PI3Kγ promotes obesity-associated hepatocellular carcinoma by regulating metabolism and inflammation.

BACKGROUND & AIMS: Phosphatidylinositides-3 kinases (PI3Ks) are promising drug targets for cancer therapy, but blockage of PI3K-AKT signalling causes hyperglycaemia, hyperinsulinaemia, and liver damage in patients, and hepatocellular carcinoma (HCC) in mice. There are 4 PI3Ks: PI3Kα, PI3Kß, PI3Kδ, and PI3Kγ. The role of PI3Kγ in HCC is unknown. METHODS: We performed histopathological, metabolic, and molecular phenotyping of mice with genetic ablation of PI3Kγ using models where HCC was initiated by the carcinogen diethylnitrosamine (DEN) and promoted by dietary or genetic obesity (ob/ob). The role of PI3Kγ in leucocytes was investigated in mice lacking PI3Kγ in haematopoietic and endothelial cells. RESULTS: Loss of PI3Kγ had no effects on the development of DEN-induced HCC in lean mice. However, in mice injected with DEN and placed on an obesogenic diet, PI3Kγ ablation reduced tumour growth, which was associated with reduced insulinaemia, steatosis, and expression of inflammatory cytokines. ob/ob mice lacking PI3Kγ, and mice with diet-induced obesity lacking PI3Kγ in leucocytes and endothelial cells did not display improved insulin sensitivity, steatosis, metabolic inflammation, or reduced tumour growth. However, these mice showed a reduced number of tumours, reduced liver infiltration by neutrophils, and reduced hepatocyte proliferation acutely induced by DEN. CONCLUSIONS: Loss of PI3Kγ reduces tumour development in obesity-promoted HCC through multiple cell types and mechanisms that include improved insulinaemia, steatosis, and metabolic inflammation as well as the regulation of acute neutrophil infiltration and compensatory hepatocyte proliferation. PI3Kγ-selective inhibition may represent a novel therapeutic approach to reduce HCC initiation and slow HCC progression. LAY SUMMARY: Class-1 phosphatidylinositides-3 kinases (PI3Ks) are critical targets in cancer therapy, but complete inhibition of all isoforms causes liver damage, hyperglycaemia, and insulinaemia. Here we show that selective ablation of the PI3Kγ isoform dampens tumour initiation and growth in a mouse model of carcinogen-initiated and obesity-promoted hepatocellular carcinoma (HCC). The effect of PI3Kγ ablation on reduced tumour growth was explained by reduced tumour cell proliferation, which was associated with reduced insulin levels, liver lipids, and reduced expression of tumour-promoting cytokines. PI3Kγ ablation in leucocytes of obese mice had no effects on tumour size. However, it reduced tumour number in association with reduced carcinogen-induced neutrophil infiltration and hepatocyte proliferation in livers of obese mice. Inhibition of PI3Kγ may thus reduce HCC initiation and growth in obese subjects by a mechanism involving reduced metabolic stress and insulinaemia and reduced carcinogen-induced neutrophil infiltration to the fatty liver.

JNK1 ablation improves pancreatic ß-cell mass and function in db/db diabetic mice without affecting insulin sensitivity and adipose tissue inflammation.

The cJun N-terminal Kinases (JNK) emerged as a major link between obesity and insulin resistance, but their role in the loss of pancreatic ß-cell mass and function driving the progression from insulin resistance to type-2 diabetes and in the complications of diabetes was not investigated to the same extent. Furthermore, it was shown that pan-JNK inhibition exacerbates kidney damage in the db/db model of obesity-driven diabetes. Here we investigate the role of JNK1 in the db/db model of obesity-driven type-2 diabetes. Mice with systemic ablation of JNK1 (JNK1-/-) were backcrossed for more than 10 generations in db/+ C57BL/KS mice to generate db/db-JNK1-/- mice and db/db control mice. To define the role of JNK1 in the loss of ß-cell mass and function occurring during obesity-driven diabetes we performed comprehensive metabolic phenotyping, evaluated steatosis and metabolic inflammation, performed morphometric and cellular composition analysis of pancreatic islets, and evaluated kidney function in db/db-JNK1-/- mice and db/db controls. db/db-JNK1-/- mice and db/db control mice developed insulin resistance, fatty liver, and metabolic inflammation to a similar extent. However, db/db-JNK1-/- mice displayed better glucose tolerance and improved insulin levels during glucose tolerance test, higher pancreatic insulin content, and larger pancreatic islets with more ß-cells than db/db mice. Finally, albuminuria, kidney histopathology, kidney inflammation and oxidative stress in db/db-JNK1-/- mice and in db/db mice were similar. Our data indicate that selective JNK1 ablation improves glucose tolerance in db/db mice by reducing the loss of functional ß-cells occurring in the db/db mouse model of obesity-driven diabetes, without significantly affecting metabolic inflammation, steatosis, and insulin sensitivity. Furthermore, we have found that, differently from what previously reported for pan-JNK inhibitors, selective JNK1 ablation does not exacerbate kidney dysfunction in db/db mice. We conclude that selective JNK1 inactivation may have a superior therapeutic index than pan-JNK inhibition in obesity-driven diabetes.

PI3Kγ ablation does not promote diabetes in db/db mice, but improves insulin sensitivity and reduces pancreatic ß-cell apoptosis.

PI3Kγ has emerged as a promising target for the treatment of obesity and insulin resistance; however, previous studies have indicated that PI3Kγ activity in pancreatic ß cells is required for normal insulin secretion in response to glucose. Hence, a possible deterioration of insulin secretion capacity in patients who are predisposed to the failure of pancreatic ß-cell function is a major concern for the pharmacologic inhibition of PI3Kγ. To address this issue, we investigated the effects of PI3Kγ ablation in db/db diabetic mice, a genetic model of obesity-driven ß-cell failure and diabetes. Mice that lacked PI3Kγ were backcrossed into db/+ mice C57BL/KS (>10 generations) to obtain db/db-PI3Kγ-/- mice. db/db-PI3Kγ-/- mice and control db/db mice were phenotyped for glucose homeostasis, insulin sensitivity, insulin secretion, steatosis, metabolic inflammation, pancreatic islet morphometry, islet cellular composition, and inflammation. Pancreatic ß-cell apoptosis and proliferation were also evaluated. db/db-PI3Kγ -/- mice and control db/db mice developed similar body weight, steatosis, glycemia, and insulin levels after a glucose load; however, db/db-PI3Kγ-/- mice displayed improved insulin tolerance, higher levels of fasting serum insulin, and lower pancreatic insulin content. In db/db-PI3Kγ-/- mice, the number of adipose tissue macrophages was similar to control, but displayed reduced adipose tissue neutrophils and M2-polarized adipose tissue gene expression. Finally, db/db-PI3Kγ-/- mice have more pancreatic ß cells and larger islets than db/db mice, despite displaying similar islet inflammation. This phenotype could be explained by reduced ß-cell apoptosis in db/db-PI3Kγ-/- mice compared with control db/db mice. Our results are consistent with the concept that the beneficial action of PI3Kγ ablation in obesity-driven glucose intolerance is largely a result of its leptin-dependent effects on adiposity and, to a lesser extent, the promotion of adipose tissue neutrophil recruitment and M1 polarization of gene expression. Of importance, our data challenge the concept that PI3Kγ is required for insulin secretion in response to glucose in vivo, and indicate that PI3Kγ ablation protects db/db mice from ß-cell apoptosis and improves fasting insulin levels. We conclude that PI3Kγ inhibition in obese patients who are predisposed to ß-cell failure is not expected to produce adverse effects on insulin secretion.-Breasson, L., Sardi, C., Becattini, B., Zani, F., Solinas, G. PI3Kγ ablation does not promote diabetes in db/db mice, but improves insulin sensitivity and reduces pancreatic ß-cell apoptosis.

PI3Kγ activity in leukocytes promotes adipose tissue inflammation and early-onset insulin resistance during obesity.

The phosphoinositide 3-kinase γ (PI3Kγ) plays a major role in leukocyte recruitment during acute inflammation and has been proposed to inhibit classical macrophage activation by driving immunosuppressive gene expression. PI3Kγ plays an important role in diet-induced obesity and insulin resistance. In seeking to determine the underlying molecular mechanisms, we showed that PI3Kγ action in high-fat diet-induced inflammation and insulin resistance depended largely on its role in the control of adiposity, which was due to PI3Kγ activity in a nonhematopoietic cell type. However, PI3Kγ activity in leukocytes was required for efficient neutrophil recruitment to adipose tissue. Neutrophil recruitment was correlated with proinflammatory gene expression in macrophages in adipose tissue, which triggered insulin resistance early during the development of obesity. Our data challenge the concept that PI3Kγ is a general suppressor of classical macrophage activation and indicate that PI3Kγ controls macrophage gene expression by non-cell-autonomous mechanisms, the outcome of which is context-dependent.

JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.

Obesity and insulin resistance are associated with oxidative stress, which may be implicated in the progression of obesity-related diseases. The kinase JNK1 has emerged as a promising drug target for the treatment of obesity and type 2 diabetes. JNK1 is also a key mediator of the oxidative stress response, which can promote cell death or survival, depending on the magnitude and context of its activation. In this article, we describe a study in which the long-term effects of JNK1 inactivation on glucose homeostasis and oxidative stress in obese mice were investigated for the first time. Mice lacking JNK1 (JNK1(-/-)) were fed an obesogenic high-fat diet (HFD) for a long period. JNK1(-/-) mice fed an HFD for the long term had reduced expression of antioxidant genes in their skin, more skin oxidative damage, and increased epidermal thickness and inflammation compared with the effects in control wild-type mice. However, we also observed that the protection from obesity, adipose tissue inflammation, steatosis, and insulin resistance, conferred by JNK1 ablation, was sustained over a long period and was paralleled by decreased oxidative damage in fat and liver. We conclude that compounds targeting JNK1 activity in brain and adipose tissue, which do not accumulate in the skin, may be safer and most effective.-Becattini, B., Zani, F., Breasson, L., Sardi, C., D'Agostino, V. G., Choo, M.-K., Provenzani, A., Park, J. M., Solinas, G. JNK1 ablation in mice confers long-term metabolic protection from diet-induced obesity at the cost of moderate skin oxidative damage.

PER2 promotes glucose storage to liver glycogen during feeding and acute fasting by inducing Gys2 PTG and G L expression.

The interplay between hepatic glycogen metabolism and blood glucose levels is a paradigm of the rhythmic nature of metabolic homeostasis. Here we show that mice lacking a functional PER2 protein (Per2 (Brdm1) ) display reduced fasting glycemia, altered rhythms of hepatic glycogen accumulation, and altered rhythms of food intake. Per2 (Brdm1) mice show reduced hepatic glycogen content and altered circadian expression during controlled fasting and refeeding. Livers from Per2 (Brdm1) mice display reduced glycogen synthase protein levels during refeeding, and increased glycogen phosphorylase activity during fasting. The latter is explained by PER2 action on the expression of the adapter proteins PTG and GL, which target the protein phosphatase-1 to glycogen to decrease glycogen phosphorylase activity. Finally, PER2 interacts with genomic regions of Gys2, PTG, and G L . These results indicate an important role for PER2 in the hepatic transcriptional response to feeding and acute fasting that promotes glucose storage to liver glycogen.

Native adipose stromal cells egress from adipose tissue in vivo: evidence during lymph node activation.

Adipose tissue (AT) has become accepted as a source of multipotent progenitor cells, the adipose stromal cells (ASCs). In this regard, considerable work has been performed to harvest and characterize this cell population as well as to investigate the mechanisms by which transplanted ASCs mediate tissue regeneration. In contrast the endogenous release of native ASCs by AT has been poorly investigated. In this work, we show that native ASCs egress from murine AT. Indeed, we demonstrated that the release of native ASCs from AT can be evidenced both using an ex vivo perfusion model that we set up and in vivo. Such a mobilization process is controlled by CXCR4 chemokine receptor. In addition, once mobilized from AT, circulating ASCs were found to navigate through lymph fluid and to home into lymph nodes (LN). Therefore, we demonstrated that, during the LN activation, the fat depot encapsulating the activated LN releases native ASCs, which in turn invade the activated LN. Moreover, the ASCs invading the LN were visualized in close physical interaction with podoplanin and ER-TR7 positive structures corresponding to the stromal network composing the LN. This dynamic was impaired with CXCR4 neutralizing antibody. Taken together, these data provide robust evidences that native ASCs can traffic in vivo and that AT might provide stromal cells to activated LNs.