To the Editor: Metformin is the most frequently used drug for the treatment of type 2 (non-insulin-dependent) diabetes mellitus. Its beneficial effects include the lowering of blood glucose levels and weight loss [1], but the mechanism by which it induces the latter effect is not known. However, it is known that metformin stimulates catabolic pathways through the activation of adenosine monophosphate–activated protein kinase (AMPK) [2] and that a synthetic activator of AMPK, 5-aminoimidazole4-carboxamide ribofuranoside (AICAR) inhibits adipogenesis in the pre-adipocyte cell line, 3T3-L1 [3]. Therefore, the present studywas designed to assess the effect of metformin on adipogenesis and the phosphorylation state of AMPK in 3T3-L1 cells. The 3T3-L1 (ATTC reference number CL-173) cells were grown at 37 °C in a humidified atmosphere of 5% CO2 in air in 25 cm 2 tissue culture flasks containing Dulbecco’s Modified Eagle’s Medium (Gibco Invitrogen, Carlsbad, CA, USA) as previously described [4]. The cells were cultured from day 0 through to day 8 in the absence (negative control) or presence of 1 mM AICAR (positive control; Sigma-Aldrich, St Louis, MO, USA) [3] or 2–16 mMmetformin (1,1-dimethylbiguanide hydrochloride, Sigma-Aldrich) [2]. Intracellular lipid accumulation was measured on days 0 and 8 using Oil Red O that was extracted from the cells into 3.5 ml of 60% isopropyl alcohol, and the absorbance of this extract was read at 510 nm on a spectrophotometer [4]. Adherent 3T3-L1 cells were harvested, lysed, centrifuged and the resulting supernatant was analysed for phosphorylated AMPK (phospho-AMPK) by Western blotting using a primary antibody that is specifically raised against phospho-AMPK and detected by enhanced chemiluminescence [2,5,6]. The intensity of the phosphorylated AMPK bands was quantified using Kodak 1D IMAGE ANALYSIS software V 3.6 (Kodak Scientific Imaging Systems, Eastman Kodak Company, Rochester, NY, USA). Student’s t-test was used to calculate the differences in means for intracellular lipid accumulation and AMPK phosphorylation of control cells against AICARor metformin-treated cells. Lipid accumulation was measured 8 days after initiation of adipogenesis. Spectrophotometric absorbance readings on day 8 were 0.086 0.022 compared with 0.030 0.008 (p < 0.05) on day 0 for cells that were not cultured in the presence of AICAR or metformin (untreated). The absorbance for cells treated with 1 mM AICAR on day 8was 0.060 0.01 (p < 0.05 vs. untreated cells). The effect of metformin treatment on lipid droplet formation is illustrated in figure 1 that shows thatmetformin concentrations 4 mM significantly (p < 0.05) reduced lipid accumulation comparedwith the untreated cells. Western blot analysis of the 3T3-L1 cell extracts 8 days after the initiation of adipogenesis showed that AMPK phosphorylation at residue Thr172 of the a-subunit was higher in the presence of 1 mM AICAR or 16 mM metformin (p < 0.05 for both treatments vs. untreated cells) (figure 2). The present and a previous study [3] have shown that AICAR inhibits adipogenesis in 3T3-L1 cells. The study byHabinowski andWitters [3] showed that this occurs by downregulation of expression of the lipogenic enzymes

Objective: Lower lipid and insulin levels are found during a glucose-tolerance test in obese black than obese white South African women. Therefore, beta-cell function and lipid metabolism were compared in these populations during a mixed meal.Research Methods and Procedures: Blood concentrations of glucose, free fatty acids (FFAs), insulin, lipograms, and in vivo FFA oxidation were determined at fasting and for 7 hours after oral administration of a mixed emulsion containing glucose-casein-sucrose-lipid and [1-C-13] palmitic acid in 8 lean black women (LBW), 10 obese black women (OBW), 9 lean white women (LWW), and 10 obese white women (OWW). Subcutaneous and visceral fat mass was assessed by computerized tomography.Results: Visceral fat area was higher in OWW (152.7 +/- 17.0 cm(2)) than OBW (80.0 +/- 6.7 cm(2); p < 0.01). In OBW, 30-minute insulin levels were higher (604.3 +/- 117.6 pM) than OWW (311.0 +/- 42.9 pM; p < 0.05). Total triglyceride was higher in OWW (706.7 +/- 96.0 mM X 7 hours) than OBW (465.7 +/- 48.2 mM X 7 hours; p < 0.05) and correlated with visceral fat area (beta = 0.38, p = 0.05). Palmitate oxidation was higher in lean than obese women in both ethnic groups and correlated negatively with fat mass (beta = -0.58, p < 0.005).Discussion: The higher 30-minute insulin response in OBW may reflect a higher insulinotropic effect of FFAs or glucose. The elevated triglyceride level of OWW may be due to their higher visceral fat mass and possibly reduced clearance by adipose tissue.

The aim of this study was to determine the contribution of birth weight and gestational age to glucose tolerance in premature neonates. The study group consisted of 100 premature and/or small-for-gestational age infants. Anthropometric measurements were performed both at birth and at the time of a standardized milk feed carried out at 19.6 +/- 12.1 d (range, 1-65 d) after birth. Fasting and postprandial glucose and insulin levels were measured. Birth weight, as a proxy mirror of the intrauterine environment, was found to influence the glucose concentration following a standardized milk feed (beta = -0.46; P = 0.01 for birth weight z-score with 60-min glucose level), whereas gestational age did not. Small-for-gestational age neonates had higher 60-min insulin levels than appropriate-for-gestational age neonates (115.4 +/- 9.5 vs. 68.4 +/- 14.2; P < 0.05) despite similar glucose levels. Neonates born of mothers who were on antihypertensive treatment were smaller and had a higher insulin secretory response than neonates from normotensive mothers. Postnatal growth velocity (kilograms per day) correlated with birth weight (beta = -0.65; P < 0.0001) and insulin resistance (beta = -0.31; P = 0.0004), independently of each other. This study shows that glucose tolerance of the neonate is determined by weight attained at birth irrespective of gestational age and that maternal blood pressure may influence insulin sensitivity of the newborn. Furthermore, catch-up growth in neonates is determined by birth weight and insulin sensitivity.

OBJECTIVE: The effects of free fatty acids (FFA), leptin, tumour necrosis factor (TNF) α and body fat distribution on in vivo oxidation of a glucose load were studied in two South African ethnic groups. DESIGN AND MEASUREMENTS: Anthropometric and various metabolic indices were measured at fasting and during a 7 h oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis and subcutaneous and visceral fat mass was assessed using a five- and two-level CT-scan respectively. Glucose oxidation was evaluated by measuring the ratio of 13 CO 2 to 12 CO 2 in breath following ingestion of 1- 13 C-labelled glucose. SUBJECTS: Ten lean black women (LBW), ten obese black women (OBW), nine lean white women (LWW) and nine obese white women (OWW) were investigated after an overnight fast. RESULTS: Visceral fat levels were significantly higher ( P <0.01) in obese white than black women, despite similar body mass indexes (BMIs). There were no ethnic differences in glucose oxidation however; in the lean subjects of both ethnic groups the area under the curve (AUC) was higher than in obese subjects ( P <0.05 for both) and was found to correlate negatively with weight ( r =−0.69, P <0.01) after correcting for age. Basal TNFα concentrations were similar in all groups. Percentage suppression of FFAs at 30 min of the OGTT was 24±12% in OWW and −38±23% ( P <0.05) in OBW, ie the 30 min FFA level was higher than the fasting level in the latter group. AUC for FFAs during the late postprandial period (120–420 min) was significantly higher in OWW than OBW ( P <0.01) and LWW ( P <0.01) and correlated positively with visceral fat mass independent of age ( r =0.78, P <0.05) in the OWW only. Leptin levels were higher ( P <0.01) both at fasting and during the course of the OGTT in obese women from both ethnic groups compared to the lean women. CONCLUSIONS: Glucose oxidation is reduced in obese subjects of both ethnic groups; inter- and intra-ethnic differences were observed in visceral fat mass and FFA production and it is possible that such differences may play a role in the differing prevalences of obesity-related disorders that have been reported in these two populations.

There is a higher prevalence of ischemic heart disease (IHD) in South African white than black women. The objective of this study was to determine biochemical explanations for this prevalence. The study group contained 15 obese black women (OBW) and 14 obese white women (OWW), all premenopausal, who were examined after an overnight fast. Anthropometric measurements and blood concentrations of glucose, non-esterified fatty acids (NEFAs), catecholamines, plasminogen activator inhibitor-1, C-peptide, proinsulin, lipograms, cortisol, growth hormone, and post-heparin lipoprotein lipase activity were measured during an oral glucose tolerance test (OGTT). Body composition was measured using bioelectrical impedance analysis, and subcutaneous and visceral fat mass were assessed with CT-scans. Visceral fat area was higher in OWW (139.7 +/- 10.7 cm(2)) than in OBW (72.3 +/- 3.9 cm(2)) (P < 0.01), as were fasting and 3 h triglyceride concentrations (P < 0.05 for all). OWW also had higher NEFA levels than OBW at 3 and 4 h compared with OBW (P < 0.05 for both). Fasting cortisol (266 +/- 24 vs. 197 +/- 19 nmol/l; P < 0.05) was higher in OWW than in OBW. These data demonstrate that OWW have higher visceral fat mass than OBW, which may lead to a more atherogenic fasting and postprandial lipid profile. The higher cortisol levels of the OWW may promote visceral fat deposition.

Abnormalities observed in intermediary metabolism may be related to the pathogenesis of obesity-related diseases such as type 2 diabetes. Glycerol and lactate production was estimated in the sc adipose tissue of two anatomical regions of 10 lean (LW), 10 obese (OW), and 10 matched diabetic (DW) black urban women. This was done with the sc microdialysis technique and combined with adipose tissue blood flow (ATBF) rates calculated from (133)Xe clearance. Biochemical measurements were made in the postabsorptive and postprandial state. Bioimpedance and computed tomography scans were used to define body composition. DW present with more visceral fat (DW, 138 +/- 5.0; OW, 66.6 +/- 5.0 cm; P < 0.01). This was associated with elevated free testosterone levels (DW, 1.21 +/- 0.1; OW, 0.75 +/- 0.1 nmol/L; P < 0.05). The fasting FFA, glycerol, and lactate levels increased across the three groups (LW < OW < DW). During the oral glucose tolerance test, glucose levels were elevated in DW, with higher insulin levels [0 h: DW, 207 +/- 8.6; OW, 100 +/- 7.2 pmol/L (P < 0.01); 1 h: DW, 410 +/- 15.2; OW, 320 +/- 10.9 pmol/L (P < 0.05)], but with a flat Cpeptide response (1 h: DW, 932 +/- 40; OW, 1764 +/- 40 pmol/L; P < 0.05). Plasma lactate levels increased significantly in LW and OW at 1 h (P < 0.001), but remained lower in LW vs. OW for all time points. ATBF was highest in LW [abdominal, 0 h: DW, 4.5 +/- 0.2; OW, 1.7 mL/100 g.min (P < 0.01); femoral, 0 h: DW, 3.4 +/- 0.2; OW, 1.8 +/- 0.3 mL/100 g.min (P < 0.01)]. ATBF did not increase in DW during the oral glucose tolerance test. Glycerol release (GR) was used to assess the lipolytic rate and was highest in LW in the abdominal area [0 h: LW, 1.7 +/- 0.2; OW, 1.1 +/- 0.2 micromol/kg.min (P < 0.05); DW, 0.78 +/- 0.05 micromol/kg.min (P < 0.05 vs. OW)]. By contrast, GR was higher in the femoral area of OW (0 h: OW, 1.6 +/- 0.2; LW, 1.15 +/- 0.1 micromol/kg.min; P < 0.05). Regional differences were observed for GR in both OW and DW (femoral > abdominal). Lactate release (LR) was low in DW [abdominal, 0 h: DW, 3.5 +/- 0.4; OW, 7.8 +/- 1.0 micromol/kg.min (P < 0.001); femoral, 0 h: DW, 3.1 +/- 0.3; OW, 9.0 +/- 0.9 micromol/kg.min (P < 0.001)]. LR was appropriately low for body fat mass in LW, with a brisk increase between 0 and 1.5 h. A negative correlation exists between GR (abdominal area) and insulin levels in the postabsorptive state (P < 0.0001). In conclusion, 1) the fasting lipolytic rate is associated with insulin levels; 2) OW and DW have more adipose tissue insulin resistance than LW; 3) OW and DW have a brisker lipolysis in the femoral area; and 4) in DW, higher visceral mass is associated with elevated free testosterone and FFA concentrations. Obesity in the black population is therefore characterized by a marked degree of adipose tissue lipolysis. This degree of resistance together with increasing body fat mass may predispose the obese women to developing type 2 diabetes. Once this disease is established, the onset of adipose tissue vascular insulin resistance will sustain ongoing insulin resistance, even in the presence of relative insulinopenia.

Objective: The goal of this study was to quantify differences in lipid metabolism and insulin sensitivity in black and white subjects to explain ethnic clinicopathological differences in type 2 diabetes.Research Methods and Procedures: The in vitro lipolytic activity of adipocytes isolated from obese black and white women was measured in the presence of insulin and isoproterenol. Insulin resistance was assessed in vivo using the euglycemic hyperinsulinemic clamp technique.Results: Easting plasma levels of insulin and nonesterified fatty acid (NEFA) in black and white women were 67 +/- 5 pM vs. 152 +/- 20 pM (p < 0.01) and 863 +/- 93 mu M vs. 412 +/- 34 mu M (p < 0.01), respectively. Euglycemic hyperinsulinemic clamp studies showed that obese black subjects were more insulin-resistant than their white counterparts (glucose infusion rates: 1.3 +/- 0.2 vs. 2.2 +/- 0.3 mg/kg per min; p < 0.05). Isolated adipocytes from white women were more responsive to insulin than those from black women with 0.7 nM insulin causing a 55 +/- 4% inhibition of isoproterenol-stimulated lipolysis compared with 27 +/- 10% in black women (p < 0.05).Discussion: The low responsiveness of adipocyte lipolytic activity to insulin in black women in the presence of a relative insulinopenia may account for the high plasma NEFA levels seen in these women, which may, in turn, account for their higher in vivo insulin resistance. High NEFA levels may also contribute to the low insulin secretory activity observed in the obese black females. These data suggest that the pathogenesis of insulin resistance and type 2 diabetes within the black obese community is strongly influenced by their adipocyte metabolism.

Aims/hypothesis. This study aimed to assess the effects of fetal and childhood growth on beta-cell activity and insulin sensitivity in 7-year-old children. Methods. Insulin, des-31,32 proinsulin, proinsulin, non-esterified fatty acids and glucose concentrations were measured in oral glucose tolerance tests in 152 South African children for whom longitudinal weight data was available. Results. Children with low weights at birth and 7 years (low–low) had relatively low beta-cell activity whereas children with low birth weight and high weight at 7 years (low–high) had relatively high beta-cell activity. The low–low group had higher 30-min glucose concentrations than children with high birth weights. When each insulin-related peptide was expressed as a percentage of all these peptides the low–low children had the highest percentage of insulin but the lowest of the prohormones. The low–high children had the lowest percentage of insulin but the highest of the prohormones. Non-esterified fatty acid concentrations were lowest and their suppression post-glucose load highest in the low–high group. Conclusion/interpretation. Poor fetal and neonatal growth give rise to low beta-cell numbers compensated for by increased efficiency of proinsulin processing to insulin. Poor fetal followed by higher postnatal growth results in low beta-cell numbers and reduced whole-body glucose uptake which leads to reduced efficiency in the processing of proinsulin. Growth in utero and postnatally therefore have profound effects on beta-cell activity and insulin sensitivity with poor fetal coupled with high postnatal growth being detrimental to these processes but not detrimental to the suppression of lipolysis. [Diabetologia (2000) 43: 978–985]

OBJECTIVE: The rate of glucose disposal was determined in 10 black and 10 white obese nondiabetic urban women from South Africa to assess insulin resistance. DESIGN AND METHODS: Euglycemic hyperinsulinemic clamp and body composition analysis. RESULTS: Age, body mass index (BMI), anthropometric measurements and body composition were similar in both groups of women. A five-level computed tomography (CT) scan showed a similar mean subcutaneous fat mass in both groups of women (black obese women 555±9.0 vs white obese women 532±6.0 cm 2 ), but less visceral fat in black obese women (90±3.0 vs 121±3.1 cm 2 ; P <0.05). Black obese women had higher fasting free fatty acid (997±69 vs 678±93 μmol/l; P <0.05) and lactate concentrations (1462±94 vs 1038±39 μmol/l; P <0.05), but lower fasting insulin levels (87±12 vs 155±9 pmol/l; P <0.001). Black obese women also had a more favorable HDL: total cholesterol ratio (30.5% vs 23.0%; P <0.04). The mean glucose disposal rate ( M ) and disposal expressed as glucose sensitivity index ( M/I ) were reduced in the black obese women vs white obese women ( M : 7.1±0.8 vs 13.7±1.0 mmol/kg·min −1 ×100; P <0.01, and M/I : 0.12±0.01 vs 0.24±0.02 mmol/kg·min −1 /pmol/l×1000; P <0.01). Only black obese women showed a significant decrease in C-peptide levels during the clamp (2.9±0.22 vs 1.2±0.12 nmol/l; P <0.001). During the euglycemic period, the black obese women had higher lactate levels at all time points, but only the white obese women had increased lactate levels (918±66 to 1300±53 μmol/l; P <0.05). CONCLUSION: Black obese women demonstrate a higher degree of insulin resistance, despite less visceral fat and a higher HDL: total-cholesterol ratio. In addition, endogenous β-cell secretory function in black obese women appears to be more sensitive to the suppressive effect of exogenous insulin administration. The significant increase in lactate levels in white obese women confirms that they are more insulin sensitive.

To measure interstitial glycerol and lactate production from the sc adipose tissue of two regions in nine black and nine white lean men, sc microdialysis was performed in combination with adipose tissue blood flow rates measured with 133Xe clearance. In the postabsorptive state, the plasma glucose and insulin levels of the black men and white men were similar. The black men had higher plasma free fatty acids (825+/-97 vs. 439+/-58 micromol/L; P < 0.005), glycerol (99.5+/-5.1 vs. 54.1+/-3.3 micromol/L; P < 0.0001), and lactate (1056+/-95 vs. 729+/-45 micromol/L; P < 0.01). Interstitial glycerol concentrations in the black and white men were 227 vs. 163 micromol/L (P < 0.01) and 230 vs. 162 micromol/L (P < 0.05) in the abdominal and femoral regions. The adipose tissue blood flow rate was higher in the black men in the abdominal (7.9+/-0.9 vs. 3.1+/-0.5 mL/100 g x min; P < 0.01) and femoral area (5.2+/-0.6 vs. 2.8+/-0.3; P < 0.01). Interstitial lactate concentrations in black and white men were 1976 vs. 1364 micromol/L (P < 0.004) and 1953 vs. 1321 micromol/L (P < 0.004) in the abdominal and femoral regions, respectively. Glycerol release was higher in black men vs. white men for abdominal (0.21+/-0.02 vs. 0.14+/-0.02 micromol/100 g x min; P < 0.02) and femoral (0.22+/-0.02 vs. 0.15+/-0.01; P < 0.05) areas. Postprandially, black men had higher plasma glucose levels [1 h, 9.6+/-0.4 vs. 8.2+/-0.5 mmol/L (P < 0.05); 2 h, 8.9+/-0.4 vs. 7.2+/-0.4 mmol/L (P < 0.01)], but lower plasma insulin levels [1 h, 173+/-13 vs. 264+/-48 pmol/L (P < 0.05); 2 h, 136+/-20 vs. 209+/-34 pmol/L (P < 0.05)]. Plasma free fatty acid, lactate, and glycerol levels remained higher in the black men. After 1 h, lactate release was higher in the black men vs. that in the white men for abdominal (20.5+/-1.6 vs. 14.7+/-2.5 micromol/100 g x min;P < 0.05) and femoral (15.6+/-1.1 vs. 12.1+/-1.8; P < 0.03) areas. We conclude that the black men, who are relatively insulinopenic postprandially, have a brisker lipolysis and also release more lactate from sc fat tissue than white men. These differences in adipose tissue metabolism may be related to differences in the lipid profiles and glucose metabolism previously documented in these ethnic groups.

OBJECTIVE: To investigate the relationship between leptin concentrations, various metabolic indices and body composition in six different groups. DESIGN AND MEASUREMENTS: Anthropometric measurements, fasting plasma glucose, serum insulin, C-peptide, FFA and leptin levels were performed. In the obese and diabetic subjects, body composition was analysed with bio-impedance equipment and as a 5 level CT scan. SUBJECTS: Five lipoatrophic diabetes mellitus (LDM) patients, five normal subjects (N), nine white and nine black obese women (WW, BW), and nine white and nine black diabetic women (DWW, DBW) were investigated after an overnight fast. RESULTS: In both ethnic groups there was a positive correlation between leptin and BMI (black group: r =0.8; P <0.0001, white group: r =0.7, P <0.002) and leptin and SC fat mass (black group: r =0.6; P <0.005, white group: r =0.6; P <0.004). CONCLUSIONS: Across the groups, there were positive linear correlations between leptin concentrations, BMI, SC fat mass and FFA levels. Leptin and FFA concentrations are higher and insulin levels lower in both groups of black women compared to the two groups of white women, despite a similar BMI and body fat mass. In the DBW the large increase in visceral fat mass may be indicative of a more complex relationship between compensatory insulin resistance, elevated FFA levels and leptin secretion.

A number of studies have shown that glucose tolerance falls with decreasing birth weight and that people with low birth weight and high body mass index (BMI) as adults are those at greatest risk of developing Type II (non-insulin-dependent) diabetes mellitus. No such studies have been carried out in African populations. Therefore we investigated the relation between glucose tolerance and birth weight in a group of 7-year-old black South Africans for whom longitudinal anthropometric data were available. Oral glucose tolerance tests (OGTTs) were carried out on 152 subjects and inverse correlations were found between birth weight and the total amount of insulin secreted during the first 30 min (r = -0.19, p = 0.04) and last 90 min (r = -0.19, p = 0.04) of the oral glucose tolerance test and also between birth weight and the 30 min glucose concentrations (r = -0.20, p = 0.02). Children born with low birth weights but who had high weights at 7 years had higher insulin concentrations and indices of obesity compared with those with low birth weights and low weights at 7 years. There were also positive correlations between weight velocity and BMI (r = 0.24, p = 0.02) and weight velocity and insulin resistance (r = 0.18, p = 0.04) as measured using homeostasis model assessment (HOMA). Thus, low birth weight in conjunction with rapid childhood gains in weight especially as subcutaneous fat, produces poor glucose tolerance in 7-year-old children and can make them susceptible to the development of Type II diabetes later in life.