabolic dysfunction is a major risk factor for metabolic diseases. The involvement of IP6K1 in this process is unknown. (2) Methods: Here, we compared body and fat mass, insulin sensitivity, energy expenditure and serum-, adipose tissue- and liver-metabolic parameters of chow-fed, aged, wild type (aWT) and whole body Ip6k1 knockout (aKO) mice. (3) Results: IP6K1 was upregulated in the adipose tissue and liver of aWT mice compared to young WT mice. Moreover, Ip6k1 deletion blocked age-induced increase in body- and fat-weight and insulin resistance in mice. aKO mice oxidized carbohydrates more efficiently. The knockouts displayed reduced levels of serum insulin, triglycerides, and non-esterified fatty acids. Ip6k1 deletion partly protected age-induced decline of the thermogenic uncoupling protein UCP1 in inguinal white adipose tissue. Targets inhibited by IP6K1 activity such as the insulin sensitivity- and energy expenditure-inducing protein kinases, protein kinase B (PKB/Akt) and AMP-activated protein kinase (AMPK), were activated in the adipose tissue and liver of aKO mice. (4) Conclusions: Ip6k1 deletion maintains healthy metabolism in aging and thus, targeting this kinase may delay the development of age-induced metabolic dysfunction.

Pleiotropic actions of IP6K1 mediate hepatic metabolic dysfunction to promote nonalcoholic fatty liver disease and steatohepatitis.

OBJECTIVE: Obesity and insulin resistance greatly increase the risk of nonalcoholic fatty liver disease and steatohepatitis (NAFLD/NASH). We have previously discovered that whole-body and adipocyte-specific Ip6k1deletion protects mice from high-fat-diet-induced obesity and insulin resistance due to improved adipocyte thermogenesis and insulin signaling. Here, we aimed to determine the impact of hepatocyte-specific and whole-body Ip6k1 deletion (HKO and Ip6k1-KO or KO) on liver metabolism and NAFLD/NASH. METHODS: Body weight and composition; energy expenditure; glycemic profiles; and serum and liver metabolic, inflammatory, fibrotic and toxicity parameters were assessed in mice fed Western and high-fructose diet (HFrD) (WD: 40% kcal fat, 1.25% cholesterol, no added choline and HFrD: 60% kcal fructose). Mitochondrial oxidative capacity was evaluated in isolated hepatocytes. RNA-Seq was performed in liver samples. Livers from human NASH patients were analyzed by immunoblotting and mass spectrometry. RESULTS: HKO mice displayed increased hepatocyte mitochondrial oxidative capacity and improved insulin sensitivity but were not resistant to body weight gain. Improved hepatocyte metabolism partially protected HKO mice from NAFLD/NASH. In contrast, enhanced whole-body metabolism and reduced body fat accumulation significantly protected whole-body Ip6k1-KO mice from NAFLD/NASH. Mitochondrial oxidative pathways were upregulated, whereas gluconeogenic and fibrogenic pathways were downregulated in Ip6k1-KO livers. Furthermore, IP6K1 was upregulated in human NASH livers and interacted with the enzyme O-GlcNAcase that reduces protein O-GlcNAcylation. Protein O-GlcNAcylation was found to be reduced in Ip6k1-KO and HKO mouse livers. CONCLUSION: Pleiotropic actions of IP6K1 in the liver and other metabolic tissues mediate hepatic metabolic dysfunction and NAFLD/NASH, and thus IP6K1 deletion may be a potential treatment target for this disease.

Targeting the Inositol Pyrophosphate Biosynthetic Enzymes in Metabolic Diseases.

In mammals, a family of three inositol hexakisphosphate kinases (IP6Ks) synthesizes the inositol pyrophosphate 5-IP7 from IP6. Genetic deletion of Ip6k1 protects mice from high fat diet induced obesity, insulin resistance and fatty liver. IP6K1 generated 5-IP7 promotes insulin secretion from pancreatic ß-cells, whereas it reduces insulin signaling in metabolic tissues by inhibiting the protein kinase Akt. Thus, IP6K1 promotes high fat diet induced hyperinsulinemia and insulin resistance in mice while its deletion has the opposite effects. IP6K1 also promotes fat accumulation in the adipose tissue by inhibiting the protein kinase AMPK mediated energy expenditure. Genetic deletion of Ip6k3 protects mice from age induced fat accumulation and insulin resistance. Accordingly, the pan IP6K inhibitor TNP [N2-(m-trifluorobenzyl), N6-(p-nitrobenzyl)purine] ameliorates obesity, insulin resistance and fatty liver in diet induced obese mice by improving Akt and AMPK mediated insulin sensitivity and energy expenditure. TNP also protects mice from bone loss, myocardial infarction and ischemia reperfusion injury. Thus, the IP6K pathway is a potential target in obesity and other metabolic diseases. Here, we summarize the studies that established IP6Ks as a potential target in metabolic diseases. Further studies will reveal whether inhibition of this pathway has similar pleiotropic benefits on metabolic health of humans.