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Obesity is a prevalent chronic condition in many developed and developing nations that raises the risk for developing heart disease, stroke, and diabetes. Previous studies have shown that consuming particular probiotic strains of Lactobacillus is associated with improvement in the obese and diabetic phenotype; however, the mechanisms of these beneficial effects are not well understood. In this study, C57BL/6J male mice were fed a lard-based high fat diet for 15 weeks with Lactobacillus plantarum supplementation NCIMB8826 (Lp) between weeks 10 and 15 ( n = 10 per group). Systemic metabolic effects of supplementation were analyzed by NMR metabolomics, protein expression assays, gene transcript quantification, and 16S rRNA marker gene sequencing. Body and organ weights were not significantly different with Lp supplementation, and no microbiota community structure changes were observed in the cecum; however, L. plantarum numbers were increased in the treatment group according to culture-based and 16S rRNA gene quantification. Significant differences in metabolite and protein concentrations (serum, liver, and colon), gene expression (ileum and adipose), and cytokines (colon) were observed between groups with increases in the gene expression of tight junction proteins in the ileum and cecum and improvement of some markers of glucose homeostasis in blood and tissue with Lp supplementation. These results indicate Lp supplementation impacts systemic metabolism and immune signaling before phenotypic changes and without large-scale changes to the microbiome. This study supports the notion that Lp is a beneficial probiotic, even in the context of a high fat diet.

The Caenorhabditis elegans model is a rapid and inexpensive method to address pharmacologic questions. We describe the use of C. elegans to explore 2 pharmacologic questions concerning candidate antiobesity drugs and illustrate its potential usefulness in pharmacologic research: (1) to determine a ratio of betahistine–olanzapine that blocks the olanzapine-induced intestinal fat deposition (IFD) as detected by Nile red staining and (2) to identify the mechanism of action of a pharmaceutical candidate AB-101 that reduces IFD. Olanzapine (53 μg/mL) increased the IFD (12.1 ± 0.1%, P < 0.02), which was blocked by betahistine (763 μg/mL, 39.3 ± 0.01%, P < 0.05) in wild-type C. elegans (N2). AB-101 (1.0%) reduced the IFD in N2 (P < 0.05), increased the pharyngeal pumping rate (P < 0.05), and reversed the elevated IFD induced by protease inhibitors atazanavir and ritonavir (P < 0.05). AB-101 did not affect IFD in a ACS null mutant strain acs-4(ok2872) III/hT2[bli-4(e937) let-?(q782) qIs48](I;III) suggesting an involvement of the lipid oxidation pathway and an upregulation of CPT-1. Our studies suggest that C. elegans may be used as a resource in pharmacologic research. This article is intended to stimulate a greater appreciation of its value in the development of new pharmaceutical interventions.

Increased dietary fiber (DF) intake elicits a wide range of physiologic effects, not just locally in the gut, but systemically. DFs can greatly alter the gut milieu by affecting the gut microbiome, which in turn influences the gut barrier, gastrointestinal immune and endocrine responses, and nitrogen cycling and microbial metabolism. These gut-associated changes can then alter the physiology and biochemistry of the body’s other main nutrient management and detoxification organs, the liver and kidneys. The molecular mechanisms by which DF alters the physiology of the gut, liver, and kidneys is likely through gut-localized events (i.e., bacterial nitrogen metabolism, microbe-microbe, and microbe–host cell interactions) coupled with specific factors that emanate from the gut in response to DF, which signal to or affect the physiology of the liver and kidneys. The latter may include microbe-derived xenometabolites, peptides, or bioactive food components made available by gut microbes, inflammation signals, and gut hormones. The intent of this review is to summarize how DF alters the gut milieu to specifically affect intestinal, liver, and kidney functions and to discuss the potential local and systemic signaling networks that are involved.

Background: Enzyme-treated wheat bran (ETWB) contains a fermentable dietary fiber previously shown to decrease liver triglycerides (TGs) and modify the gut microbiome in mice. It is not clear which mechanisms explain how ETWB feeding affects hepatic metabolism, but factors (i.e., xenometabolites) associated with specific microbes may be involved. Objective: The objective of this study was to characterize ETWB-driven shifts in the cecal microbiome and to identify correlates between microbial changes and diet-related differences in liver metabolism in diet-induced obese mice that typically display steatosis. Methods: Five-week-old male C57BL/6J mice fed a 45%-lard based fat diet supplemented with ETWB (20% wt:wt) or rapidly digestible starch (control) (n = 15/group) for 10 wk were characterized by using a multi-omics approach. Multivariate statistical analysis was used to identify variables that were strong discriminators between the ETWB and control groups. Results: Body weight and liver TGs were decreased by ETWB feeding (by 10% and 25%, respectively; P

Author(s): Peterson, Courtney M; Beyl, Robbie A; Marlatt, Kara L; Martin, Corby K; Aryana, Kayanush J; Marco, Maria L; Martin, Roy J; Keenan, Michael J; Ravussin, Eric | Abstract: BackgroundType 2 resistant starch (RS2) has been shown to improve glycemic control and some cardiovascular endpoints in rodent and human studies.ObjectiveThe aim of this study was to perform one of the first randomized clinical trials in adults with prediabetes and one of the longest trials to test whether RS2 can improve cardiometabolic health.Design68 overweight [body mass index (BMI) ≥27 kg/m2] adults aged 35-75 y with prediabetes were randomized to consume 45 g/d of high-amylose maize (RS2) or an isocaloric amount of the rapidly digestible starch amylopectin (control) for 12 wk. At baseline and postintervention, ectopic fat depots (visceral adipose tissue, intrahepatic lipids, and intramyocellular lipids) were measured by magnetic resonance imaging/spectroscopy, energy metabolism by respiratory chamber, and carbohydrate metabolism by glycated hemoglobin (HbA1c), an intravenous glucose tolerance test, and a meal tolerance test. Cardiovascular risk factors-serum lipids, blood pressure, heart rate, and inflammatory markers (high-sensitivity C-reactive protein [hs-CRP], interleukin-6, and tumor necrosis factor [TNF]-α)-were also measured. The primary endpoints were insulin sensitivity, insulin secretion, ectopic fat, and markers of inflammation. Data were primarily analyzed as treatment effects via a linear mixed model both with and without the addition of covariates.ResultsRelative to the control group, RS2 lowered HbA1c by a clinically insignificant 0.1 ± 0.2% (Δ = -1 ± 2 mmol/mol; P = 0.05) but did not affect insulin secretion, insulin sensitivity, the disposition index, or glucose or insulin areas under the curve relative to baseline (P ≥ 0.23). RS2 decreased heart rate by 5 ± 9 beats/min (P = 0.02) and TNF-α concentrations by 2.1 ± 2.7 pg/mL (P = 0.004), relative to the control group. Ectopic fat, energy expenditure, substrate oxidation, and all other cardiovascular risk factors were unaffected (P ≥ 0.06).Conclusions12 wk of supplementation with resistant starch reduced the inflammatory marker TNF-α and heart rate, but it did not significantly improve glycemic control and other cardiovascular disease risk factors, in adults with prediabetes. This trial was registered at clinicaltrials.gov as NCT01708694.

BackgroundResistant starch is a prebiotic metabolized by the gut bacteria. It has been shown to attenuate chronic kidney disease (CKD) progression in rats. Previous studies employed taxonomic analysis using 16S rRNA sequencing and untargeted metabolomics profiling. Here we expand these studies by metaproteomics, gaining new insight into the host-microbiome interaction.MethodsDifferences between cecum contents in CKD rats fed a diet containing resistant starch with those fed a diet containing digestible starch were examined by comparative metaproteomics analysis. Taxonomic information was obtained using unique protein sequences. Our methodology results in quantitative data covering both host and bacterial proteins.Results5,834 proteins were quantified, with 947 proteins originating from the host organism. Taxonomic information derived from metaproteomics data surpassed previous 16S RNA analysis, and reached species resolutions for moderately abundant taxonomic groups. In particular, the Ruminococcaceae family becomes well resolved – with butyrate producers and amylolytic species such as R. bromii clearly visible and significantly higher while fibrolytic species such as R. flavefaciens are significantly lower with resistant starch feeding. The observed changes in protein patterns are consistent with fiber-associated improvement in CKD phenotype. Several known host CKD-associated proteins and biomarkers of impaired kidney function were significantly reduced with resistant starch supplementation. Data are available via ProteomeXchange with identifier PXD008845.Conclusions- Metaproteomics analysis of cecum contents of CKD rats with and without resistant starch supplementation reveals changes within gut microbiota at unprecedented resolution, providing both functional and taxonomic information. Proteins and organisms differentially abundant with RS supplementation point toward a shift from mucin degraders to butyrate producers.

© 2017 Dietary resistant starch (RS) might alter gastrointestinal tract function in a manner that improves human health, particularly among adults at risk for diabetes. Here, we report the design and baseline results (with emphasis on race differences) from the STARCH trial, the first comprehensive metabolic phenotyping of people with prediabetes enrolled in a randomized clinical trial testing the effect of RS on risk factors for diabetes. Overweight/obese participants (BMI ≥ 27 kg/m2 and weight ≤ 143 kg), age 35–75 y, with confirmed prediabetes were eligible. Participants were randomized to consume 45 g/day of RS (RS = amylose) or amylopectin (Control) for 12 weeks. The study was designed to evaluate the effect of RS on insulin sensitivity and secretion, ectopic fat, and inflammatory markers. Secondary outcomes included energy expenditure, substrate oxidation, appetite, food intake, colonic microbial composition, fecal and plasma levels of short-chain fatty acids, fecal RS excretion, and gut permeability. Out of 280 individuals screened, 68 were randomized, 65 started the intervention, and 63 were analyzed at baseline (mean age 55 y, BMI 35.6 kg/m2); 2 were excluded from baseline analyses due to abnormal insulin and diabetes. Sex and race comparisons at baseline were reported. African-Americans had higher baseline acute insulin response to glucose (AIRg measured by frequently sampled intravenous glucose tolerance test) compared to Caucasians, despite having less visceral adipose tissue mass and intrahepatic lipid; all other glycemic variables were similar between races. Sleep energy expenditure was ~ 90–100 kcal/day lower in African-Americans after adjusting for insulin sensitivity and secretion. This manuscript provides an overview of the strategy used to enroll people with prediabetes into the STARCH trial and describes methodologies used in the assessment of risk factors for diabetes. Clinicaltrials.gov identifier: STARCH (NCT01708694). The present study reference can be found here: https://clinicaltrials.gov/ct2/show/NCT01708694. Submission Category: “Study Design, Statistical Design, Study Protocols”.

Resistant starch (RS) is a dietary fermentable fiber that decreases body fat accumulation, and stimulates the secretion of glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) in rodents. GLP-1 and PYY are gut secreted hormones with anti-obesity effect. Thus, blocking the signals of increased GLP-1 and PYY may also block the effect of dietary RS on body fat. In a ten-week study, C57BL/6J and GLP-1 receptor null (GLP-1R KO) mice were fed control or 30% RS diet, and received daily intraperitoneal injection of either saline or PYY receptor antagonist (BIIE 0246, 20ug/kg body weight). Dietary RS significantly decreased body fat accumulation only in wild type mice that has saline injection, but not in GLP-1R KO mice. PYY receptor antagonist diminished RS action on body fat in wild type mice, but did not interfere with GLP-1R KO mice response to RS. Regardless of genotype and injection received, all RS fed mice had increased cumulative food intake, cecal fermentation, and mRNA expression of proglucagon and PYY. Thus, our results suggest that increased GLP-1 and PYY is important in RS effects on body fat accumulation.

The realization that low–glycemic index diets were formulated using resistant starch led to more than a decade of research on the health effects of resistant starch. Determination of the metabolizable energy of the resistant starch product allowed for the performance of isocaloric studies. Fermentation of resistant starch in rodent studies results in what appears to be a healthier gut, demonstrated by increased amounts of short-chain fatty acids, an apparent positive change in the microbiota, and increased gene expression for gene products involved in normal healthy proliferation and apoptosis of potential cancer cells. Additionally, consumption of resistant starch was associated with reduced abdominal fat and improved insulin sensitivity. Increased serum glucagon-like peptide 1 (GLP-1) likely plays a role in promoting these health benefits. One rodent study that did not use isocaloric diets demonstrated that the use of resistant starch at 8% of the weight of the diet reduced body fat. This appears to be approximately equivalent to the human fiber requirement. In human subjects, insulin sensitivity is increased with the feeding of resistant starch. However, only 1 of several studies reports an increase in serum GLP-1 associated with resistant starch added to the diet. This means that other mechanisms, such as increased intestinal gluconeogenesis or increased adiponectin, may be involved in the promotion of improved insulin sensitivity. Future research may confirm that there will be improved health if human individuals consume the requirement for dietary fiber and a large amount of the fiber is fermentable.

Background: High-amylose-maize resistant starch type 2 (HAMRS2) is a fermentable dietary fiber known to alter the gut milieu, including the gut microbiota, which may explain the reported effects of resistant starch to ameliorate obesity associated metabolic dysfunction.Objective: Our working hypothesis was that HAMRS2-induced microbiome changes alter gut-derived signals (i.e., xenometabolites) reaching the liver via the portal circulation, in turn altering liver metabolism by regulating gene expression and other pathways.Methods: We used a multi-omics systems biology approach to characterize HAMRS2-driven shifts to the cecal microbiome, liver metabolome, and transcriptome, identifying correlates between microbial changes and liver metabolites under obesogenic conditions that, to our knowledge, have not previously been recognized. Five-week-old male C57BL/6J mice were fed an energy-dense 45% lard-based-fat diet for 10 wk supplemented with either 20% HAMRS2 by weight (n = 14) or rapidly digestible starch (control diet; n = 15).Results: Despite no differences in food intake, body weight, glucose tolerance, fasting plasma insulin, or liver triglycerides, the HAMRS2 mice showed a 15-58% reduction in all measured liver amino acids, except for Gln, compared with control mice. These metabolites were equivalent in the plasma of HAMRS2 mice compared with controls, and transcripts encoding key amino acid transporters were not different in the small intestine or liver, suggesting that HAMRS2 effects were not simply due to lower hepatocyte exposure to systemic amino acids. Instead, alterations in gut microbial metabolism could have affected host nitrogen and amino acid homeostasis: HAMRS2 mice showed a 62% increase (P Conclusion: Together, these differences indicate that HAMRS2 dramatically alters hepatic metabolism and gene expression concurrent with shifts in specific gut bacteria in C57BL/6J mice.

Objective Obesity is a health concern. Resistant starch (RS) type 2 from high-amylose maize (HAM-RS2) and dietary sodium butyrate (SB) reduce abdominal fat in rodents. RS treatment is associated with increased gut hormones peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), but it is not known if SB increases these hormones. Design and Methods This was investigated in a 2 × 2 rat study with HAM-RS2 (0 or 28% weight) and dietary sodium butyrate (0 and 3.2%) resulting in isocaloric treatments: energy control (EC), sodium butyrate (SB), HAM-RS2 (RS), and the combination (SBRS). Results RS and SB reduced abdominal fat and the combination reduced abdominal fat compared to SB and RS. RS was associated with increased fermentation in the cecum. Serum PYY and GLP-1 total were increased with RS treatment. RS treatment was associated with increased cecal butyrate produced from fermentation of RS, but there was no cecal increase for dietary SB. Conclusions SB after its absorption into the blood appears to not affect production of PYY and GLP-1, while butyrate from fermentation in the cecum promotes increased PYY and GLP-1. Future studies with lower doses of RS and SB are warranted and the combination may be beneficial for human health.

Objective The effects of type 2 resistant starch from high-amylose maize (HAM-RS2) in rodents fed with low-fat diets were demonstrated in previous studies. Fish oil is also reported to reduce body fat. In the current study, the effects of high fat and fish oil on HAM-RS2 feeding in rats were investigated. Design and Methods Rats were fed 0 or 27% (weight) HAM-RS2 with low (15% energy) or high fat (42% energy) diets that included 0 or 10% (energy) tuna oil to test the effect of HAM-RS2 in diet-induced obesity and effects of tuna oil. Data were analyzed as 2 × 2 × 2 factorial. Results Rats fed HAM-RS2 had decreased cecal contents pH, increased cecal and cecal contents weight, increased cecal contents acetate, propionate, and butyrate, increased GLP-1 and PYY, and decreased abdominal fat. However, high fat partially attenuated effects of HAM-RS2, but increased GLP-1 active. Dietary tuna oil had limited effects at concentration used. Conclusions Results demonstrated that a high fat diet partially attenuates the response to HAM-RS2. The mechanism may center on reduced levels of cecal contents propionate and butyrate and reduced serum PYY. This study demonstrated that with consumption of high fat, HAM-RS2 produces fermentation but results in partial attenuation of effects.

Objective: Obesity after menopause is a health concern for older females. Changes in the microbiota are likely to occur with this condition. Modifying the microbiota with a prebiotic is a plausible strategy for improving the health of menopausal females. Design and Methods: Resistant starch type 2 from high-amylose maize (HAM-RS2) was used as a prebiotic in rats in a 2 × 2 factorial study with two levels of HAM-RS2 (0 or 29.7% of weight of diet) referred to as energy control (EC) and HAM-RS2 diets, respectively; and two levels of surgery, ovariectomized (OVX) and sham. Results: In a 6-week, postsurgery recovery period, OVX rats gained more body weight with consumption of a similar amount of food. Subsequently, consumption of HAM-RS2 versus EC diets resulted in reduced abdominal fat in both OVX and sham rats; but when normalized for disemboweled body weight (body weight minus GI tract), there was no effect of surgery, only reduction with HAM-RS2. Targeted bacterial populations were estimated that are known to ferment HAM-RS2 or metabolize the products of that initial fermentation. OVX and sham rats demonstrated increased bacterial levels with dietary HAM-RS2 for all bacteria. Additionally, culture techniques and qPCR provided similar results. Conclusion: This study shows that, as expected, OVX increases adiposity. However, contrary to previous effects seen in obese mice, this did not prevent fermentation of HAM-RS2 and consequently, the fat gain associated with OVX was attenuated.

© 2016 the American Physiological Society. Patients and animals with chronic kidney disease (CKD) exhibit profound alterations in the gut environment including shifts in microbial composition, increased fecal pH, and increased blood levels of gut microbe-derived metabolites (xenometabolites). The fermentable dietary fiber high amylose maize-resistant starch type 2 (HAMRS2) has been shown to alter the gut milieu and in CKD rat models leads to markedly improved kidney function. The aim of the present study was to identify specific cecal bacteria and cecal, blood, and urinary metabolites that associate with changes in kidney function to identify potential mechanisms involved with CKD amelioration in response to dietary resistant starch. Male Sprague-Dawley rats with adenine-induced CKD were fed a semipurified low-fiber diet or a high-fiber diet [59% (wt/wt) HAMRS2] for 3 wk (n = 9 rats/group). The cecal microbiome was characterized, and cecal contents, serum, and urine metabolites were analyzed. HAMRS2-fed rats displayed decreased cecal pH, decreased microbial diversity, and an increased Bacteroidetes-to-Firmicutes ratio. Several uremic retention solutes were altered in the cecal contents, serum, and urine, many of which had strong correlations with specific gut bacteria abundances, i.e., serum and urine indoxyl sulfate were reduced by 36% and 66%, respectively, in HAMRS2-fed rats and urine p-cresol was reduced by 47% in HAMRS2-fed rats. Outcomes from this study were coincident with improvements in kidney function indexes and amelioration of CKD outcomes previously reported for these rats, suggesting an important role for microbial-derived factors and gut microbe metabolism in regulating host kidney function.

Dietary interventions might prevent or reverse age-related declines in health through modification of the activity and composition of the intestinal microbiota. As a first step toward more comprehensive evaluations of single dietary components on healthy aging, 16S rRNA gene amplicon sequencing was applied to determine the structure of the bacterial communities in the ceca of 20-month-old healthy mice fed energy-controlled diets containing 0, 18, or 36% type 2 resistant starch (RS) from high-amylose maize (HAM-RS2). The cecal microbiota of mice fed a diet depleted in RS and containing the readily digestible carbohydrate amylopectin were dominated by bacteria in the Firmicutes phylum and contained low levels of Bacteroidetes and Actinobacteria. In contrast, mice fed diets containing HAM-RS2 were colonized by higher levels of Bacteroidetes and Bifidobacterium, Akkermansia, and Allobaculum species in proportions that were dependent on the concentration of the dietary fiber. The proportions of Bifidobacterium and Akkermansia were positively correlated with mouse feeding responses, gut weight, and expression levels of proglucagon, the precursor of the gut anti-obesity/diabetic hormone GLP-1. This study showed that aging mice harbor a distinct microbiota, which can be modulated by RS and enriched for bacteria that are associated with improved health.

Dietary resistant starch impact on intestinal microbiome and improving healthspan is the topic of this review. In the elderly population, dietary fiber intake is lower than recommended. Dietary resistant starch as a source of fiber produces a profound change in gut microbiota and fermentation in animal models of aging. Dietary resistant starch has the potential for improving healthspan in the elderly through multiple mechanisms as follows: (1) enhancing gut microbiota profile and production of short-chain fatty acids, (2) improving gut barrier function, (3) increasing gut peptides that are important in glucose homeostasis and lipid metabolism, and (4) mimicking many of the effects of caloric restriction including upregulation of genes involved in xenobiotic metabolism.

We investigated the effects of dietary whey protein on food intake, body fat, and body weight gain in rats. Adult (11-12 week) male Sprague-Dawley rats were divided into three dietary treatment groups for a 10-week study: control. Whey protein (HP-W), or high-protein content control (HP-S). Albumin was used as the basic protein source for all three diets. HP-W and HP-S diets contained an additional 24% (wt/wt) whey or isoflavone-free soy protein, respectively. Food intake, body weight, body fat, respiratory quotient (RQ), plasma cholecystokinin (CCK), glucagon like peptide-1 (GLP-1), peptide YY (PYY), and leptin were measured during and/or at the end of the study. The results showed that body fat and body weight gain were lower (P < 0.05) at the end of study in rats fed HP-W or HP-S vs. control diet. The cumulative food intake measured over the 10-week study period was lower in the HP-W vs. control and HP-S groups (P < 0.01). Further, HP-W fed rats exhibited lower N(2) free RQ values than did control and HP-S groups (P < 0.01). Plasma concentrations of total GLP-1 were higher in HP-W and HP-S vs. control group (P < 0.05), whereas plasma CCK, PYY, and leptin did not differ among the three groups. In conclusion, although dietary HP-W and HP-S each decrease body fat accumulation and body weight gain, the mechanism(s) involved appear to be different. HP-S fed rats exhibit increased fat oxidation, whereas HP-W fed rats show decreased food intake and increased fat oxidation, which may contribute to the effects of whey protein on body fat.

This study examined a typology of adaptive and maladaptive behaviors of 2,552 ethnically diverse early adolescents who attended sixth grade in public schools and lived in disadvantaged communities. Behavioral adjustment of adolescents was measured by teacher ratings using the Behavior Assessment System for Children (BASC). Although typologies of children based on the BASC have been available in the literature for the past decade, little research exists on typologies of adolescents using the same instrument. The present study extended into early adolescence previous classification works focusing on childhood. A series of cluster analyses supported a seven-cluster solution: well-adapted, average, adaptive skills deficits, internalizing problems, mildly disruptive, disruptive behavior problems, and severe problems. Results appear similar to prior investigations with unique features reflecting the high-risk characteristics of the current sample. Implications of this research are discussed with regard to prevention and intervention efforts implemented within the schools.

Use of fenugreek, a food with demonstrated efficacy in lowering blood sugar, is limited by its bitter taste and strong flavor. A bread incorporating fenugreek using a proprietary process was tested for its taste acceptability and its effect on carbohydrate metabolism. We developed a fenugreek bread formula that was produced in a commercial bakery by incorporating fenugreek flour into a standard wheat bread formula. Whole wheat bread was prepared by the same formula in the same bakery using wheat flour. Eight diet-controlled diabetic subjects were served two slices (56 g) and 5% fenugreek. Blood glucose and insulin were tested periodically over a 4-hour period after consumption. The tests were run on two occasions 1 week apart, once with the fenugreek bread and once with regular bread. The study was double-blind, and the order was randomized and balanced. Fenugreek and whole wheat bread samples were evaluated for sensory attributes and nutrient composition. There was no statistically significant difference in proximate composition, color, firmness, texture, and flavor intensity between the fenugreek and wheat bread (P > .05). The area under the curve for glucose and insulin was lower in the fenugreek condition, but only reached significance with insulin (P < .05). The fenugreek-containing bread was indistinguishable from the whole wheat bread control. Normally, fenugreek flour impacts bread quality negatively. The bread maintained fenugreek's functional property of reducing insulin resistance. Acceptable baked products can be prepared with added fenugreek, which will reduce insulin resistance and treat type 2 diabetes.

OBJECTIVE We examined the role of butyric acid, a short-chain fatty acid formed by fermentation in the large intestine, in the regulation of insulin sensitivity in mice fed a high-fat diet. RESEARCH DESIGN AND METHODS In dietary-obese C57BL/6J mice, sodium butyrate was administrated through diet supplementation at 5% wt/wt in the high-fat diet. Insulin sensitivity was examined with insulin tolerance testing and homeostasis model assessment for insulin resistance. Energy metabolism was monitored in a metabolic chamber. Mitochondrial function was investigated in brown adipocytes and skeletal muscle in the mice. RESULTS On the high-fat diet, supplementation of butyrate prevented development of insulin resistance and obesity in C57BL/6 mice. Fasting blood glucose, fasting insulin, and insulin tolerance were all preserved in the treated mice. Body fat content was maintained at 10% without a reduction in food intake. Adaptive thermogenesis and fatty acid oxidation were enhanced. An increase in mitochondrial function and biogenesis was observed in skeletal muscle and brown fat. The type I fiber was enriched in skeletal muscle. Peroxisome proliferator–activated receptor-γ coactivator-1α expression was elevated at mRNA and protein levels. AMP kinase and p38 activities were elevated. In the obese mice, supplementation of butyrate led to an increase in insulin sensitivity and a reduction in adiposity. CONCLUSIONS Dietary supplementation of butyrate can prevent and treat diet-induced insulin resistance in mouse. The mechanism of butyrate action is related to promotion of energy expenditure and induction of mitochondria function.

Resistant starch (RS) is fermentable dietary fiber. Inclusion of RS in the diet causes decreased body fat accumulation and altered gut hormone profile. This study investigates the effect of feeding RS on the neuropeptide messenger RNA (mRNA) expressions in the arcuate nucleus (ARC) of the hypothalamus and whether vagal afferent nerves are involved. The rats were injected intraperitoneally with capsaicin to destroy unmyelinated small vagal afferent nerve fibers. The cholecystokinin (CCK) food suppression test was performed to validate the effectiveness of the capsaicin treatment. Then, capsaicin-treated rats and vehicle-treated rats were subdivided into a control diet or a RS diet group, and fed the corresponding diet for 65 days. At the end of study, body fat, food intake, plasma peptide YY (PYY) and glucagon-like peptide 1 (GLP-1), and hypothalamic pro-opiomelanocortin (POMC), neuropeptide Y (NPY), agouti-related peptide (AgRP) gene expressions were measured. RS-fed rats had decreased body fat, increased POMC expression in the hypothalamic ARC, and elevated plasma PYY and GLP-1 in both the capsaicin and vehicle-treated rats. Hypothalamic NPY and AgRP gene expressions were not changed by RS or capsaicin. Therefore, destruction of the capsaicin-sensitive afferent nerves did not alter the response to RS in rats. These findings suggest that dietary RS might reduce body fat through increasing the hypothalamic POMC expression and vagal afferent nerves are not involved in this process. This is the first study to show that dietary RS can alter hypothalamic POMC expression.

Glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) are anti-diabetes/obesity hormones secreted from the gut after meal ingestion. We have shown that dietary-resistant starch (RS) increased GLP-1 and PYY secretion, but the mechanism remains unknown. RS is a fermentable fiber that lowers the glycemic index of the diet and liberates short-chain fatty acids (SCFAs) through fermentation in the gut. This study investigates the two possible mechanisms by which RS stimulates GLP-1 and PYY secretion: the effect of a meal or glycemic index, and the effect of fermentation. Because GLP-1 and PYY secretions are stimulated by nutrient availability in the gut, the timing of blood sample collections could influence the outcome when two diets with different glycemic indexes are compared. Thus we examined GLP-1 and PYY plasma levels at various time points over a 24-h period in RS-fed rats. In addition, we tested proglucagon (a precursor to GLP-1) and PYY gene expression patterns in specific areas of the gut of RS-fed rats and in an enteroendocrine cell line following exposure to SCFAs in vitro. Our findings are as follows. 1) RS stimulates GLP-1 and PYY secretion in a substantial day-long manner, independent of meal effect or changes in dietary glycemia. 2) Fermentation and the liberation of SCFAs in the lower gut are associated with increased proglucagon and PYY gene expression. 3) Glucose tolerance, an indicator of increased active forms of GLP-1 and PYY, was improved in RS-fed diabetic mice. We conclude that fermentation of RS is most likely the primary mechanism for increased endogenous secretions of total GLP-1 and PYY in rodents. Thus any factor that affects fermentation should be considered when dietary fermentable fiber is used to stimulate GLP-1 and PYY secretion.

Metabolic fuels act on hypothalamic neurons to regulate feeding behavior and energy homeostasis, but the signaling mechanisms mediating these effects are not fully clear. Rats placed on a low-protein diet (10% of calories) exhibited increased food intake ( P < 0.05) and hypothalamic Agouti-related protein ( Agrp) gene expression ( P = 0.002). Direct intracerebroventricular injection of either an amino acid mixture (RPMI 1640) or leucine alone (1 μg) suppressed 24-h food intake ( P < 0.05), indicating that increasing amino acid concentrations within the brain is sufficient to suppress food intake. To define a cellular mechanism for these direct effects, GT1–7 hypothalamic cells were exposed to low amino acids for 16 h. Decreasing amino acid availability increased Agrp mRNA levels in GT1–7 cells ( P < 0.01), and this effect was attenuated by replacement of the amino acid leucine ( P < 0.05). Acute exposure to elevated amino acid concentrations increased ribosomal protein S6 kinase phosphorylation via a rapamycin-sensitive mechanism, suggesting that amino acids directly stimulated mammalian target of rapamycin (mTOR) signaling. To test whether mTOR signaling contributes to amino acid inhibition of Agrp gene expression, GT1–7 cells cultured in either low or high amino acids for 16 h and were also treated with rapamcyin (50 nM). Rapamycin treatment increased Agrp mRNA levels in cells exposed to high amino acids ( P = 0.01). Taken together, these observations indicate that amino acids can act within the brain to inhibit food intake and that a direct, mTOR-dependent inhibition of Agrp gene expression may contribute to this effect.

The rising prevalence of obesity and the vulnerability of the pediatric age group have highlighted the critical need for a careful consideration of effective, safe, remedial and preventive dietary interventions. Amylose starch (RS2) from high-amylose maize (HAM) ferments in the gut and affects body weight. One hundred and ten children, of 7-8 (n=91) or 13-14 (n=19) years of age scored the sensory qualities of a yogurt supplemented with either HAM-RS2 or an amylopectin starch. The amylopectin starch yogurt was preferred to the HAM-RS2-enriched yogurt by 7-8 year old panelists (P<0.0001). Appearance, taste, and sandiness scores given by 13- to 14-year-old panelists were more favorable for the amylopectin starch yogurt than for HAM-RS2-enriched yogurt (P<0.05). HAM-RS2 supplementation resulted in acceptable (≥6 on a 1-9 scale) sensory and hedonic ratings of the yogurt in 74% of subjects. Four children consumed a HAM-RS2-enriched yogurt for four weeks to test its fermentability in a clinical trial. Three adolescents, but not the single pre-pubertal child, had reduced stool pH (P=0.1) and increased stool short-chain fatty acids (SCFAs) (P<0.05) including increased fecal acetate (P=0.02), and butyrate (P=0.089) from resistant starch (RS) fermentation and isobutyrate (P=0.01) from protein fermentation post-treatment suggesting a favorable change to the gut microbiota. HAM-RS2 was not modified by pasteurization of the yogurt, and may be a palatable way to increase fiber intake and stimulate colonic fermentation in adolescents. Future studies are planned to determine the concentration of HAM-RS2 that offers the optimal safe and effective strategy to prevent excessive fat gain in children.

Intestinal microbes have important roles in the regulation of energy homeostasis, lipid and glucose metabolism, and immune system responses. Dysbiotic gut microbiota is associated with obesity, typ...

Chronic kidney disease (CKD) is characterized by impaired urinary excretion and accumulation of waste products in the body. Recent observations found patients with CKD have an altered gut microbiom...

Incidence of obesity and type 2 diabetes mellitus (T2DM) is steadily increasing, and the contributions of diet and the gastrointestinal microbiota to these diseases are only partially understood. E...

Sitagliptin (SG) increases serum GLP-1 (Glucagon-like peptide-1) through inhibition of the hormone degradation. Resistant starch (RS) induces GLP-1 expression by stimulating L-cells in the intestine. Sitagliptin and resistant starch may have a synergistic interaction in the induction of GLP-1. This possibility was tested in current study in a mouse model of type 2 diabetes. Hyperglycemia was induced in the diet-induced obese mice by a signal injection of streptozotocin (STZ). Sitagliptin (0.4g/100g diet) was tested in the mice (n = 55) with dietary RS (HAM-RS2) at three dosages (0, 15, or 28g/100g diet). Energy and glucose metabolism were monitored in the evaluation of synergistic activity, and GLP-1 activity was determined in the GLP-1 receptor knockout (KO) mice. In the wild type mice, body weight and adiposity were reduced by sitagliptin, which was enhanced by RS (28g). Serum GLP-1 was induced and energy expenditure was enhanced by sitagliptin. Fasting glucose, insulin, and leptin levels were decreased by sitagliptin. The sitagliptin effects were lost in the KO mice (n = 25) although induction of serum GLP-1 by sitagliptin was even stronger in KO mice. The data suggests that sitagliptin is able to reduce adiposity and insulin resistance through induction of energy expenditure. The effect of sitagliptin is partially enhanced by RS. GLP-1 receptor may regulate serum GLP-1 by facilitating the hormone clearance.

Glucose transporter 2 (GLUT2) has been proposed as a glucose sensor in pancreatic β cells. GLUT2 has been found widely expressed in the brain and GLUT2 in the hypothalamus and hindbrain has been suggested to be involved in the central glucose sensing and regulation of glucose homeostasis and food intake. In this study, we overexpressed GLUT2 in GT1-7 neuroblastoma cells and investigated the effect of GLUT2 overexpression on cellular energy status in these cells. Compared with control cells, GLUT2 overexpression resulted in significantly increased cellular ATP levels at 5 and 25 mM glucose concentrations, more inhibition of agouti-related peptide (AgRP) mRNA and AMP-activated protein kinase (AMPK) phosphorylation by glucose, and attenuated stimulation of AgRP mRNA and AMPK by 2-deoxy- d -glucose (2DG), implicating that brain GLUT2 may be important in the regulation of food intake.

The objective of this work was to test the hypothesis that a somatotropin (STH)-induced reduction in body fat would prolong the life span of the obese Zucker rat. Two experiments were conducted. In the first experiment, male and female, lean and obese Zucker rats were treated with STH (0 or 2 mg/d bovine STH) for 4 weeks, beginning at 7 months of age. Across phenotypes, STH treatment increased the growth rate by 159%, muscle weights by 14%, and circulating insulin-like growth factor (IGF)-1 by 23%, and decreased carcass fat by 21% (P < 0.05). The second experiment was a longevity trial to determine whether these changes in body composition would increase the life span of the obese rat. Beginning at 7 months of age, individually housed, male and female, lean and obese rats were assigned to daily STH treatments (0 or 2 mg/d). Rats were monitored daily, and sick or moribund rats were euthanized and necropsied to determine existing pathologies. The average life span of the lean rats was 661 days and was unaffected by STH treatment (639 days, NS) or gender. Average life span of the vehicle-injected obese rats (435 days) was less than that of the lean group (P < 0.001). STH treatment of the obese rats resulted in a further reduction of life span (349 days, P < 0.02). The predominant pathology observed across the treatment groups was renal disease, characterized by progressive glomerulonephropathy. Thus, although exogenous STH was able to reduce carcass lipid and to increase lean tissue mass in obese rats, there was no improvement in longevity. In contrast to the hypothesis, STH actually reduced the life span of the obese rat. It is likely that STH treatment accelerated the development of progressive glomerulonephropathy in the obese rat.

It has been suggested that certain cells in the brain, like pancreatic beta-cells, use glucose transporter-2 (GLUT-2), glucokinase and glucagon-like peptide-1 receptor (GLP-1R) to sense glucose in the service of multiple aspects of energy balance. The obese Zucker rat displays numerous disturbances in energy homeostasis and may provide a model of dysfunctional expression of genes related to nutrient control systems. Using real-time RT-PCR we measured gene expression for three of the pancreatic glucose-sensing markers and neuropeptide Y (NPY) in the medial, lateral hypothalamus and hindbrain of lean and obese Zucker rats of both genders. Additionally, we measured circulating levels of glucose, leptin, insulin, corticosterone and glucagon. The results indicate that GLUT-2 mRNA expression is decreased, whereas glucokinase is increased in the hindbrain of obese rats. NPY mRNA level is significantly higher, whereas GLP-1R is significantly lower in the medial hypothalamus in obese individuals. Gender-related differences were found in the hindbrain and medial hypothalamus for GLUT-2 and in the lateral hypothalamus for GLP-1R and they may be related to the fact that the female Zucker rats do not develop diabetes as readily as males. Furthermore, the hindbrain may be an important site for glucose-sensing where major phenotypic changes occur for glucose-sensing genes expression.

Glucokinase (GK), glucose transporter GLUT2, sulfonylurea receptor-1 (SUR1), glucagon-like peptide-1 receptor (GLP-1R) and neuropeptide Y (NPY) have been proposed to be involved in central glucose sensing or regulation of food intake. In this study, we combined tissue micropunch and real time reverse transcription polymerase chain reaction (RT-PCR), and measured GK, GLUT2, SUR1, GLP-1R and NPY mRNA expression in discrete areas in the hypothalamus and the hindbrain.

cope : We examined the intestinal and systemic responses to incorporating a type 2 resistant starch (RS) into a high fat diet fed to obese mice. Methods and results : Diet-induced obese, C57BL/6J mice were fed a HF diet without or with 20% (by weight) high-amylose maize resistant starch (HF-RS) for six weeks. Serum adiponectin levels were higher with RS consumption, but there were no differences in weight gain and adiposity. With HF-RS, the expression levels of ileal TLR2 and Reg3g and cecal occludin, TLR2, TLR4, NOD1 and NOD2 were induced; whereas colonic concentrations of the inflammatory cytokine IL-17A declined. The intestinal, serum, liver, and urinary metabolomes were also altered. HF-RS resulted in lower amino acid concentrations, including lower serum branched chain amino acids, and increased quantities of urinary di/trimethylamine, 3-indoxylsulfate, and phenylacetylglycine. Corresponding to these changes were enrichments in Bacteroidetes (S24-7 family) and certain Firmicutes taxa (Lactobacillales and Erysipelotrichaceae) with the HF-RS diet. Parabacteroides and S24-7 positively associated with cecal maltose concentrations. These taxa and Erysipelotrichaceae, Allobaculum, and Bifidobacterium were directly correlated with uremic metabolites. Conclusion : Consumption of RS modified the intestinal microbiota, stimulated intestinal immunity and endocrine-responses, and modified systemic metabolomes in obese mice consuming an otherwise obesogenic diet. This article is protected by copyright. All rights reserved

International audience; This paper is based on a workshop held in Oslo, Norway in November 2013, in which experts discussed how to reach consensus on the healthiness of red and processed meat. Recent nutritional recommendations include reducing intake of red and processed meat to reduce cancer risk, in particular colorectal cancer (CRC). Epidemiological and mechanistic data on associations between red and processed meat intake and CRC are inconsistent and underlying mechanisms are unclear. There is a need for further studies on differences between white and red meat, between processed and whole red meat and between different types of processed meats, as potential health risks may not be the same for all products. Better biomarkers of meat intake and of cancer occurrence and updated food composition databases are required for future studies. Modifying meat composition via animal feeding and breeding, improving meat processing by alternative methods such as adding phytochemicals and improving our diets in general are strategies that need to be followed up.

The gut microbiota affects health. Research with antibiotics (ampicillin 1 g/L, and neomycin 0.5 g/L in drinking water) reduced bacterial DNA and endotoxemia in mice fed high fat diet or ob/ob mice fed chow. The antibiotics reduced Lactobacillus, Bifidobacterium, and Bacteroides spp. Several of these ferment HAMRS2. We tested if these antibiotics prevented fermentation of HAMRS2 in rats. Phase 1 consisted of feeding two groups of rats with either HAMRS2 diet without antibiotics (n=10) or a diet with no fermentable carbohydrates with antibiotics (n=19). After 3.5 weeks, 5 rats from each group were euthanized for cecal content pH and short chain fatty acid (SCFA) analysis. An additional 5 rats on the HAMRS2 diet were euthanized where their cecal contents were pooled. The cecal pH was significantly lower (p

Two thirds of the US population is overweight or obese and childhood obesity is increasing. Growing evidence supports a connection between obesity, diabetes, metabolic syndrome and environmental chemical exposure. Effects of imidacloprid, a neonicotinoid insecticide, on intestinal fat deposition and lifespan were tested in wild type (N2) and daf-16 deficient (daf-16(mgDf50)I) Caenorhabditis elegans (C. elegans) in liquid culture supplied with 5-fluoro-2’-deoxyuridine (0.24mM). Control group received E. coli (OP50). Experimental group was treated with additional imidacloprid (0.1-1,000nM). Half of the nematodes were fixed in 4% paraformaldehyde, washed with PBS, stained with Nile red and fluorescent spectrometry determined intensity. Imidacloprid dose-dependently reduced mean survival time during 22 day study and increased intestinal fat deposition in N2. The reduced fat deposition was diminished and the lifespan was mildly decreased by imidacloprid in daf-16(mgDf50)I (0.1nM, P=0.003). These data prove tha...

Resistant starch (RS) is a fermentable dietary fiber shown to alter gut hormones and modify the gut microbiome. Because RS impacts the gut microbiota, we hypothesize that RS changes gut-derived signals reaching the liver by altering the gut microbiota and, in turn altering liver metabolism. To assess the effects of RS on the liver metabolome under obesogenic conditions, male C57Bl/6J mice were fed a 45% fat diet for 10 weeks supplemented with type 2 RS (10% by wt) or maltodextrin (n=15/group). As expected, increased cecal weights (by 55%, p

Objective: Our objective in this study was to measure the expression of cyclin D1 in pig primary stromal-vascular (S-V) cells as they differentiate into adipose cells and to identify which factors may alter cyclin D1 expression. Research Methods and Procedures: Western blot analysis was performed on cultured S-V cells using 8% sodium dodecyl sulfate-polyacrylamide gels, mouse monoclonal cyclin D1 antibody, and anti-mouse IgG secondary labeled with horseradish peroxidase. For immunocytochemistry, cultures were fixed with 4% paraformaldehyde and incubated with anti-CCAAT/enhancer binding protein (C/EBPα) and anti-cyclin D1 together. Cyclin D1 expression was evaluated in 105-day fetal dorsal subcutaneous tissues using paraffin sections. Results: Our results with Western blot analysis showed that cyclin D1 was found in freshly isolated S-V cells and continued to be expressed during the first 3 days of adipose cell development with a significant increase in late development at day 9. Elevated cyclin D1 levels were colocalized with C/EBPα beginning at day 3 and remained colocalized with C/EBPα through day 9. Removing insulin from cultures resulted in a reduction in differentially elevated levels of cyclin D1. Discussion: The elevated level of cyclin D1 expression colocalized with C/EBPα expression is unexpected because differentiated adipocytes would be expected to have reduced proliferative potential. The elevated levels of cyclin D1 expression we observed in mature adipocytes depend on insulin. In addition, cyclin D1 is absent from lipid-filled fetal adipose cells in vivo, where insulin levels are very low. The activity of cyclin D1 in differentiated adipocytes may be directed toward proteins outside of the cell cycle.

Kidney dysfunction and mesangial enlargement are consequences of obesity found in Zucker rats. This study examines some of the early mechanisms by which the kidneys of Zucker rats undergo these changes. Our study shows that the glomerular planar area in the genetically obese Zucker rat undergo enlargement as early as 12 weeks of life, compared to the lean controls. This suggests mesangial proliferation is already occurring at this time, earlier than previously shown. The mRNA expression for IGF-I, and GHR in the kidney and liver of the obese Zucker rats were significantly reduced compared to the lean controls. However, the mRNA of the IGF-IR was significantly elevated in the kidney of the obese Zucker rats. The increase in kidney IGF-1R mRNA in the obese Zucker rat may suggest an increase in IGF-1 binding leading to the kidney hypertrophy observed in these rats.

Recent studies have demonstrated a reduction in body fat in growing animals fed conjugated linoleic acid (CLA). Two experiments were conducted to extend these observations to obese rats so that the mechanism of the actions of CLA might be more easily elucidated. In experiment 1, male lean and obese Zucker rats were fed diets containing either 0 or 0.5% CLA for 5 wk. There was no effect of diet on growth rate or food intake. Dietary CLA reduced retroperitoneal and inguinal fat pad weights in the lean rats but increased fat pad weights in the obese genotype (diet x genotype interaction; P < 0.05). Determination of fat pad cellularity indicated that these changes in fat pad weight were due to a reduction or increase in average fat cell size for the lean and obese Zucker rats, respectively. In experiment 2, we sought to reproduce these effects on fat pad size, as well as to determine the effect of dietary CLA on the catabolic response to bacterial endotoxin injection in obese Zucker rats. Growing female lean and obese Zucker rats were fed diets containing 0 or 0.5% CLA for 8 wk. On d 28, each rat was injected intraperitoneally with lipopolysaccharide from Escherichia coli serotype 055:B5 (1 mg/kg body weight) and body weight was determined over the next 96 h. There was a diet x genotype interaction (P < 0.05) for the body weight response to lipopolysaccharide 24 h postinjection. Lean rats fed CLA lost less weight than did lean controls, but obese rats fed CLA lost more weight than did obese controls. As in the first experiment, there was a diet x genotype (P < 0.05) for the effect of treatment on retroperitoneal fat pad weights determined at the end of the experiment. Lean rats fed CLA had smaller RP fat pads than did lean controls, but obese rats fed CLA once again had heavier RP fat pads than did obese controls. These results indicate that CLA reduces body fat and catabolic response to endotoxin injection in lean Zucker rats but not in the obese genotype. The observed interaction between diet and genotype warrants additional investigation into the specific mechanism(s) of the biological activities of CLA.

cope Antibiotics ampicillin 1g/L and neomycin 0.5g/L were added to drinking water before or during feeding of resistant starch (RS) to rats to inhibit fermentation. Methods and Results In a preliminary study antibiotics and no RS were given prior to rats receiving a transplant of cecal contents via gavage from donor rats fed RS (without antibiotics) or a water gavage before feeding resistant starch to both groups. Antibiotics given prior to feeding RS did not prevent later fermentation of RS regardless of either type of gavage. In the second study antibiotics were given simultaneously with feeding of RS. This resulted in inhibition of fermentation of RS with cecal contents pH >8 and low amounts of acetate and butyrate. Rats treated with antibiotics had reduced Bifidobacteria spp., but similar Bacteroides spp. to control groups to reduce acetate and butyrate and preserve the production of propionate. Despite reduced fermentation, rats given antibiotics had increased glucagon-like peptide 1 (GLP-1) and cecum size, measures that are usually associated with fermentation. Conclusions A simultaneous delivery of antibiotics inhibited fermentation of RS. However, increased GLP-1 and cecum size would be confounding effects in assessing the mechanism for beneficial effects of dietary RS by knocking out fermentation. This article is protected by copyright. All rights reserved

OBJECTIVE Elevated levels of tumor necrosis factor-alpha (TNF-alpha) protein and mRNA have been reported in adipose tissue from obese humans and rodents. However, TNF-alpha has catabolic and antiadipogenic effects on adipocytes. Addressing this paradox, we tested the hypothesis that paracrine levels of TNF-alpha, alone or together with insulin-like growth factor-I (IGF-I), support preadipocyte development. RESEARCH METHODS AND PROCEDURES Cultured stromal-vascular cells from rat inguinal fat depots were exposed to serum-free media containing insulin and 0.2 nM TNF-alpha, 2.0 nM TNF-alpha, or 0.2 nM TNF-alpha + 1.0 nM IGF-I at different times during 7 days of culture. RESULTS TNF-alpha inhibited adipocyte differentiation as indicated by a reduction in both immunocytochemical reactivity for the preadipocyte-specific antigen (AD3; early differentiation marker) and glycerol-3-phosphate dehydrogenase activity (late differentiation marker). Early exposure (Days 1 through 3 of culture) to 0.2 nM TNF-alpha did not have a long term effect on inhibiting differentiation. Continuous exposure to 0.2 nM TNF-alpha from Days 1 through 7 of culture resulted in a 75% increase in cell number from control. There was a synergistic effect of 0.2 nM TNF-alpha + 1 nM IGF-I on increasing cell number by Day 7 of culture to levels greater than those observed with either treatment applied alone. DISCUSSION These data suggest that paracrine levels (0.2 nM) of TNF-alpha alone or in combination with IGF-I may support adipose tissue development by increasing the total number of stromal-vascular and/or uncommitted cells within the tissue. These cells may then be recruited to become preadipocytes or may alternatively serve as infrastructure to support adipose tissue growth.

To determine if high-fat (HF) diet-induced changes in adipose tissue cellularity are associated with the presence of paracrine growth factor(s) that alter preadipocyte proliferation, Osborne-Mendel rats were fed either a HF (76% energy) or a low-fat (LF, 12% energy) diet for 85 days. HF-fed rats had greater ( P < 0.05) fat pad size, total fat cell number, number of small (30–70 μm) and large (80–140 μm) adipocytes, and percentage of 100- to 140-μm adipocytes compared with LF-fed rats. Preadipocytes in primary cell culture treated with inguinal adipose tissue conditioned medium (ATCM) prepared from HF-fed rats had greater ( P < 0.05) proliferation compared with cultures treated with ATCM from LF-fed rats. Proliferative capacity of ATCM prepared from HF-fed rats was attenuated after the stripping of the medium of insulin-like growth factor I using an immunomagnetic bead separation system. These data are consistent with the concept that insulin-like growth factor I is involved in the paracrine regulation of adipogenesis.

Control pig fetuses and fetuses hypophysectomized (hypox) at 70 days of gestation were treated with hormones on day 90. At 105 days of gestation, subcutaneous adipose tissue was prepared for morphological studies, leptin Northern blot and Western blot analysis. Fetal ontogeny studies showed that leptin mRNA in adipose tissue increased with morphological development, and the highest level of leptin mRNA was observed in 105-day fetal pigs. In hypox fetuses, levels of leptin mRNA were similar to those in controls. Treatment with either hydrocortisone (HC) or thyroxine (T4) resulted in a slight increase in leptin mRNA levels in hypox fetuses but not in intact fetuses. Supplementation with both HC and T4 markedly stimulated leptin mRNA expression in both hypox and intact fetuses. Morphological data showed that hypox slightly enhanced lipid accretion and treatment of hypox fetuses with HC and T4 increased lipid accretion to a greater degree than did either HC or T4 alone. However, serum leptin levels were not influenced by age, hypox or hormone treatment. Leptin protein expression was not detected in adipose tissue of hypox or intact fetuses regardless of hormone treatment. Leptin protein was detected in adipose tissue of 7-day-old pigs and placenta. As compared to 7-day postnatal adipose tissue, placenta showed a higher level of leptin protein expression. Leptin mRNA expression in fetal adipose tissue was not correlated with body weight and organ weight. The expression of long-form leptin receptor mRNA was detected in fetal adipose tissue. Our results indicate that adipose tissue leptin may not be the main source of serum leptin in the fetus and may not be involved in general prenatal growth and development. But adipose tissue leptin may act as an autocrine or paracrine factor in the development of fetal adipose tissue.

Inguinal, epididymal, and retroperitoneal adipose tissue from lean and obese Zucker rats, 3–15 wk of age, was used to determine the association among adipocyte size distribution, the presence of paracrine growth factors in adipose tissue, and subsequent changes in adipocyte number. For each specific depot and time point, obese rats had a greater percentage of large adipocytes than did lean rats. A positive correlation ( P < 0.02) was found in obese rats between the percentage of inguinal and epididymal adipocytes in the 140- to 180-μm size range and the ability of conditioned medium prepared from these depots to stimulate cellular proliferation in a bioassay system utilizing preadipocytes from inguinal fat pads of normal rats. Proliferative activity of the conditioned medium from all depots in obese rats was positively correlated ( P < 0.01) to subsequent changes in fat cell number. The data presented here for the inguinal and epididymal depot of obese Zucker rats are consistent with the hypothesis that enlarged adipocytes secrete growth factors that induce preadipocyte proliferation.