KK Mice Research Articles

Altered Expression of Insulin Signaling Proteins in Islets from T2DM Female Mice

With development and progression of T2DM in KKAy mice, pancreatic islets alter their expression of GLUT2, insulin and insulin receptor. The study’s goal was to identify glucoregulatory proteins in the insulin receptor signaling pathway that were early markers of islet dysfunction after 4 weeks of uncontrolled hyperglycemia. Isolated islets were pooled from 2 groups of 12 week old mice: insulin-resistant, euglycemic KK mice and insulin-resistant, hyperglycemic KKAy T2DM mice. Samples were analyzed for 40 proteins using 221 antibodies in an insulin phospho antibody array. After 4 weeks of hyperglycemia in KKAy mice, many proteins were not altered as compared to KK mice (ratio: 0.94-1.02) including insulin receptor (Phospho-Tyr1355), FOXO1A (Phospho-Ser329), PI3-kinase p85-alpha (Phospho-Tyr607), ERK1/2, PP1-alpha, and AMPK1. The phosphorylation states of some proteins were altered including AKT1 (Phosphor-Ser124) (0.77) and IRS-1 (Phosphor-Ser636) (1.25). Three proteins were then selected for ELISA analysis in islets isolated from 4, 8 (onset of T2DM), and 12 week old mice: GLUT2, iNOS (1.41) and CBL (Phospho-Tyr700) (0.78), an E3 ubiquitin ligase. GLUT2 was lower in KKAy mice at 4 weeks (KK, 4.0±0.5; KKAy, 2.3±0.3, p<0.001), but not at 8 or 12 weeks, because GLUT2 decreased with age in both groups (12 weeks: KK, 1.0±0.1; KKAy, 1.4±0.1, p=0.31) [age*mouse: F(2,18)=11.104, p=0.001]. CBL-b was higher in KKAy mice (KK, 0.50±0.09; KKAy, 0.65±0.07) [F(1,17)=4.527, p=0.048] and decreased with age in both groups (12 week: KK, 0.30±0.02; KKAy, 0.45±0.13) [F(2,17)=12.231, p=0.001]. In contrast, iNOS was lower at 4 week (KK, 3.0±0.3; KKAy, 2.0±0.4; p=0.029), then higher at 8 and 12 week (KK, 0.8±0.4; KKAy, 2.1±0.2; p=0.007) with iNOS steady in KKAy mice [age*mouse: F(2,16)=10.902, p=0.001]. With short-term hyperglycemia in KKAy mice, many insulin signaling proteins were not altered as compared to diabetes susceptible KK mice. At onset on T2DM in KKAy mice, sustained iNOS and elevated CBL-b were early markers of islet dysfunction. Disclosure K.L. Sondgeroth: None. K. LePard: None.

Metabolomic and microarray analyses of adipose tissue of dapagliflozin-treated mice, and effects of 3-hydroxybutyrate on induction of adiponectin in adipocytes

Sodium/glucose cotransporter 2 (SGLT2) inhibitor improves systemic glucose metabolism. To clarify the effect of dapagliflozin, we performed gene expression microarray and metabolomic analyses of murine adipose tissue. Three groups of mice were used; non-diabetic control KK mice (KK), diabetic KKAy mice (KKAy), and KKAy mice treated with dapagliflozin (KKAy + Dapa). Plasma glucose levels were significantly reduced in KKAy + Dapa compared with KKAy. Food consumption was larger in KKAy + Dapa than KKAy, and there were no significant differences in body and adipose tissue weight among the groups. Metabolomic analysis showed higher levels of many intermediate metabolites of the glycolytic pathway and TCA cycle in KKAy than KK, albeit insignificantly. Dapagliflozin partially improved accumulation of glycolytic intermediate metabolites, but not intermediate metabolites of the TCA cycle, compared with KKAy. Interestingly, dapagliflozin increased plasma and adipose 3-hydroxybutyric acid (3-HBA) levels. Microarray analysis showed that adipocytokines were downregulated in KKAy compared with KK mice, and upregulated by dapagliflozin. In vitro, 3-HBA induced β-hydroxybutyrylation of histone H3 at lysine 9 and upregulation of adiponectin in 3T3-L1 adipocytes independent of their acetylation or methylation. Our results suggest that 3-HBA seems to provide protection through epigenetic modifications of adiponectin gene in adipocytes.

The Effects of Dietary Calcium Levels on Pancreatic Function in KK Mice

The Effects of Dietary Calcium Levels on Pancreatic Function in KK Mice

Treatment with FGFR2-IIIc monoclonal antibody suppresses weight gain and adiposity in KKAy mice.

Expression of β-Kotho, fibroblast growth factor receptor (FGFR)-1c and 2c, which bind FGF21, is decreased in the white adipose tissue of obese mice. The aim of the present study was to determine the role of FGFR2c in the development of obesity and diabetes in KKAy mice. Treatment with mouse monoclonal FGFR2-IIIc antibody (0.5 mg kg−1) significantly suppressed body weight gain and epididymal white adipose tissue weight in individually housed KKAy mice while having no effect on daily food intake. In addition, treatment with FGFR2-IIIc antibody significantly increased plasma-free fatty acid levels while having no effect on blood glucose or plasma FGF21 levels. Moreover, treatment with FGFR2-IIIc antibody had no significant effect on the expression of uncoupling protein-1, uncoupling protein-2 or peroxisome proliferator-activated receptor-γ coactivator 1α in the epididymal white adipose tissue. The treatment with FGFR2-IIIc antibody had no significant effects on daily food intake and body weight gain in individually housed KK mice. These findings suggest that FGFR2-IIIc upregulates the adiposity induced by social isolation in KKAy mice, and that decreased expression and/or function of FGFR2c might be a compensatory response to enhanced adiposity. Inhibition of FGFR2-IIIc function might be a novel therapeutic approach for obesity.

Protective effects of fish oil and pioglitazone on pancreatic tissue in obese KK mice with type 2 diabetes

n-3 Polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), have protective effects against the pancreatic β-cell dysfunction through several mechanisms. Thiazolidines are insulin sensitizers and are used in treating patients with type 2 diabetes. Our previous study demonstrated that a combination of fish oil, which is rich with EPA and DHA, and pioglitazone exerts beneficial effects on obesity and diabetes through their actions on the liver and adipose tissue. However, it remains largely unknown whether such combination therapy affects the pancreas. To answer this question, KK mice, which serve as a model for obesity and type 2 diabetes, were treated for 8 weeks with fish oil and pioglitazone. The combined regimen suppressed pancreatic islet hypertrophy (mean islet area decreased by an average of 49% vs. control) compared with mice treated with fish oil or pioglitazone alone (decreased by an average of 21% and 32% vs. control, respectively). Compared with the controls, individual or combined treatment significantly increased the percentage of β-cell area in the pancreatic islets, significantly decreased endoplasmic reticulum stress, and reduced the percentage of apoptotic cell death in the pancreatic islets. These findings suggest that fish oil and/or pioglitazone prevents β-cell dysfunction by improving the insulin resistance and decreasing the ER stress.

Combination of Astragalus membranaceus and Euonymus alatus (THUNB.) SIEB. for treatment of diabetes: A network-based pharmacology research

ObjectiveTo find the target genes in combination of astragalus root (Astragalus membranaceus) and spindle tree wings (Euonymus alatus (THUNB.) SIEB.) (Qijian Heji) with the function of regulating blood glucose levels in kk mice by real-time fluorescence quantitative PCR (RT-PCR). To achieve the primary exploration on the material basis for Qijian Heji to improve the blood glucose levels of type 2 diabetic kk mice. MethodsWe applied RT-PCR to detecting the expression of gene targets in kidney tissues of type 2 diabetes model kk mice. The detected gene targets were predicted by the network pharmacology method in the early stage of the project. Through the observation of the hypoglycemic effect of different ratios of Qijian Heji, the optimal proportion was determined and then used to conduct research of material basis. ResultsContinuous administration of Qijian Heji (astragalus root: spindle tree wings = 3:1) for six weeks resulted in significantly lowering blood glucose levels in mice in the fourth week, dropping from 33.3 to 21.9 mmol/L; this lower level was maintained in the fifth to sixth weeks. Blood glucose values in the positive drug group decreased from 33.3 to 21.85 mmol/L in the fourth week and maintained steady decreasing in the fifth and sixth weeks. ConclusionQijian Heji, with proportion of 3:1, could significantly reduce the blood glucose values in kk mice, animal experiment results verified the accurate prediction of network pharmacology, and at the same time, it is verified there was a certain therapeutic effect about the combination of the two herbs on reducing the glucose value. Animal experiments and RT-PCR results showed that Qijian Heji conducted a hypoglycemic effect by modulating estrogen receptor (ESR1) and renin (REN) expression.

Repeated ozone exposure exacerbates insulin resistance and activates innate immune response in genetically susceptible mice

Background: Inhaled ozone (O3) has been demonstrated as a harmful pollutant and associated with chronic inflammatory diseases such as diabetes and vascular disorders. However, the underlying mechanisms by which O3 mediates harmful effects are poorly understood.Objectives: To investigate the effect of O3 exposure on glucose intolerance, immune activation and underlying mechanisms in a genetically susceptible mouse model.Methods: Diabetes-prone KK mice were exposed to filtered air (FA), or O3 (0.5 ppm) for 13 consecutive weekdays (4 h/day). Insulin tolerance test (ITT) was performed following the last exposure. Plasma insulin, adiponectin, and leptin were measured by ELISA. Pathologic changes were examined by H&E and Oil-Red-O staining. Inflammatory responses were detected using flow cytometry and real-time PCR.Results: KK mice exposed to O3 displayed an impaired insulin response. Plasma insulin and leptin levels were reduced in O3-exposed mice. Three-week exposure to O3 induced lung inflammation and increased monocytes/macrophages in both blood and visceral adipose tissue. Inflammatory monocytes/macrophages increased both systemically and locally. CD4 + T cell activation was also enhanced by the exposure of O3 although the relative percentage of CD4 + T cell decreased in blood and adipose tissue. Multiple inflammatory genes including CXCL-11, IFN-γ, TNFα, IL-12, and iNOS were up-regulated in visceral adipose tissue. Furthermore, the expression of oxidative stress-related genes such as Cox4, Cox5a, Scd1, Nrf1, and Nrf2, increased in visceral adipose tissue of O3-exposed mice.Conclusions: Repeated O3 inhalation induces oxidative stress, adipose inflammation and insulin resistance.

Fish oil and fenofibrate inhibit pancreatic islet hypertrophy, and improve glucose and lipid metabolic dysfuntions with different ways in diabetic KK mice

We examined the effects of fish oil and fenofibrate (FF) on the pancreatic islet hypertrophy, and on the modification of glucose and lipid metabolic dysfunctions in KK mice with insulin resistance. The mice were fed one of four diets [25en% lard/safflower oil (LSO), 25en% fish oil (FO), or each of these diets plus 0.1wt% FF (LSO/FF, FO/FF)] for 9 weeks. FO group and both FF groups had significantly lower final body and adipose tissue weights than LSO group. Pancreatic islet hypertrophy was observed only in LSO group but not in the other groups with fish oil or FF. And, it is likely that fish oil has a stronger therapeutic effect on islet hypertrophy. Plasma adiponectin level was significantly higher in FO group but not in both FF groups. Expression of hepatic lipogenic enzyme genes such as fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD-1) was lower in FO groups with or without FF, whereas fatty acid oxidation-related mRNAs such as acyl-CoA oxidase (AOX) and uncoupling protein-2 (UCP-2) were more abundant in FF groups with or without fish oil. Our results suggest that both fish oil and FF improve pancreatic islet hypertrophy with the amelioration of insulin resistance. Fish oil enhances insulin sensitivity by increasing plasma adiponectin; however, the beneficial effect of FF on insulin resistance seems to be independent of the plasma adiponectin level. These results mean that improvement of glucose and lipid metabolic dysfuctions in diabetic KK mice are independently approached by fish oil and FF.

Hepatic lipid accumulation is ameliorated in obese KK mice by dietary sei whale oil

Whale oil (WO) contains n-3 polyunsaturated fatty acids (PUFAs), but the effects of whale oil intake on lipid metabolism have remained unclear. We examined the influence of WO on lipid metabolism in obese KK mice. Male KK mice were fed a high-fat diet for 12 weeks to induce obesity. The mice were then given free access to a different diet for 10 weeks: the lard/safflower oil (LSO) diet consisted of 25 energy% (en%) LSO (6:4), the WO diet consisted of 25 en% WO, and the fish oil (FO) diet consisted of 11 en% FO plus 14 en% LSO. The n-3 PUFA content of the FO diet was the same as that in the WO diet. In the WO and FO groups, the total plasma cholesterol level was significantly decreased compared with that in the LSO group. The number of lipid droplets in liver specimens was also lower in the WO and FO groups, and the hepatic triglyceride and total cholesterol levels were decreased in these groups compared with the LSO group. Hepatic mRNA levels of fatty acid synthase, and stearoyl-CoA desaturase 1, which are fatty acid synthesis-related genes, were decreased in the WO and FO groups. No significant differences were observed between the WO and FO groups. Taken together, these results indicate that WO intake inhibits lipid accumulation in the liver by reducing fatty acid biosynthesis.

Fish oil prevents excessive accumulation of subcutaneous fat caused by an adverse effect of pioglitazone treatment and positively changes adipocytes in KK mice.

Pioglitazone, a thiazolidinedione (TZD), is widely used as an insulin sensitizer in the treatment of type 2 diabetes. However, body weight gain is frequently observed in TZD-treated patients. Fish oil improves lipid metabolism dysfunction and obesity. In this study, we demonstrated suppression of body weight gain in response to pioglitazone administration by combination therapy of pioglitazone and fish oil in type 2 diabetic KK mice. Male KK mice were fed experimental diets for 8 weeks. In safflower oil (SO), safflower oil/low-dose pioglitazone (S/PL), and safflower oil/high-dose pioglitazone (S/PH) diets, 20% of calories were provided by safflower oil containing 0%, 0.006%, or 0.012% (wt/wt) pioglitazone, respectively. In fish oil (FO), fish oil/low-dose pioglitazone (F/PL), and fish oil/high-dose pioglitazone (F/PH) diets, 20% of calories were provided by a mixture of fish oil and safflower oil. Increased body weight and subcutaneous fat mass were observed in the S/PL and S/PH groups; however, diets containing fish oil were found to ameliorate these changes. Hepatic mRNA levels of lipogenic enzymes were significantly decreased in fish oil-fed groups. These findings demonstrate that the combination of pioglitazone and fish oil decreases subcutaneous fat accumulation, ameliorating pioglitazone-induced body weight gain, through fish oil-mediated inhibition of hepatic de novo lipogenesis.

Nicotinamide Riboside Ameliorates Hepatic Metaflammation by Modulating NLRP3 Inflammasome in a Rodent Model of Type 2 Diabetes

Low-grade chronic inflammation (metaflammation) is a major contributing factor for the onset and development of metabolic diseases, such as type 2 diabetes, obesity, and cardiovascular disease. Nicotinamide riboside (NR), which is present in milk and beer, is a functional vitamin B3 having advantageous effects on metabolic regulation. However, the anti-inflammatory capacity of NR is unknown. This study evaluated whether NR modulates hepatic nucleotide binding and oligomerization domain-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome. Male, 8-week-old KK/HlJ mice were allocated to the control or NR group. NR (100 mg/kg/day) or vehicle (phosphate-buffered saline) was administrated by an osmotic pump for 7 days. Glucose control, lipid profiles, NLRP3 inflammasome, and inflammation markers were analyzed, and structural and histological analyses were conducted. NR treatment did not affect body weight gain, food intake, and liver function. Glucose control based on the oral glucose tolerance test and levels of serum insulin and adiponectin was improved by NR treatment. Among tested lipid profiles, NR lowered the total cholesterol concentration in the liver. Histological and structural analysis by hematoxylin and eosin staining and transmission electron microscopy, respectively, showed that NR rescued the disrupted cellular integrity of the mitochondria and nucleus in the livers of obese and diabetic KK mice. In addition, NR treatment significantly improved hepatic proinflammatory markers, including tumor necrosis factor-alpha, interleukin (IL)-6, and IL-1. These ameliorations were accompanied by significant shifts of NLRP3 inflammasome components (NLRP3, ASC, and caspase1). These results demonstrate that NR attenuates hepatic metaflammation by modulating the NLRP3 inflammasome.

A combination of glucosyl hesperidin and caffeine exhibits an anti-obesity effect by inhibition of hepatic lipogenesis in mice.

To develop an anti-obesity agent, we examined the combination effect of glucosyl hesperidin (G-hesperidin) and caffeine on obesity in mice. High-fat diet-induced obese KK mice were fed a low-fat diet with or without G-hesperidin, caffeine, or their combination for 2 weeks. Decreases in body weight and significantly lower adipose tissue weight were observed in the combination-fed mice but not in the G-hesperidin-fed or caffeine-fed mice. DNA microarray analysis of mouse liver suggested that the feeding of G-hesperidin + caffeine was associated with lower lipogenesis. Therefore, we examined the anti-lipogenic effect of G-hesperidin + caffeine in fasted-refed KK mice. Hepatic triglyceride levels were significantly lower in the mice fed G-hesperidin + caffeine during the refeeding period but not in the mice fed each alone. In addition, hepatic expressions of genes related to lipogenesis, such as sterol regulatory element-binding protein-1c or fatty acid synthase, were significantly lower in the mice fed G-hesperidin + caffeine compared with that in the control mice. These results suggested that G-hesperidin + caffeine is effective for controlling obesity partly by the inhibition of hepatic lipogenesis.

Mo1777 Location of Glucose Transporter GLUT2 in Beta Cell Membrane, Not Beta Cell Mass, Determines Glycemic Control in 4 and 16 Week Old Female KK and KKAY Mice

Mo1777 Location of Glucose Transporter GLUT2 in Beta Cell Membrane, Not Beta Cell Mass, Determines Glycemic Control in 4 and 16 Week Old Female KK and KKAY Mice

Novel Indole-N-glucoside, TA-1887 As a Sodium Glucose Cotransporter 2 Inhibitor for Treatment of Type 2 Diabetes.

Inhibition of the renal sodium glucose cotransporter (SGLT) increases urinary glucose excretion (UGE) and thus reduces blood glucose levels during hyperglycemia. To explore the potential of new antihyperglycemic agents, we synthesized and determined the human SGLT2 (hSGLT2) inhibitory potential of novel substituted 3-benzylindole-N-glucosides 6. Optimization of 6 resulted in the discovery of 3-(4-cyclopropylbenzyl)-4-fluoroindole-N-glucoside 6a-4 (TA-1887), a highly potent and selective hSGLT2 inhibitor, with pronounced antihyperglycemic effects in high-fat diet-fed KK (HF-KK) mice. Our results suggest the potential of indole-N-glucosides as novel antihyperglycemic agents through inhibition of renal SGLT2.

Skeletal myoblast transplantation on gene expression profiles of insulin signaling pathway and mitochondrial biogenesis and function in skeletal muscle

AimThe study aims to investigate the gene expression profiling of insulin signaling pathway and mitochondrial biogenesis and function in the skeletal muscle of KK mice. MethodsKK mice were divided into the following groups: KK control group, basal medium (M199) only; KK fibroblast group, with human fibroblast transplantation; KK myoblast group, with human skeletal myoblast transplantation. C57BL mice received hSkM transplantation as a normal control. Cells were transplanted into mice hind limb skeletal muscle. All animals were treated with cyclosporine for 6 weeks only. The mice were sacrificed in a fasting state at 12 weeks after treatment. Hind limb skeletal muscle was harvested and used for study of gene expression profiling. ResultshSkMs survived extensively in mice skeletal muscle at 12 weeks after cell transplantation. Glucose tolerance test showed a significant decrease of blood glucose in the mice of KK myoblast group compared to the KK control and fibroblast groups. Transcriptional patterns of insulin signaling pathway showed alterations in KK myoblast as compared with KK control group (23 genes), KK fibroblast group (7 genes), and C57BL group (8 genes). Transcriptional patterns of mitochondrial biogenesis and function also had alterations in KK myoblast as compared with KK control group (27 genes), KK fibroblast group (9 genes), and C57BL group (6 genes). ConclusionsThese data demonstrated for the first time that hSKM transplantation resulted in a change of gene transcript in multiple genes involved in insulin signaling pathway and mitochondrial biogenesis and function.

C-Glucosides with heteroaryl thiophene as novel sodium-dependent glucose cotransporter 2 inhibitors

Canagliflozin (1), a novel inhibitor for sodium-dependent glucose cotransporter 2 (SGLT2), has been developed for the treatment of type 2 diabetes. To investigate the effect of replacement of the phenyl ring in 1 with heteroaromatics, C-glucosides 2 were designed, synthesized, and evaluated for their inhibitory activities against SGLT2. Of these, 3-pyridyl, 2-pyrimidyl or 5-membered heteroaryl substituted derivatives showed highly potent inhibitory activity against SGLT2, while 5-pyrimidyl substitution was associated with slightly reduced activity. In particular, 2g (TA-3404) had remarkable anti-hyperglycemic effects in high-fat diet fed KK (HF-KK) mice.

Lack of GPR40/FFAR1 does not induce diabetes even under insulin resistance condition

G protein-coupled receptor/free fatty acid receptor 1 (GPR40/FFAR1 ) regulates free fatty acid-induced insulin secretion. This study has been performed to clarify whether or not loss of GPR40/FFAR1 function exacerbates diabetes, that is, whether GPR40 has an essential physiological role in the development of diabetes or not. We generated GPR40/FFAR1 knockout (KO) mice and analysed their phenotypes in vitro and in vivo under the condition of dietary or genetically induced insulin resistance. GPR40/FFAR1 KO mice kept on a high-fat diet became obese, developed glucose intolerance to a similar degree as GPR40/FFAR1 wild-type (WT) mice. In addition, the phenotype of KO mice harbouring diabetogenic KK background genes showed glucose intolerance at a level similar to level for control KK mice. In both mouse models with insulin resistance, insulin secretion after oral glucose load and homeostasis model assessment-insulin resistance (HOMA-IR) did not change between GPR40/FFAR1 KO and WT mice. Although glucose-induced insulin secretion under high palmitate concentration was significantly lower in KO than in WT islets, pancreatic insulin content and insulin secretion stimulated with glucose alone were not different between KO and WT mice. GPR40/FFAR1 has a major role in regulating fatty-acid-mediated insulin secretion, but the lack of GPR40/FFAR1 does not exacerbate glucose intolerance and insulin resistance induced by high-fat diet or diabetogenic KK gene. Our findings indicate that loss of GPR40/FFAR1 function does not play an important role in inducing or exacerbating diabetes.

Age-dependent slowing of enteric axonal transport in insulin-resistant mice

To investigate retrograde tracer transport by gastric enteric neurons in insulin resistant mice with low or high glycosylated hemoglobin (Hb). Under anesthesia, the retrograde tracer fluorogold was superficially injected into the fundus or antrum using a microsyringe in KK Cg-Ay/J mice prior to onset of type 2 diabetes mellitus (T2DM; 4 wk of age), at onset of T2DM (8 wk of age), and after 8, 16, or 24 wk of untreated T2DM and in age-matched KK/HIJ mice. Six days later, mice were sacrificed by CO₂ narcosis followed by pneumothorax. Stomachs were removed and fixed. Sections from fundus, corpus and antrum were excised and mounted on a glass slide. Tracer-labeled neurons were viewed using a microscope and manually counted. Data were expressed as the number of neurons in short and long descending and ascending pathways and in local fundus and antrum pathways, and the number of neurons in all regions labeled after injection of tracer into either the fundus or the antrum. By 8 wk of age, body weights of KKAy mice (n = 12, 34 ± 1 g) were heavier than KK mice (n = 17, 29 ± 1 g; F (4, 120) = 4.414, P = 0.002] and glycosylated Hb was higher [KK: (n = 7), 4.97% ± 0.04%; KKAy: (n = 6), 6.57% ± 0.47%; F (1, 26) = 24.748, P < 0.001]. The number of tracer labeled enteric neurons was similar in KK and KKAy mice of all ages in the short descending pathway [F (1, 57) = 2.374, P = 0.129], long descending pathway [F (1, 57) = 0.922, P = 0.341], local fundus pathway [F (1, 53) = 2.464, P = 0.122], local antrum pathway [F (1, 57) = 0.728, P = 0.397], and short ascending pathway [F (1, 53) = 2.940, P = 0.092]. In the long ascending pathway, fewer tracer-labeled neurons were present in KKAy as compared to KK mice [KK: (n = 34), 302 ± 17; KKAy: (n = 29), 230 ± 15; F (1, 53) = 8.136, P = 0.006]. The number of tracer-labeled neurons was decreased in all mice by 16 wk as compared to 8 wk of age in the short descending pathway [8 wk: (n = 15), 305 ± 26; 16 wk: (n = 13), 210 ± 30; F (4, 57) = 9.336, P < 0.001], local antrum pathway [8 wk: (n = 15), 349 ± 20; 16 wk: (n = 13), 220 ± 33; F (4, 57) = 8.920, P < 0.001], short ascending pathway [8 wk: (n = 14), 392 ± 15; 16 wk: (n = 14), 257 ± 33; F (4, 53) = 17.188, P < 0.001], and long ascending pathway [8 wk: (n = 14), 379 ± 39; 16 wk: (n = 14), 235 ± 26; F (4, 53) = 24.936, P < 0.001. The number of tracer-labeled neurons decreased at 24 wk of age in the local fundus pathway [8 wk: (n = 14), 33 ± 11; 24 wk: (n = 12), 3 ± 2; F (4, 53) = 5.195, P = 0.001] and 32 wk of age in the long descending pathway [8 wk: (n = 15), 16 ± 3; 32 wk: (n = 12), 3 ± 2; F (4, 57) = 2.944, P = 0.028]. The number of tracer-labeled enteric neurons was correlated to final body weight for local fundus and ascending pathways [KK: (n = 34), r = -0.746, P < 0.001; KKAy: (n = 29), r = -0.842, P < 0.001] as well as local antrum and descending pathways [KK (n = 36), r = -0.660, P < 0.001; KKAy (n = 31), r = -0.622, P < 0.001). In contrast, glycosylated Hb was not significantly correlated to number of tracer-labeled neurons [KK (n = 17), r = -0.164, P = 0.528; KKAy (n = 16), r = -0.078, P = 0.774]. Since uncontrolled T2DM did not uniformly impair tracer transport in gastric neurons, long ascending neurons may be more susceptible to persistent hyperglycemia and low effective insulin.

Regulation of TNFα converting enzyme activity in visceral adipose tissue of obese mice

Tumor necrosis factor α (TNFα) is a pro-inflammatory cytokine and one of the major mediators of obesity-induced insulin resistance. TNFα is generated through TNFα converting enzyme (TACE)-mediated cleavage of the transmembrane precursor pro-TNFα. Inhibition of TACE resulted in the improvement in glucose and insulin levels in diabetic animals, suggesting a crucial role of TACE activity in glucose metabolism. However, the regulation of TACE activity in insulin-sensitive tissues has not been fully determined. This study aimed to investigate the impact of TACE in insulin-sensitive tissues in the early stage of the development of obesity. C57BL6 mice were fed standard chow (B6-SC) or high-fat/high-sucrose diet (B6-HF/HS). KK-Ay mice were fed SC ad libitum (Ay-AL) or fed reduced amounts of SC (caloric restriction (CR); Ay-CR). As control for Ay-AL, KK mice fed SC ad libitum (KK-AL) were used. TACE activity in visceral adipose tissue (VAT), but not in liver or skeletal muscle, was significantly elevated in B6-HF/HS and Ay-AL compared with B6-SC and KK-AL, respectively. Phosphorylation of JNK and p38MAPK, but not ERK, in VATs from B6-HF/HS and Ay-AL was also significantly elevated. Ay-CR showed significantly lower TACE, JNK and p38MAPK activities in VAT and serum TNFα level compared with those of Ay-AL. In contrast, intraperitoneal injection of TNFα activated TACE, JNK and p38MAPK activities in VAT in KK mice. In conclusion, during the development of obesity, TACE activity is elevated only in VAT, and CR effectively reduced TACE activity and TACE-mediated pro-TNFα shedding in VAT.

Autosomal recessive retinitis pigmentosa E150K opsin mice exhibit photoreceptor disorganization

The pathophysiology of the E150K mutation in the rod opsin gene associated with autosomal recessive retinitis pigmentosa (arRP) has yet to be determined. We generated knock-in mice carrying a single nucleotide change in exon 2 of the rod opsin gene resulting in the E150K mutation. This novel mouse model displayed severe retinal degeneration affecting rhodopsin's stabilization of rod outer segments (ROS). Homozygous E150K (KK) mice exhibited early-onset retinal degeneration, with disorganized ROS structures, autofluorescent deposits in the subretinal space, and aberrant photoreceptor phagocytosis. Heterozygous (EK) mice displayed a delayed-onset milder retinal degeneration. Further, mutant receptors were mislocalized to the inner segments and perinuclear region. Though KK mouse rods displayed markedly decreased phototransduction, biochemical studies of the mutant rhodopsin revealed only minimally affected chromophore binding and G protein activation. Ablation of the chromophore by crossing KK mice with mice lacking the critical visual cycle protein LRAT slowed retinal degeneration, whereas blocking phototransduction by crossing KK mice with GNAT1-deficient mice slightly accelerated this process. This study highlights the importance of proper higher-order organization of rhodopsin in the native tissue and provides information about the signaling properties of this mutant rhodopsin. Additionally, these results suggest that patients heterozygous for the E150K mutation should be periodically reevaluated for delayed-onset retinal degeneration.