Kehao Zhang

Activation of KATP channels suppresses glucose production in humans

Increased endogenous glucose production (EGP) is a hallmark of type 2 diabetes mellitus. While there is evidence for central regulation of EGP by activation of hypothalamic ATP-sensitive potassium (K(ATP)) channels in rodents, whether these central pathways contribute to regulation of EGP in humans remains to be determined. Here we present evidence for central nervous system regulation of EGP in humans that is consistent with complementary rodent studies. Oral administration of the K(ATP) channel activator diazoxide under fixed hormonal conditions substantially decreased EGP in nondiabetic humans and Sprague Dawley rats. In rats, comparable doses of oral diazoxide attained appreciable concentrations in the cerebrospinal fluid, and the effects of oral diazoxide were abolished by i.c.v. administration of the K(ATP) channel blocker glibenclamide. These results suggest that activation of hypothalamic K(ATP) channels may be an important regulator of EGP in humans and that this pathway could be a target for treatment of hyperglycemia in type 2 diabetes mellitus.

Reduced Adipose Tissue Macrophage Content Is Associated With Improved Insulin Sensitivity in Thiazolidinedione-Treated Diabetic Humans

Obesity is associated with increased adipose tissue macrophage (ATM) infiltration, and rodent studies suggest that inflammatory factors produced by ATMs contribute to insulin resistance and type 2 diabetes. However, a relationship between ATM content and insulin resistance has not been clearly established in humans. Since thiazolidinediones attenuate adipose tissue inflammation and improve insulin sensitivity, we examined the temporal relationship of the effects of pioglitazone on these two parameters. The effect of 10 and 21 days of pioglitazone treatment on insulin sensitivity in 26 diabetic subjects was assessed by hyperinsulinemic-euglycemic clamp studies. Because chemoattractant factors, cytokines, and immune cells have been implicated in regulating the recruitment of ATMs, we studied their temporal relationship to changes in ATM content. Improved hepatic and peripheral insulin sensitivity was seen after 21 days of pioglitazone. We found early reductions in macrophage chemoattractant factors after only 10 days of pioglitazone, followed by a 69% reduction in ATM content at 21 days and reduced ATM activation at both time points. Although markers for dendritic cells and neutrophils were reduced at both time points, there were no significant changes in regulatory T cells. These results are consistent with an association between adipose macrophage content and systemic insulin resistance in humans.

Adipocyte-Derived Factors Potentiate Nutrient-Induced Production of Plasminogen Activator Inhibitor–1 by Macrophages

In response to free fatty acids and factors from fat cells, macrophages within the fat tissue of obese individuals release a hormone that contributes to atherosclerosis and insulin resistance. Deleterious Cytokines from Adipose Tissue Obesity is bad for your metabolism. One possible contributor to obesity-induced diabetes and atherosclerosis is plasminogen activator inhibitor-1 or PAI-1, a hormone secreted from fat cells. PAI-1, a protease inhibitor and hemostatic agent, is correlated with cardiovascular events and atherosclerosis, and mice engineered without this hormone are protected from these diseases. Free fatty acids in blood, which are derived from the diet, are also correlated with PAI-1, with higher fatty acid concentrations resulting in more blood PAI-1. Kishore et al. tested whether diet-derived free fatty acids cause increased PAI-1 production in human adipose tissue, focusing on macrophages, immune cells resident in fat that secrete PAI-1. By delivering free fatty acids directly to the bloodstream of their subjects, the authors elicited increased serum PAI-1 concentrations, as well as decreased glucose uptake into tissues. This increased PAI-1 in blood was accompanied by PAI-1 gene expression in macrophages from adipose tissue, although similar cells in the bloodstream did not respond in this way to the fatty acids. Suspecting that the fat cells surrounding the adipose tissue macrophages were somehow sensitizing the macrophages, the authors added adipocyte-conditioned medium to macrophages. This treatment augmented the response of the macrophages to fatty acids, boosting their production of PAI-1. Thus the adipose-tissue macrophages seemed to be uniquely stimulated by their surrounding environment to respond to free fatty acids by secreting the undesirable hormone PAI-1, which can increase propensity to cardiovascular disease. Adipose tissue is gaining a reputation for being more than an inert fat depot. The results from this study by Kishore et al. reinforce this idea, suggesting that in humans adipose tissue delivers elevated concentrations of PAI-1 by creating a unique, local paracrine environment to promote its synthesis. Macrophages are more abundant in adipose tissue from obese individuals than from those of normal weight and may contribute to the metabolic consequences of obesity by producing various circulating factors. One of these factors is plasminogen activator inhibitor–1 (PAI-1), which contributes to both atherosclerosis and insulin resistance. Because nutritional factors appear to regulate PAI-1 expression, we hypothesized that exposure to fatty acids and adipocyte secretory products could stimulate production of PAI-1 by adipose macrophages. Increased free fatty acid (FFA) concentrations in blood for 5 hours in nondiabetic, overweight subjects markedly suppressed insulin-stimulated glucose uptake and raised circulating PAI-1 concentrations, with a concomitant increase in the expression of the PAI-1 gene in adipose tissue. FFAs also rapidly increased PAI-1 gene expression in adipose macrophages and PAI-1 protein immunofluorescence surrounding these cells. By contrast, PAI-1 expression in circulating monocytes was very low and was not affected by raising the concentration of FFAs. Medium from cultured adipocytes stimulated PAI-1 expression in cultured macrophages and potentiated the increase in PAI-1 messenger RNA expression in response to FFAs. Together, our data suggest that adipocyte-derived factors prime adipose macrophages so that they respond to nutritional signals (FFAs) by releasing a key inflammatory adipokine, PAI-1.

Insulin sensitizing and anti-inflammatory effects of thiazolidinediones are heightened in obese patients.

Objective The American Diabetes Association has called for further research on how patients’ demographics should determine drug choices for individuals with type 2 diabetes mellitus (T2DM). Here, using in-depth physiology studies, we investigate whether obese patients with T2DM are likely to benefit from thiazolidinediones, medications with a known adverse effect of weight gain. Materials and Methods Eleven obese and 7 nonobese individuals with T2DM participated in this randomized, placebo-controlled, double-blind, crossover study. Each subject underwent a pair of “stepped” pancreatic clamp studies with subcutaneous adipose tissue biopsies after 21 days of pioglitazone (45 mg) or placebo. Results Obese subjects demonstrated significant decreases in insulin resistance and many adipose inflammatory parameters with pioglitazone relative to placebo. Specifically, significant improvements in glucose infusion rates, suppression of hepatic glucose production, and whole fat expression of certain inflammatory markers (IL-6, IL-1B, and inducible nitric oxide synthase) were observed in the obese subjects but not in the nonobese subjects. Additionally, adipose tissue from the obese subjects demonstrated reduced infiltration of macrophages, dendritic cells, and neutrophils as well as increased expression of factors associated with fat “browning” (peroxisome proliferator–activated receptor gamma coactivator-1α and uncoupling protein-1). Conclusions These findings support the efficacy of pioglitazone to improve insulin resistance and reduce adipose tissue inflammation in obese patients with T2DM.

Resveratrol Improves Vascular Function and Mitochondrial Number but Not Glucose Metabolism in Older Adults

Background Resveratrol, a plant-derived polyphenol, has been reported to improve glucose metabolism and vascular function and to extend life span in animal models, but studies in humans have been inconclusive. Methods In a randomized, double-blind crossover study, we treated older glucose-intolerant adults (n = 30) with resveratrol (2-3 g/daily) or placebo, each for 6 weeks. A standard mixed-meal test was used to assess insulin sensitivity (Matsuda index) and secretion (C-peptide deconvolution) and vascular function by reactive hyperemia peripheral arterial tonometry. Skeletal muscle samples were obtained for gene expression using RNA-Seq analysis and to assess mitochondrial morphology. Results There were no changes in glucose tolerance, insulin sensitivity, weight, blood pressure, or lipid profile following resveratrol treatment. Fasting reactive hyperemia index improved with resveratrol (2.02 ± 0.2 vs 1.76 ± 0.02, p = .002). RNA-Seq analysis yielded 140 differentially expressed transcripts (corrected p-value ≤ .05), predominantly associated with mitochondrial genes and noncoding RNA. Ingenuity Pathway Analysis confirmed that mitochondrial dysfunction (p = 2.77 × 10-12) and oxidative phosphorylation (p = 1.41 × 10-11) were the most significantly perturbed pathways. Mitochondrial number, but not size, was increased. Conclusions Resveratrol treatment of older adults with impaired glucose regulation may have beneficial effects on vascular function, but not glucose metabolism or insulin sensitivity. Changes in gene expression suggest effects similar to those observed with caloric restriction, which has been shown to increase life and health span in animal models, although its significance for humans is uncertain. Future human studies should address the appropriate dose range and low bioavailability of resveratrol.

Fatty Acid-Induced Production of Plasminogen Activator Inhibitor-1 by Adipose Macrophages Is Greater in Middle-Aged Versus Younger Adult Participants

BACKGROUND Human aging is associated with heightened risk of diabetes and cardiovascular disease. Increased fat mass may contribute to age-related diseases by harboring inflammatory macrophages that produce metabolically important proteins such as plasminogen activator inhibitor-1 (PAI-1). Elevated PAI-1 concentrations have been implicated in the pathogenesis of such aging-related conditions as insulin resistance, obesity, and atherosclerosis. We have previously reported that increased plasma free fatty acid (FFA) concentrations augment both circulating PAI-1 concentrations and PAI-1 production by adipose tissue macrophages (ATMs). METHODS Because increasing age is associated with increased infiltration and reactivity of adipose macrophages, we performed euglycemic-hyperinsulinemic clamp studies and adipose tissue biopsies with and without elevated FFA concentrations in 31 nondiabetic participants stratified by age, to determine whether middle-aged individuals manifest heightened insulin resistance and PAI-1 production by ATMs in response to elevated nutrient signals relative to their young adult peers. RESULTS We observed that elevating FFA concentrations under euglycemic-hyperinsulinemic clamp conditions induced the same degree of insulin resistance in both middle-aged and younger body mass index-matched adults, whereas systemic PAI-1 concentrations were significantly increased in the middle-aged group. Likewise, elevated FFA and insulin concentrations induced larger increases in PAI-1 gene expression in the whole fat and ATMs of middle-aged compared with younger adult participants. CONCLUSIONS These studies reveal a heightened adipose inflammatory response to increased FFA and insulin availability in middle-aged individuals relative to younger adults, suggesting that increased susceptibility to the effects of fatty acid excess may contribute to the pathogenesis of age-related diseases.

Metformin regulates metabolic and nonmetabolic pathways in skeletal muscle and subcutaneous adipose tissues of older adults

Administration of metformin increases healthspan and lifespan in model systems, and evidence from clinical trials and observational studies suggests that metformin delays a variety of age‐related morbidities. Although metformin has been shown to modulate multiple biological pathways at the cellular level, these pleiotropic effects of metformin on the biology of human aging have not been studied. We studied ~70‐year‐old participants (n = 14) in a randomized, double‐blind, placebo‐controlled, crossover trial in which they were treated with 6 weeks each of metformin and placebo. Following each treatment period, skeletal muscle and subcutaneous adipose tissue biopsies were obtained, and a mixed‐meal challenge test was performed. As expected, metformin therapy lowered 2‐hour glucose, insulin AUC, and insulin secretion compared to placebo. Using FDR<0.05, 647 genes were differentially expressed in muscle and 146 genes were differentially expressed in adipose tissue. Both metabolic and nonmetabolic pathways were significantly influenced, including pyruvate metabolism and DNA repair in muscle and PPAR and SREBP signaling, mitochondrial fatty acid oxidation, and collagen trimerization in adipose. While each tissue had a signature reflecting its own function, we identified a cascade of predictive upstream transcriptional regulators, including mTORC1, MYC, TNF, TGFß1, and miRNA‐29b that may explain tissue‐specific transcriptomic changes in response to metformin treatment. This study provides the first evidence that, in older adults, metformin has metabolic and nonmetabolic effects linked to aging. These data can inform the development of biomarkers for the effects of metformin, and potentially other drugs, on key aging pathways.

Central Regulation of Glucose Production May Be Impaired in Type 2 Diabetes.

The challenges of achieving optimal glycemic control in type 2 diabetes highlight the need for new therapies. Inappropriately elevated endogenous glucose production (EGP) is the main source of hyperglycemia in type 2 diabetes. Because activation of central ATP-sensitive potassium (KATP) channels suppresses EGP in nondiabetic rodents and humans, this study examined whether type 2 diabetic humans and rodents retain central regulation of EGP. The KATP channel activator diazoxide was administered in a randomized, placebo-controlled crossover design to eight type 2 diabetic subjects and seven age- and BMI-matched healthy control subjects. Comprehensive measures of glucose turnover and insulin sensitivity were performed during euglycemic pancreatic clamp studies following diazoxide and placebo administration. Complementary rodent clamp studies were performed in Zucker Diabetic Fatty rats. In type 2 diabetic subjects, extrapancreatic KATP channel activation with diazoxide under fixed hormonal conditions failed to suppress EGP, whereas matched control subjects demonstrated a 27% reduction in EGP (P = 0.002) with diazoxide. Diazoxide also failed to suppress EGP in diabetic rats. These results suggest that suppression of EGP by central KATP channel activation may be lost in type 2 diabetes. Restoration of central regulation of glucose metabolism could be a promising therapeutic target to reduce hyperglycemia in type 2 diabetes.

Skeletal muscle fibrosis is associated with decreased muscle inflammation and weakness in patients with chronic kidney disease

Muscle dysfunction is an important cause of morbidity among patients with chronic kidney disease (CKD). Although muscle fibrosis is present in a CKD rodent model, its existence in humans and its impact on physical function are currently unknown. We examined isometric leg extension strength and measures of skeletal muscle fibrosis and inflammation in vastus lateralis muscle from CKD patients ( n = 10) and healthy, sedentary controls ( n = 10). Histochemistry and immunohistochemistry were used to assess muscle collagen and macrophage and fibro/adipogenic progenitor (FAP) cell populations, and RT-qPCR was used to assess muscle-specific inflammatory marker expression. Muscle collagen content was significantly greater in CKD compared with control (18.8 ± 2.1 vs. 11.7 ± 0.7% collagen area, P = 0.008), as was staining for collagen I, pro-collagen I, and a novel collagen-hybridizing peptide that binds remodeling collagen. Muscle collagen was inversely associated with leg extension strength in CKD ( r = -0.74, P = 0.01). FAP abundance was increased in CKD, was highly correlated with muscle collagen ( r = 0.84, P < 0.001), and was inversely associated with TNF-α expression ( r = -0.65, P = 0.003). TNF-α, CD68, CCL2, and CCL5 mRNA were significantly lower in CKD than control, despite higher serum TNF-α and IL-6. Immunohistochemistry confirmed fewer CD68+ and CD11b+ macrophages in CKD muscle. In conclusion, skeletal muscle collagen content is increased in humans with CKD and is associated with functional parameters. Muscle fibrosis correlated with increased FAP abundance, which may be due to insufficient macrophage-mediated TNF-α secretion. These data provide a foundation for future research elucidating the mechanisms responsible for this newly identified human muscle pathology.

42 ADIPONECTIN RECEPTORS ARE NOT MODULATED BY SHORT-TERM THIAZOLIDINEDIONE THERAPY.

The fat-derived protein adiponectin enhances insulin action in rodents and its plasma levels are correlated with insulin sensitivity in humans. Adiponectin levels are reduced in obesity and type 2 diabetes mellitus (T2DM) and increased by thiazolidinediones (TZDs) in concert with improved insulin action. Two adiponectin receptors (AdipoR1 and AdipoR2) have recently been cloned. We examined the expression of these receptors in human adipose tissue and whether they are also regulated by TZDs. Gene expression was quantified by real-time rt-PCR in subcutaneous (Sub) and omental (Om) adipose tissue from 10 nondiabetic patients (7 F/3 M, age 38 x 3 years, BMI 31.6 ± 1.5 kg/m2, range 23-38) undergoing elective abdominal surgery. In a subgroup (n = 5), adipocytes and stromal cells were immediately separated by collagenase. RNA copy numbers (plasmid standard curves, normalized to GAPDH) of AdipoR1 were universally {223}10-fold higher than AdipoR2, and expression of AdipoR1 was similar in Sub and Om, while AdipoR2 expression was higher in Om. While adiponectin gene expression was specific to adipocytes, the receptors were expressed in both adipocytes and stromal cells (adipocytes:stromal cell ratio = 5:4). We then studied the effects of short-term pioglitazone (45 mg daily for 21 days) vs placebo on AdipoR1/R2 gene expression and insulin action (insulin clamp studies) in 11 T2DM subjects (9 M/2 F, age 49 ± 3 years, HbA1C 9.7 ± 0.7%, BMI 32 ± 2 kg/m2). There was no correlation between baseline insulin action and expression of AdipoR1/R2 in muscle or fat. While pioglitazone increased adiponectin expression 2-fold in Sub fat and improved both hepatic and peripheral insulin action, it did not affect expression of either receptor in fat or muscle. There were also no correlations between changes in insulin action and changes in receptor expression with pioglitazone in individual subjects. This was in contrast with a remarkably tight correlation (r2 = .93) between improved hepatic insulin action and increase in the active high-molecular-weight form of adiponectin. Conclusions Both adiponectin receptor subtypes are expressed in human fat, with AdipoR1 being much more abundant. Unlike adiponectin, the receptors are expressed in both adipocytes and stromal adipose cells and are not affected by TZDs. Additionally; receptor expression did not correlate with insulin action at baseline or in response to pioglitazone. Further study is warranted to determine the physiologic significance of these receptors in humans.