Brain-Wide Insulin Resistance, Tau Phosphorylation Changes, and Hippocampal Neprilysin and Amyloid-ß Alterations in a Monkey Model of Type 1 Diabetes.

Epidemiological findings suggest that diabetic individuals are at a greater risk for developing Alzheimer's disease (AD). To examine the mechanisms by which diabetes mellitus (DM) may contribute to AD pathology in humans, we examined brain tissue from streptozotocin-treated type 1 diabetic adult male vervet monkeys receiving twice-daily exogenous insulin injections for 8-20 weeks. We found greater inhibitory phosphorylation of insulin receptor substrate 1 in each brain region examined of the diabetic monkeys when compared with controls, consistent with a pattern of brain insulin resistance that is similar to that reported in the human AD brain. Additionally, a widespread increase in phosphorylated tau was seen, including brain areas vulnerable in AD, as well as relatively spared structures, such as the cerebellum. An increase in active ERK1/2 was also detected, consistent with DM leading to changes in tau-kinase activity broadly within the brain. In contrast to these widespread changes, we found an increase in soluble amyloid-ß (Aß) levels that was restricted to the temporal lobe, with the greatest increase seen in the hippocampus. Consistent with this localized Aß increase, a hippocampus-restricted decrease in the protein and mRNA for the Aß-degrading enzyme neprilysin (NEP) was found, whereas various Aß-clearing and -degrading proteins were unchanged. Thus, we document multiple biochemical changes in the insulin-controlled DM monkey brain that can link DM with the risk of developing AD, including dysregulation of the insulin-signaling pathway, changes in tau phosphorylation, and a decrease in NEP expression in the hippocampus that is coupled with a localized increase in Aß. SIGNIFICANCE STATEMENT: Given that diabetes mellitus (DM) appears to increase the risk of developing Alzheimer's disease (AD), understanding the mechanisms by which DM promotes AD is important. We report that DM in a nonhuman primate brain leads to changes in the levels or posttranslational processing of proteins central to AD pathobiology, including tau, amyloid-ß (Aß), and the Aß-degrading protease neprilysin. Additional evidence from this model suggests that alterations in brain insulin signaling occurred that are reminiscent of insulin signaling pathway changes seen in human AD. Thus, in an in vivo model highly relevant to humans, we show multiple alterations in the brain resulting from DM that are mechanistically linked to AD risk.

Altered expression of glial markers, chemokines, and opioid receptors in the spinal cord of type 2 diabetic monkeys.

Neuroinflammation is a pathological condition that underlies diabetes and affects sensory processing. Given the high prevalence of pain in diabetic patients and crosstalk between chemokines and opioids, it is pivotal to know whether neuroinflammation-associated mediators are dysregulated in the central nervous system of diabetic primates. Therefore, the aim of this study was to investigate whether mRNA expression levels of glial markers, chemokines, and opioid receptors are altered in the spinal cord and thalamus of naturally occurring type 2 diabetic monkeys (n=7) compared with age-matched non-diabetic monkeys (n=6). By using RT-qPCR, we found that mRNA expression levels of both GFAP and IBA1 were up-regulated in the spinal dorsal horn (SDH) of diabetic monkeys compared with non-diabetic monkeys. Among all chemokines, expression levels of three chemokine ligand-receptor systems, i.e., CCL2-CCR2, CCL3-CCR1/5, and CCL4-CCR5, were up-regulated in the SDH of diabetic monkeys. Moreover, in the SDH, seven additional chemokine receptors, i.e., CCR4, CCR6, CCR8, CCR10, CXCR3, CXCR5, and CXCR6, were also up-regulated in diabetic monkeys. In contrast, expression levels of MOP, KOP, and DOP, but not NOP receptors, were down-regulated in the SDH of diabetic monkeys, and the thalamus had fewer changes in the glial markers, chemokines and opioids. These findings indicate that neuroinflammation, manifested as glial activation and simultaneous up-regulation of multiple chemokine ligands and receptors, seems to be permanent in type 2 diabetic monkeys. As chemokines and opioids are important pain modulators, this first-in-primate study provides a translational bridge for determining the functional efficacy of spinal drugs targeting their signaling cascades.

Condylomatous genital lesions in cynomolgus macaques from Mauritius.

Genital condyloma-like lesions were observed on male and female cynomolgus macaque monkeys (Macaca fascicularis) originating from the island of Mauritius. Cytobrush and/or biopsy samples were obtained from lesions of 57 affected macaques. Primary histologic features included eosinophilic, neutrophilic, and lymphoplasmacytic penile and vulvar inflammation, epidermal hyperplasia with acanthosis, and increased collagenous stroma. Polymerase chain reaction-based assays to amplify viral DNA revealed the presence of macaque lymphocryptovirus (LCV) DNA but not papillomavirus or poxvirus DNA. Subsequent DNA analyses of 3 genomic regions of LCV identified isolates associated with lesions in 19/25 (76%) biopsies and 19/57 (33%) cytology samples. Variable immunolabeling for proteins related to the human LCV Epstein Barr Virus was observed within intralesional plasma cells, stromal cells, and epithelial cells. Further work is needed to characterize the epidemiologic features of these lesions and their association with LCV infection in Mauritian-origin macaques.

Life stage differences in mammary gland gene expression profile in non-human primates.

Breast cancer (BC) is the most common malignancy of women in the developed world. To better understand its pathogenesis, knowledge of normal breast development is crucial, as BC is the result of disregulation of physiologic processes. The aim of this study was to investigate the impact of reproductive life stages on the transcriptional profile of the mammary gland in a primate model. Comparative transcriptomic analyses were carried out using breast tissues from 28 female cynomolgus macaques (Macaca fascicularis) at the following life stages: prepubertal (n = 5), adolescent (n = 4), adult luteal (n = 5), pregnant (n = 6), lactating (n = 3), and postmenopausal (n = 5). Mammary gland RNA was hybridized to Affymetrix GeneChip(®) Rhesus Macaque Genome Arrays. Differential gene expression was analyzed using ANOVA and cluster analysis. Hierarchical cluster analysis revealed distinct separation of life stage groups. More than 2,225 differentially expressed mRNAs were identified. Gene families or pathways that changed across life stages included those related to estrogen and androgen (ESR1, PGR, TFF1, GREB1, AR, 17HSDB2, 17HSDB7, STS, HSD11B1, AKR1C4), prolactin (PRLR, ELF5, STAT5, CSN1S1), insulin-like growth factor signaling (IGF1, IGFBP1, IGFBP5), extracellular matrix (POSTN, TGFB1, COL5A2, COL12A1, FOXC1, LAMC1, PDGFRA, TGFB2), and differentiation (CD24, CD29, CD44, CD61, ALDH1, BRCA1, FOXA1, POSTN, DICER1, LIG4, KLF4, NOTCH2, RIF1, BMPR1A, TGFB2). Pregnancy and lactation displayed distinct patterns of gene expression. ESR1 and IGF1 were significantly higher in the adolescent compared to the adult animals, whereas differentiation pathways were overrepresented in adult animals and pregnancy-associated life stages. Few individual genes were distinctly different in postmenopausal animals. Our data demonstrate characteristic patterns of gene expression during breast development. Several of the pathways activated during pubertal development have been implicated in cancer development and metastasis, supporting the idea that other developmental markers may have application as biomarkers for BC.

Hydrogen and oxygen isotope ratios in body water and hair: modeling isotope dynamics in nonhuman primates.

The stable isotopic composition of drinking water, diet, and atmospheric oxygen influence the isotopic composition of body water ((2)H/(1)H, (18)O/(16)O expressed as δ(2) H and δ(18)O). In turn, body water influences the isotopic composition of organic matter in tissues, such as hair and teeth, which are often used to reconstruct historical dietary and movement patterns of animals and humans. Here, we used a nonhuman primate system (Macaca fascicularis) to test the robustness of two different mechanistic stable isotope models: a model to predict the δ(2)H and δ(18)O values of body water and a second model to predict the δ(2)H and δ(18)O values of hair. In contrast to previous human-based studies, use of nonhuman primates fed controlled diets allowed us to further constrain model parameter values and evaluate model predictions. Both models reliably predicted the δ(2)H and δ(18)O values of body water and of hair. Moreover, the isotope data allowed us to better quantify values for two critical variables in the models: the δ(2)H and δ(18)O values of gut water and the (18)O isotope fractionation associated with a carbonyl oxygen-water interaction in the gut (α(ow)). Our modeling efforts indicated that better predictions for body water and hair isotope values were achieved by making the isotopic composition of gut water approached that of body water. Additionally, the value of α(ow) was 1.0164, in close agreement with the only other previously measured observation (microbial spore cell walls), suggesting robustness of this fractionation factor across different biological systems.

Development of a sensitive ELISA to quantify apolipoprotein CIII in nonhuman primate serum.

Apolipoprotein CIII (apoCIII), a major constituent of triglyceride-rich lipoprotein, has been proposed as a key contributor to hypertriglyceridemia on the basis of its inhibitory effects on lipoprotein lipase. Many immunochemical methods have been developed for human apoCIII quantification, including ELISA. However, a sensitive and quantitative assay for nonhuman primates is not commercially available. We developed a sensitive, quantitative, and highly specific sandwich ELISA to measure apoCIII in both nonhuman primate and human serum. Our assay generates a linear calibration curve from 0.01 µg/ml to 10 µg/ml using an apoCIII standard that was purified from cynomolgus monkey serum. It is highly reproducible (intra- and interplate CV < 5% and < 8%, respectively), sensitive enough to distinguish 10% difference of apoCIII present in serum, and has no interference from purified human apolipoprotein AI, AII, B, CI, CII, or E. The same assay can also be used to measure human apoCIII with a linear calibration curve from 0.005 µg/ml to 1 µg/ml using purified human apoCIII as the standard. This fast and highly sensitive ELISA could be a useful tool to investigate the role of apoCIII in lipoprotein transport and cardiovascular disease.

Restoring HSP70 deficiencies improves glucose tolerance in diabetic monkeys.

We evaluated heat shock protein 70 (HSP70) changes in diabetes mellitus (DM) in a nonhuman primate model. To this end, two studies were conducted in DM vervet monkeys. 1) Normal control and streptozotocin-induced DM monkeys (Stz-DM) that were differentiated into moderately or poorly controlled DM by judicious insulin administration were evaluated. Liver was collected at 4, 8, 12, 16, and 20 wk after streptozotocin, exposed to ex vivo heat shock at 42°C, and immunoblotted for heat shock factor 1 (HSF1), HSP70, and phosphorylated HSF1. 2) Spontaneous DM monkeys that were not pharmacologically induced were included in a crossover study of the HSP70-inducing drug geranylgeranylacetone (GGA). GGA at 20 mg/kg was given for 14 days with a 6-wk washout period. Glucose tolerance testing and plasma and muscle HSP70 were the primary outcome measurements. In Stz-DM, hyperglycemia reduced hepatic HSP70 in a dose-dependent fashion. HSF1 was increased in livers of monkeys with Stz-DM, but responses to ex vivo heat shock were impaired vs. normal monkeys. Activation of HSF1 appears to be important, because the phosphorylation change with heat stress was nearly perfectly correlated with HSP70 increases. Impaired HSF1 activation was also seen in Stz-DM after chronic hyperglycemia (>12 wk). In naturally occurring DM, increased circulating HSP70 resulted in significantly improved glucose tolerance and significant, positive trends in other measurements of insulin resistance. No change in muscle HSP70 content was observed. We conclude that increasing HSP70, potentially through targeting hyperglycemia-related deficits in HSF1 induction and activation in the liver, is a potent and viable strategy to improve glucose tolerance.

A selective cannabinoid-1 receptor antagonist, PF-95453, reduces body weight and body fat to a greater extent than pair-fed controls in obese monkeys.

Cannabinoid-1 (CB(1)) receptor antagonists exhibit pharmacological properties favorable to treatment of obesity, caused by both centrally mediated effects on appetite and peripherally mediated effects on energy metabolism. However, the relative contribution of these effects to the weight loss produced by CB(1) receptor antagonists remains unclear. Here, we compare food intake-related and independent effects of the CB(1)-selective antagonist 1-(7-(2-chlorophenyl)-8-(4-chlorophenyl)-2-methylpyrazolo[1,5-a][1,3,5]triazin-4-yl)-3-(methylamino) azetidine-3-carboxamide (PF-95453) in obese cynomolgus monkeys. Monkeys were divided into three study groups (n = 10 each) and treated once daily for 8 weeks with either vehicle or PF-95453 as follows: 1, fed ad libitum and dosed orally with vehicle; 2, fed ad libitum and dosed orally with PF-95453 (0.5 mg/kg weeks 1-3, 1.0 mg/kg weeks 4-8); and 3, fed an amount equal to the amount consumed by the drug-treated group and dosed orally with vehicle (pair-fed). PF-95453 treatment significantly reduced food consumption by 23%, body weight by 10%, body fat by 39%, and leptin by 34% while increasing adiponectin by 78% relative to vehicle-treated controls. Pair-fed animals did not exhibit reductions in body weight or leptin but did show significantly reduced body fat (11%) and increased adiponectin (15%) relative to vehicle-treated controls but markedly less than after PF-95453 treatment. Indeed, significant differences were noted between the drug-treated and pair-fed groups with respect to body weight reduction, body fat reduction, increased adiponectin, and leptin reduction. Similar to humans, monkeys treated with the CB(1) receptor antagonist exhibited decreased body weight and body fat, a substantial portion of which seemed to be independent of the effects on food intake.

A selective peroxisome proliferator-activated receptor alpha agonist, CP-900691, improves plasma lipids, lipoproteins, and glycemic control in diabetic monkeys.

Peroxisome proliferator-activated receptors (PPARs) are involved in the regulation of lipid and glucose metabolism. PPARgamma agonists improve insulin sensitivity and hyperglycemia and are effective in treating type 2 diabetes mellitus (T2DM), whereas PPARalpha agonists are used to treat dyslipidemia and atherosclerosis. The goal here was to examine the efficacy of a selective PPARalpha agonist {(S)-3-[3-(1-carboxy-1-methyl-ethoxy)-phenyl]-piperidine-1-carboxylic acid 4-trifluoromethyl-benzyl ester; CP-900691} on lipid, glycemic, and inflammation indices in 14 cynomolgus monkeys with spontaneous T2DM maintained on daily insulin therapy. Monkeys were dosed orally with either vehicle (n = 7) or CP-900691 (3 mg/kg, n = 7) daily for 6 weeks. CP-900691 treatment increased plasma high-density lipoprotein cholesterol (HDLC) (33 +/- 3 to 60 +/- 4 mg/dL, p < 0.001) and apolipoprotein A1 (96 +/- 5 to 157 +/- 5 mg/dL, p < 0.001), reduced plasma triglycerides (547 +/- 102 to 356 +/- 90 mg/dL, p < 0.01), and apolipoprotein B (62 +/- 3 to 45 +/- 3 mg/dL, p < 0.01), improved the lipoprotein index (HDL to non-HDLC ratio; 0.28 +/- 0.06 to 0.79 +/- 0.16, p < 0.001), decreased body weight (p < 0.01) and C-reactive protein (CRP) (1700 +/- 382 to 304 +/- 102 ng/ml, p < 0.01), and increased adiponectin (1697 +/- 542 to 4242 +/- 1070 ng/ml, p < 0.001) compared with baseline. CP-900691 treatment reduced exogenous insulin requirements by approximately 25% (p < 0.04) while lowering plasma fructosamine from 2.87 +/- 0.09 to 2.22 +/- 0.17 mM (p < 0.05), indicative of improved glycemic control. There were no changes in any of the aforementioned parameters in the vehicle group. Because low HDLC and high triglycerides are well established risk factors for cardiovascular disease, the marked improvements in these parameters, and in glycemic control, body weight, and CRP, suggest that CP-900691 may be of benefit in diabetic and obese or hyperlipidemic populations.

Estrogen decreases atherosclerosis in part by reducing hepatic acyl-CoA:cholesterol acyltransferase 2 (ACAT2) in monkeys.

OBJECTIVE: Estrogens decrease atherosclerosis progression, mediated in part through changes in plasma lipids and lipoproteins. This study aimed to determine estrogen-induced changes in hepatic cholesterol metabolism, plasma lipoproteins, and the relationship of these changes to atherosclerosis extent. METHODS AND RESULTS: Ovariectomized monkeys (n=34) consumed atherogenic diets for 30 months which contained either no hormones (control, n=17) or conjugated equine estrogens (CEE, n=17) at a human dose equivalent of 0.625 mg/d. Hepatic cholesterol content, low-density lipoprotein (LDL) receptor expression, cholesterol 7 alpha-hydroxylase and acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity, and expression levels were determined. CEE treatment resulted in lower plasma concentrations of very-low- and intermediate- density lipoprotein cholesterol (V+IDLC; P=0.01), smaller LDL particles (P=0.002), and 50% lower hepatic cholesterol content (total, free, and esterified; P<0.05 for all). Total ACAT activity was significantly lower (P=0.01), explained primarily by reductions in the activity of ACAT2. Estrogen regulation of enzymatic activity was at the protein level as both ACAT1 and 2 protein, but not mRNA levels, were lower (P=0.02 and <0.0001, respectively). ACAT2 activity was significantly associated with hepatic total cholesterol, plasma V+IDLC cholesterol, and atherosclerosis. CONCLUSIONS: Atheroprotective effects of estrogen therapy may be related to reduced hepatic secretion of ACAT2-derived cholesteryl esters in plasma lipoproteins.

Tissue-specific regulation and expression of heat shock proteins in type 2 diabetic monkeys.

The chaperone protein heat shock protein (HSP) 70 has been shown to protect against obesity-associated insulin resistance. Induction of HSPs is thus considered an exciting therapeutic strategy for diabetes (DM). The aims of this study were to (1) determine HSP levels in plasma, hepatic, and pancreatic tissues of type 2 DM primates and (2) assess the relationship between chaperone proteins of the HSP family and cellular protection. We collected plasma from 24 type 2 DM and 25 normoglycemic control (CTL) cynomolgus macaques. A subset of DM monkeys had liver and pancreas samples available which were compared to a second group of CTL monkeys. We found that DM monkeys had 32% lower HSP70 in circulation which remained significant even after adjustment for the greater age and bodyweight of these monkeys (p < 0.001). The liver demonstrated a similar reductions in both HSP70 and 90 that was related to 50% lower levels of the transcription factor, heat shock factor 1 (HSF1; p = 0.03). Pancreatic tissue had the opposite expression pattern with significantly higher HSF1 (p = 0.004) and accordingly higher HSP70 and 90. Pancreas from DM monkeys had less nitrosative oxidation (p = 0.03) which was unaccounted for by superoxide dismutases and was negatively associated with HSP levels (r = -0.57, p = 0.009). HSF1/HSP deficiency exists in DM liver which may contribute to hepatic insulin resistance and this deficiency was reflected in lower circulating concentrations. Pancreas maintains HSP levels despite hyperglycemia, likely in an attempt to protect vulnerable beta cells from exocrine pancreatic damage and from stress associated with insulin hypersecretion.

Enhanced cholinergic response in pancreata of nonhuman primates with impaired glucose tolerance shown on [18F]fluorobenzyltrozamicol positron emission tomography.

BACKGROUND: Islet cell adaptation to insulin resistance in type 2 diabetes mellitus may be due in part to increased stimulation of beta cells by the autonomic nervous system. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor correlates with cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[18F]fluorobenzyltrozamicol ([18F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. In this study, we utilize [18F]FBT PET to demonstrate pancreatic cholinergic activity before and after dextrose infusion in nonhuman primates with normal (NGT) and impaired (IGT) glucose tolerance. METHODS: Seven adult female vervet (Chlorocebus aethiops) monkeys were maintained on an atherogenic Western diet. They were divided into two groups: four with NGT and three with IGT. Each subject underwent [18F]FBT PET twice: first, a baseline PET under fasting conditions; and second, PET under fasting conditions but after intravenous infusion of dextrose solution. Quantitative analysis of pancreatic uptake at 60 min post-injection was performed. RESULTS: There was no difference in pancreatic uptake of [18F]FBT on baseline scans between the two groups. Pancreatic uptake of [18F]FBT increased in every subject after dextrose infusion (P = 0.03). On post-dextrose PET scans, pancreatic uptake of [18F]FBT was significantly higher in IGT subjects compared with NGT subjects (P = 0.03). The post-dextrose to pre-dextrose uptake ratios were higher in IGT subjects (P = 0.08). CONCLUSIONS: Acute increases in pancreatic cholinergic activity in vivo were detected in the pancreata of nonhuman primates with NGT and IGT after intravenous dextrose infusion on [18F]FBT PET. In subjects with IGT, this activity was significantly higher, suggesting increased autonomic nervous system stimulation of the pancreatic islets in insulin-resistant subjects.

Dual radiotracer analysis of cholinergic neuronal changes in prediabetic mouse pancreas.

BACKGROUND: Pancreatic neuronal changes associated with beta cell loss in type 1 diabetes mellitus are complex, involving, in part, parasympathetic mechanisms to compensate for preclinical hyperglycemia. The parasympathetic neurotransmitter acetylcholine (ACh) mediates insulin release via M3 muscarinic receptors on islet beta cells. The vesicular ACh transporter (VAChT) receptor has been shown to be a useful marker of cholinergic activity in vivo. The positron emission tomography (PET) radiotracer (+)-4-[(18)F]fluorobenzyltrozamicol ([(18)F]FBT) binds to the VAChT receptor on presynaptic cholinergic neurons and can be quantified by PET. The compound 4-diphenylacetoxy-N-methylpiperidine (4-DAMP), available in a tritiated form, binds to M3 muscarinic receptors on beta cells and is a potential target for assessing pancreatic beta cell mass. In this study, we investigate the feasibility of dual radiotracer analysis in identifying neurofunctional changes that may signify type 1 diabetes mellitus in its early preclinical state. METHODS: Ex vivo determinations of pancreatic uptake were performed in prediabetic nonobese diabetic mice and controls after intravenous injection of [(18)F]FBT or 4-[(3)H]DAMP. Beta cell loss in prediabetic mice was confirmed using immunohistochemical methods. RESULTS: [(18)F]FBT uptake was significantly higher in prediabetic pancreata than controls: 3.22 +/- 0.81 and 2.51 +/- 1.04, respectively (P < 0.03). 4-[(3)H]DAMP uptake was significantly lower in prediabetic pancreata than controls: 0.612 +/- 0.161 and 0.968 +/- 0.364, respectively (P = 0.01). CONCLUSIONS: These data suggest that a combination of radiotracer imaging agents that bind to neuronal elements intimately involved in insulin production may be an effective method of evaluating changes associated with early beta cell loss using PET.

Effects of soy vs. casein protein on body weight and glycemic control in female monkeys and their offspring.

Nutritional interventions are important for reducing obesity and related conditions. Soy is a good source of protein and also contains isoflavones that may affect plasma lipids, body weight, and insulin action. Described here are data from a monkey breeding colony in which monkeys were initially fed a standard chow diet that is low fat with protein derived from soy. Monkeys were then randomized to a defined diet with a fat content similar to the typical American diet (TAD) containing either protein derived from soy (TAD soy) or casein-lactalbumin (TAD casein). The colony was followed for over two years to assess body weight, and carbohydrate and lipid measures in adult females (n=19) and their offspring (n=25). Serum isoflavone concentrations were higher with TAD soy than TAD casein, but not as high as when monkey chow was fed. Offspring consuming TAD soy had higher serum isoflavone concentrations than adults consuming TAD soy. Female monkeys consuming TAD soy had better glycemic control, as determined by fructosamine concentrations, but no differences in lipids or body weight compared with those consuming diets with TAD casein. Offspring born to dams consuming TAD soy had similar body weights at birth but over a two-year period weighed significantly less, had significantly lower triglyceride concentrations, and like adult females, had significantly lower fructosamine concentrations compared to TAD casein. Glucose tolerance tests in adult females were not significantly different with diet, but offspring eating TAD soy had increased glucose disappearance with overall lower glucose and insulin responses to the glucose challenge compared with TAD casein. Potential reasons for the additional benefits of TAD soy observed in offspring but not in adults may be related to higher serum isoflavone concentrations in offspring, presence of the diet differences throughout more of their lifespan (including gestation), or different tissue susceptibilities in younger animals.

Effects of soy protein and isoflavones on insulin resistance and adiponectin in male monkeys.

Isoflavones may influence insulin action by means of their well-known receptor-mediated estrogenic activity. However, isoflavones also bind to peroxisome proliferator-activated receptors (PPARs) that are strongly associated with insulin action. Soy protein with its isoflavones has previously been shown to improve glycemic control in diabetic postmenopausal women and to improve insulin sensitivity in ovariectomized monkeys. The purpose of the current report was to extend our studies of dietary soy protein to male monkeys and determine effects of the soy isoflavones on insulin resistance. Two studies are reported here. Study one involved 91 male monkeys consuming 3 diets differing only by the source of protein (casein-lactalbumin, soy protein with a low isoflavone concentration, or soy protein with a high isoflavone concentration). Intravenous glucose tolerance tests were done, and plasma adiponectin and lipoprotein concentrations were determined after 25 months of study. Samples of visceral fat were obtained at 31 months for assessment of adiponectin and PPARgamma expression. The second study involved 8 monkeys in a Latin-square design that compared the effects of diets with casein/lactalbumin, soy protein with a high isoflavone concentration, or soy protein that was alcohol-washed to deplete the isoflavones. After 8 weeks of treatment, insulin sensitivity and plasma lipoproteins were assessed. At 10 weeks, a biopsy of the skeletal muscle was performed for determination of insulin receptor, PPARalpha, and PPARgamma content. The major findings were that consumption of isoflavone-containing soy protein dose-dependently increased insulin responses to the glucose challenge and decreased plasma adiponectin, whereas isoflavone-depleted soy protein decreased body weight and had no effect on plasma adiponectin concentrations. Muscle PPARalpha and gamma expression was also increased with the isoflavone-depleted soy relative to either casein or soy protein containing the isoflavones. Further studies are needed to determine the mechanisms involved in these effects of a high-soy isoflavone diet and to optimize dietary isoflavone content for maximal health benefits in male subjects.

Addition of medroxyprogesterone acetate to conjugated equine estrogens results in insulin resistance in adipose tissue.

The purpose of this study was to determine if the insulin resistance we have previously reported in surgically postmenopausal primates treated with combined hormone therapy (HT) is due in part to effects on adipose tissue. Eighty-seven ovariectomized monkeys were fed a moderately atherogenic diet (0.28 mg cholesterol per kilocalorie [0.07 mg/kJ]) and randomized to receive no hormones (control, n = 29), estrogen therapy (ET, conjugated equine estrogens, 0.625 mg/d human equivalent; n = 29), or HT (ET + medroxyprogesterone acetate, 2.5 mg/d human equivalent; n = 29) in the diet for 2 years. Fasting glycemic measures were made at baseline and at the end of treatment. Circulating adiponectin measures, insulin tolerance tests, glucose tolerance tests, and isolated adipocyte glucose uptake assays were performed at the end of the trial. Hormone therapy-treated animals were insulin resistant, as determined by greater fasting insulin concentrations (P = .008), greater homeostasis model assessment of insulin resistance (HOMA-R) value (P = .005) and slower glucose disposal after insulin administration (K(ITT); P = .02) when compared with controls. Subcutaneous adipocytes from HT-treated monkeys had a greater ED(50) for insulin (P = .04) and lower maximal glucose uptake per cell (P < .001) compared with controls, suggesting impaired adipocyte insulin sensitivity. Adipocytes were smaller (P = .001) and adiponectin concentrations were greatest in the ET group (P = .02), with no difference between controls and HT-treated monkeys. In conclusion, estrogen therapy resulted in smaller adipocyte size and greater adiponectin concentrations than control or HT. Hormone therapy resulted in impaired insulin sensitivity and adipocyte glucose uptake compared with controls, whereas there was no difference between ET and controls. Because no adverse effects were found with ET alone, it is likely that the progestin, medroxyprogesterone acetate, resulted in the negative effects of the combined HT regimen on whole-body insulin sensitivity, which were mediated, in part, by reductions in adipose tissue responses to insulin.

Diverse Non-genetic, Allele-Specific Expression Effects Shape Genetic Architecture at the Cellular Level in the Mammalian Brain.

Interactions between genetic and epigenetic effects shape brain function, behavior, and the risk for mental illness. Random X inactivation and genomic imprinting are epigenetic allelic effects that are well known to influence genetic architecture and disease risk. Less is known about the nature, prevalence, and conservation of other potential epigenetic allelic effects in vivo in the mouse and primate brain. Here we devise genomics, in situ hybridization, and mouse genetics strategies to uncover diverse allelic effects in the brain that are not caused by imprinting or genetic variation. We found allelic effects that are developmental stage and cell type specific, that are prevalent in the neonatal brain, and that cause mosaics of monoallelic brain cells that differentially express wild-type and mutant alleles for heterozygous mutations. Finally, we show that diverse non-genetic allelic effects that impact mental illness risk genes exist in the macaque and human brain. Our findings have potential implications for mammalian brain genetics. VIDEO ABSTRACT.

Type 2 diabetes-an introduction to the development and use of animal models.

Although the epidemiology of type 2 diabetes (T2D) has been well described, there is much about the disease that remains unclear. For example, lifestyle factors-including increased body weight with visceral fat deposition and insufficient physical activity-are thought to be primary contributors to the adverse changes in the metabolism of muscle and fat cells that comprise the first stage of the disease. However, the precise mechanisms underlying these initial alterations are incompletely understood. Other, less obvious questions relate to the presence of sex differences in the development and health consequences of T2D, the etiological role of the central nervous system ("stress"), and the potential evolutionary origins of T2D susceptibility. Some of these issues can be resolved by further study of human populations. However, many questions can be answered only through the kinds of controlled prospective studies that are conducted with appropriate animal models. The use of such models can be an invaluable part of an overall strategy designed to elucidate the mechanisms underlying the development of T2D, understand the natural history of the disease, identify targets for therapy, and evaluate interventions. Current evidence indicates that no single animal model replicates the development of human T2D in all of its details. Nonetheless, the existing models (e.g., naturally occurring and genetically modified rodents, cats, pigs, and nonhuman primates) offer researchers a rich array of opportunities to investigate the myriad complexities of T2D. The individual contributions comprising this issue of ILAR Journal review the research that has been conducted on many of these animals.

Effects of soy isoflavones and conjugated equine estrogens on inflammatory markers in atherosclerotic, ovariectomized monkeys.

The effects of dietary soy isoflavones (IF) and conjugated equine estrogens (CEE) on circulating inflammatory markers were determined at the end of a 3-yr study of ovariectomized monkeys consuming a moderately atherogenic diet. Treatments were: 1) control, receiving alcohol-extracted soy-protein-based diet with low IF content (comparable to approximately 5 mg/d); 2) CEE, added to the control diet at a dose comparable to 0.625 mg/d; and 3) IF, consumed as a part of unextracted soy protein isolate at a dose comparable to 129 mg/d. Serum soluble vascular cell adhesion molecule-1 (sVCAM-1) was reduced by both IF (P < 0.006) and CEE (P < 0.0001) relative to controls. Serum monocyte chemoattractant protein (MCP)-1 was reduced by CEE (P < 0.0001) but not by IF (P = 1.00). Treatments did not affect serum IL-6 (P = 0.40), soluble E-selectin (P = 0.17), or C-reactive protein (P = 0.15). Serum MCP-1 and, to a lesser extent, IL-6 significantly correlated with atherosclerosis (plaque area) in the iliac and carotid arteries (all P < 0.05). Serum MCP-1 was also strongly associated with coronary artery atherosclerosis and with indices of plaque inflammation and matrix remodeling (matrix metalloproteinase-9) in the coronary artery intima (all P < 0.01). We conclude that, in this well-established nonhuman primate model of atherosclerosis, this dose of soy IF provided an antiinflammatory effect specific for sVCAM-1, whereas the effects of CEE extended to both sVCAM-1 and MCP1. It is possible that the atheroprotective effects of IF and CEE are mediated, at least in part, by effects on VCAM-1. The sites of IF inhibitory effects on sVCAM-1 production are not known, but likely candidates include the liver and/or the cardiovascular system.

The applicability of hormonal effects on atherosclerosis in animals to heart disease in postmenopausal women.

The discrepancy between estrogen's beneficial cardiovascular effects found in animal studies and observational studies in women compared with the recently published randomized clinical trials have stimulated a great deal of controversy. Possibilities for the discrepancy include the age of the women, number of years postmenopausal, and amount of atherosclerotic complication (necrosis and inflammation) present when hormone therapy is initiated. Many of the previous benefits of estrogens noted in both animal studies and observational studies were found in primary prevention studies that experimentally refer to a decrease in the progression of atherosclerosis extent rather than prevention of clinical events, which often occur in women with undiagnosed atherosclerotic plaques. In support of this notion, animal studies in which artery damage was present prior to hormone treatment, due to either consumption of an atherogenic diet or balloon injury of the endothelium, found no benefit with estrogen treatment. These animal studies are consistent with the lack of protection found in secondary prevention studies in women. Other areas of concern deal with the route of hormone delivery or dose of hormones used. Higher doses of oral estrogens may result in increased risk of inflammation and thrombosis. Future studies should be directed at studying hormone therapy in relevant ages of women (perimenopausal women) using the lowest effective doses of hormones and comparing oral and parenteral forms of delivery.