PANDER KO mice on high-fat diet are glucose intolerant yet resistant to fasting hyperglycemia and hyperinsulinemia

The recent creation of the PANDER (pancreatic-derived factor) knockout (PANKO) and acute mouse models have revealed a biological function in the regulation of glycemic levels via promotion of hepatic glucose production (HGP) and pancreatic β-cell insulin secretion. Therefore, we hypothesized that the absence of PANDER may afford some degree of protection from high-fat diet (HFD) induced fasting hyperglycemia. On HFD, fasting glycemic levels were significantly lower in the PANKO mice. Also, fasting insulin levels and the in vivo insulin response following glucose injection were inhibited in PANKO mice. The lowered fasting glycemic levels are attributed to decreased HGP due to the absence of PANDER. Overall, our findings further indicate PANDER impacts glycemic levels and may represent a potential but complicated therapeutic target.     

F3MB(PANDER) Decreases Mice Hepatic Triglyceride and Is Associated with Decreased DGAT1 Expression

ObjectivePancreatic-derived factor (PANDER, also named as FAM3B) is secreted by pancreatic α and β cells. Increasing evidence suggests that it may serve a hormonal function related to glycemic and lipid metabolism. In this study, we investigated the effects of PANDER overexpression on hepatic and adipose triglyceride metabolism in high-fat diet-fed male C57BL/6 mice.MethodsPANDER overexpression was achieved by tail-vein injection of recombinant Ad-PANDER and Ad-GFP injected mice served as a control. The TG metabolism in both groups were compared.ResultsAdenoviral-mediated overexpression of PANDER did not affect body weight, food consumption, or liver enzymes. The triglyceride (TG) content of both liver and adipose tissue was significantly decreased in Ad-PANDER mice (liver: 6.16±1.89 mg/g vs. control 14.95±2.27 mg/g, P<0.05; adipose: 39.31±1.99 mg/100mg vs. 47.22±2.21 mg/100mg, P<0.05). The free fatty acid (FFA) content of adipose tissue in Ad-PANDER mice was also decreased (1.38±0.18 mg/g vs. 2.77±0.31 mg/g, P<0.01). The investigation of key enzymes of triglyceride hydrolysis and FFA oxidation in liver and adipose tissue showed that p-HSL/HSL was significantly increased and that DGAT1 gene and protein expression were significantly reduced in the liver of PANDER-overexpressing mice. PKA phosphorylation was also significantly increased in the livers of Ad-PANDER mice. No differences in ATGL, CPT1, ACOX1, or DGAT2 expression were observed.ConclusionOverexpression of PANDER is associated with observable decreases in TG, increases in PKA phosphorylation, and decreased DGAT1 expression, suggesting a possible interrelationship. The mechanisms by which this occurs remain to be elucidated.     

PANcreatic-DERived factor: novel hormone PANDERing to glucose regulation

PANcreatic-DERived factor (PANDER, FAM3B) is a member of the FAM3 family of cytokine molecules that were initially described in 2002. PANDER expression is primarily localized to the endocrine pancreas and is secreted from both pancreatic α and β-cells. Initial characterization of PANDER revealed a potential role in pancreatic islet apoptosis. However, recent animal models have indicated PANDER functions as a hormone by regulating glucose levels via interaction with both the liver and the endocrine pancreas. An understanding of the function of PANDER can further the insight into the mechanisms of glucose regulation and potentially provide additional therapeutic targets for the treatment of diabetes. This review details the supporting data demonstrating PANDER has a biological function in glycemic regulation.     

T cell autoimmunity in Ig transgenic mice.

Autoantibodies directed at a diverse group of proteins of the U1/Sm ribonucleoprotein (snRNP) are characteristic of systemic lupus erythematosus and are found in the MRL murine model of this disease. This study examines the role of transgenic B lymphocytes in the regulation of autoreactive T cells to the snRNP autoantigen. Transgenic mice were developed bearing an Ig heavy chain gene specific for the D protein component of murine snRNP. B lymphocytes in these mice are neither deleted nor anergic and are of an immature (heat-stable Aghigh) phenotype. T lymphocytes from anti-snRNP transgenic mice were examined using a recombinant form of the D protein of the murine snRNP complex. Our results revealed that transgenic anti-snRNP B cell APCs stimulated CD4 T cells from wild-type C57BL/6 and MRL lpr/lpr mice, while nonspecific APCs failed to stimulate CD4 T cells. This study demonstrates that autoreactive T cells are not deleted from wild-type mice, although their activation is facilitated by autoantigen-specific APCs. The snRNP-reactive T cells in C57BL/6 transgenic mice are tolerized, in contrast to those T cells from MRL lpr/lpr transgenic mice. These studies implicate a role for autoreactive B lymphocytes in the in vivo activation and/or diversification of autoreactive T cells.     

Template to improve glycemic control without reducing adiposity or dietary fat

Drugs that improve chronic hyperglycemia independently of insulin signaling or reduction of adiposity or dietary fat intake may be highly desirable. Ad36, a human adenovirus, promotes glucose uptake in vitro independently of adiposity or proximal insulin signaling. We tested the ability of Ad36 to improve glycemic control in vivo and determined if the natural Ad36 infection in humans is associated with better glycemic control. C57BL/6J mice fed a chow diet or made diabetic with a high-fat (HF) diet were mock infected or infected with Ad36 or adenovirus Ad2 as a control for infection. Postinfection (pi), systemic glycemic control, hepatic lipid content, and cell signaling in tissues pertinent to glucose metabolism were determined. Next, sera of 1,507 adults and children were screened for Ad36 antibodies as an indicator of past natural infection. In chow-fed mice, Ad36 significantly improved glycemic control for 12 wk pi. In HF-fed mice, Ad36 improved glycemic control and hepatic steatosis up to 20 wk pi. In adipose tissue (AT), skeletal muscle (SM), and liver, Ad36 upregulated distal insulin signaling without recruiting the proximal insulin signaling. Cell signaling suggested that Ad36 increases AT and SM glucose uptake and reduces hepatic glucose release. In humans, Ad36 infection predicted better glycemic control and lower hepatic lipid content independently of age, sex, or adiposity. We conclude that Ad36 offers a novel tool to understand the pathways to improve hyperglycemia and hepatic steatosis independently of proximal insulin signaling, and despite a HF diet. This metabolic engineering by Ad36 appears relevant to humans for developing more practical and effective antidiabetic approaches.     

Vitamin K1 inversely correlates with glycemia and insulin resistance in patients with type 2 diabetes (T2D) and positively regulates SIRT1/AMPK pathway of glucose metabolism in liver of T2D mice and hepatocytes cultured in high glucose

There is no previous study in the literature that has examined the relationship between circulating vitamin K1 (VK1) with glycemic status in type 2 diabetes (T2D). Moreover, scientific explanation for the beneficial role of VK1 supplementation in lowering glycemia in diabetes is yet to be determined. This study for the first time demonstrated that circulating VK1 was significantly lower in T2D patients compared to age-matched control subjects, and VK1 levels in T2D were significantly and inversely associated with fasting glucose and insulin resistance [homeostatic model assessment of insulin resistance (HOMA-IR)], which suggest that boosting plasma VK1 may reduce the fasting glucose and insulin resistance in T2D patients. Using high-fat-diet-fed T2D animal model, this study further investigated the positive effect of VK1 supplementation on glucose metabolism and examined the underlying molecular mechanism. Results showed that VK1 supplementation [1, 3, 5 μg/kg body weight (BW), 8 weeks] dose dependently improved the glucose tolerance; decreased BW gain, fasting glucose and insulin, glycated hemoglobin, HOMA-IR and cytokine secretion (monocyte chemoattractant protein-1 and interleukin-6); and regulated the signaling pathway of hepatic glucose metabolism [sirtuin 1 (SIRT1)/AMP-activated protein kinase (AMPK)/phosphoinositide 3-kinase/phosphatase and tensin homolog/glucose transporter 2/glucokinase/glucose 6 phosphatase], lipid oxidation (peroxisome proliferator-activated receptor alpha/carnitine palmitoyltransferase 1A) and inflammation (nuclear factor kappa B) in T2D mice. Comparative signal silencing studies also depicted the role of SIRT1/AMPK in mediating the effect of VK1 on glucose metabolism, lipid oxidation and inflammation in high-glucose-treated cultured hepatocytes. In conclusion, this study demonstrates that circulating VK1 has a positive effect on lowering fasting glucose and insulin resistance in T2D via regulating SIRT1/AMPK signaling pathway.     

Regulation of Insulin Degrading Enzyme Activity by Obesity-Associated Factors and Pioglitazone in Liver of Diet-Induced Obese Mice

Insulin degrading enzyme (IDE) is a potential drug target in the treatment of type 2 diabetes (T2D). IDE controls circulating insulin through a degradation-dependent clearance mechanism in multiple tissues. However, there is not sufficient information about IDE regulation in obesity. In this study, we test obesity-associated factors and pioglitazone in the regulation of IDE in diet-induced obese (DIO) C57BL/6 mice. The enzyme activity and protein level of IDE were increased in the liver of DIO mice. Pioglitazone (10 mg/kg/day) administration for 2 months significantly enhanced the enzyme activity (75%), protein (180%) and mRNA (100%) of IDE in DIO mice. The pioglitazone-induced changes were coupled with 50% reduction in fasting insulin and 20% reduction in fasting blood glucose. The mechanism of IDE regulation in liver was investigated in the mouse hepatoma cell line (Hepa 1c1c7 cells), in which pioglitazone (5 µM) increased IDE protein and mRNA in a time-dependent manner in an 8 h study. Free fatty acid (palmitate 300 µM) induced IDE protein, but reduced the mRNA. Glucagon induced, and TNF-α decreased IDE protein. Insulin did not exhibit any activity in the same condition. In summary, pioglitazone, FFA and glucagon directly increased, but TNF-α decreased the IDE activity in hepatocytes. The results suggest that IDE activity is regulated in liver by multiple factors in obesity and pioglitazone may induce IDE activity in the control of T2D.     

Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

Aims

The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting).

Methods and Results

Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle.

Conclusion

Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.     

Structure-function studies of PANDER, an islet specific cytokine inducing cell death of insulin-secreting beta cells

PANDER (pancreatic derived factor, FAM3B) is a novel cytokine, present in insulin secretory granules, that induces apoptosis of alpha and beta cells of mouse, rat, and human islets in a dose- and time-dependent manner, and may be implicated in diabetes. PANDER has the predicted secondary structure of 4 alpha-helical bundles with an up-up-down-down topology, and two disulfide bonds. Eleven mutated PANDERs were constructed and expressed in beta-TC3 cells to identify the essential region of PANDER involved in beta-cell death. Beta-cell function was assessed by assays of cell viability and insulin secretion. Based on quantitative real-time RT-PCR all mutant PANDERs had similar mRNA expression levels in beta-TC3 cells. Immunoblotting showed that ten of eleven mutant PANDER proteins were synthesized and detected in beta-TC3 cells. A mutant PANDER with no signal peptide, however, was not expressed. Truncation of helix D alone caused a 40-50% decrease in PANDER's activity, while truncation of both helices C and D resulted in a 75% loss of activity. In contrast, truncation of the N-terminus of PANDER (helix A, the loop between helices A and B, and the first two cysteines) had no effect on PANDER-induced beta-cell death. The third and fourth cysteines of PANDER, C91 and C229, were shown to form one disulfide bond and be functionally important. Finally, the region between Cys91 and Phe152 constitutes the active part of PANDER, based on the demonstration that mutants with truncation of helix B or C caused decreased beta-cell death and did not inhibit insulin secretion, as compared to wild-type PANDER. Hence, helices B and C and the second disulfide bond of PANDER are essential for PANDER-induced beta-cell death.     

PANDER binds to the liver cell membrane and inhibits insulin signaling in HepG2 cells

PANDER is a cytokine co-secreted with insulin from islet β-cells. To date, the physiological function of PANDER remains largely unknown. Here we show that PANDER binds to the liver membrane by ¹²⁵I-PANDER saturation and competitive binding assays. In HepG2 cells, pre-treatment with PANDER ranging from 4 pM to 4 nM for 8 h resulted in a maximal inhibition of insulin-stimulated activation of insulin receptor and insulin receptor substrate 1 by 52% and 63%, respectively. Moreover, PANDER treatment also reduced insulin-stimulated PI3K and pAkt levels by 55% and 48%, respectively. In summary, we have identified the liver as a novel target for PANDER, and PANDER may be involved in the progression of diabetes by regulating hepatic insulin signaling pathways.     

Effect of TRB3 on insulin and nutrient-stimulated hepatic p70 S6 kinase activity

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.     

Accelerated Fatty Acid Oxidation in Muscle Averts Fasting-induced Hepatic Steatosis in SJL/J Mice

The accumulation of triglycerides (TG) in the liver, designated hepatic steatosis, is characteristically associated with obesity and insulin resistance, but it can also develop after fasting. Here, we show that fasting-induced hepatic steatosis is under genetic control in inbred mice. After a 24-h fast, C57BL/6J mice and SJL/J mice both lost more than 20% of body weight and ∼60% of total body TG. In C57BL/6J mice, TG accumulated in liver, producing frank steatosis. In striking contrast, SJL/J mice failed to accumulate any hepatic TG even though they lost nearly as much adipose tissue mass as the C57BL/6J mice. Mice from five other inbred strains developed fasting-induced steatosis like the C57BL/6J mice. Measurements of the uptake of free fatty acids (FA) in vivo and in vitro demonstrated that SJL/J mice were protected from steatosis because their heart and skeletal muscle took up and oxidized twice as much FA as compared with C57BL/6J mice. As a result of this muscle diversion, serum-free FA and ketone bodies rose much less after fasting in SJL/J mice as compared with C57BL/6J mice. When livers of SJL/J and C57BL/6J mice were perfused with similar concentrations of FA, the livers took up and esterified similar amounts. We conclude that SJL/J mice express one or more variant genes that lead to enhanced FA uptake and oxidation in muscle, thereby sparing the liver from FA overload in the fasting state.Liver and adipose tissue coordinate metabolic responses to oscillations in nutrient availability (1, 2). In the postprandial state, the liver secretes triglycerides (TG)⁴ into the blood in very low-density lipoproteins (VLDL). In adipose tissue, lipoprotein lipase hydrolyzes the TG, producing fatty acids (FA) and monoglycerides that enter fat cells for reesterification and storage as TG (1). The activity of adipose tissue lipoprotein lipase is enhanced by the postprandial rise in insulin. At the same time, insulin inhibits lipolysis of stored TG in fat cells, assuring that the TG will be retained in the cells (3).Under fasting conditions, insulin falls and the inhibitory effect of insulin on adipose tissue lipolysis is diminished. The released FA enters the blood and is used as an energy source in liver, heart, and skeletal muscle. In the liver, excess FA are either re-esterified into TG for intracellular storage or oxidized and secreted as ketone bodies, which become the main energy source for the brain. In skeletal muscle during fasting, FA are oxidized to CO₂ (1, 2).We (4–6) and others (7) previously reported that livers of mice accumulate large amounts of TG after fasting for 6–24 h. In the current study, we screened 7 strains of inbred mice to study the genetic control of fasting-induced hepatic TG accumulation. Mice from 6 of 7 strains exhibited fasting-induced fatty liver. In the unique mouse strain (SJL/J), hepatic TG failed to accumulate after a 24-h fast even though the SJL/J mice lost amounts of body weight and adipose tissue that were similar to those of the other 6 strains. To trace the mechanism for the difference in hepatic TG accumulation, we conducted extensive comparisons of SJL/J mice and C57BL/6J mice. We provide evidence that mice from both strains release comparable amounts of FA from adipose tissue into blood after fasting. In the SJL/J mice, the bulk of these FA are taken up by muscle and oxidized. In C57BL/6J mice, FA uptake in muscle is comparatively low, and the excess FA are taken up by the liver where they are converted to TG. Thus, genetic control of muscle FA uptake determines the level of hepatic TG accumulation in fasted mice.     

The SHP-1 protein tyrosine phosphatase negatively modulates glucose homeostasis

The protein tyrosine phosphatase SHP-1 is a well-known inhibitor of activation-promoting signaling cascades in hematopoietic cells but its potential role in insulin target tissues is unknown. Here we show that Ptpn6(me-v/me-v) (also known as viable motheaten) mice bearing a functionally deficient SHP-1 protein are markedly glucose tolerant and insulin sensitive as compared to wild-type littermates, as a result of enhanced insulin receptor signaling to IRS-PI3K-Akt in liver and muscle. Downregulation of SHP-1 activity in liver of normal mice by adenoviral expression of a catalytically inert mutant of SHP-1, or after small hairpin RNA-mediated SHP-1 silencing, further confirmed this phenotype. Tyrosine phosphorylation of CEACAM1, a modulator of hepatic insulin clearance, and clearance of serum [125I]-insulin were markedly increased in SHP-1-deficient mice or SHP-1-deficient hepatic cells in vitro. These findings show a novel role for SHP-1 in the regulation of glucose homeostasis through modulation of insulin signaling in liver and muscle as well as hepatic insulin clearance.     

Enhanced gluconeogenesis and hepatic insulin resistance in insulin-like growth factor binding protein-1 transgenic mice

Fasting hyperglycemia is observed in transgenic mice which overexpress insulin-like growth factor binding protein-1. In an attempt to understand the mechanisms underlying this observation we have examined glycogenolysis and gluconeogenesis in isolated hepatocytes from wild-type and transgenic mice. Glucose production from pyruvate was significantly less responsive to inhibition by insulin in hepatocytes from transgenic mice compared to hepatocytes from wild-type mice. Serum from transgenic mice resulted in more glucose production by hepatocytes than serum from wild-type mice. Serum alanine was increased while serum lactate was significantly reduced in transgenic mice compared to wild-type mice. Serum free fatty acids and beta-hydroxybutyrate were similar in both groups of mice. These data suggest that fasting hyperglycemia is due to enhanced gluconeogenesis, hepatic insulin resistance and increased serum gluconeogenic substrate in transgenic mice.     

Diet Is Critical for Prolonged Glycemic Control after Short-Term Insulin Treatment in High-Fat Diet-Induced Type 2 Diabetic Male Mice

Background

Clinical studies suggest that short-term insulin treatment in new-onset type 2 diabetes (T2DM) can promote prolonged glycemic control. The purpose of this study was to establish an animal model to examine such a “legacy” effect of early insulin therapy (EIT) in long-term glycemic control in new-onset T2DM. The objective of the study was to investigate the role of diet following onset of diabetes in the favorable outcomes of EIT.

Methodology

As such, C57BL6/J male mice were fed a high-fat diet (HFD) for 21 weeks to induce diabetes and then received 4 weeks of daily insulin glargine or sham subcutaneous injections. Subsequently, mice were either kept on the HFD or switched to a low-fat diet (LFD) for 4 additional weeks.

Principal Findings

Mice fed a HFD gained significant fat mass and displayed increased leptin levels, increasing insulin resistance (poor HOMA-IR) and worse glucose tolerance test (GTT) performance in comparison to mice fed a LFD, as expected. Insulin-treated diabetic mice but maintained on the HFD demonstrated even greater weight gain and insulin resistance compared to sham-treated mice. However, insulin-treated mice switched to the LFD exhibited a better HOMA-IR compared to those mice left on a HFD. Further, between the insulin-treated and sham control mice, in spite of similar HOMA-IR values, the insulin-treated mice switched to a LFD following insulin therapy did demonstrate significantly better HOMA-B% values than sham control and insulin-treated HFD mice.

Conclusion/Interpretation

Early insulin treatment in HFD-induced T2DM in C57BL6/J mice was only beneficial in animals that were switched to a LFD after insulin treatment which may explain why a similar legacy effect in humans is achieved clinically in only a portion of cases studied, emphasizing a vital role for diet adherence in diabetes control.     

Uncoupling protein-2 modulates the lipid metabolic response to fasting in mice

Uncoupling protein-2 (UCP2) regulates insulin secretion by controlling ATP levels in beta-cells. Although UCP2 deficiency improves glycemic control in mice, increased expression of UCP2 interferes with glucose-stimulated insulin secretion. These observations link UCP2 to beta-cell dysfunction in type 2 diabetes with a perplexing evolutionary role. We found higher residual serum insulin levels and blunted lipid metabolic responses in fasted ucp2(-/-) mice, supporting the concept that UCP2 evolved to suppress insulin effects and to accommodate the fuel switch to fatty acids during starvation. In the absence of UCP2, fasting initially promotes peripheral lipolysis and hepatic fat accumulation at less than expected rates but culminates in protracted steatosis, indicating diminished hepatic utilization and clearance of fatty acids. We conclude that UCP2-mediated control of insulin secretion is a physiologically relevant mechanism of the metabolic response to fasting.