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Nonalcoholic fatty liver disease (NAFLD) is a major cause of chronic liver abnormalities and has been linked with metabolic syndrome hallmarks. Unfortunately, current treatments are limited. This work aimed to elucidate the effects of three cannabis extracts on metabolic alteration and gut microbiota composition in a mouse model of NAFLD and obesity. Male mice were fed with a high-fat diet (HFD) for 12 weeks. Following the establishment of obesity, the HFD-fed group was subdivided into HFD or HFD that was supplemented with one of three cannabis extracts (CN1, CN2, and CN6) for additional 8 weeks. Metabolic parameters together with intestinal microbiota composition were evaluated. Except for several minor changes in gene expression, no profound metabolic effect was found due to cannabis extracts addition. Nevertheless, marked changes were observed in gut microbiota diversity and composition, with CN1 and CN6 exhibiting microbial abundance patterns that are associated with more beneficial outcomes. Taken together, specific cannabis extracts’ addition to an HFD results in more favorable modifications in gut microbiota. Although no marked metabolic effect was disclosed, longer treatments duration and/or higher extracts concentrations may be needed. More research is required to ascertain this conjecture and to establish the influence of various cannabis extracts on host health in general and NAFLD in particular.

Background Dietary oils differ in their fatty acid composition and the presence of additional microcomponents (antioxidants, etc.). These differences are thought to invoke different biochemical pathways, thus affecting fats and carbohydrates metabolism differently. Olive oil (OO) and soybean oil (SO) are common vegetable oils in the local cuisine. Peanuts oils of local varieties are viewed as potential sources of dietary vegetable oils, especially in the food industry. Objective We examined the effect of four different dietary vegetable oils on carbohydrate and lipid metabolism in mice. The selected oils were OO, high in oleic acid, extracted from cultivated high oleic acid peanut (C-PO), regular peanut oil (PO), and SO. Design In this study, 32 male C57BL/6J mice were randomly divided into four groups (n = 8 in each group) and were fed with four different diets enriched with 4% (w/w) dietary vegetable oils (OO, C-PO, PO, or SO). After 10 weeks, the mice were sacrificed. Western blot was used to examine proteins such as phospho-AMP-activated protein kinase (p-AMPK), ace-tyl-CoA carboxylase (ACC), cluster of differentiation 36 (CD36), and Sirtuin 1 (SIRT1), whereas real-time polymerase chain reaction (PCR) was used to examine the expression of sterol regulatory element-binding protein-1c (SREBP-1C), fatty acid synthase (FAS), glucose-6-phosphatase (G6Pase), and CD36 transcripts. Results In mice-fed SO, lipid accumulation was predominately in adipose tissue, accompanied a tendency decrease in insulin sensitivity. Mice-fed OO had lower plasma triglycerides (TG) and increased hepatic CD36 gene expression. The C-PO group presented lower messenger RNA (mRNA) levels in the liver for all examined genes: SREBP-1c, FAS, G6Pase, and CD36. There were no significant differences in weight gain, plasma cholesterol and high-density lipoprotein (HDL) cholesterol levels, hepatic ACC, SIRT1, AMPK, and CD36 protein levels or in liver function among the diets. Discussion It seems that as long as fat is consumed in moderation, oil types may play a lesser role in the metabolism of healthy individuals. Conclusion This finding has the potential to increase flexibility in choosing oil types for consumption.

Background: Oleic-acid consumption can possibly prevent or delay metabolic diseases. In Israel, a Virginia-type peanut cultivar with a high content of oleic acid has been developed. Objective: This study examined the effect of consuming high oleic peanuts (D7) on the development of fatty liver compared to the standard HN strain. Design: The two peanut cultivars were added to normal diet (ND) and high-fat (HF) mouse diet. Male C57BL/6 mice were fed for 8 and 10 weeks on a 4% D7, 4% HN, or control diet. At the end of the experiments, blood and tissues were collected. Triglyceride, lipid levels, histology, and protein expression were examined. The diets’ effects on intestinal microbiota were also evaluated. Results: Both D7 and HFD7 led to a reduction in plasma triglycerides. Lipids, triglycerides, and free fatty acids in the liver were low in diets containing D7. Additionally, CD36 expression decreased in the D7 group. Consumption of D7 led to higher Prevotella levels, and consumption of ND that contained HN or D7 led to a lower Firmicutes/Bacteroidetes ratio. Conclusion: These findings suggest that consumption of peanuts high in oleic acid (D7) may have the potential to delay primary fatty liver symptoms.

Quantification of fatty vacuoles in the liver, with differentiation from lumina of liver blood vessels and bile ducts, is an example where the traditional semiquantitative pathology assessment can be enhanced with artificial intelligence (AI) algorithms. Using glass slides of mice liver as a model for nonalcoholic fatty liver disease, a deep learning AI algorithm was developed. This algorithm uses a segmentation framework for vacuole quantification and can be deployed to analyze live histopathology fields during the microscope-based pathology assessment. We compared the manual semiquantitative microscope-based assessment with the quantitative output of the deep learning algorithm. The deep learning algorithm was able to recognize and quantify the percent of fatty vacuoles, exhibiting a strong and significant correlation ( r = 0.87, P < .001) between the semiquantitative and quantitative assessment methods. The use of deep learning algorithms for difficult quantifications within the microscope-based pathology assessment can help improve outputs of toxicologic pathology workflows.

Background: Nonalcoholic fatty-liver disease (NAFLD) is a global health problem associated with gut dysbiosis and intestinal permeability. Broccoli is a natural source of bioactive phytochemicals, characterized by health-promoting properties. Objective: This study evaluated the effect of broccoli florets and stalks on liver fat accumulation, inflammation, gut microbiome, and intestinal barrier integrity. Design: Male C57BL/6J mice (n = 32, 8-week-old) were fed with a high-fat high-cholesterol diet (HFCD) with/without 15% broccoli (florets or stalks) for 7 weeks. Liver damage was evaluated by changes in glucose response and histological and biochemical parameters. Protein and gene expressions related to liver inflammation were examined. The effect of broccoli on microbiota population together with genes related to barrier integrity in the gut was investigated. Results: Dietary broccoli improved the glycemic response assessed by oral glucose tolerance test (OGTT). Histological evaluation showed no change in hepatic steatosis. Broccoli consumption also attenuated inflammation as revealed by lower inducible nitric oxide synthase (iNOS) and serum amyloid A1 (SAA1) expression levels in broccoli-supplemented groups. Gut microbiota analysis demonstrated elevated Acidifaciens and reduced Mucispirillum schaedleri abundance in the stalks group, whereas Proteobacteria strains abundance was increased in the florets group. Gut integrity remained unchanged. Conclusion: Broccoli supplementation improves glucose tolerance, attenuates liver inflammation, and alters microbial composition, but does not affect gut integrity. This research provides new evidence on the effects of dietary broccoli under HFCD.

Nonalcoholic fatty liver disease (NAFLD) is linked to obesity, type 2 diabetes, hyperlipidemia, and gut dysbiosis. Gut microbiota profoundly affects the host energy homeostasis, which, in turn, is affected by a high-fat diet (HFD) through the liver-gut axis, among others. Broccoli contains beneficial bioactive compounds and may protect against several diseases. This study aimed to determine the effects of broccoli supplementation to an HFD on metabolic parameters and gut microbiome in mice. Male (7–8 weeks old) C57BL/J6 mice were divided into four groups: normal diet (ND), high-fat diet (HFD), high-fat diet+10% broccoli florets (HFD + F), and high-fat diet + 10% broccoli stalks (HFD + S). Liver histology and serum biochemical factors were evaluated. Alterations in protein and gene expression of the key players in lipid and carbohydrate metabolism as well as in gut microbiota alterations were also investigated. Broccoli florets addition to the HFD significantly reduced serum insulin levels, HOMA-IR index, and upregulated adiponectin receptor expression. Conversely, no significant difference was found in the group supplemented with broccoli stalks. Both broccoli stalks and florets did not affect fat accumulation, carbohydrate, or lipid metabolism-related parameters. Modifications in diversity and in microbial structure of proteobacteria strains, Akermansia muciniphila and Mucispirillum schaedleri were observed in the broccoli-supplemented HFD-fed mice. The present study suggests that dietary broccoli alters parameters related to insulin sensitivity and modulates the intestinal environment. More studies are needed to confirm the results of this study and to investigate the mechanisms underlying these beneficial effects.

Background Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome, which often includes obesity, diabetes, and dyslipidemia. Several studies in mice and humans have implicated the involvement of the gut microbiome in NAFLD. While cannabis and its phytocannabinoids may potentially be beneficial for treating metabolic disorders such as NAFLD, their effects on liver diseases and gut microbiota profile have yet to be addressed. In this study, we evaluated the therapeutic effects of the two major cannabinoids, delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD), on NAFLD progression. Methods NAFLD was induced by feeding mice a high fat-cholesterol diet (HFCD) for 6 weeks. During this period, the individual cannabinoids, THC or CBD, were added to the experimental diets at a concentration of 2.5 or 2.39 mg/kg. Profile of lipids, liver enzymes, glucose tolerance, and gene expression related to carbohydrate lipids metabolism and liver inflammation was analyzed. The effect of THC or CBD on microbiota composition in the gut was evaluated. Results While not alleviating hepatic steatosis, THC or CBD treatment influenced a number of parameters in the HFCD mouse model. CBD increased food intake, improved glucose tolerance, reduced some of the inflammatory response including TNFa and iNOS, and partially mitigated the microbiome dysbiosis observed in the HFCD fed mice. THC produced a much weaker response, only slightly reducing inflammatory-related gene expression and microbiome dysbiosis. Conclusions The results of this study indicate the potential therapeutic effects of individual phytocannabinoids are different from the effects of the cannabis plant possessing a mixture of compounds. While CBD may help ameliorate symptoms of NAFLD, THC alone may not be as effective. This disparity can putatively be explained based on changes in the gut microbiota.

Introduction: Nonalcoholic fatty liver disease (NAFLD) is associated with metabolic syndrome, which often includes obesity, diabetes, and dyslipidemia. Several studies in mice and humans have implicated the involvement of the gut microbiome in NAFLD. While cannabis may potentially be beneficial for treating metabolic disorders such as NAFLD, the effects of cannabis on liver diseases and gut microbiota profile are yet to be addressed. In this study, we evaluated the therapeutic effects of cannabis strains with different cannabinoid profiles on NAFLD progression. Materials and Methods: NAFLD was induced by feeding mice a high-fat/cholesterol diet (HFCD) for 6 weeks. During this period, cannabis extracts were administrated orally at a concentration of 5 mg/kg every 3 days. Profile of lipids, liver enzymes, glucose tolerance, and gene expression related to carbohydrate lipid metabolism and liver inflammation were analyzed. The effect of cannabis strains on microbiota composition in the gut was evaluated. Results: A cannabidiol (CBD)-rich extract produced an increase in inflammatory related gene expression and a less diverse microbiota profile, associated with increased fasting glucose levels in HFCD-fed mice. In contrast, mice receiving a tetrahydrocannabinol (THC)-rich extract exhibited moderate weight gain, improved glucose response curves, and a decrease in liver enzymes. Conclusions: The results of this study indicate that the administration of cannabis containing elevated levels of THC may help ameliorate symptoms of NAFLD, whereas administration of CBD-rich cannabis extracts may cause a proinflammatory effect in the liver, linked with an unfavorable change in the microbiota profile. Our preliminary data suggest that these effects are mediated by mechanisms other than increased expression of the endocannabinoid receptors cannabinoid receptor 1 (CB1) and CB2.

Nonalcoholic steatohepatitis (NASH) is currently one of the most common liver diseases worldwide. The toxic effects of lipids and bile acids contribute to NASH. The regenerative pathway in response to damage to the liver includes activation of the inflammatory process and priming of hepatocytes to proliferate to restore tissue homeostasis. However, the effects of cholesterol on bile acid toxicity, inflammation, and fibrosis remain unknown. We have used two mouse models of bile acid toxicity to induce liver inflammation and fibrosis. A three-week study was conducted using wild-type mice receiving an atherogenic diet (1% (w/w) cholesterol and 0.5% (w/w) cholic acid) and its separate constituents. Mdr2-/- mice were fed a high-cholesterol-enriched diet or standard AIN-93 diet for 6 weeks. We measured serum transaminase levels to assess liver tissue necrosis and fibrosis; iNOS, SAA1, SAA2, and F4/80 levels to determine liver inflammation; PCNA and HGF levels to evaluate proliferative response; and Nrf-2, HIF-1α, and downstream gene expression to establish protective responses. In both studies, high bile acid levels increased serum transaminases and liver fibrosis, whereas cholesterol supplementation attenuated these effects. Cholesterol supplementation activated survival and the robustness of HIF-1α and Nrf-2 gene expression in hepatocytes, induced liver inflammation and hepatocyte proliferation, and inhibited stellate cell hyperplasia and fibrosis. In conclusion, our data show for the first time that cholesterol intake protects against bile acid liver toxicity. The balance between hepatic cholesterol and bile acid levels may be of prognostic value in liver disease progression and trajectory.

Nonalcoholic steatohepatitis (NASH) is currently one of the most common liver diseases worldwide. The toxic effects of lipids and bile acids contribute to NASH. The regenerative pathway in response to damage to the liver includes activation of the inflammatory process and priming of hepatocytes to proliferate to restore tissue homeostasis. However, the effects of cholesterol on bile acid toxicity, inflammation, and fibrosis remain unknown. We have used two mouse models of bile acid toxicity to induce liver inflammation and fibrosis. A three-week study was conducted using wild-type mice receiving an atherogenic diet (1% (w/w) cholesterol and 0.5% (w/w) cholic acid) and its separate constituents. Mdr2-/- mice were fed a high-cholesterol-enriched diet or standard AIN-93 diet for 6 weeks. We measured serum transaminase levels to assess liver tissue necrosis and fibrosis; iNOS, SAA1, SAA2, and F4/80 levels to determine liver inflammation; PCNA and HGF levels to evaluate proliferative response; and Nrf-2, HIF-1¿, and downstream gene expression to establish protective responses. In both studies, high bile acid levels increased serum transaminases and liver fibrosis, whereas cholesterol supplementation attenuated these effects. Cholesterol supplementation activated survival and the robustness of HIF-1¿ and Nrf-2 gene expression in hepatocytes, induced liver inflammation and hepatocyte proliferation, and inhibited stellate cell hyperplasia and fibrosis. In conclusion, our data show for the first time that cholesterol intake protects against bile acid liver toxicity. The balance between hepatic cholesterol and bile acid levels may be of prognostic value in liver disease progression and trajectory, The manuscript was supported by the Israel Science Foundation grant 371/12 to ZM and OT.

Objective Our goal was to evaluate the effect of breakfast size and composition on body weight, glycemic control, and metabolic markers in adults with type 2 diabetes mellitus (T2DM). Methods 59 overweight/obese adults with T2DM were randomized to one of two isocaloric diabetic diets for 3 months; big breakfast (BB), breakfast was rich in fat and protein and provided 33% of total daily energy or small breakfast (SB), breakfast was rich in carbohydrates and provided 12.5% of total daily energy. Results Although body weight was reduced similarly in both groups, the BB group showed greater HbA1c and systolic blood pressure reductions (HbA1c: −4.62% vs. −1.46%, p = 0.047; SBP −9.58 vs. −2.43 mmHg; p = 0.04). T2DM medication dose was reduced in a greater proportion of the BB participants (31% vs. 0%; p = 0.002) while in the SB, a greater proportion of participants had a dose increases (16.7% vs. 3.4%; p = 0.002). Hunger scores were lower in the BB group and greater improvements in fasting glucose were observed in the BB group. Conclusions A simple dietary manipulation enriching breakfast with energy as protein and fat appears to confer metabolic benefits and might be a useful alternative for the management of T2DM.

This study was designed to investigate the effect of a low-calorie diet with carbohydrates eaten mostly at dinner on anthropometric, hunger/satiety, biochemical, and inflammatory parameters. Hormonal secretions were also evaluated. Seventy-eight police officers (BMI >30) were randomly assigned to experimental (carbohydrates eaten mostly at dinner) or control weight loss diets for 6 months. On day 0, 7, 90, and 180 blood samples and hunger scores were collected every 4 h from 0800 to 2000 hours. Anthropometric measurements were collected throughout the study. Greater weight loss, abdominal circumference, and body fat mass reductions were observed in the experimental diet in comparison to controls. Hunger scores were lower and greater improvements in fasting glucose, average daily insulin concentrations, and homeostasis model assessment for insulin resistance (HOMA(IR)), T-cholesterol, low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) levels were observed in comparison to controls. The experimental diet modified daily leptin and adiponectin concentrations compared to those observed at baseline and to a control diet. A simple dietary manipulation of carbohydrate distribution appears to have additional benefits when compared to a conventional weight loss diet in individuals suffering from obesity. It might also be beneficial for individuals suffering from insulin resistance and the metabolic syndrome. Further research is required to confirm and clarify the mechanisms by which this relatively simple diet approach enhances satiety, leads to better anthropometric outcomes, and achieves improved metabolic response, compared to a more conventional dietary approach.

Accumulating evidence indicates that mitochondria have a key role in non-alcoholic fatty liver disease (NAFLD). C57BL/6J mice were fed a choline-deficient, ethionine-supplemented (CDE) diet. Histological studies demonstrated accumulation of fat vacuoles in up to 90% of hepatocytes in mice fed the CDE diet for 14 days. In addition, a decrease in mitochondrial levels, together with an increase in superoxide radicals' levels were observed, indicating elevation of oxidative stress in hepatocytes. ATP levels were decreased in livers from CDE-fed mice after overnight fasting. This was accompanied by a compensative and significant increase in peroxisome-proliferator-activated receptor-γ coactivator 1α (PGC1α) mRNA levels in comparison to control livers. However, there was a reduction in PGC1α protein levels in CDE-treated mice. Moreover, the expression of mitochondrial biogenesis genes nuclear respiratory factor 1 (NRF-1), mitochondrial transcription factor A (TFAM), mitochondrial transcription factor B1 (TFB1M) and mitochondrial transcription factor B2 (TFB2M), which are all regulated by PGC1α activity, remained unchanged in fasted CDE-treated mice. These results indicate impaired activity of PGC1α. The impaired activity was further confirmed by chromatin immunoprecipitation analysis, which demonstrated decreased interaction of PGC1α with promoters containing NRF-1 and NRF-2 response elements in mice fed the CDE diet. A decrease in PGC1α ability to activate the expression of the gluconeogenic gene phosphoenol-pyruvate carboxykinase was also observed. This study demonstrates, for the first time, that attenuated mitochondrial biogenesis in steatotic livers is associated with impaired biological activity of PGC1α.

The circadian clock controls energy homeostasis by regulating circadian expression of proteins involved in metabolism. Disruption of circadian rhythms leads to obesity and metabolic disorders. Little is known regarding the control of the biological clock over adiponectin signaling pathway in adipose tissue, the adiponectin producer, and muscle, an adiponectin target tissue under fasting, low-fat (LF), or high-fat (HF) diet. Mice were fed LF or HF diet for 7 weeks and fasted on the last day. The circadian mRNA expression of clock genes and components of adiponectin metabolic pathway (mAdipoR1, mAdipoR2, mPparalpha, mPpargamma, mAmpk, and mAcc) in the muscle and adipose tissue were tested. Using average daily levels of multiple time points around the circadian cycle, we assessed mRNA levels of the different adiponectin signaling components. In addition, serum glucose, adiponectin, and insulin were measured. Under LF diet, adiponectin signaling pathway components exhibited circadian rhythmicity at the mRNA levels. Fasting and HF diet followed by fasting disrupted this circadian expression causing a phase advance or delay, respectively. Changes were also found in the expression levels of adiponectin receptor, mAmpk, mAcc, mPparalpha, and mPpargamma reflecting a defect in adiponectin signaling. As both peroxisome proliferator-activated receptor alpha (PPARalpha) and mAMPK are linked to the core clock mechanism, they could mediate the disruptions seen in clock gene expression under HF diet. In turn, the circadian clock affects the daily rhythm of these adiponectin signaling components.

Summary Background & aims Fatty liver, inflammation, fibrosis and cirrhosis are associated with portal hypertension. Nitric oxide (NO) is a universal messenger molecule that plays diverse and essential physiological roles in multiple organ systems. A decrease in NO production can lead to disruption in blood flow and portal hypertension. We therefore sought to evaluate the in vivo effect of lipid accumulation in the liver on NO production, endothelial nitric oxide synthase (eNOS) expression and liver function. Methods Rats were intravenously infused with lipid emulsion (20% w/w triglycerides) for 1, 2, 4 and 6 days. Liver histology, blood parameters, and liver eNOS protein expression were studied. Results Histological evaluation and blood liver enzymes showed liver damage after 6 days of infusion, while a decrease in eNOS levels began at day 4. The latter was accompanied by an increase in 4-hydroxynonenal-protein adducts, a marker of oxidative stress. Conclusions These findings support the hypothesis that downregulation of eNOS in steatotic livers is an early event in non-alcoholic fatty liver disease progression.

Effective nutritional guidelines for reducing abdominal obesity and metabolic syndrome are urgently needed. Over the years, many different dietary regimens have been studied as possible treatment alternatives. The efficacy of low-calorie diets, diets with different proportions of fat, protein, and carbohydrates, traditional healthy eating patterns, and evidence-based dietary approaches were evaluated. Reviewing literature published in the last 5 y reveals that these diets may improve risk factors associated with obesity and metabolic syndrome. However, each diet has limitations ranging from high dropout rates to maintenance difficulties. In addition, most of these dietary regimens have the ability to attenuate some, but not all, of the components involved in this complicated multifactorial condition. Recently, interest has arisen in the time of day foods are consumed (food timing). Studies have examined the implications of eating at the right or wrong time, restricting eating hours, time allocation for meals, and timing of macronutrient consumption during the day. In this paper we review new insights into well-known dietary therapies as well as innovative time-associated dietary approaches for treating obesity and metabolic syndrome. We discuss results from systematic meta-analyses, clinical interventions, and animal models.

Background Interventional studies have shown that increased intake of fruit and vegetables reduces blood pressure (BP). However, the contribution of specific dietary components has not been evaluated. The aim of the present study was to determine, in patients with stage I hypertension, the antihypertensive effect of juice of the so-called sweetie fruit (a hybrid between grapefruit and pummelo) with and without high flavonoid content. Methods A double-blind, cross-over study was conducted in 12 patients. Each patient received alternately high-flavonoid (HF) sweetie juice and low-flavonoid (LF) sweetie juice, each for a 5-week period. The LF sweetie juice had 25% of naringin and 30% of narirutin content compared with the original HF sweetie juice. Results The HF sweetie juice was more effective than LF sweetie juice in reducing diastolic blood pressure (P = .04). Systolic blood pressure declined in both groups; however there was no significant difference between subjects given HF sweetie versus those given LF sweetie juice. Conclusions In this study HF sweetie juice was shown to have a significant beneficial effect in reducing diastolic blood pressure, compared with the effect observed with LF sweetie juice, in patients with stage I hypertension. These data suggest that the active ingredients associated with the antihypertensive effect of sweetie juice are the flavonoids naringin and narirutin.

This study was designed to evaluate the effects of triacylglycerol (TG) on nitric oxide (NO) production, expression of endothelial (e) and inducible (i) nitric oxide synthase (NOS) and variables related to oxidative stress in rat isolated hepatocytes. Hepatocytes were isolated and exposed to TG in the form of a lipid emulsion (0.01-0.1% LE). Exposure to LE dose dependently decreased nitrite levels. Nitrite levels were inhibited 67% and intracellular reactive oxygen species (ROS) levels were increased 250% at 0.1% LE. The decline in nitrite levels was accompanied by 37 and 67% reductions in iNOS and eNOS expressions, respectively. To evaluate whether the increased oxidative stress inhibited NOS synthesis, cells were treated for 48 h with rotenone (a mitochondrial complex 1 inhibitor) or buthionine sulfoximine (a glutathione synthesis inhibitor). Both compounds elevated ROS production, which was followed by inhibition of nitrite production. To determine whether there is an association between LE-mediated ROS production and the inhibition of NO synthesis by the LE, hepatocytes were treated with antioxidants. N-Acetyl-l-cysteine (NAC), ascorbate, and resveratrol attenuated the reduction of nitrite levels due to LE alone. NAC inhibited the reductions in eNOS and iNOS transcription and protein levels. Nuclear factor-kappaB (NF-kappaB), one of the transcription factors involved in eNOS and iNOS transcriptional regulation, was decreased 15% in the nucleus by LE treatment. These results suggest that TG reduces nitrite production by elevating intracellular ROS levels (prolonged oxidative stress), and the downregulation of NOS enzymes may occur at least in part via the NFkappaB pathway.

Hypoxia inducible factor-1 (HIF-1) and cholesterol have been separately implicated in the development of several liver pathologies. The aim of the present study was to investigate possible activation of HIF-1 pathway by cholesterol in AML12-hepatocytes. Increased HIF-1α protein accumulation was evident in hepatocytes after 2h of incubation with cholesterol while enhanced HIF-1 transcriptional activity was observed after 6h. Investigating the molecular mechanism revealed that cholesterol inhibited HIF-1α proteasomal degradation. Hepatocytes incubated with cholesterol exhibited enhanced Nitric oxide (NO) levels, which corresponded with increased iNOS induction. Inhibition of iNOS had no effect on HIF-1α stabilization at 2h but effectively attenuated cholesterol-induced HIF-1 accumulation and transcriptional activity at 6h. Mitochondrial dysfunction and enhanced mitochondrial ROS generation were observed in 2h cholesterol-treated cells. In order to establish a role for mitochondria-derived ROS in HIF-1α prot...

The present study investigated the effects of the red microalgaPorphyridiumsp. on gastrointestinal physiology and lipid metabolism in male Sprague-Dawley rats. Diets containing dietary fibre from pelleted red microalgal cells (biomass) or their sulfated polysaccharide, pectin or cellulose (control) were fed to rats for a period of 30 d. All three fibre-supplemented diets increased the length of both the small intestine and colon, with a significantly greater effect in rats fed the algal polysaccharide. The polysaccharide also increased mucosa and muscularis cross-sectional area of the jejunum, and caused hypertrophy in the muscularis layer. The algal biomass significantly lowered gastrointestinal transit time by 44 % in comparison with the control rats. Serum and mucosal cholecystokinin levels were lower in rats on the pectin and polysaccharide diets, while cholecystokinin levels in rats fed algal biomass were not different from those in the control animals. In comparison with the control diet, all the experimental diets significantly lowered serum cholesterol levels (22–29 %). Feeding of non-fermentable algal polysaccharide or biomass significantly increased faecal weight and bile acid excretion compared with pectin-fed or control rats. The algal polysaccharide and biomass were thus shown to be potent hypocholesterolaemic agents active at low concentrations in the diet. Both metabolic and morphological changes were observed following consumption of algae, suggesting several possible mechanisms by which the alga affects lipid metabolism. The results presented in the present study encourage the use of red microalga as a functional food.

Glucose metabolism was studied in 31 pregnant ewes fed mixtures (50:50) of chopped alfalfa hay and corn grain given in whole (HWC) or extruded form (HEC). Number of fetuses was 1 (n = 10), 2 (n = 11) or 3 (n = 10). Diets were supplied at three dietary levels: maintenance (1.0 M), 1.25 x maintenance (1.25 M) or twice maintenance (2.0 M). Glucose metabolism was estimated by a double isotope dilution procedure at days 97-121 of pregnancy. Glucose entry rate (GER) was higher (P < 0.04), and glucose irreversible loss rate (GILR) tended to be higher (P < 0.07) in ewes given HEC than HWC. Dietary level (P < 0.001) and the number of fetuses (P < 0.005) affected GER and GILR positively. These results provide a probable explanation for higher birth weights previously found in lambs born to HEC-, compared with HWC-fed dams.

Fenugreek gum was extracted from fenugreek seeds and evaluated for its surface activity. This unique galactomannan has a mannose backbone grafted with galactose units at an average ratio of one. The purified fenugreek gum was found to reduce surface tension to values lower than guar gum (42 and 55 mN/m, respectively). The interfacial activity was surprisingly better than other galactomannans (interfacial tension was reduced to 2 mN/m in vegetable oils) which led to the formation of oil-in-water emulsions with small droplet size (2–3 μm) and long-term stability. The fenugreek gum was found to adsorb (or ‘precipitate’) on the oil interface forming a relatively thick interfacial film. The emulsions are more stable than any equivalent emulsions stabilized by other galactomannan gums. No flocculation was observed in emulsions stabilized with fenugreek concentrations sufficient for a good coverage of the oil interface as expressed by the coverage index (Rc) correlated to the gum/oil weight ratio (Rc>12).

As part of a program aimed at increasing lamb survival by maximizing lamb birth weight, the relationship between corn starch degradability in late pregnancy diets and lambing performance was studied in 60 Finn–Awassi ewes fed diets supplemented with 500 g d−1 of whole (WC), extruded (EC) or ground corn (GC). Lamb birth- weights ranked as follows: single lambs: EC > WC > GC (NS at 2nd parity, P WC = GC (P

Our goal was to evaluate the effect of breakfast size and composition on body weight, glycemic control, and metabolic markers in adults with type 2 diabetes mellitus (T2DM).59 overweight/obese adults with T2DM were randomized to one of two isocaloric diabetic diets for 3 months; big breakfast (BB), breakfast was rich in fat and protein and provided 33% of total daily energy or small breakfast (SB), breakfast was rich in carbohydrates and provided 12.5% of total daily energy.Although body weight was reduced similarly in both groups, the BB group showed greater HbA1c and systolic blood pressure reductions (HbA1c: -4.62% vs. -1.46%, p = 0.047; SBP -9.58 vs. -2.43 mmHg; p = 0.04). T2DM medication dose was reduced in a greater proportion of the BB participants (31% vs. 0%; p = 0.002) while in the SB, a greater proportion of participants had a dose increases (16.7% vs. 3.4%; p = 0.002). Hunger scores were lower in the BB group and greater improvements in fasting glucose were observed in the BB group.A simple dietary manipulation enriching breakfast with energy as protein and fat appears to confer metabolic benefits and might be a useful alternative for the management of T2DM.

Structural modifications of the intestine following fiber intake may play an important role in the physiological effects induced by dietary fiber. The aim of this study was to examine possible mechanisms for the hypocholesterolemic effects of dietary fibers by evaluating both biochemical and morphometric parameters. Thirty male Sprague-Dawley rats were divided into 3 groups and fed either a fiber-free, 15% fiber high methoxy pectin, or 15% cellulose diet for a 4-wk period. Plasma cholesterol levels, hepatic enzyme activity, and fecal bile acid excretion were determined, and changes in the intestinal structure were evaluated. Daily fecal bile acid excretion was significantly higher in rats fed the pectin diet accompanied by elevated HMG-CoA reductase activity. Pectin feeding led to significant elongation of both the small and large intestine whereas cellulose only affected colon length. Computerized image analysis of intestinal cross-sections showed enlarged muscle area in the ileum and midcolon of pectin-fed rats and greater mucosal area in the colon. Circular muscle cell hypertrophy was also observed in the colon length. Computerized image analysis of intestinal cross-sections showed enlarged muscle area in the ileum and midcolon of pectin-fed rats and greater mucosal area in the colon. Circular muscle cell hypertrophy was also observed in the colon. In contrast, the effects of cellulose feeding were limited to elongation of the colon accompanied by muscle cell hypertrophy, and little effect on ileal morphology was documented. These data indicate that the hypocholestrolemic effects of pectin may be due to increased bile acid secretion accompanied by significant morphological changes in the ileum and colon.

The effects of various sources of dietary fibre on the high glycaemic index of an Israeli ethnic food, melawach, were investigated in subjects with non-insulin-dependent diabetes mellitus (NIDDM). Locust-bean (Ceratonia siliqua) gum significantly decreased the glucose response to, and glycaemic index of, melawach in these diabetic subjects (P < 0·05). It also tended to decrease their insulinaemic response and insulinaemic index, but differences were not significant. Dietary fibre from lupin (Lupinus albus) and insoluble maize-cob fibre did not affect glucose and insulin levels in NIDDM volunteers. Subjects with a BMI < 30 kg/m2 exhibited similar glucose, but not insulin, responses to fibre. Locust-bean gum had no significant effect on glycaemic response in NIDDM subjects with a BMI > 30 kg/m2, whereas insulinaemic response decreased. The results indicate that foods containing the same nutrients in almost the same amounts, but differing in added dietary fibre, lead to different physiological responses in diabetic subjects. Furthermore, insulin response should be considered when fibre is incorporated into the diabetic's diet.

Olive tree (Olea europaea L.) leaves have been widely used in traditional remedies in European and Mediterranean countries as extracts, herbal teas, and powder. They contain several potentially bioactive compounds that may have hypoglycemic properties. To examine the efficacy of 500 mg oral olive leaf extract taken once daily in tablet form versus matching placebo in improving glucose homeostasis in adults with type 2 diabetes (T2DM). In this controlled clinical trial, 79 adults with T2DM were randomized to treatment with 500 mg olive leaf extract tablet taken orally once daily or matching placebo. The study duration was 14 weeks. Measures of glucose homeostasis including Hba1c and plasma insulin were measured and compared by treatment assignment. In a series of animal models, normal, streptozotocin (STZ) diabetic, and sand rats were used in the inverted sac model to determine the mechanism through which olive leaf extract affected starch digestion and absorption. In the randomized clinical trial, the subjects treated with olive leaf extract exhibited significantly lower HbA1c and fasting plasma insulin levels; however, postprandial plasma insulin levels did not differ significantly by treatment group. In the animal models, normal and STZ diabetic rats exhibited significantly reduced starch digestion and absorption after treatment with olive leaf extract compared with intestine without olive leaf treatment. Reduced digestion and absorption was observed in both the mucosal and serosal sides of the intestine. Though reduced, the decline in starch digestion and absorption did not reach statistical significance in the sand rats. Olive leaf extract is associated with improved glucose homeostasis in humans. Animal models indicate that this may be facilitated through the reduction of starch digestion and absorption. Olive leaf extract may represent an effective adjunct therapy that normalizes glucose homeostasis in individuals with diabetes.

The hypocholesterolaemic properties of an ethanol extract from defatted fenugreek (Trigonella foenumgraecum) seeds were investigated. Purification of the crude extract by dialysis produced an isolated component with haemolytic properties. The dialysate was also found to contain saponins demonstrated by thin-layer chromatography. Experimentsin vitroemploying the everted-sac technique showed that the ethanol extract had the ability to inhibit taurocholate and deoxycholate absorption in a dose-dependent manner. In two separate feeding experiments, hypercholesterolaemic rats were fed on 30 or 50 g ethanol extract/kg for a 4-week period. Reductions in plasma cholesterol levels ranged from 18 to 26% and a tendency for lower concentrations of liver cholesterol was observed. These results indicate that the ethanol extract from fenugreek seeds contained hypocholesterolaemic components which appear to be saponins that interact with bile salts in the digestive tract.

The primary objective of this study was to investigate the impact of lipid oversupply on the AMPK pathway in skeletal muscle, liver, and adipose tissue. Male Wistar rats were infused with lipid emulsion (LE) or phosphate-buffered saline for 5 h/day for 6 days. Muscles exposed to LE for 6 days exhibited increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation, along with a greater association between AMPK and Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK). No differences in muscle protein phosphatase 2C (PP2C) activity, LKB1 phosphorylation or AMPK and LKB1 association were observed. Muscle ACCbeta, and adiponectin receptor 1 (AdipoR1) mRNA levels and PPARgamma-co-activator 1alpha (PGC1alpha) protein levels were also increased in LE-treated rats. In contrast, AMPK and ACC phosphorylation decreased and PP2C activity increased in rat livers exposed to LE. Hepatic mRNA levels of ACCalpha, PPARalpha, AdipoR1, AdipoR2, and sterol regulatory element-binding protein-1c (SREBP1c) were also reduced after LE infusion. In adipose tissue, there was no significant alteration in AMPK or ACC phosphorylation. These results demonstrate that following lipid oversupply the AMPK pathway was enhanced in rat skeletal muscle while diminished in the liver and was unchanged in adipose tissue. CaMKK in skeletal muscle and PP2C in the liver, at least in part, appear to mediate these alterations. Alterations in AMPK pathway in the liver induced metabolic defects associated with lipid oversupply.

Red microalgae contain functional sulfated polysaccharides (containing dietary fibers), polyunsaturated fatty acids, zeaxanthin, vitamins, minerals, and proteins. Studies in rat models support the therapeutic properties of algal biomass and isolated polysaccharides. Algal products incorporated into rat diets were found to significantly improve total serum cholesterol, serum triglycerides, hepatic cholesterol levels, HDL/LDL ratios and increased fecal excretion of neutral sterols and bile acids. Morphological and metabolic changes were induced by consumption of algal products. These results suggest that red microalgae can be used as potent hypocholesterolemic agents, and they support the potential use of red microalgae as novel nutraceuticals.

Glycogen synthase (GS) activity was characterized in rat brown adipose tissue (BAT) and the activity was found to be much higher than that in white adipose tissue. Prolonged starvation had no effect on the active form of GS, as found in the liver and muscle. The GS activity was similar in BAT of rats housed in an animal room (21+± 1°) whether they were fed on high-carbohydrate, high-fat, or stock diets. Acclimatizatioin of rats to cold (4+± 1°) for 2 weeks significantly increased GS activity. This increase in the cold was fivefold greater when rats were fed on high-carbohydrate diets than in control rats at room temperature fed on an identical diet. The increase was accompanied by a large accumulation of glycogen in BAT. It was concluded that GS may play an important role in BAT and may contribute to the control of blood glucose in a cold environment. Its relevance to thermogenesis requires further elucidation.

Background: The use of Web-based apps to promote a healthy lifestyle is increasing, although most of these programs were not assessed using suitable epidemiological methods. We evaluated the effectiveness of a newly developed Web-based app in promoting a healthy lifestyle and educating adults on such lifestyles. We also analyzed predictors for success in acquiring and maintaining a healthy lifestyle. Objective: Our aim was to compare people receiving a new Web-based app with people who got an introductory lecture alone on healthy lifestyle, weight change, nutritional knowledge, and physical activity, and to identify predictors of success for maintaining a healthy lifestyle. Methods: Subjects were recruited from the community and were randomized into intervention and control groups. The intervention subjects received access to the app without any face-to-face support; the control subjects continued their standard lifestyle. Measurements were taken by the researcher at baseline and after 14 weeks and included weight and waist circumference. Nutritional knowledge, diet quality, and physical activity duration were obtained using online questionnaires. The new Web-based app was developed based on current US Department of Agriculture and Israel Ministry of Health recommendations for healthy lifestyle. The app provides tools for monitoring diet and physical activity while instructing and encouraging healthy diet and physical activity. Results: Out of 99 subjects who were randomized into app and control groups, 85 participants (86%) completed the study, 56 in the intervention and 29 in the control group. The mean age was 47.9 (SD 12.3) years, and mean Body Mass Index was 26.2 (SD 3.9). Among the intervention group only, frequency of app use was 2.7 (SD 1.9) days/week. The mean change in physical activity was 63 (SD 20.8) minutes in the app group and -30 (SD 27.5) minutes in the control group ( P =.02). The mean weight change was -1.44 (SD 0.4) kg in the app group and -0.128 (SD 0.36) kg in the control group ( P =.03). Knowledge score increased significantly in the app group, 76 (SD 7.5) to 79 (SD 8.7) at the end of the study ( P =.04) compared with the control group. Diet quality score also increased significantly at the end of the study, from 67 (SD 9.8) to 71 (SD 7.6; P

BARNEA, MAAYAN, AVI SHAMAY, ALIZA H. STARK, AND ZECHARIA MADAR. A high-fat diet has a tissue-specific effect on adiponectin and related enzyme expression. Obesity. 2006;14:2145–2153. Objective: This study was designed to test whether adiponectin plays a role in diet-induced obesity and insulin resistance and acts as a mediator to induce or inhibit specific metabolic pathways involved in lipid metabolism Research Methods and Procedures: Forty C57BL/6J male mice were fed either a high-fat (HF) or control diet for 4 months, and adiponectin, its receptors, and enzyme expression in liver and muscle tissue were measured. Results: Mice fed the HF diet exhibited significantly greater weight gain, abnormal oral glucose tolerance test curves, and elevated homeostasis model assessment of insulin resistance (5.3 0.89 vs. 2.8 0.39). A significant reduction of adiponectin RNA expression (51%) and protein levels (15%) was observed in the adipose tissue of HF animals; however, serum adiponectin levels did not differ between groups (7.12 0.34 g/mL vs. 6.44 0.38 g/mL). Expression of hepatic mRNA of AdipoR1 and AdipoR2 was reduced by 15% and 25%, respectively, in animals fed the HF diet. In contrast, receptor mRNA expression of AdipoR1 and AdipoR2 increased by 25% and 30%, respectively, in muscle tissue. No effect was found on hepatic adenosine monophosphate-activated protein kinase expression; however, a significant reduction of phosphoadenosine monophosphate kinase levels in muscles was observed. Hepatic acetyl-coenzyme A carboxylase was similar between groups, but in muscles, the inactive form phosphoacetylcoenzyme A carboxylase was significantly reduced (p 0.05). Discussion: The HF diet led to decreased insulin sensitivity accompanied by impaired activity of adiponectin-related enzymes in skeletal muscles but not in the liver. These results suggest that the HF diet has a tissue-specific effect on adiponectin and associated enzyme expression.

The innate immunity, utilizes a battery of broad-spectrum antibacterial cationic polypeptides (3 - 5 kDa) named α- and β-defensins. Several β-defensins have been isolated and shown to play a role in the defense of various tissues. Herein, we report the expression pattern of two rat β-defensins, rBD-1 and rBD-2, in liver, kidney, lung, spleen, and brain using RT-PCR. To study polymorphism and verify gene identity, all cDNA products were sequenced. rBD-1 was expressed in the kidney, lung, brain, but not in spleen or liver, whereas rBD-2 was expressed in the lung, but not in the kidney or spleen. In addition, rBD-2 was expressed in the brain and liver. No polymorphism was found in the genes encoding rat β-defensins. These findings demonstrate a different expression pattern for rBD-2 than what has been reported. We conclude that the rat may be a useful model to investigate the function and contribution of β-defensins to host defense. IUBMB Life, 57: 41-43, 2005

Phospholipase A2 (PLA2) hydrolyzes cell membrane phospholipids (PL) to produce arachidonic acid and lyso-PL. The PLA2 enzymes include the secretory (sPLA2) and cytosolic (cPLA2) isoforms, which are assumed to act synergistically in production of eicosanoids that are involved in inflammatory processes. However, growing evidence raises the possibility that in airways and asthma-related inflammatory cells (eosinophils, basophils), the production of the bronchoconstrictor cysteinyl leukotrienes (CysLT) is linked exclusively to sPLA2, whereas the bronchodilator prostaglandin PGE2 is produced by cPLA2. It has been further reported that the capacity of airway epithelial cells to produce CysLT is inversely proportional to PGE2 production. This seems to suggest that sPLA2 and cPLA2 play opposing roles in asthma pathophysiology and the possibility of a negative feedback between the two isoenzymes. To test this hypothesis, we examined the effect of a cell-impermeable extracellular sPLA2 inhibitor on bronchoconstriction and PLA2 expression in rats with ovalbumin (OVA)-induced asthma. It was found that OVA-induced bronchoconstriction was associated with elevation of lung sPLA2 expression and CysLT production, concomitantly with suppression of cPLA2 expression and PGE2 production. These were reversed by treatment with the sPLA2 inhibitor, resulting in amelioration of bronchoconstriction and reduced CysLT production and sPLA2 expression, concomitantly with enhanced PGE2 production and cPLA2 expression. This study demonstrates, for the first time in vivo, a negative feedback between sPLA2 and cPLA2 and assigns opposing roles for these enzymes in asthma pathophysiology: sPLA2 activation induces production of the bronchoconstrictor CysLT and suppresses cPLA2 expression and the subsequent production of the bronchodilator PGE2.

This review evaluates the potential health benefits of three legume sources that rarely appear in Western diets and are often overlooked as functional foods. Fenugreek (Trigonella foenum graecum) and isolated fenugreek fractions have been shown to act as hypoglycaemic and hypocholesterolaemic agents in both animal and human studies. The unique dietary fibre composition and high saponin content in fenugreek appears to be responsible for these therapeutic properties. Faba beans (Vicia faba) have lipid-lowering effects and may also be a good source of antioxidants and chemopreventive factors. Mung beans (Phaseolus aureus, Vigna radiatus) are thought to be beneficial as an antidiabetic, low glycaemic index food, rich in antioxidants. Evidence suggests that these three novel sources of legumes may provide health benefits when included in the daily diet.

Cell-cell interaction via gap junctions is considered to be a key factor in tissue homeostasis, and its alteration is associated with the neoplastic phenotype. Experimental and epidemiologic data suggest that carotenoids, particularly lycopene and beta-carotene, can reduce the risk of certain cancers. The aim of this study was to assess whether lycopene and beta-carotene interfere at some stage with the carcinogenic processes in human cancer cells derived from the oral cavity. KB-1 cells, originating from a human oral cavity tumor, were incubated with different concentrations of lycopene or beta-carotene delivered via the cell culture media from stock solutions in tetrahydrofuran. Lycopene strongly and dose dependently inhibited proliferation of KB-1 human oral tumor cells. beta-Carotene was a far less effective growth inhibitor. Lycopene (3 and 7 micro mol/L) significantly upregulated both the transcription (P < 0.005) and the expression (P < 0.05) of connexin 43, a key protein in the formation of gap-junctional communication. beta-Carotene (3 micro mol/L) tended to upregulate connexin 43 expression (P = 0.07) and significantly affected transcription of connexin 43 at 7 micro mol/L (P < 0.05). Gap-junctional communication measured by scrape-loading dye transfer and electron microscopy showed that lycopene enhanced gap-junctional communication between the cancer cells, whereas beta-carotene was less effective in this regard. The pattern of cellular uptake and incorporation into cancer KB-1 cells differed significantly between the carotenoids. beta-Carotene was avidly and rapidly incorporated into KB-1 cells, whereas lycopene uptake into the cells took place after longer incubation periods and only at the highest concentrations. The results of the present study further support the hypothesis that carotenoids in general, and lycopene in particular, may be effective anticarcinogenic agents in oral carcinogenesis.

We studied whether feeding pregnant female rats different high fat diets affects structural zones in the spleen and lymph nodes, involved in production of T and B cells, as well as cell kinetics and apoptosis in some offspring with mammary glands tumors. Rat mothers were fed either a 7% or 15% corn-oil or a 7% or 15% olive-oil diet. At four weeks of age, female offspring (n=10-15 per group) were transferred to 7% corn oil diet. Five-week old offspring were exposed twice to the carcinogen, dimethylbenz(a)antracene (10 mg/rat/week). Three months later, tumors were counted and sized, and samples from the spleen, axillary lymph nodes and tumors collected for immunohistochemical analyses. Feeding the mothers with both the 7% and 15% olive-oil diets significantly increased the number of tumor-free rats in offspring. Tumors were characterized with active mitosis, intensive lymphoid infiltration inside a knot and high rates of apoptosis, particularly in tumors obtained from rats whose mothers were fed the 15% olive-oil diet. In the spleen, the 15% olive-oil diet significantly increased the areas of the follicles and germinal centers but only in tumor-free rats. In tumor-bearing rats, areas of germinal centers increased compared to the 7% olive-oil diet. The 15% olive-oil diet increased all areas of the lymph nodes in tumor-free rats, while in tumor-bearing rats, this diet increased the areas of the cortex and mantle layer. We conclude that exposure to various diets in utero and during lactation affects the immune system. In addition, the promotion of apoptosis may play a key role in the mechanisms involved in the transplacental effects on mammary tumor development as seen using a 15% olive-oil diet, similar to the high fat diets of Mediterranean countries.

alpha-Lipoic acid (LA) is currently being investigated as a glucose-lowering agent for diabetes control; it is also considered a powerful dietary antioxidant. The objective of this study was to investigate the fate of glucose in isolated rat muscles incubated with LA and determine its effects on intramuscular redox status. Rat soleus muscles were incubated for up to 60 min with 2.4 mmol/L LA in the presence or absence of insulin. Intramuscular concentrations of LA were evaluated (uptake and reduction), and glycogen synthesis, glucose oxidation, intramuscular reactive oxygen species (ROS) production and mitochondrial membrane potential investigated. Insulin enhanced glycogen synthesis, whereas LA decreased rates by >50%. LA elevated ROS production and in combination with t-butylhydroperoxide, an oxidant, additively inhibited glycogen synthesis rates by 80%. Insulin acted as an antioxidant and attenuated ROS production by 30%. LA uncoupled the mitochondria and accelerated glucose oxidation 1.5-fold relative to the control. The glycogen synthesis pathway was found to be dependent on mitochondrial function because treatment with mitochondrial inhibitors eliminated the majority of glycogen synthesis. These data show that in this model, LA acts as a mild prooxidant, causing mitochondrial uncoupling and inhibition of glycogen synthesis. It appears that LA regulates glucose metabolism in the muscle differently than insulin.