Diabetic nephropathy (DN) is a major cause of end-stage renal disease, and therapeutic options for preventing its progression are limited. To identify novel therapeutic strategies, we studied protective factors for DN using proteomics on glomeruli from individuals with extreme duration of diabetes (≥50 years) without DN and those with histologic signs of DN. Enzymes in the glycolytic, sorbitol, methylglyoxal and mitochondrial pathways were elevated in individuals without DN. In particular, pyruvate kinase M2 (PKM2) expression and activity were upregulated. Mechanistically, we showed that hyperglycemia and diabetes decreased PKM2 tetramer formation and activity by sulfenylation in mouse glomeruli and cultured podocytes. Pkm-knockdown immortalized mouse podocytes had higher levels of toxic glucose metabolites, mitochondrial dysfunction and apoptosis. Podocyte-specific Pkm2-knockout (KO) mice with diabetes developed worse albuminuria and glomerular pathology. Conversely, we found that pharmacological activation of PKM2 by a small-molecule PKM2 activator, TEPP-46, reversed hyperglycemia-induced elevation in toxic glucose metabolites and mitochondrial dysfunction, partially by increasing glycolytic flux and PGC-1a mRNA in cultured podocytes. In intervention studies using DBA2/J and Nos3 (eNos) KO mouse models of diabetes, TEPP-46 treatment reversed metabolic abnormalities, mitochondrial dysfunction and kidney pathology. Thus, PKM2 activation may protect against DN by increasing glucose metabolic flux, inhibiting the production of toxic glucose metabolites and inducing mitochondrial biogenesis to restore mitochondrial function.

Insulin and insulin-like growth factor 1 (IGF1) are ubiquitous growth factors that regulate proliferation in most mammalian tissues including pancreatic islets. To explore the specificity of insulin receptors in compensatory ␤-cell growth, we examined two models of insulin resistance. In the first model, we used liverspecific insulin receptor knockout (LIRKO) mice, which exhibit hyperinsulinemia without developing diabetes due to a compensatory increase in ␤-cell mass. LIRKO mice, also lacking functional insulin receptors in ␤-cells (␤IRKO/LIRKO), exhibited severe glucose intolerance but failed to develop compensatory islet hyperplasia, together leading to early death. In the second model, we examined the relative significance of insulin versus IGF1 receptors in islet growth by feeding high-fat diets to ␤IRKO and ␤-cell-specific IGF1 receptor knockout (␤IGFRKO) mice. Although both groups on the high-fat diet developed insulin resistance, ␤IRKO, but not ␤IG-FRKO, mice exhibited poor islet growth consistent with insulinstimulated phosphorylation, nuclear exclusion of FoxO1, and reduced expression of Pdx-1. Together these data provide direct genetic evidence that insulin/FoxO1/Pdx-1 signaling is one pathway that is crucial for islet compensatory growth response to insulin resistance.␤-cell growth ͉ compensatory hyperplasia ͉ ␤-cell apoptosis I nsulin resistance is a common feature of type 2 diabetes, obesity, and hyperlipidemias (1). In patients with type 2 diabetes and mouse models of diabetes and obesity, the ␤-cells compensate for the insulin resistance by increasing their mass (2, 3). One of these models, the liver-specific insulin receptor knockout (LIRKO) mouse, manifests severe insulin resistance and glucose intolerance, but the mutants do not become overtly diabetic due, in part, to a significant increase in ␤-cell mass (4). Although lineage tracing experiments in normal mice and studies in cyclin D2 knockouts indicate that ␤-cell replication is a major mechanism for regeneration of adult ␤-cells (5, 42), the signals that induce ␤-cell replication are not fully defined.Insulin and IGFI and the proteins in their signaling pathways regulate cell growth and function (6), but their specificity in modulating ␤-cell proliferation is not fully explored. Thus, ␤-cellspecific insulin receptor (␤IRKO) (7), IGF1 receptor knockout (␤IGFRKO) (8, 9), or double mutants (10) surprisingly exhibited normal growth and development of ␤-cells consistent with data from global knockouts of insulin receptor or IGF1 receptor genes (11). Mice with global knockout of IRS1 (12, 13) or IRS2 genes (12, 14) also do not manifest defects in the development of islet cells, whereas overexpression of Akt in ␤-cells promotes an increase in cell size (15). Together these studies indicate that insulin/IGF1 signaling is not critical for early development and growth of ␤-cells.Our follow-up studies showed that ␤IRKO, but not ␤IGFRKO, mice developed an age-dependent decrease in ␤-cell mass and a susceptibility to develop overt diabetes (7,16,17). F...

During fasting, increased concentrations of circulating catecholamines promote the mobilization of lipid stores from adipose tissue in part by phosphorylating and inactivating acetyl-coenzyme A carboxylase (ACC), the rate-limiting enzyme in fatty acid synthesis. Here, we describe a parallel pathway, in which the pseudokinase Tribbles 3 (TRB3), whose abundance is increased during fasting, stimulates lipolysis by triggering the degradation of ACC in adipose tissue. TRB3 promoted ACC ubiquitination through an association with the E3 ubiquitin ligase constitutive photomorphogenic protein 1 (COP1). Indeed, adipocytes deficient in TRB3 accumulated larger amounts of ACC protein than did wild-type cells. Because transgenic mice expressing TRB3 in adipose tissue are protected from diet-induced obesity due to enhanced fatty acid oxidation, these results demonstrate how phosphorylation and ubiquitination pathways converge on a key regulator of lipid metabolism to maintain energy homeostasis.

Summary Glucagon plays an important role in glucose homeostasis by regulating hepatic glucose output in both normo- and hypo-glycemic conditions. In this study, we created and characterized α-cell specific insulin receptor knockout (αIRKO) mice to directly explore the role of insulin signaling in the regulation of glucagon secretion in vivo. Adult male αIRKO mice exhibited mild glucose intolerance, hyperglycemia and hyperglucagonemia in the fed state, and enhanced glucagon secretion in response to L-Arginine stimulation. Hyperinsulinemic-hypoglycemic clamp studies revealed an enhanced glucagon secretory response and an abnormal norepinephrine response to hypoglycemia in αIRKO mice. The mutants also exhibited an age-dependent increase in β-cell mass. Furthermore, siRNA-mediated knockdown of insulin receptor in glucagon-secreting InR1G cells promoted enhanced glucagon secretion and complemented our in vivo findings. Together, these data indicate a significant role for intra-islet insulin signaling in the regulation of α-cell function in both normo- and hypo-glycemic conditions.

An appropriate beta cell mass is pivotal for the maintenance of glucose homeostasis. Both insulin and IGF-1 are important in regulation of beta cell growth and function (reviewed in ref. 2). To define the roles of these hormones directly, we created a mouse model lacking functional receptors for both insulin and IGF-1 only in beta cells (betaDKO), as the hormones have overlapping mechanisms of action and activate common downstream proteins. Notably, betaDKO mice were born with a normal complement of islet cells, but 3 weeks after birth, they developed diabetes, in contrast to mild phenotypes observed in single mutants. Normoglycemic 2-week-old betaDKO mice manifest reduced beta cell mass, reduced expression of phosphorylated Akt and the transcription factor MafA, increased apoptosis in islets and severely compromised beta cell function. Analyses of compound knockouts showed a dominant role for insulin signaling in regulating beta cell mass. Together, these data provide compelling genetic evidence that insulin and IGF-I-dependent pathways are not critical for development of beta cells but that a loss of action of these hormones in beta cells leads to diabetes. We propose that therapeutic improvement of insulin and IGF-I signaling in beta cells might protect against type 2 diabetes.

LKB1 is a tumor suppressor that may also be fundamental to cell metabolism, since LKB1 phosphorylates and activates the energy sensing enzyme AMPK. We generated muscle-specific LKB1 knockout (MLKB1KO) mice, and surprisingly, found that a lack of LKB1 in skeletal muscle enhanced insulin sensitivity, as evidenced by decreased fasting glucose and insulin concentrations, improved glucose tolerance, increased muscle glucose uptake in vivo, and increased glucose utilization during a hyperinsulinemic-euglycemic clamp. MLKB1KO mice had increased insulin-stimulated Akt phosphorylation and a >80% decrease in muscle expression of TRB3, a recently identified Akt inhibitor. Akt/TRB3 binding was present in skeletal muscle, and overexpression of TRB3 in C2C12 myoblasts significantly reduced Akt phosphorylation. These results demonstrate that skeletal muscle LKB1 is a negative regulator of insulin sensitivity and glucose homeostasis. LKB1-mediated TRB3 expression provides a novel link between LKB1 and Akt, critical kinases involved in both tumor genesis and cell metabolism.LKB1 is a serine/threonine kinase that links a diverse array of cellular processes, including cancer, cellular polarity, and metabolism. Originally identified as the tumor suppressor protein mutated in Peutz-Jeghers syndrome (17, 21), LKB1 has since been shown to regulate polarity in a number of systems, including Caenorhabditis elegans (48), Drosophila melanogaster (28), Xenopus (33), and mammalian cells (3). Biochemically, LBK1 can phosphorylate and activate at least 13 members of the AMP-activated protein kinase (AMPK) subfamily of protein kinases (20, 27) when associated with two regulatory proteins essential for catalytic activity, STE20-related adapter protein and mouse protein 25 (MO25) (15). AMPK, the most studied of LKB1's downstream substrates, is a conserved serine/threonine kinase that functions in the regulation of energy metabolism (16,18,29). Studies with cell culture (15,40,51) or conditional knockouts of LKB1 in skeletal muscle (38), cardiac muscle (39), or liver (41) have found that LKB1 regulates AMPK activity both in vitro and in vivo. Other than AMPK and the microtubule affinity-regulating kinase (MARK) proteins, which have been implicated in the control of cellular polarity (9), relatively little is known about the function of the AMPK-related kinases. The well-established role of AMPK in metabolism, however, directly implicates LKB1 in the maintenance of energy balance.The protein kinase Akt functions both in the control of cell proliferation and as a critical node in insulin signaling, appearing to mediate most of the metabolic effects of insulin (44). Akt is activated by phosphorylation of Thr 308 within the T loop of the catalytic domain and Ser 473 , located in a C-terminal, noncatalytic region of the enzyme (1). A mammalian homolog of D. melanogaster tribbles, TRB3, was recently identified as a negative regulator of Akt activity in human embryonic kidney 293 (HEK293) cells and mouse liver (10). In HEK293 cells and liver, TRB3 bind...

Summary Although compensatory islet hyperplasia in response to insulin resistance is a recognized feature in diabetes, the factor(s) that promote β-cell proliferation have been elusive. We previously reported that the liver is a source for such factors in the liver insulin receptor knockout (LIRKO) mouse, an insulin resistance model which manifests islet hyperplasia. Using proteomics we show that serpinB1, a protease inhibitor, which is abundant in the hepatocyte secretome and sera derived from LIRKO mice, is the liver-derived secretory protein that regulates β-cell proliferation in humans, mice and zebrafish. Small molecule compounds, that partially mimic serpinB1 effects of inhibiting elastase activity, enhanced proliferation of β-cells, and mice lacking serpinB1 exhibit attenuated β-cell compensation in response to insulin resistance. Finally, SerpinB1-treatment of islets modulated proteins in growth/survival pathways. Together, these data implicate serpinB1 as an endogenous protein that can potentially be harnessed to enhance functional β-cell mass in patients with diabetes.

Patients with type 2 diabetes lose beta cells, but the underlying mechanisms are incompletely understood. Glucose-6-phosphate dehydrogenase (G6PD) is the principal source of the major intracellular reductant, NADPH, which is required by many enzymes, including enzymes of the antioxidant pathway. Previous work from our laboratory has shown that high glucose impairs G6PD activity in endothelial and kidney cells, which leads to decreased cell survival. Pancreatic beta cells are highly sensitive to increased ROS. This study aimed to determine whether G6PD and NADPH play central roles in beta-cell survival. Human and mouse islets, MIN6 cell line, and G6PD deficient mice were studied. High glucose inhibited G6PD expression and activity. Inhibition of G6PD with siRNA led to increased ROS and apoptosis, decreased proliferation, and impaired insulin secretion. High glucose decreased insulin secretion, which was improved by overexpressing G6PD. G6PD-deficient mice had smaller islets and impaired glucose tolerance compared with control mice, which suggests that G6PD deficiency per se leads to beta-cell dysfunction and death. G6PD plays an important role in beta-cell function and survival. High-glucose-mediated decrease in G6PD activity may provide a mechanistic explanation for the gradual loss of beta cells in patients with diabetes.